1. An amphiphilic poly aggregation-induced emission polymer, wherein the amphiphilic poly aggregation-induced emission polymer has a structural formula shown in any one of the following two formulas:

1) structural formula I:

2) structural formula II:

wherein n is 7-659; r₁Selected from the group consisting of oxygen, -OCH₂CH₂Any one of O < - >; r₂Selected from any one of hydrogen and hydroxyl.

2. The method of claim 1 for preparing an amphiphilic aggregation-induced emission polymer, comprising the steps of:

(1) synthesis of hyaluronic acid derivative 1: dissolving hyaluronic acid in solvent, adding catalyst, catalyzing at 10-70 deg.C for 0.5-24 hr, adding saturated monohydric aliphatic alcohol, stirring at 10-70 deg.C for 0.5-72 hr, dialyzing, purifying, and lyophilizing to obtain hyaluronic acid derivative 1;

(2) synthesis of hyaluronic acid derivative 2: dissolving the hyaluronic acid derivative 1 in a solvent, adding hydrazine hydrate, reacting at 10-70 ℃ for 2-48h, dialyzing, purifying and freeze-drying a reaction product to obtain a hyaluronic acid derivative 2;

(3) synthesis of tetraphenylethylene derivatives: dissolving 4-formyl benzoic acid in a solvent, adding a catalyst, catalyzing for 0.5-48h at 10-70 ℃, then adding substituent modified tetraphenylethylene, stirring and reacting for 0.5-96h at 10-70 ℃, washing a reaction product by dichloromethane after dialysis and purification, and freeze-drying to obtain a tetraphenylethylene derivative;

(4) synthesis of amphiphilic aggregation-induced emission polymer: respectively dissolving hyaluronic acid derivative 2 and tetraphenylethylene derivative in a solvent, mixing the solution containing hyaluronic acid derivative 2 and the solution containing tetraphenylethylene derivative at 10-70 ℃, reacting for 2-48h, dialyzing, purifying and freeze-drying the reaction product to obtain the amphiphilic aggregation-induced emission polymer.

3. The method of claim 2, wherein in step (1), the hyaluronic acid has an average molecular weight of 5306-499522 Da.

4. The method of claim 2, wherein the solvent is selected from the group consisting of water, formamide, dimethylsulfoxide, N-dimethylformamide, tetrahydrofuran, and N, N-dimethylacetamide.

5. The method of claim 2 wherein the catalyst is selected from the group consisting of EDC HCl, NHS, DMAP, DCC.

6. The method for preparing an amphiphilic aggregation-inducing luminescent polymer according to claim 2, wherein the molar ratio of the carboxyl groups in the hyaluronic acid to the hydroxyl groups in the saturated monohydric aliphatic alcohol in step (1) is (1:10) - (10: 1); the molar ratio of carboxyl in hyaluronic acid to total dosage of EDC & HCl and NHS is (1:5) - (5: 1); EDC. HCl and NHS are used in a molar ratio of (1:10) - (10: 1).

7. The method for preparing an amphiphilic aggregation-inducing luminescent polymer according to claim 2, wherein the molar ratio of ester groups to hydrazine hydrate in the hyaluronic acid derivative 1 in the step (2) is (1:10) - (10: 1).

8. The method for preparing an amphiphilic poly-aggregation-inducing luminescent polymer according to claim 2, wherein the molar ratio of the carboxyl groups in the 4-formylbenzoic acid to the hydroxyl groups in the substituent-modified tetraphenylethylene in the step (3) is (1:15) to (15: 1); the molar ratio of carboxyl groups in the 4-formylbenzoic acid to the total amount of EDC. HCl and NHS is (1:5) - (5: 1); EDC. HCl and NHS are used in a molar ratio of (1:10) - (10: 1).

9. The method for preparing an amphiphilic aggregation-inducing luminescent polymer according to claim 2, wherein the molar ratio of the hydrazide groups in the hyaluronic acid derivative 2 to the aldehyde groups in the tetraphenylethylene derivative in step (4) is (1:10) to (10: 1).

10. The amphiphilic poly-aggregation-inducing luminescent polymer of any one of claims 1-9, for use in encapsulating anti-tumor drugs or labeling tumor cells for fluorescence imaging.

