CN107115527B

CN107115527B - Photosensitizer compound and preparation method and application thereof

Info

Publication number: CN107115527B
Application number: CN201710312002.8A
Authority: CN
Inventors: 李斯文; 赵磊
Original assignee: Individual
Current assignee: Individual
Priority date: 2017-05-05
Filing date: 2017-05-05
Publication date: 2020-06-30
Anticipated expiration: 2037-05-05
Also published as: CN107115527A

Abstract

The invention relates to a photosensitizer compound and a preparation method and application thereof, belonging to the technical field of photodynamic therapy. The photosensitizer compound provided by the invention is of a shell-core structure, chitosan is used as a hydrophilic framework, a hydrophobic compound is coupled inside the hydrophilic framework of the chitosan through an amido bond, and the hydrophobic compound is coupled with a photosensitizer to form a core structure; the outer part of the chitosan hydrophilic skeleton acts with polar water molecules, and is modified by a specific antibody to form a shell structure. The photosensitizer compound has good targeting property and high safety, can ensure that the selectivity of photodynamic therapy does not depend on a light region, ensures that the photodynamic therapy has cell selectivity, can only generate phototoxicity by the photosensitizer taken by cells, can be well applied to the treatment of hemangioma, and has wide application prospect in the preparation of acute B lymphocyte leukemia medicaments.

Description

Photosensitizer compound and preparation method and application thereof

Technical Field

The invention belongs to the technical field of photodynamic therapy, and particularly relates to a photosensitizer compound and a preparation method and application thereof.

Background

The principle of photodynamic therapy (PDT) is that after a photosensitizer absorbs light of a specific wavelength, the ability to absorb photons is transferred to surrounding oxygen atoms, which oxidize the oxygen atoms into active oxygen, which reoxidizes lipid, protein, and nucleic acid biomolecules, thereby killing cells. Oxygen, photosensitizer, light of a certain wavelength are 3 major elements of PDT treatment.

The existing photosensitizer compound prepared by taking chitosan as a carrier is a photosensitizer used for photodynamic therapy. Although the photosensitizer can obviously prolong the circulation time of the photosensitizer in vivo, reduce the dosage of the photosensitizer per se and improve the proportion of the photosensitizer actually gathered in target cells, the existing photosensitizer can generate a photodynamic killing effect under the action of specific exciting light no matter whether the photosensitizer is taken up by cells or not, and therefore, the photosensitizer is only suitable for treating solid tumors with a limited illumination area. Because lymphoma cells are dispersed in blood, PDT for treating blood tumor needs to irradiate the blood, and the illumination area selectivity is lost, so that the photosensitizer using chitosan as a carrier is not suitable for treating the blood tumor even if the photosensitizer has targeting property and good controlled release property.

Disclosure of Invention

The invention aims to provide a photosensitizer compound and a preparation method and application thereof. The photosensitizer compound provided by the invention has good targeting property and high safety, can ensure that the selectivity of photodynamic therapy does not depend on an illumination area, ensures that the photodynamic therapy has cell selectivity, and can be well applied to the treatment of hemangioma.

The invention provides a photosensitizer compound, which is of a shell-core structure, takes chitosan as a hydrophilic skeleton, and is internally coupled with a hydrophobic compound through amido bonds, wherein the hydrophobic compound is coupled with a photosensitizer to form a core structure; the outer part of the chitosan hydrophilic skeleton acts with polar water molecules, and is modified by a specific antibody to form a shell structure.

Preferably, the hydrophobic compound is a polyene hydrophobic compound having two adjacent carboxyl groups.

Preferably, the polyene hydrophobic compound contains conjugated double bonds.

Preferably, the polyene hydrophobic compound is a carotenoid compound.

Preferably, the polyene hydrophobic compound is a 2-carboxycarotenoid.

Preferably, the photosensitizer is Ce 6.

Preferably, the specific antibody is an anti-CD 19 single-chain antibody, and the nucleotide sequence of a gene encoding the anti-CD 19 single-chain antibody is shown as SEQ ID NO. 3.

The invention also provides a preparation method of the photosensitizer compound in the technical scheme, which comprises the following steps:

1) mixing chitosan and a hydrophobic compound according to a mass ratio of (20-30): 4 for esterification reaction to obtain a graft polymer, and removing the residual hydrophobic compound through dialysis;

2) dissolving the graft polymer obtained in the step 1) in a polar solution to obtain a graft polymer solution with the concentration of 0.5-1.5 mg/mL, and dripping a photosensitizer into the graft polymer solution at the speed of 20-40 mu L/min to obtain a micelle loaded with the photosensitizer;

3) and (3) carrying out esterification reaction on the micelle loaded with the photosensitizer in the step 2) and a specific antibody to obtain a photosensitizer compound.

