CN106967167B

CN106967167B - Protein/polypeptide-polymer conjugate with fluorescence emission property and preparation method and application thereof

Info

Publication number: CN106967167B
Application number: CN201710317186.7A
Authority: CN
Inventors: 刘世勇; 刘固寰; 姜琰琰
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2017-05-08
Filing date: 2017-05-08
Publication date: 2020-05-05
Anticipated expiration: 2037-05-08
Also published as: CN106967167A

Abstract

The invention provides a protein/polypeptide-polymer conjugate with fluorescence emission property, which has a structure shown in a formula I. The protein/polypeptide-polymer conjugate bridges a protein/polypeptide and a polymer by a bifunctional fluorescent molecule, the bifunctional fluorescent molecule has no or only weak fluorescence emission capability, and the fluorescent molecule has strong fluorescence emission only after the protein/polypeptide-polymer conjugate is conjugated, so that the conjugation process of the protein/polypeptide-polymer conjugate can be monitored in situ through fluorescence change, and the protein/polypeptide-polymer conjugate can be applied to the transmission of therapeutic polypeptides and anticancer drugs.

Description

Protein/polypeptide-polymer conjugate with fluorescence emission property and preparation method and application thereof

Technical Field

The invention relates to the technical field of organic bridging molecules, in particular to a protein/polypeptide-polymer conjugate with fluorescence emission property and a preparation method and application thereof.

Background

Covalent functionalization of polypeptides, proteins and antibodies with synthetic polymers, drugs and imaging probes form important bioconjugates for clinical applications, with protein-polymer conjugates, antibody-drug conjugates, as typical examples. Protein-polymer conjugates date back to 1970 where Davis, Abuchowski and co-workers reported the conjugation of polyethylene glycol (PEG) to bovine serum albumin. This technology is now known as PEGylation and extends to many polymer types, such as responsive polymers and zwitterionic polymers. The attachment of synthetic polymers to proteins such as PEGylation can provide a number of advantages including enhanced protein solubility and stability, reduced immunogenicity, and increased blood circulation half-life, at least PEGylated proteins are currently being approved by the U.S. Food and Drug Administration (FDA). On the other hand, the binding of Antibody Drug Conjugates (ADCs) to monoclonal antibodies that specifically target the diseased site kills cancer cells. Two FDA-approved ADCs, brentuximabvedodin (trade name), and trastuzumab (trade name, Kadcyla), are currently commercially available, with about 40 ADCs currently in clinical use.

Protein-polymer conjugates are prepared by the methods of growing branches (graft from), grafting branches (graft to), and macromonomer copolymerisation grafting (graft through). The preparation of both antibody-drug conjugates and protein-polymers relies on the selection of appropriate efficient conjugation reactions and linking moieties. For protein/antibody conjugates, it is best to choose so as not to affect protein/drug activity and antibody function. Typically, this can be achieved by modification of specific sites of native proteins (e.g., reduction of disulfide bonds, modification of carbon termini, or oxidation of polysaccharides) by protein/antibody tissue engineering, and specific reactions directly using specific amino acids. Covalent attachment of synthetic functional polymers/drugs to proteins/antibodies mainly utilizes orthogonal click reactions such as the Staudinger reaction, copper-catalyzed azide-alkyne cycloaddition (CuAAC), strain-promoted azide-cycloalkyne cycloaddition (SPAAC), D-a addition, michael addition, and oxime/hydrazone formation from aldehydes and ketones. The introduction of new design principles, such as modular design, gentle synthesis, optical tracking and the ability to integrate multiple functions, further advance the field.

It is noteworthy that even with optimally designed protein-polymer conjugates, significant impairment of protein function and activity is inevitable. One solution is to prepare cleavable bioconjugates that release the native protein in vivo over time. During cellular internalization, the ADCs should be able to efficiently release their active drug load to exhibit cytotoxicity. However, monitoring reveals that the extent of conjugation of protein-polymer conjugates and antibody-drug conjugates and subsequent release of the protein/drug depends largely on traditional ex situ techniques, such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Mass Spectrometry (MS), High Performance Liquid Chromatography (HPLC), and volume exclusion chromatography (SEC). This prevents real-time monitoring of the release process of the polymer/drug conjugate in vitro and at the cellular level.