Background

Cancer is a serious disease threatening human health, and the treatment and diagnosis thereof has been troubling many biologists, chemists and medical scientists. The current methods for treating cancer mainly comprise surgery, radiotherapy, chemotherapy, hormone therapy, immunotherapy and the like, wherein chemotherapy is one of the most important cancer treatment strategies in clinic. However, conventional chemotherapy can only deliver drugs to cancer cells, and cannot monitor the distribution of drug delivery vehicles in cancer cells.

In recent years, aggregation-induced emission materials have increasingly gained wide applications in the biomedical field as a new generation of fluorescent materials. In contrast to conventional aggregation-induced quenching materials, aggregation-induced emission materials have unique characteristics due to their high emission efficiency in the aggregated state, low background noise and large stokes shift, and thus have been widely used for biosensing and bioimaging. Because the intramolecular movement is limited, the aggregation-induced luminescent material such as tetraphenylethylene has high luminescent efficiency in an aggregation state, and can be combined with other high molecular materials to obtain a novel amphiphilic aggregation-induced luminescent polymer with a specific fluorescence effect.

Hyaluronic acid is used as a naturally-occurring linear macromolecular polysaccharide, is widely distributed on each part of a human body, and has the advantages of good biocompatibility, biodegradability and the like; the hyaluronic acid structure contains active groups such as carboxyl, hydroxyl and the like, and corresponding derivatives can be easily obtained through chemical modification; in addition, the hyaluronic acid binding receptor CD44 is over-expressed in many tumor cells, and can be used for the targeted therapy of tumors. Therefore, chemical modification of hyaluronic acid as a hydrophilic moiety of an amphiphilic polymer is a good choice.

For example, patent publication No. CN104312577B discloses a fluorescent compound composed of 1, 2-bis {4- [4- (N, N, N-trimethylammonium) butoxy ] phenyl } -1, 2-diphenylethylene dibromide aqueous solution and hyaluronic acid aqueous solution, and a preparation method and application thereof, the compound overcomes the disadvantage of quenching of aggregated fluorescence of the traditional fluorescent material, and has the function of targeting cancer cells highly expressed by CD44 receptors; the preparation method is simple, the conditions are mild, the fluorescence emission is realized by one step method, and substances with targeting effect on cancer cells are loaded. The compound also relates to the application of targeting and recognizing tumor cells, but the scheme has the defect that the tumor part cannot be released sensitively.

In order to further improve the antitumor efficacy, precise release of antitumor drugs is one of the effective strategies, which makes the research of various tumor environment-responsive amphiphilic polymers urgent.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an amphiphilic poly-aggregation-induced luminescent polymer and a preparation method and application thereof.

The method is realized by the following technical scheme:

an amphiphilic poly aggregation-inducing luminescent polymer, the amphiphilic poly aggregation-inducing luminescent polymer having a structural formula of either of:

1) structural formula I:

2) structural formula II:

wherein n is 7-659; r₁Selected from the group consisting of oxygen, -OCH₂CH₂Any one of O < - >; r₂Selected from any one of hydrogen and hydroxyl.

A preparation method of an amphiphilic poly-aggregation-induced luminescent polymer comprises the following steps:

(1) synthesis of hyaluronic acid derivative 1: dissolving hyaluronic acid in solvent, adding catalyst, catalyzing at 10-70 deg.C for 0.5-24 hr, adding saturated monohydric aliphatic alcohol, stirring at 10-70 deg.C for 0.5-72 hr, dialyzing, purifying, and lyophilizing to obtain hyaluronic acid derivative 1;

(2) synthesis of hyaluronic acid derivative 2: dissolving the hyaluronic acid derivative 1 in a solvent, adding hydrazine hydrate, reacting at 10-70 ℃ for 2-48h, dialyzing, purifying and freeze-drying a reaction product to obtain a hyaluronic acid derivative 2;

(3) synthesis of tetraphenylethylene derivatives: dissolving 4-formyl benzoic acid in a solvent, adding a catalyst, catalyzing for 0.5-48h at 10-70 ℃, then adding substituent modified tetraphenylethylene, stirring and reacting for 0.5-96h at 10-70 ℃, washing a reaction product by dichloromethane after dialysis and purification, and freeze-drying to obtain a tetraphenylethylene derivative;

(4) synthesis of amphiphilic aggregation-induced emission polymer: respectively dissolving hyaluronic acid derivative 2 and tetraphenylethylene derivative in a solvent, mixing the solution containing hyaluronic acid derivative 2 and the solution containing tetraphenylethylene derivative at 10-70 ℃, reacting for 2-48h, dialyzing, purifying and freeze-drying the reaction product to obtain the amphiphilic aggregation-induced emission polymer.

