阅读说明：本技术 连接子，载药连接子、细胞穿透肽偶联药物、抗体偶联药物及其制备方法 (Linker, drug-loaded linker, cell-penetrating peptide conjugate drug, antibody conjugate drug, and method for producing same ) 是由 周传政 臧传龙 于 2019-08-13 设计创作，主要内容包括：本发明涉及药物递送领域,公开了连接子,载药连接子、细胞穿透肽偶联药物、抗体偶联药物及其制备方法,该连接子具有式(I)所示的结构,本发明提供的光响应自催化断裂的连接子和载药连接子能够将药物偶联至多肽或蛋白质类递送载体上,能够实现药物向细胞内的可控递送。<Image he="423" wi="700" file="DDA0002165101590000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>(The invention relates to the field of drug delivery, and discloses a linker, a drug-loaded linker, a cell-penetrating peptide conjugated drug, an antibody conjugated drug and a preparation method thereof, wherein the linker has a structure shown in formula (I), and the photoresponse autocatalytically cleaved linker and the drug-loaded linker provided by the invention can couple the drug to a polypeptide or protein delivery carrier, so that the controllable delivery of the drug to cells can be realized.)

1. A photo-responsive autocatalytic cleavage type linker having a structure represented by formula (I),

wherein, in the formula (I),

R₁is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl;

R₂is a chain alkyl group containing or not containing at least one heteroatom selected from N, O, S to form R₂The number of atoms in the main chain of the chain alkyl group is 10 to 60, the main chain of the chain alkyl group contains or does not contain a saturated ring structure, contains or does not contain an unsaturated ring structure, contains or does not contain an alkenyl group, contains or does not contain an alkynyl group, and the main chain of the chain alkyl group is an unsubstituted group or is selected from C_1-3Or a group substituted with at least one of the alkyl group and the halogen of (a), or the chain alkyl groupAt least one C atom and O atom on the main chain of (1) form a double bond structure.

2. The linker of claim 1, wherein the linker has a structure represented by formula (I1),

wherein, in the formula (I1),

R₁is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl;

n is a positive integer greater than or equal to 2;

preferably, in formula (I1),

R₁is 4, 5-dimethoxy-2-nitrobenzyl, and n is 3;

preferably, the linker has a structure represented by formula (I2), and in formula (I2), R₁Is 4, 5-dimethoxy-2-nitrobenzyl;

3. a drug-loaded linker of a photoresponsive autocatalytic cleavage type, characterized in that the drug-loaded linker has a structure represented by formula (II), wherein in formula (II), R is₁And R₂Is as defined in claim 1 or 2, M is a group containing a group selected from-NH₂At least one group capable of reacting among-SH, -OH, Y is-NH-, -S-or-O-provided by M;

preferably, the drug-carrying linker has a structure represented by formula (II1), in formula (II1), DOX is a group provided by doxorubicin,

4. a method of preparing a drug-loaded linker of formula (II1), comprising:

(1) reacting a compound shown in a formula (1) with triphosgene in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (2);

(2) reacting the compound shown in the formula (2) with adriamycin in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (3);

(3) reacting the compound shown in the formula (3) with azido polyethylene glycol maleimide shown in the formula (4) and ascorbate in the presence of copper sulfate, dimethylformamide and water to obtain the drug-loaded linker;

wherein, in the formula (II1), the formula (1), the formula (2) and the formula (3), R₁Is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl.

5. The method according to claim 4, wherein in the step (1), the compound represented by the formula (1) is used in a molar ratio of 1: (2-3): (1-1.5);

preferably, in step (1), the solvent is tetrahydrofuran;

preferably, in step (1), the reaction conditions include: the temperature is 10-40 ℃, the time is 4-24h, and the stirring speed is 50-1000 rpm.