Disclosure of Invention

In view of the above, the technical problem to be solved by the present invention is to provide a protein/polypeptide-polymer conjugate with fluorescence emission property, and a preparation method and an application thereof, which can monitor the conjugation process in situ by a fluorescence monitoring method, and can be applied to the delivery of therapeutic polypeptides and anticancer drugs.

The invention provides a protein/polypeptide-polymer conjugate with fluorescence emission property, which has a structure shown in a formula I:

wherein POI is a protein or polypeptide; polymer is a Polymer.

The invention provides a protein-polymer conjugate with fluorescence emission property, which has a structure shown in a formula I-a:

wherein, the POI is bovine serum albumin or salmon calcitonin, and the Polymer is polyethylene glycol.

The invention provides a preparation method of the protein-polymer conjugate, which comprises the following steps:

carrying out Michael reaction and click reaction on bovine serum albumin containing sulfydryl, polyethylene glycol with azide end group and a compound B to prepare a protein-polymer conjugate shown in a formula I-a;

wherein POI is bovine serum albumin, and n is 23-445;

or comprises the following steps:

carrying out Michael reaction and click reaction on salmon calcitonin containing sulfhydryl groups, polyethylene glycol with azide end groups and a compound B to prepare a protein-polymer conjugate shown in a formula I-a;

wherein POI is salmon calcitonin; n is 23 to 445.

The invention provides a polypeptide-polymer conjugate with fluorescence emission property, which has a structure shown in a formula I-b:

wherein the POI is a matrix metalloproteinase cleavable polypeptide and the Polymer is polytrimethylene carbonate.

The invention provides a preparation method of the polypeptide-polymer conjugate, which comprises the following steps:

carrying out Michael reaction and click reaction on the polypeptide, the azide-terminated polytrimethylene carbonate and the compound B shown in the formula D through the matrix metalloproteinase cleavable shown in the formula C to prepare a polypeptide-polymer conjugate shown in the formula I-B;

wherein m is 10-55.

Preferably, the conjugation efficiency of the conjugate is monitored in situ by fluorescence emission intensity.

The invention provides a polymersome composed of the polypeptide-polymer conjugate or the polypeptide-polymer conjugate prepared by the preparation method.

Preferably, the particle size of the polymersome is 60-150 nm.

Preferably, the polymersome has matrix metalloenzyme response characteristics.

The invention provides an application of the polymer vesicle as a drug carrier or an application of the polymer vesicle as a fluorescence indicator for monitoring drug release in real time.

Compared with the prior art, the invention provides a protein/polypeptide-polymer conjugate with fluorescence emission property, which has a structure shown in a formula I. The protein/polypeptide-polymer conjugate bridges a protein/polypeptide and a polymer by a bifunctional fluorescent molecule, the bifunctional fluorescent molecule has no or only weak fluorescence emission capability, and the fluorescent molecule has strong fluorescence emission only after the protein/polypeptide-polymer conjugate is conjugated, so that the conjugation process of the protein/polypeptide-polymer conjugate can be monitored in situ through fluorescence change, and the protein/polypeptide-polymer conjugate can be applied to the transmission of therapeutic polypeptides and anticancer drugs.

Drawings

FIG. 1 is a graph showing the fluorescence change process of the conjugation process for preparing bovine serum albumin-polyethylene glycol conjugate in example 2;

FIG. 2 is a graph showing the relationship between the fluorescence change and the conjugation efficiency in the preparation of bovine serum albumin-polyethylene glycol conjugate in example 2;

FIG. 3 is a diagram of sodium dodecasulfonate-polyacrylamide gel electrophoresis of the bovine serum albumin-polyethylene glycol conjugate prepared in example 2;

FIG. 4 is a graph of the fluorescence change process of the conjugation process for preparing salmon calcitonin-polyethylene glycol conjugate in example 3;

FIG. 5 is a volume exclusion chromatogram of the preparation of salmon calcitonin-polyethylene glycol conjugate in example 3;

FIG. 6 is a diagram of sodium dodecasulfonate-polyacrylamide gel electrophoresis of the preparation of salmon calcitonin-polyethylene glycol conjugate in example 3;

FIG. 7 is a transmission electron micrograph of the vesicles prepared in example 6 in water;

FIG. 8 is a graph of the controlled release of the drug from example 7.

Detailed Description

wherein POI is a protein or polypeptide; polymer is a Polymer.