In the step (1), the average molecular weight of the hyaluronic acid is 5306-499522 Da.

The solvent is selected from one or more of water, formamide, dimethyl sulfoxide, N-dimethylformamide, tetrahydrofuran and N, N-dimethylacetamide.

The catalyst is selected from any one or more of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl), N-hydroxysuccinimide (NHS), 4-Dimethylaminopyridine (DMAP) and Dicyclohexylcarbodiimide (DCC).

Preferably, the catalyst is a composition of EDC & HCl and NHS in a molar ratio of (1:10) - (10: 1).

Further preferably, the catalyst is a composition of EDC & HCl and NHS in a molar ratio of (1:5) - (5: 1).

In the step (1), the saturated monohydric aliphatic alcohol is any one or more of methanol, ethanol, n-propanol and n-butanol.

The molar ratio of carboxyl in hyaluronic acid to hydroxyl in saturated monohydric aliphatic alcohol in the step (1) is (1:10) - (10: 1); the molar ratio of carboxyl in hyaluronic acid to total dosage of EDC & HCl and NHS is (1:5) - (5: 1); EDC. HCl and NHS are used in a molar ratio of (1:10) - (10: 1).

The hyaluronic acid derivative 1 has a general formula:

wherein R is any one of methyl, ethyl, n-propyl and n-butyl.

The molar ratio of the ester group to the hydrazine hydrate in the hyaluronic acid derivative 1 in the step (2) is (1:10) - (10: 1).

The structural formula of the hyaluronic acid derivative 2 is as follows:

in the step (3), the substituent modified tetraphenylethylene is Any one or more of them.

The molar ratio of carboxyl in the 4-formylbenzoic acid to hydroxyl in the substituent-modified tetraphenylethylene in the step (3) is (1:15) - (15: 1); the molar ratio of carboxyl groups in the 4-formylbenzoic acid to the total amount of EDC. HCl and NHS is (1:5) - (5: 1); EDC. HCl and NHS are used in a molar ratio of (1:10) - (10: 1).

In the step (4), the molar ratio of the hydrazide groups in the hyaluronic acid derivative 2 to the aldehyde groups in the tetraphenylethylene derivative is (1:10) - (10: 1).

The amphiphilic poly aggregation-induced emission polymer is used as a drug carrier.

The amphiphilic poly-aggregation-induced emission polymer is used for encapsulating anti-tumor drugs and is prepared into an anti-tumor drug preparation.

The antitumor drug is one or more of docetaxel, paclitaxel, adriamycin and camptothecin.

The amphiphilic poly aggregation-induced emission polymer antitumor pharmaceutical preparation is prepared by the following steps: dissolving an anti-tumor drug in an organic solvent to prepare an anti-tumor drug solution; then in the process of dispersing the amphiphilic poly aggregation-induced emission polymer by ultrasound, the anti-tumor drug solution is dripped into the amphiphilic poly aggregation-induced emission polymer solution, after the dripping is finished, the ultrasound dispersion is continued for 3min, and the preparation is prepared after the dialysis treatment.

The amphiphilic poly-aggregation induced emission polymer is used for marking tumor cell fluorescence imaging and monitoring the distribution condition of an anti-tumor drug carrier in tumor cells in real time.

The technical principle of the invention is as follows:

the phenylglyoxaline bond is an acid-sensitive chemical bond, has the characteristics of stable chemical bond under normal physiological conditions and chemical bond breakage under tumor tissue acidic conditions, and can realize the accurate release of the antitumor drug by introducing the phenylglyoxaline bond into the amphiphilic polymer. The invention grafts the tetraphenyl ethylene derivative on the hyaluronic acid skeleton through the benzoyl imine bond to prepare the aggregation-induced emission polymer with amphipathy, and the polymer can target tumor parts and break under the condition of low pH value in tumor tissues, thereby releasing the encapsulated anti-tumor drug and realizing accurate and targeted release of the anti-tumor drug at the tumor parts. Meanwhile, the amphiphilic poly-aggregation induced luminescent polymer has high luminescent efficiency in an aggregation state, so that the fluorescent imaging can be carried out on tumor cells, and the distribution condition of the antitumor drug carrier in the tumor cells can be monitored in real time.