6. The method according to claim 4, wherein in the step (2), the molar ratio of the compound shown in the formula (2) to the diisopropylethylamine and the drug is 1: (2-3): (1-1.5);

preferably, in step (2), the solvent is dichloromethane;

preferably, in step (2), the reaction conditions include: the temperature is 10-40 ℃, the time is 1-15h, and the stirring speed is 50-1000 rpm;

preferably, in the step (3), the compound shown in the formula (3) and the azido polyethylene glycol maleimide, ascorbate and copper sulfate shown in the formula (4) are used in a molar ratio of 1: (1-1.5): (0.8-1.2): (0.4-0.6);

preferably, in step (3), the reaction conditions include: the temperature is 10-40 ℃, the time is 2-30h, and the stirring speed is 50-1000 rpm.

7. A method of preparing a cell-penetrating peptide-conjugated drug, the method comprising:

(1) dissolving the drug-loaded linker of claim 3 in a solvent to obtain a drug-loaded linker stock;

(2) carrying out addition reaction on the drug-loaded linker storage solution and cell penetrating peptide H3-V35C to obtain the cell penetrating peptide coupling drug;

preferably, in the step (2), the molar ratio of the drug-loaded linker stock to the cell penetrating peptide H3-V35C is (1-1.5): 1;

preferably, in step (2), the conditions of the addition reaction include: the reaction temperature is 20-60 ℃, and the reaction time is 0.5-8 h;

preferably, in step (2), the addition reaction is carried out in the presence of HEPES buffer.

8. A cell-penetrating peptide-conjugated drug produced by the method of claim 7.

9. A method of preparing an antibody-conjugated drug, the method comprising:

(1) dissolving the drug-loaded linker of claim 3 in a solvent to obtain a drug-loaded linker stock; and carrying out reduction reaction on the monoclonal antibody and a reducing agent solution to obtain a solution after the antibody is reduced;

(2) carrying out addition reaction on the drug-loaded linker storage solution and the solution obtained after the reduction of the antibody to obtain the antibody conjugate drug;

preferably, in step (1), the monoclonal antibody is a trastuzumab monoclonal antibody.

10. An antibody-conjugated drug prepared by the method of claim 9.

Technical Field

The invention relates to the field of drug delivery, in particular to a photoresponse autocatalytic rupture type connector, a photoresponse autocatalytic rupture type drug-loaded connector and a preparation method thereof, a method for preparing a cell penetrating peptide coupled drug, a cell penetrating peptide coupled drug prepared by the method, a method for preparing an antibody coupled drug and the antibody coupled drug prepared by the method.

Background

Prodrug strategies are widely used in drug design and development with the aim of improving the pharmacokinetic properties, especially the targeted delivery capacity, of the parent drug (nat. rev. drug Discov.2108,17, 559-.

In general, prodrugs are constructed by coupling a drug molecule to a carrier via a linker containing a "trigger switch". After the prodrug is delivered to target cells or tissues, the linker is cleaved under the action of endogenous stimuli (pH value change, enzymes, redox reactions) or exogenous stimuli (small-molecule photoinitiators), so that the active drug is released and the drug effect is exerted. Therefore, rational design of the linker is key to achieving efficient, controlled delivery of the prodrug.

In some cases, the spatial distance between the drug and the carrier may disrupt cleavage of the linker, resulting in incomplete release of the drug.

To overcome this problem, researchers developed "self-immolative" linkers (Angew. chem. int. Ed.2003,42, 4494-. The self-sacrifice linker can efficiently release active drugs through a series decomposition reaction triggered by a stimulus factor.

To date, only two classes of "self-immolative" linkers have been widely used; one is via a tandem elimination mechanism and the other is via a cyclization mechanism (Angew. chem. int. Ed.2015,54, 7492-7509; chem. Eng. J.2018,340, 24-31). However, both types of linkers may produce products with toxic side effects, such as quinone methides, during cleavage, resulting in additional side effects.

Therefore, the development of new, biocompatible, highly efficient "self-immolative" linkers is essential for the design and development of prodrugs.

Disclosure of Invention

One of the objectives of the present invention is to overcome the drawbacks of the prior art and to provide a novel photoresponsive autocatalytic cleavage type linker and a drug-loaded linker.

The invention also aims to provide a cell penetrating peptide coupling drug and an antibody coupling drug, so as to realize the targeted and controllable delivery of the anticancer drug to cancer cells.