The protein/polypeptide-polymer conjugate bridges a protein/polypeptide and a polymer by a bifunctional fluorescent molecule which has no or only weak fluorescence emission capability, and has strong fluorescence emission only after the protein/polypeptide-polymer conjugate is conjugated, so that the conjugation process of the protein/polypeptide-polymer conjugate can be monitored in situ by fluorescence change, and the protein/polypeptide-polymer conjugate can be applied to the delivery of therapeutic polypeptides and anticancer drugs.

In certain embodiments of the invention, the POI is bovine serum albumin and the Polymer is polyethylene glycol having the structure according to formula I-a:

wherein n is 23 to 445.

The above protein-polymer conjugate is preferably prepared according to the following method:

in the present invention, the raw material ratio and reaction conditions of the above reaction are not particularly limited, and may be those of Michael reaction and click reaction which are conventional in the art.

Preferably, the conjugation efficiency of the protein-polymer conjugates described above is monitored in situ by fluorescence emission intensity.

In other embodiments of the invention, the POI is salmon calcitonin and the Polymer is polyethylene glycol having the structure of formula I-a:

wherein n is 23 to 445.

carrying out Michael reaction and click reaction on salmon calcitonin containing sulfhydryl groups, polyethylene glycol with azide end groups and a compound B to prepare the protein-polymer conjugate shown in the formula I-a.

In other embodiments of the invention, the POI is a matrix metalloproteinase cleavable polypeptide having the sequence β APVGLIG β AC-SH, wherein SH is a sulfhydryl group (the sulfhydryl group is located at a carbon-terminal cysteine residue), the polypeptide is obtained from Shanghai Qianzhiya Biotech company, and the Polymer is polytrimethylene carbonate having the structure shown in formula I-b:

wherein m is 10-55.

The preparation method is preferably as follows:

The invention also discloses a vesicle which consists of the polypeptide-polymer conjugate or the polypeptide-polymer conjugate prepared by the preparation method.

The method for producing the vesicle of the present invention is not particularly limited, and may be a method known to those skilled in the art. In the preferred embodiment of the present invention, the polypeptide-polymer conjugate is dissolved in DMSO, added with deionized water, and then dialyzed with deionized water.

The polymer vesicle provided by the invention is uniformly dispersed, has uniform particle size distribution, and has particle size distribution of 60-150 nm.

The polymersome has matrix metalloenzyme response characteristics, so that the polymersome can be used as a drug carrier or a fluorescent indicator for monitoring drug release in real time.

To further illustrate the present invention, the protein/polypeptide-polymer conjugates with fluorescence emission property provided by the present invention, and the preparation method and application thereof are described in detail below with reference to examples.

The synthesis route of the bifunctional fluorescent molecule B is as follows:

example 1 Synthesis of c1

4-Bromomalicylaldehyde (1; 3.11g,15.4mmol), Pd (PPh)₃)₂Cl₂(0.22g,0.31mmol),PPh₃(0.061g,0.23mmol), dry tetrahydrofuran (50mL) was added to a reaction flask containing a magnetic stirrer, the reaction was degassed for 30min with a dry nitrogen sparge, and then freshly distilled dry Et₃N (3.05g,30.0mmol) and trimethylethynylsilicon (1.67g,17.0mmol) were added under a nitrogen atmosphere and the solution turned orange. After stirring for 20min, the cocatalyst CuI (0.088g,0.46mmol) was addedThe reaction mixture was added to the reaction system under nitrogen atmosphere, and the solution became dark brown. Stirring overnight at room temperature, then the solvent was removed to give a dark brown solid, which was dissolved in n-pentane and filtered to give a yellow solution. All solvents were removed by rotation, and finally, recrystallization was carried out twice in n-hexane to obtain yellow crystal 2(2.96g, yield: 88.2%,>95％purity by HPLC)。

¹H NMR(CDCl₃delta, ppm, TMS) 11.0(s,1H, benzene-O)H),9.87(s,1H,-CHO),7.48(d, J ═ 8.4Hz,1H, aromatic hydrogen), 7.07(m,2H, aromatic hydrogen), 0.26(s,9H, -Si (C)H ₃)₃)。

Dissolve 2(2.85g,13.1mmol) in dry THF (40mL), then add 20mL MeOH solution containing KOH (0.74g,13.2mmol), stir the reaction at room temperature overnight, then spin off all solvent, redisperse the residue in water, add 1.0mL acetic acid and extract with 3X200mL chloroform. The organic phases were combined and dried over anhydrous magnesium sulfate, filtered and all solvents removed to give a brown solid, which was recrystallized twice from n-hexane to give 3 as a yellow solid (1.02g, yield: 53.2%, > 95% purity by HPLC).