Has the advantages that:

the amphiphilic aggregation-induced emission polymer has good aggregation-induced emission characteristics, can be used as a drug carrier to entrap various anti-tumor drugs, and has good biocompatibility; meanwhile, the amphiphilic polymeric aggregation-induced emission polymer has good targeting and pH-sensitive drug release characteristics, realizes the accurate release of the antitumor drug in tumor cells, and can monitor the distribution condition of the drug carrier in the tumor cells in real time.

(1) The invention takes natural macromolecular hyaluronic acid as a basic raw material, and is safe and nontoxic. The good biocompatibility and biodegradability of the hyaluronic acid are widely used in the medical field, and meanwhile, the hyaluronic acid binding receptor CD44 is over-expressed in a plurality of tumor cells, so that the hyaluronic acid binding receptor CD44 can be used for the targeted therapy of tumors.

(2) The amphiphilic poly-aggregation induced emission polymer is prepared by bridging a tetraphenylethylene derivative and a hyaluronic acid derivative through a benzimide bond based on a low-pH microenvironment in a tumor tissue. The benzoylimine bond in the polymer can be broken in a special micro-environment of the tumor, and the accurate release of the antitumor drug at the tumor part can be realized.

(3) The preparation method is simple, the raw materials are rich and easily available, and the tetraphenylethylene derivative is connected with the hyaluronic acid derivative in a chemical bonding mode, so that on one hand, the dispersibility and the biocompatibility of the tetraphenylethylene derivative are effectively improved; on the other hand, the high aggregation state fluorescence characteristic of the amphiphilic aggregation-induced emission polymer is utilized, so that the uptake condition and the distribution condition of the carrier in cancer cells can be monitored in real time.

Drawings

FIG. 1: example 1 ir spectra of amphiphilic aggregation-induced emission polymer 1.

Detailed Description

The following is a detailed description of the embodiments of the present invention, but the present invention is not limited to these embodiments, and any modifications or substitutions in the basic spirit of the embodiments are included in the scope of the present invention as claimed in the claims.

Example 1

A synthetic method of an amphiphilic poly-aggregation induced luminescent polymer 1 comprises the following steps:

(1) synthesis of hyaluronic acid derivative 1

Weighing 200mg of hyaluronic acid and a catalyst, wherein the catalyst consists of 293mg of EDC & HCl and 176mg of NHS; dissolving hyaluronic acid in a solvent and adding a catalyst in a round-bottom flask, stirring and activating at 30 ℃ for 4 hours, then adding 0.09mL of saturated monohydric aliphatic alcohol, stirring and reacting at 30 ℃ for 24 hours, dialyzing, purifying and freeze-drying the reaction product to obtain a hyaluronic acid derivative 1;

the structural formula of the hyaluronic acid derivative 1 is as follows:

when hyaluronic acid derivative 1 was synthesized, the average molecular weight of the hyaluronic acid was 9000 Da; the solvent is distilled water; the saturated monohydric aliphatic alcohol is ethanol;

(2) synthesis of hyaluronic acid derivative 2

Weighing 100mg of hyaluronic acid derivative 1, dissolving in distilled water, adding 0.04mL of hydrazine hydrate, and stirring at 40 ℃ for reaction for 12 hours; after the reaction is finished, dialyzing, purifying and freeze-drying the reaction product to obtain a hyaluronic acid derivative 2;

the structural formula of the hyaluronic acid derivative 2 is as follows:

(3) synthesis of tetraphenylethylene derivative 1

Weighing 20mg of 4-formylbenzoic acid and a catalyst, wherein the catalyst consists of 76mg of EDC & HCl and 46mg of NHS; dissolving 4-formylbenzoic acid in a solvent in a round-bottom flask, adding a catalyst, stirring and activating at 40 ℃ for 24 hours, then adding 30mg of substituent modified tetraphenylethylene, and continuing to stir and react at 40 ℃ for 48 hours; dialyzing and purifying the reaction product, washing by dichloromethane, and freeze-drying to obtain a tetraphenylethylene derivative 1;

in the synthesis of the tetraphenylethylene derivative 1, the solvent is N, N-dimethylformamide; the substituent modified tetraphenylethylene is