In order to achieve the above object, a first aspect of the present invention provides a linker of a photoresponsive autocatalytic cleavage type, the linker having a structure represented by formula (I),

wherein, in the formula (I),

R₁is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl;

R₂is a chain alkyl group containing or not containing at least one heteroatom selected from N, O, S to form R₂The number of atoms in the main chain of the chain alkyl group is 10 to 60, the main chain of the chain alkyl group contains or does not contain a saturated ring structure, contains or does not contain an unsaturated ring structure, contains or does not contain an alkenyl group, contains or does not contain an alkynyl group, and the main chain of the chain alkyl group is an unsubstituted group or is selected from C_1-3Or a double bond structure is formed between at least one C atom and an O atom on the main chain of the chain alkyl group.

The second aspect of the present invention provides a drug-carrying linker of a photoresponsive autocatalytic cleavage type, which has a structure represented by formula (II), wherein, in formula (II), R₁And R₂Is as defined in claim 1 or 2, M is a group containing a group selected from-NH₂At least one group capable of reacting among-SH, -OH, Y is-NH-, -S-or-O-provided by M;

a third aspect of the invention provides a method of preparing a drug-carrying linker of formula (II1), the method comprising:

(1) reacting a compound shown in a formula (1) with triphosgene in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (2);

(2) reacting the compound shown in the formula (2) with adriamycin in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (3);

(3) reacting the compound shown in the formula (3) with azido polyethylene glycol maleimide shown in the formula (4) and ascorbate in the presence of copper sulfate, dimethylformamide and water to obtain the drug-loaded linker;

wherein, in the formula (II1), the formula (1), the formula (2) and the formula (3), R₁Is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl.

In a fourth aspect, the present invention provides a method for preparing a cell-penetrating peptide-conjugated drug, the method comprising:

(1) dissolving a drug-loaded linker described herein in a solvent to obtain a drug-loaded linker stock;

(2) and carrying out addition reaction on the drug-loaded linker storage solution and cell penetrating peptide H3-V35C to obtain the cell penetrating peptide coupling drug.

A fifth aspect of the invention provides a cell penetrating peptide conjugate drug prepared by the method as hereinbefore described.

A sixth aspect of the present invention provides a method for preparing an antibody-conjugated drug, the method comprising:

(1) dissolving the drug-loaded linker in a solvent to obtain a drug-loaded linker storage solution; and carrying out reduction reaction on the monoclonal antibody and a reducing agent solution to obtain a solution after the antibody is reduced;

(2) and carrying out addition reaction on the drug-carrying linker storage solution and the solution obtained after the reduction of the antibody to obtain the antibody conjugated drug.

The seventh aspect of the present invention provides an antibody-conjugated drug prepared by the method described above.

The photoresponse autocatalytic cleavage linker and the drug-loaded linker provided by the invention can couple a drug to a polypeptide or protein delivery carrier, and can realize controllable delivery of the drug to cells.

Drawings

FIG. 1 is a confocal laser microscopy analysis of the localization of the drug Doxorubicin (DOX) in HeLa cells;

FIG. 2 is a toxicity study of cell penetrating peptide coupling drug H3-PC4AP-DOX in HeLa cells;

FIG. 3 shows the efficiency of the antibody-conjugated drug Trastuzumab-PC4P-DOX to release drug DOX in serum after light irradiation;

FIG. 4 is a confocal laser microscopy analysis of the binding affinity of Trastuzumab and the antibody conjugate Trastuzumab-PC4P-DOX to breast cancer cells with different expression levels of HER2, wherein FIG. 4a shows that both Trastuzumab and the antibody conjugate Trastuzumab-PC4P-DOX are able to recognize and bind to SK-RB-3 cells with high expression of HER2, and FIG. 4b shows that Trastuzumab and the antibody conjugate Trastuzumab-PC4P-DOX are unable to recognize and bind to MCF7 cells with low expression of HER 2;