¹H NMR(CDCl₃Delta, ppm, TMS) 11.0(s,1H, benzene-O)H),9.89(s,1H,-CHO),7.52(d, J ═ 8.4Hz,1H, aromatic hydrogens), 7.12(m,2H, aromatic hydrogens), 3.29(s,1H, -C ≡ C)H)。

Benzenesulfonyl chloride (2.51g,14.3mmol), Et₃N (2.15g,21.3mmol) and N-acetylglycine (0.88g,7.5mmol) were dissolved in THF and stirred at room temperature overnight. After removing insoluble salts, compound 3(1.02g,7.0mmol) was added to the reaction system and stirred at 80 ℃ for 10h, then cooled to 0 ℃ to give precipitate 4(1.09g, yield: 68.5%,>95% HPLC purity).

¹H NMR(CDCl₃,δ,ppm,TMS):9.80(s,1H,-CO-NH-),8.57(s,1H, aromatic hydrogen), 7.67(d, J ═ 8.4Hz,1H, aromatic hydrogen), 7.35(m,2H, aromatic hydrogen), 4.40(s,1H, -C ≡ C)H),2.14(s,3H,-COCH ₃)。

Compound 4(1.09g,4.8mmol), 4-Dimethylaminopyridine (DMAP) (0.11g,0.96mmol), (Boc)₂O (2.11g,9.8mmol) was dissolved in 40mL THF, and the reaction was stirred at 70 deg.C for 4h, then cooled to room temperature and addedMeOH (10mL) and NH were added₂NH₂·H₂O (0.96g,19.2mmol), and the reaction was stirred for an additional 4 h. Adding CH₂Cl₂The reaction mixture was then washed with 1MHCl solution, the organic phase was collected and anhydrous MgSO₄And (5) drying. Filtration to remove insoluble MgSO₄All solvents were removed by rotary evaporation and the residue was dissolved in CH₂Cl₂(40mL) and TFA (10mL) for 2h with NaHCO₃The solution was washed with water and the organic phase was MgSO₄Drying, filtering to remove MgSO₄After this time, the solvent was removed by rotary evaporation to give the desired product 5(0.76g), which was immediately used for the synthesis of C1 as follows:

compound 5(0.76g,4.1mmol) and maleic anhydride (2.01g,20.5mmol) were dissolved in 100mL acetone, refluxed overnight, and cooled to 0 ℃ to give a yellow solid precipitate. The yellow solid precipitate (1.03g) and p-toluenesulfonic acid monohydrate (154mg) were dissolved in 30mL methanol at reflux overnight and cooled to 0 ℃ to give the crude product as a yellow precipitate which was further purified by column chromatography using EtOAc/DCM (v/v ═ 1/2) as eluent to give C1(480mg, yield: 39.4%, > 95% HPLC purity) as a yellow solid.

¹H NMR(d₆-DMSO,δ,ppm,TMS):10.28(s,1H,-CONH-),8.65(s,1H, aromatic hydrogen), 7.73(d, J ═ 8.1Hz,1H, aromatic hydrogen), 7.49(s,1H, aromatic hydrogen), 7.38(s,1H, aromatic hydrogen), 6.74(d, J ═ 9.3Hz,1H, -nhoc —,1HH＝CH-),6.51(d,J＝8.7Hz,1H,-CH＝CH-COO-),4.43(s,1H,-C≡CH),3.66(s,3H,-COO-CH ₃)。

¹³C NMR(CDCl₃,δ,ppm TMS):167.5,164.2,157.6,149.9,131.9,129.7,128.8,128.6,125.3,124.0,123.1,120.7,119.2,83.8,83.0,52.1。

RP-HPLC analysis 4.4min (mobile phase: MeOH/H)₂O v/v 4/1)。

ESI-MS:m/z calc.for C₁₆H₁₂NO₅:298.06[M+H]⁺；found:298.0705。

Example 2 bifunctional fluorescent molecules c1 mediated BSA and PEG₂₂₇-N₃Fluorescent conjugates of (a)

Bovine serum albumin BSA (498mg, 7.5. mu. mol) was dissolved in phosphorusAcid buffer solution (PBS) (70mL, pH 6.5,0.1M, containing 1mM EDTA), while tris (2-chloroethyl) phosphate hydrochloride (TCEP. HCl) (21.5mg, 75. mu. mol) was dissolved in PBS (2.5mL), and then added dropwise to the above BSA solution, after 4h, the solution was dialyzed with deionized water for 24h (2.0kDa cut-off), and then lyophilized to obtain BSA_red。