The structural formula of the tetraphenylethylene derivative 1 is as follows:

(4) synthesis of amphiphilic aggregation-induced emission polymer 1

Weighing 100mg of hyaluronic acid derivative 2, dissolving in water, then weighing 127mg of tetraphenylethylene derivative, dissolving in dimethyl sulfoxide, mixing the two solutions at 40 ℃ for reaction for 12 hours, and dialyzing, purifying and freeze-drying the reaction product to obtain an amphiphilic aggregation-induced luminescent polymer 1;

the structural formula of the amphiphilic aggregation-induced emission polymer 1 is as follows:

example 2

The procedure was carried out in substantially the same manner as in example 1 except that the production methods of steps (3) and (4) were as follows:

(3) synthesis of tetraphenylethylene derivative 2

20mg of 4-formylbenzoic acid, a catalyst consisting of 76mg of EDC. HCl and 46mg of NHS, were weighed out. In a round-bottom flask, 4-formylbenzoic acid is dissolved in a solvent and added with a catalyst, stirred and activated at 40 ℃ for 36h, then 23mg of substituent modified tetraphenylethylene is added, and the reaction is stirred at 40 ℃ for 72 h. Dialyzing and purifying the reaction product, washing by dichloromethane, and freeze-drying to obtain a tetraphenylethylene derivative 2;

when the tetraphenylethylene derivative 2 is synthesized, the adopted solvent is N, N-dimethylformamide; the substituent modified tetraphenylethylene is

The structural formula of the tetraphenylethylene derivative 2 is as follows:

(4) synthesis of amphiphilic poly-aggregation-induced luminescent polymer 2

Weighing 100mg of hyaluronic acid derivative 2, dissolving in water, then weighing 104mg of tetraphenylethylene derivative, dissolving in dimethyl sulfoxide, mixing the two solutions at 40 ℃ for reaction for 12 hours, and dialyzing, purifying and freeze-drying the reaction product to obtain an amphiphilic aggregation-induced luminescent polymer 2;

the structural formula of the amphiphilic aggregation-induced emission polymer 2 is as follows:

example 3

The procedure was carried out in substantially the same manner as in example 1 except that the production methods of steps (3) and (4) were as follows:

(3) synthesis of tetraphenylethylene derivative 3

Weighing 20mg of 4-formylbenzoic acid and a catalyst, wherein the catalyst consists of 76mg of EDC & HCl and 46mg of NHS; dissolving 4-formylbenzoic acid in a solvent in a round-bottom flask, adding a catalyst, stirring and activating at 40 ℃ for 36h, then adding 26mg of substituent modified tetraphenylethylene, and continuing to stir and react at 40 ℃ for 72 h; dialyzing and purifying the reaction product, washing by dichloromethane, and freeze-drying to obtain a tetraphenylethylene derivative 3;

when the tetraphenylethylene derivative 3 is synthesized, the adopted solvent is N, N-dimethylformamide; the substituent modified tetraphenylethylene is

The structural formula of the tetraphenylethylene diffractant 3 is as follows:

(4) synthesis of amphiphilic poly-aggregation-induced luminescent polymer 3

Weighing 100mg of hyaluronic acid derivative 2, dissolving in water, then weighing 114mg of tetraphenylethylene derivative, dissolving in dimethyl sulfoxide, mixing the two solutions at 40 ℃ for reaction for 12 hours, and dialyzing, purifying and freeze-drying the reaction product to obtain an amphiphilic aggregation-induced luminescent polymer 3;

the structural formula of the amphiphilic aggregation-induced emission polymer 3 is as follows:

comparative example 1

Synthesis of amphiphilic poly-aggregation-induced luminescent polymer 4

Weighing 200mg of hyaluronic acid and a catalyst, wherein the catalyst consists of 293mg of EDC & HCl and 176mg of NHS; dissolving hyaluronic acid in a solvent in a round-bottom flask, adding a catalyst, stirring and activating at 30 ℃ for 4 hours, then adding 231mg of substituent-modified tetraphenylethylene, stirring and reacting at 30 ℃ for 48 hours, dialyzing, purifying and freeze-drying a reaction product to obtain an amphiphilic aggregation-induced emission polymer 4;

the hyaluronic acid has an average molecular weight of 9000Da when an amphiphilic aggregation-inducing luminescent polymer is synthesized; the solvent is formamide; the substituent modified tetraphenylethylene is