FIG. 5 is a graph of the toxicity of the antibody conjugate drug Trastuzumab-PC4AP-DOX in breast cancer cells with different levels of HER2 expression, wherein FIG. 5a shows that the toxicity of the antibody conjugate drug Trastuzumab-PC4AP-DOX is dose-dependent in SK-BR-3 cells with high expression of HER2, and FIG. 5b compares the toxicity difference of the antibody conjugate drug Trastuzumab-PC4AP-DOX in SK-BR-3 cells with high expression of HER2 with MCF7 cells with low expression of HER 2.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

As described above, the first aspect of the present invention provides a linker of a photoresponsive autocatalytic cleavage type having a structure represented by formula (I),

wherein, in the formula (I),

R₁is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl;

R₂is a chain alkyl group containing or not containing at least one heteroatom selected from N, O, S to form R₂The number of atoms in the main chain of the chain alkyl group is 10 to 60, the main chain of the chain alkyl group contains or does not contain a saturated ring structure, contains or does not contain an unsaturated ring structure, contains or does not contain an alkenyl group, contains or does not contain an alkynyl group, and the main chain of the chain alkyl group is an unsubstituted group or is selected from C_1-3Or a double bond structure is formed between at least one C atom and an O atom on the main chain of the chain alkyl group.

According to a preferred embodiment, in formula (I), the linker has a structure represented by formula (I1),

wherein, in the formula (I1),

R₁is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl;

n is a positive integer of 2 or more.

According to another preferred embodiment, in formula (I1),

R₁is 4, 5-dimethoxy-2-nitrobenzyl, and n is 3.

According to a particularly preferred embodiment, the linker has the structure shown in formula (I2), and in formula (I2), R₁Is 4, 5-dimethoxy-2-nitrobenzyl;

as mentioned above, the second aspect of the present invention provides a drug-carrying linker of photoresponsive autocatalytic cleavage type, which has a structure represented by formula (II), wherein in formula (II), R₁And R₂Is as defined in claim 1 or 2, M is a group containing a group selected from-NH₂At least one group capable of reacting among-SH, -OH, Y is-NH-, -S-or-O-provided by M;

according to a preferred embodiment, the drug-carrying linker has the structure shown in formula (II1), in formula (II1) DOX is a group provided by doxorubicin,

as mentioned above, the third aspect of the present invention provides a method for preparing a drug-carrying linker of formula (II1), the method comprising:

(1) reacting a compound shown in a formula (1) with triphosgene in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (2);

(2) reacting the compound shown in the formula (2) with adriamycin in the presence of diisopropylethylamine and a solvent to obtain a compound shown in a formula (3);

(3) reacting the compound shown in the formula (3) with azido polyethylene glycol maleimide shown in the formula (4) and ascorbate in the presence of copper sulfate, dimethylformamide and water to obtain the drug-loaded linker;

wherein, in the formula (II1), the formula (1), the formula (2) and the formula (3), R₁Is at least one photosensitive protecting group selected from 4, 5-dimethoxy-2-nitrobenzyl, 2-nitrobenzyl and 1- (2-nitrobenzyl) ethyl.

Preferably, in the step (1), the compound represented by the formula (1) is used in a molar ratio of 1: (2-3): (1-1.5).

Preferably, in step (1), the solvent is tetrahydrofuran.

Preferably, in step (1), the reaction conditions include: the temperature is 10-40 ℃, the time is 4-24h, and the stirring speed is 50-1000 rpm.

Preferably, in the step (2), the molar ratio of the compound shown in the formula (2) to the diisopropylethylamine and the drug is 1: (2-3): (1-1.5).

Preferably, in step (2), the solvent is dichloromethane.

Preferably, in step (2), the reaction conditions include: the temperature is 10-40 ℃, the time is 1-15h, and the stirring speed is 50-1000 rpm.

Preferably, in the step (3), the compound shown in the formula (3) and the azido polyethylene glycol maleimide, ascorbate and copper sulfate shown in the formula (4) are used in a molar ratio of 1: (1-1.5): (0.8-1.2): (0.4-0.6).

Preferably, in step (3), the reaction conditions include: the temperature is 10-40 ℃, the time is 2-30h, and the stirring speed is 50-1000 rpm.

The aforementioned step (1), step (2) and step (3) of the present invention may be further combined with various post-treatment means conventionally used in the art to perform post-treatment on the obtained intermediates and the like, and the present invention is not described in detail herein, and those skilled in the art should not be construed as limiting the present invention.