BSA or BSA_red(4mg, 0.06. mu. mol) in PBS (0.9mL, pH 7.0,50mM), C1 (0.6. mu. mol, dissolved in 0.1mL DMSO), PEG₂₂₇-N₃(6mg, 0.6. mu. mol) and CuSO₄the/Na-ascorbate (1/5 molar ratio) was added to the solution and stirred at 25 ℃ for various periods of time, and the progress of conjugation was detected by in situ observation of the change in fluorescence emission intensity at about 420 nm. After the set time was reached, 80 μ L of the sample solution was removed, then diluted with 2mL PBS, quickly added copper ion adsorption resin (american marine chemical, 100mg), removed copper ions, shaken for 5min, and after 5min, the supernatant was filtered through a 0.22 μm sterile syringe filter before further SDS-PAGE testing.

The fluorescence change during the reaction is shown in FIG. 1, and the relationship between the fluorescence change and the conjugation efficiency is shown in FIG. 2.

Gel electrophoresis experiment: SDS-PAGE experiments were performed on a gel electrophoresis apparatus (Bio-Rad) and the BSA-PEG conjugate containing solution (80. mu.L) was mixed with 20. mu.L SDS-PAGE loading buffer and the bands were visualized directly by irradiation with UV light (365nm) or white light from a UVP EC3 imaging system (Coomassie brilliant blue staining) using 15.0 wt% polyacrylamide gel according to standard protocols.

The gel electrophoresis pattern is shown in FIG. 3.

Example 3 bifunctional fluorescent molecule c1 mediated Salmon calcitonin (sCT) and PEG₂₂₇-N₃Fluorescent conjugates of (a)

TCEP HCl (14.2mg, 50. mu. mol) was dissolved in PBS (2mL, pH 7.0,50mM), 40. mu.L of the above solution (containing 1. mu. mol of TCEP HCl) was added to a vial containing salmon calcitonin (sCT,1.7mg, 0.5. mu. mol PBS (9mL, pH 7.0,0.05M)), stirred at room temperature and RP-HPLC analysis showed Cys¹-Cys⁷The disulfide bond is quantitatively reduced within 30 min.

200μDMSO solution of LC1 (containing 0.2. mu. molC1), PEG₂₂₇-N₃(10.0mg, 1.0. mu. mol) and CuSO₄Na-ascorbate (1/5 molar ratio) was added to reduced sCT (0.1. mu. mol, dissolved in 1.8mL PBS buffer), stirred at 25 ℃ for various times, the progress of conjugation was detected by in situ observation of the change in fluorescence emission intensity at-420 nm, after 4h copper ion was removed by rapid addition of copper ion adsorption resin (100 mg, ocean chemical Co., USA), and after 5min shaking, the supernatant was filtered through a 0.22 μm sterile syringe filter. 80 μ L of the solution was used for SDS-PAGE experiments, and the remaining solution was dialyzed with deionized water for 24h before RP-HPLC experiments.

The fluorescence change during the reaction is shown in FIG. 4.

The molecular weight of the prepared protein-polymer conjugate was determined by volume exclusion chromatography, and the volume exclusion chromatogram thereof is shown in fig. 5.

Gel electrophoresis experiment: SDS-PAGE experiments were performed on a gel electrophoresis apparatus (Bio-Rad), and the sCT-PEG conjugate-containing solution (80. mu.L) was mixed with 20. mu.L SDS-PAGE loading buffer using 15.0 wt% polyacrylamide gel according to standard protocols, and the bands were visualized directly by irradiation with ultraviolet light (365nm) or white light from a UVP EC3 imaging system (Coomassie Brilliant Blue staining).

The gel electrophoresis pattern is shown in FIG. 6.