The structural formula of the amphiphilic aggregation-induced emission polymer 4 is as follows:

test example 1: aggregation induced emission performance test

In order to research the aggregation-induced emission performance of the amphiphilic aggregation-induced emission polymer, the research is carried out by gradually changing the water content in a mixed solvent system; firstly, preparing a mixed solvent system of formamide and water (wherein the water content is increased from 0 to 100 percent in sequence), and then respectively adding the amphiphilic aggregation-inducing luminescent polymer prepared by any one of the methods of examples 1-3 and comparative example 1 into the mixed solvent system, wherein the concentration of the amphiphilic aggregation-inducing luminescent polymer is fixed to be 0.25 mg/mL; measuring the fluorescence intensity by fluorescence spectrum (excitation wavelength is 345nm, emission wavelength is 470 nm);

the results show that the amphiphilic aggregation-induced emission polymers prepared by any one of the methods of examples 1 to 3 and comparative example 1 all have the following rule: when the water content in the mixed solvent system is lower than 70%, the amphiphilic aggregation-induced emission polymer has no obvious fluorescence, the amphiphilic aggregation-induced emission polymer is in a dispersed state in the mixed solvent system, when the water content exceeds 70%, the fluorescence intensity is remarkably increased, when the water content reaches 100%, the fluorescence intensity is highest, and the amphiphilic aggregation-induced emission polymer is in an aggregated state; aggregation-induced emission performance tests show that the amphiphilic polymeric aggregation-induced emission polymer has good aggregation-induced emission performance.

Test example 2: test for pH sensitivity

In order to determine the pH sensitivity of the amphiphilic aggregation-induced emission polymer solution, the amphiphilic aggregation-induced emission polymer prepared by any one of the methods of examples 1 to 3 and comparative example 1 was selected for the experiment, specifically as follows: adding 2mg of amphiphilic aggregation-induced emission polymer into a certain amount of phosphate buffer solution with different pH values (5.0 and 7.4) to prepare an amphiphilic aggregation-induced emission polymer solution; then, oscillating the amphiphilic poly aggregation-induced emission polymer solution in a water bath constant temperature oscillator at 37 ℃ for 12 h; measuring a change in particle diameter of the polymer solution by a dynamic light scattering method (DLS); in addition, the amphiphilic poly aggregation-induced emission polymers are respectively added into mixed solvent systems with different proportions (pH values of 5.0 and 7.4 are also kept), and after oscillation is carried out for 12 hours at 37 ℃ in a water bath constant temperature oscillator, the fluorescence intensity of different solutions is measured;

the results show that the amphiphilic aggregation-induced emission polymers prepared in examples 1 to 3 all have the following rule: after 12h of shaking in phosphate buffer solution with pH 7.4, no significant change in the particle size of the amphiphilic aggregation-inducing luminescent polymer solution was observed. However, when the pH is lowered to 5.0, the particle size of the amphiphilic aggregation-inducing luminescent polymer solution increases significantly and the size distribution is not uniform. In addition, when the fluorescence intensity of the amphiphilic aggregation-inducing luminescent polymer solution was measured, it was found that the phosphate buffer solution at pH 7.4 of the amphiphilic aggregation-inducing luminescent polymer prepared in examples 1 to 3 was selected to have a lower fluorescence intensity than the phosphate buffer solution at pH 5.0 in the mixed solvent system of the same ratio. In contrast, comparative example 1 showed no significant change in particle size and fluorescence intensity in phosphate buffered saline at different pH values. The above test results show that the amphiphilic aggregation-inducing luminescent polymers having a benzimide bond prepared in examples 1 to 3 have pH sensitivity, whereas the amphiphilic aggregation-inducing luminescent polymer having no benzimide bond prepared in comparative example 1 has no pH sensitivity.