The method for preparing the compound represented by formula (1) is not particularly limited in the present invention, and those skilled in the art can obtain the compound according to the structural formula of the compound represented by formula (1) by a conventional preparation method in the field of organic synthesis. The following exemplary embodiment of the present invention provides a preferred embodiment for preparing the compound represented by formula (1), and those skilled in the art should not be construed as limiting the present invention.

(1) Carrying out first mixing on the compound shown in the formula (a1), imidazole, tert-butyldiphenylchlorosilane and dimethylformamide to form a first mixed solution, washing, drying, filtering and carrying out column chromatography on an organic phase obtained after reaction to obtain the compound shown in the formula (a2), wherein R is R₂Radicals provided for tert-butyldiphenylchlorosilane, R₃Is benzoyl;

(2) secondly mixing the compound shown as the formula (a2) with sodium methoxide methanol solution and dichloromethane to form a second mixed solution, then performing rotary evaporation on the second mixed solution to remove the solvent, and performing column chromatography to obtain the compound shown as the formula (a 3);

(3) carrying out third mixing on the compound shown in the formula (a3), sodium hydride and tetrahydrofuran, and then cooling to form a third mixed solution; reacting the third mixed solution with propargyl bromide; then washing, drying, filtering and carrying out column chromatography on an organic phase obtained after the reaction to obtain a compound shown as a formula (a 4);

(4) reacting the compound shown as the formula (a4) with boron trifluoride diethyl etherate, propanedithiol and dichloromethane, and then washing, drying, filtering and carrying out column chromatography on an organic phase obtained after the reaction to obtain a compound shown as a formula (a 5);

(5) reacting the compound shown as the formula (a5) with oxalyl chloride, triethylamine and dimethyl sulfoxide, and then washing, drying, filtering and carrying out column chromatography on an organic phase obtained after the reaction to obtain a compound shown as a formula (a 6);

(6) reacting the compound shown as the formula (a6) with N-bromosuccinimide, R₁Mixing the alcohol compound of the protective group and acetonitrile to form a mixed solution, then performing rotary evaporation on the mixed solution to remove the solvent, and performing column chromatography to obtain a compound shown as a formula (a 7);

(7) and (2) reacting the compound shown as the formula (a7) with tetra-n-butylammonium fluoride and tetrahydrofuran, then performing rotary evaporation on the solution obtained after the reaction to remove the solvent, and performing column chromatography to obtain the compound shown as the formula (1).

Preferably, in the aforementioned preferred embodiment of the preparation of the compound represented by the formula (1), in the step (1), the compound represented by the formula (a1), imidazole, t-butyldiphenylchlorosilane and dimethylformamide are used in a ratio of 1mmol (3-4) mmol (1.2-1.5) mmol (50-70) mL; more preferably, the conditions of the first mixing include: the temperature is 18-25 ℃, the time is 10-12 hours, and the stirring speed is 400-600 revolutions per minute.

Preferably, in the aforementioned preferred embodiment of the preparation of the compound represented by the formula (1), in the step (2), the amount ratio of the compound represented by the formula (a2) to the sodium methoxide methanol solution and dichloromethane is 1mmol (0.3-0.4) mL (4-7) mL; more preferably, the conditions of the second mixing include: the temperature is 18-25 ℃, the time is 0.5-1 h, and the stirring speed is 400-600 r/min.

Preferably, in the aforementioned preferred embodiment for preparing the compound represented by the formula (1), in the step (3), the compound represented by the formula (a3) is used in an amount ratio to the propargyl bromide, the sodium hydride and the tetrahydrofuran of 1mmol (2-3) mmol: (3-5) mL. More preferably, the third mixing conditions include: the temperature is between 10 ℃ below zero and 10 ℃, the time is between 15 and 30 minutes, and the stirring speed is 400-600 revolutions per minute. Preferably, the conditions for reacting the third mixed solution with propargyl bromide include: the temperature is 18-25 ℃, the time is 4-6 hours, and the stirring speed is 400-600 revolutions per minute.