EXAMPLE 4 Synthesis of azide-functionalized PTMC (PTMC-N)₃)

Recrystallized trimethylene carbonate (TMC) monomer (500mg,4.9mmol) was dissolved in dry CH₂Cl₂To (1mL), then 3-azido-1-propanol (24mg,0.24mmol) and 1, 3-dicyclohexylurea (DCU,8mg,0.05mmol) were added dropwise to dry CH₂Cl₂Stirring the reaction mixture at room temperature under nitrogen atmosphere for 12h, quenching the reaction with acetic acid, diluting with tetrahydrofuran, precipitating with excessive cold methanol for three times, and drying in vacuum drying oven to obtain white powder PTMC-N₃(160mg, yield 30.5%), actual degree of polymerization of PTMC was determined by¹H NMR calculated as 14 and hence abbreviated as PTMC₁₄-N₃。

Example 5 bifunctional fluorescencePhotomolecule C1 mediated PVGLIG polypeptides and PTMC₁₄-N₃Fluorescent conjugates of (a)

PTMC₁₄-N₃(7.5mg, 5.0. mu. mol) and C1(1.5mg, 5.1. mu. mol) were dissolved in DMSO (1.8mL), PVGLIG (8.1mg, 10.1. mu. mol) and CuSO₄Ascorbic acid (1/5 mol ratio) was dissolved in 0.2mL deionized water and added, the reaction system was stirred at 25 ℃ and the conjugation process was detected by fluorescence in situ. After co-culturing for 3h, rapidly adding copper ion adsorption resin (100 mg, ocean chemical company, USA) to remove copper ions, shaking for 5min, filtering out supernatant through a 0.22 μm sterile syringe filter, precipitating filtrate with excessive cold acetonitrile, centrifuging, collecting precipitate, and drying in a vacuum drying oven overnight.

Example 6 preparation of PVGLIG-C1-PTMC₁₄Polymersome

PVGLIG-C1-PTMC₁₄(2mg) was dissolved in 1mL DMSO and added to a 15mL vial containing a magnetic stir bar and stirred at room temperature for 3h, 9mL deionized water (-500 rpm) was added over-20 s with stirring, and after stirring for 5h dialysis (3.5 kDa cut-off) was performed with deionized water for 24 h.

A transmission electron micrograph of the prepared vesicles in water is shown in FIG. 7.

Example 7PVGLIG-C1-PTMC₁₄Polymer vesicle-embedded adriamycin hydrochloride

PVGLIG-C1-PTMC₁₄(2mg) was dissolved in 1mL DMSO and added to a 15mL vial containing a magnetic stir bar, followed by addition of DOX HCl in deionized water (5g/L,2mL) with stirring, followed by addition of the remaining 7mL portions at the same rate, stirring for 5h and dialysis against deionized water (3.5 kDa cut-off) for 24 h. The load content of DOX is 8.0 wt%.

The graph of the controlled drug release application is shown in fig. 8.

As can be seen from the above examples, the above protein/polypeptide-polymer conjugates prepared according to the present invention can monitor conjugation efficiency in situ by fluorescence emission intensity, and are applied to delivery of therapeutic polypeptides and anticancer drugs.

The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.

Claims

1. A protein/polypeptide-polymer conjugate having fluorescence emission properties, having the structure of formula i:

formula I;

wherein POI is a protein or polypeptide; polymer is a Polymer.

2. A protein-polymer conjugate having fluorescence emission properties, having the structure of formula i-a:

formula I-a;

3. A method of preparing the protein-polymer conjugate of claim 2, comprising the steps of:

B；

formula I-a;

wherein POI is bovine serum albumin, and n is 23-445;

or comprises the following steps:

wherein POI is salmon calcitonin; n is 23 to 445.

4. A polypeptide-polymer conjugate having fluorescence emission properties, having the structure of formula i-b:

formula I-b;

5. The method of preparing the polypeptide-polymer conjugate of claim 4, comprising the steps of:

carrying out Michael reaction and click reaction on the polypeptide which can be cut by the matrix metalloproteinase shown in the formula C, the azide-terminated polytrimethylene carbonate shown in the formula D and the compound B to prepare the polypeptide-polymer conjugate shown in the formula I-B;

formula C;

formula D;

B；

formula I-b;

wherein m is 10-55.

6. The preparation method according to claim 3 or 5, characterized in that the conjugation efficiency of the conjugate is monitored in situ by fluorescence emission intensity.

7. A polymersome composed of the polypeptide-polymer conjugate of claim 4 or the polypeptide-polymer conjugate prepared by the preparation method of claim 5.

8. The polymersome according to claim 7, wherein the polymersome has a particle size of 60 to 150 nm.

9. The polymersome of claim 7, wherein the polymersome has a matrix metalloenzyme response characteristic.

10. Use of polymersomes according to any one of claims 7 to 9 as a pharmaceutical carrier.