Test example 3: hemolysis test

In order to evaluate the biocompatibility and safety of amphiphilic aggregation-inducing luminescent polymers, in vitro hemolysis tests were performed. Fresh 4T1 mouse blood was placed in a centrifuge tube modified with heparin sodium, centrifuged at 3000rpm for 5min, the upper serum was decanted, and the lower red blood cells were washed with normal saline for 3 additional times until the supernatant was colorless. Finally, the red blood cells are collected and diluted into a red blood cell suspension with the concentration of 2% by using a 0.9% NaCl solution for standby. The amphiphilic poly-aggregation-induced emission polymers prepared in examples 1 to 3, the amphiphilic poly-aggregation-induced emission polymer prepared in comparative example 1, the tetraphenylene derivatives prepared in examples 1 to 3, and the substituent-modified tetraphenylene in comparative example 1 were weighed out and diluted with physiological saline to a series of solutions of different concentrations, respectively. A series of 2.5mL solutions were placed in a centrifuge tube and 2.5mL red blood cell suspensions were added and mixed well to obtain a final solution with a concentration range of 10-200. mu.g/mL. After incubating all samples at 37 ℃ for 2h, centrifugation was carried out at 3000rpm for 10min, the supernatant was aspirated and the absorbance was measured at 540nm using an ultraviolet spectrophotometer. The same procedure was followed using physiological saline and distilled water as negative and positive controls to determine the hemolysis rate. Hemolysis rate ═ 100% (sample absorbance-negative control absorbance)/(positive control absorbance-negative control absorbance).

TABLE 1 hemolysis rate of erythrocytes in different samples with a final concentration of 200. mu.g/mL

Sample (I)	Haemolysis rate/% of erythrocytes
		Example 1	4.6
Example 2	4.7
		Example 3	4.8
Comparative example 1	4.3
		Tetraphenylethylene derivative 1 obtained in example 1	8.7
Tetraphenylethylene derivative 2 obtained in example 2	9.0
		Tetraphenylethylene derivative 3 obtained in example 3	9.1
Substituent-modified tetraphenylethylene of comparative example 1	8.1

The results show that the hemolytic activity of the amphiphilic aggregation-inducing luminescent polymers prepared in examples 1-3 and comparative example 1 is almost negligible and the hemolytic rate is not more than 5% in the concentration range of 10-200. mu.g/mL, which indicates that the amphiphilic aggregation-inducing luminescent polymers have good hemolytic safety. The hemolysis rates of the tetraphenylethylene derivatives prepared in examples 1-3 and the substituent-modified tetraphenylethylene derivative used in comparative example 1 both exceeded 5% and had a certain hemolysis property, which indicates that the hyaluronic acid derivative effectively improved the biocompatibility of the tetraphenylethylene derivative and the substituent-modified tetraphenylethylene.

Test example 4: cytotoxicity assays

Cytotoxicity of the amphiphilic aggregation-induced emission polymer on MCF-7 cells was evaluated by using an in vitro MTT method. MCF-7 cells were plated at 1.0X 10 per well⁴The individual concentrations were plated on 96-well plates and incubated in an incubator for one day. Next, the original culture medium was replaced with 200. mu.L of DMEM medium containing the amphiphilic aggregation-inducing luminescent polymers prepared in examples 1-3 and comparative example 1 at different concentrations. After an additional 24h of incubation, the medium was removed, then 20. mu.L of MTT solution (5mg/mL) was added and the cells were incubated for 4 h. Finally, the medium was removed and 200. mu.L of DMSO was added to dissolve formazan crystals. The absorbance at 490nm was measured for each well using a microplate reader. Cell viability was calculated according to the following formula:

cell viability (%) — absorbance of cells exposed to the sample/absorbance of untreated cells × 100%.

TABLE 2 cell viability of MCF-7 cells in different examples, comparative example 1, at a final concentration of 50. mu.g/mL

Examples	Cell viability/%
		Example 1	100.3
Example 2	99.9
		Example 3	100.1
Comparative example 1	99.6

The results show that the toxicity of the amphiphilic aggregation-inducing luminescent polymer prepared by any one of the methods of examples 1-3 and comparative example 1 to MCF-7 cells within the concentration of 0.5-50 mug/mL is almost negligible, and the amphiphilic aggregation-inducing luminescent polymer prepared by any one of the methods of examples 1-3 and comparative example 1 has good cell safety.