Preferably, in the aforementioned preferred embodiment for preparing the compound represented by the formula (1), in the step (4), the ratio of the compound represented by the formula (a4) to the amounts of propanedithiol, boron trifluoride etherate and dichloromethane is 1mmol (1.0-1.5) mmol (2.0-2.5) mmol (4-6) mL; preferably, the conditions of the reaction in step (4) include: the temperature is between-5 and 0 ℃, the time is between 10 and 30 minutes, the stirring speed is 400-600 revolutions per minute, the boron trifluoride diethyl etherate is added into the system in a dropwise manner, and the dropwise adding speed is between 10 and 15 drops per minute.

Preferably, in the aforementioned preferred embodiment of the preparation of the compound represented by the formula (1), in the step (5), the ratio of the amount of the compound represented by the formula (a5), oxalyl chloride, dimethyl sulfoxide, triethylamine and dichloromethane is 1mmol (1.0-1.5) mmol (2.0-2.5) mmol (4.0-6.0) mmol (4-6) mL; preferably, in step (5), the reaction process comprises: the reaction conditions of the first stage comprise that the temperature is-78 ℃, the time is 40-60 minutes, the stirring speed is 400-600 revolutions per minute, the dimethyl sulfoxide is added into the system in a dropwise adding mode, and the dropwise adding speed is 10-15 drops per minute; the reaction conditions of the second stage comprise that the temperature is-78 ℃, the time is 40-60 minutes, the stirring speed is 400-600 r/min, the compound shown in the formula (a5) is added into the system in a dropping way, and the dropping speed is 10-15 r/min; the reaction conditions of the third stage comprise that the temperature is-78 ℃, the time is 20-40 minutes, the stirring speed is 400-600 revolutions per minute, and the triethylamine is added into the system in a dropwise manner.

Preferably, in the aforementioned preferred embodiment of the preparation of the compound represented by formula (1), in step (6), the compound represented by formula (a6) is reacted with N-bromosuccinimide, R₁Show guarantorThe dosage ratio of the alcohol compound of the protecting group to the acetonitrile is 1mmol, (5-6) mmol, (4-6) mmol, (20-30) mL; preferably, in step (6), the mixing conditions include: the temperature is-10 ℃, the time is 40-60 minutes, and the stirring speed is 400-600 revolutions per minute.

Preferably, in the aforementioned preferred embodiment for preparing the compound represented by the formula (1), in the step (7), the ratio of the compound represented by the formula (a7) to the amount of tetra-n-butylammonium fluoride and tetrahydrofuran is 1mmol (2-3) mL (8-12) mL; preferably, in step (7), the reaction conditions include: the temperature is 18-25 ℃, the time is 1-3 hours, and the stirring speed is 400-600 revolutions per minute.

As previously described, a fourth aspect of the present invention provides a method for preparing a cell-penetrating peptide-conjugated drug, the method comprising:

(1) dissolving the drug-loaded linker in a solvent to obtain a drug-loaded linker storage solution;

(2) and carrying out addition reaction on the drug-loaded linker storage solution and cell penetrating peptide H3-V35C to obtain the cell penetrating peptide coupling drug.

Preferably, in the step (2), the molar ratio of the drug-loaded linker stock to the cell penetrating peptide H3-V35C is (1-1.5): 1.

preferably, in step (2), the conditions of the addition reaction include: the reaction temperature is 20-60 ℃, and the reaction time is 0.5-8 h.

Preferably, in step (2), the addition reaction is carried out in the presence of a HEPES buffer, preferably having a pH of 7.5.

The method of the invention preferably further comprises the step of further purifying the obtained cell penetrating peptide coupled drug, preferably by PD-MiniTrap-G25 gravity centrifugal desalination, collecting the required component peak NanoDrop concentration and storing at-80 ℃ for later use.

The cell penetrating peptide conjugate drug can optically control release of a loaded antitumor drug in a tumor cell, the tumor cell is preferably a HeLa cell, and the optically controlled condition is preferably 7mW/cm²The illumination time is preferably 5 to 7 minutes.

As mentioned above, a fifth aspect of the present invention provides a cell penetrating peptide conjugate drug prepared by the method as described above.

Preferably, the cell-penetrating peptide-conjugated drug has a structure represented by formula (5):

wherein, in the formula (5), Nv is 4, 5-dimethoxy-2-nitrobenzyl.

As previously described, a sixth aspect of the present invention provides a method for preparing an antibody-conjugated drug, the method comprising:

(1) dissolving the drug-loaded linker in a solvent to obtain a drug-loaded linker storage solution; and carrying out reduction reaction on the monoclonal antibody and a reducing agent solution to obtain a solution after the antibody is reduced;

(2) and carrying out addition reaction on the drug-carrying linker storage solution and the solution obtained after the reduction of the antibody to obtain the antibody conjugated drug.

Preferably, in step (1), the monoclonal antibody is a trastuzumab monoclonal antibody.

Preferably, the time of the reduction reaction is 2-3 hours, and the reaction temperature is 35-40 ℃, and more preferably 37 ℃.

Preferably, the temperature of the addition reaction is 35-40 ℃, and more preferably 37 ℃.

Preferably, the molar amount of the reducing agent is 4 times the molar amount of the antibody based on the molar amount of the antibody.

Preferably, the molar amount of the drug-carrying linker is 8 times the molar amount of the antibody based on the molar amount of the antibody.

The method of the invention preferably further comprises the step of further purifying the obtained antibody-conjugated drug, preferably by means of PD-MiniTrap-G25 gravity centrifugation for desalting, collecting the required component peak NanoDrop for concentration determination, and storing at-80 ℃ for later use.

As mentioned above, a seventh aspect of the present invention provides an antibody-conjugated drug prepared by the method described above.

The antibody-conjugated drug of the present invention preferably has a structure represented by formula (6):

wherein, in the formula (6), the mAb is Trastuzumab (Trituzumab monoclonal antibody), n is an integer more than 1 and less than or equal to 8, and Nv is 4, 5-dimethoxy-2-nitrobenzyl.

In the invention, the Trastuzumab monoclonal antibody is a recombinant humanized antibody which specifically binds to human HER2, and the molecular weight of the antibody is about 150 kDa. M is preferably anticancer drug adriamycin DOX, and the antibody conjugate drug is prepared by the Michael addition reaction of a maleimide group on a linker and an interchain disulfide bond of a Trastuzumab antibody.

The antibody conjugated drug can realize the targeted delivery of the drug in tumor cells. Wherein the tumor cells comprise HER2 positive SK-BR-3 cells and HER2 negative MCF7 cells.

The invention also has the following specific advantages:

(1) the drug-loaded linker has good stability under physiological conditions, and has low side effect caused by off-target release;

(2) after illumination, the drug can be efficiently and quickly released from the carrier, and the linker is cracked in an autocatalytic manner, so that toxic byproducts are not generated;

(3) the linker is photo-responsive, and has the advantages of high spatial and temporal resolution and non-invasiveness.

Based on the above, the novel photoresponsive autocatalytic cleavage type linker has a wide application prospect in the aspect of controllable drug delivery.

The present invention will be described in detail below by way of examples. In the following examples, various raw materials used are commercially available without specific description.

4, 5-dimethoxy-2-nitrobenzyl alcohol (as manufactured by Korea), tert-butyldiphenylchlorosilane (carbofuran, cat # 362315), imidazole (Adamas, cat # 45081D), propargyl bromide (Innochem, cat # A64659), sodium hydride (Adamas, cat # 81778A), boron trifluoride etherate (TCI, cat # A15275), propanedithiol (Adamas, cat # 13966C), N-bromosuccinimide (Allatin, cat # B105057), methylene chloride (Tianjin chemical reagent supplier, cat # 3975), tetrahydrofuran (Tianjin chemical reagent supplier, cat # 2287), diisopropylethylamine (Alfa, cat # A11801), doxorubicin hydrochloride (Soulebao, cat # D8740-25), triphosgene (Innochem, cat # A57532), sodium ascorbate (Micheln, cat # S8135), pentahydrate Michelin (Michelle # 807656, cat # C5356).