Test example 5: cancer cell fluorescence imaging and tumor targeting assays

Human normal mammary gland cells (MCF-10A) and human breast cancer cells (MCF-7) are utilized to explore the tumor targeting property of the amphiphilic aggregation-inducing luminescent polymer. First, MCF-10A cells and MCF-7 cells were passaged to six-well plates, respectively, and 3mL of the culture solution was added for culture at 37 ℃ and 5% CO₂Culturing for 24h under the condition of content, removing the culture solution, adding a certain volume of amphiphilic poly-aggregation-induced emission polymer solution and fresh culture solution (ensuring that the total volume is 3mL), and finally ensuring that the concentration of the amphiphilic poly-aggregation-induced emission polymer solution is 50 mu g/mL. Then, respectively culturing the MCF-10A cells and the MCF-7 cells in the incubator for 1,2 and 4 hours; thereafter, the culture solution was removed, the cells were washed three times with phosphate buffer solution and fixed with 4% paraformaldehyde solution, and fluorescence imaging was performed by laser confocal scanning microscopy (CLSM) under excitation at 345 nm.

The results show that the amphiphilic aggregation-induced emission polymers prepared by any one of the methods of examples 1 to 3 and comparative example 1 all have the following rule: after the amphiphilic aggregate induced emission polymer solution is cultured with the MCF-10A cells and the MCF-7 cells for 1 hour, no obvious blue fluorescence is observed in the two cells, and after the MCF-7 cells are cultured for 2 hours, some weak blue fluorescence can be observed in the MCF-10A cells, but the blue fluorescence of the MCF-10A cells is not obvious; after further culturing for 4h, obvious blue fluorescence can be observed by MCF-7 cells, and weak blue fluorescence can be observed by MCF-10A cells. According to the results, the amphiphilic aggregate induced emission polymer has certain tumor targeting characteristics, can be applied to fluorescence imaging of cancer cells, and can monitor the cell uptake condition of the carrier in real time.

Test example 6: in vivo antitumor assay

For in vivo anti-tumor testing, pathogen-free female Balb/c mice were selected and inoculated with 4T1 cells in the right axilla of each mouse to create a mouse tumor model. When the tumor volume grows to 100mm³When the mice are divided into five groups at random, each group of 6 mice is injected with physiological saline, taxol, an amphiphilic poly aggregation induced emission polymer pharmaceutical preparation carrying paclitaxel, an amphiphilic poly aggregation induced emission polymer pharmaceutical preparation carrying doxorubicin and an amphiphilic poly aggregation induced emission polymer pharmaceutical preparation carrying camptothecin respectively. The day on which the mice received the injection of the first dose was set as day 1, and each sample was injected through the tail vein on days 1, 4, 7, and 10, respectively. After 12 days of treatment, on day 13, animals were sacrificed and tumors were excised, photographed and weighed. Tumor inhibition (TIR%) (saline group tumor weight-treatment group tumor weight)/saline group tumor weight × 100%.

TABLE 3 tumor inhibition ratio of mice injected with different formulations

As can be seen from the table, the normal saline group had no antitumor effect, and the tumor inhibition rate was 0%; the commercial taxol prescription preparation shows a certain anti-tumor effect, but the tumor inhibition rate is only 63.5%; in contrast, example 1 loaded with paclitaxel achieved a tumor growth inhibition rate of 73.0%, whereas comparative example 1 loaded with paclitaxel achieved a tumor growth inhibition rate of 68.0%, indicating that the lack of a benzoylimine bond results in a decrease in the pH sensitivity of the drug delivery system, affecting the antitumor effect. The tumor growth inhibition ratio of example 2 loaded with paclitaxel was 71.8%, and the tumor growth inhibition ratio of example 3 loaded with paclitaxel was 70.9%. The examples and comparative examples 1 loaded with doxorubicin and camptothecin had similar trends as the examples and comparative examples 1 loaded with paclitaxel. The test results show that the amphiphilic poly-aggregation induced emission polymer is expected to become a novel drug delivery system for tumor treatment.

It should be noted that the above examples and experimental examples are only for further illustration and understanding of the technical solutions of the present invention, and should not be understood as further limitation of the technical solutions of the present invention, and the invention with insubstantial features and significant improvements made by those skilled in the art still belongs to the protection scope of the present invention.