The invention also provides application of the photosensitizer compound in the technical scheme or the photosensitizer compound obtained by the preparation method in the technical scheme in preparation of a medicine for preventing and/or treating acute B lymphocyte leukemia.

The present invention provides a photosensitizer complex. The photosensitizer compound is of a shell-core structure, chitosan is used as a hydrophilic framework, a hydrophobic compound is coupled inside the hydrophilic framework of the chitosan through an amido bond, and the hydrophobic compound is coupled with a photosensitizer to form a core structure; the outer part of the chitosan hydrophilic skeleton reacts with polar water molecules to form a shell structure; the exterior of the chitosan hydrophilic skeleton is modified by a specific antibody. The photosensitizer compound provided by the invention enhances the stability of the photosensitizer through the arrangement of a shell-core structure; the modification of the specific antibody outside the hydrophilic chitosan skeleton improves the targeting property of the photosensitizer. In the whole blood circulation with neutral pH, the photosensitizer compound always keeps a stable structure, a hydrophobic compound forming an internal nuclear structure is contacted with the photosensitizer, and the action of the photosensitizer is inhibited through singlet oxygen quenching; when the chitosan-chitosan composite photosensitizer enters a cell, a hydrophobic group can be dissociated from chitosan in an acidic environment (pH 5-6) of a lysosome, and the inhibition effect on the photosensitizer is relieved. Test results show that the photosensitizer compound can circulate in blood for a long time and has cell dependence, namely, only the photosensitizer compound entering cells can generate singlet oxygen to cause toxicity to the cells under the excitation of the blood in vitro at specific wavelength, and the photosensitizer compound which is not absorbed in the blood and remains cannot cause damage to a blood system, so that the safety is high.

The photosensitizer compound provided by the invention does not depend on an illumination area, only the photosensitizer taken by cells can generate phototoxicity through the cell dependence of the photosensitizer compound, and the photosensitizer compound provided by the invention can realize the prevention and/or treatment of acute B lymphocyte leukemia.

Drawings

FIG. 1 is a nucleic acid electrophoresis diagram of recombinant vector construction expression provided in example 1 of the present invention; wherein Lane1 is a single chain antibody (MEDI-551-scFv); lane2 is a recombinant expression vector (pET-SUMO-MEDI-551-scFv);

FIG. 2 is a diagram showing the excision of the tags carried by WB detection before and after the enzyme digestion provided in embodiment 2 of the present invention; 2a is his tag in the product detected with anti-his antibody; 2b is the detection of the SUMO tag in the product with an anti-SUMO antibody; lane 1: after enzyme digestion, the effluent liquid of the product purified by a Ni column; lane 2: purifying a product by using a Ni column before enzyme digestion;

FIG. 3 is a SDS-PAGE electrophoresis result before and after the enzyme digestion provided in example 2 of the present invention; m: marker; lane 1: purifying a product by using a Ni column before enzyme digestion; lane 2: after enzyme digestion, the effluent liquid of the product purified by a Ni column;

FIG. 4 is an elution profile of the reaction product of the conjugated single-chain antibody provided in example 5 of the present invention on Sephadex G200 Sephadex;

fig. 5 is a schematic diagram of the preparation process and pH stability of the photosensitizer complex (anti cd19-CAR-CS-Ce6) provided in example 5 of the present invention: 5a is a preparation route of a photosensitizer complex (anti CD19-CAR-CS-Ce 6); 5b is a schematic diagram of photosensitizer complex (anti CD19-CAR-CS-Ce6) hydrolyzed under acidic conditions to release free Ce 6; 5c is a structural schematic diagram of the graft copolymer;

FIG. 6 is a graph of UV-vis absorption spectra of photosensitizer complex (anti CD19-CAR-CS-Ce6) provided in example 6 of the present invention;

FIG. 7 is a transmission electron microscope observation result graph of the photosensitizer complex (anti CD19-CAR-CS-Ce6) provided in example 7 of the present invention;

FIG. 8 is a standard curve of Ce6 concentration-fluorescence intensity provided in example 8 of the present invention;

FIG. 9 is a graph of the determination of Critical Micelle Concentration (CMC) provided in example 10 of the present invention;

FIG. 10 shows the variation of the release amount of Ce6 of the photosensitizer complex (anti CD19-CAR-CS-Ce6) according to example 11 of the present invention as a function of pH;

FIG. 11 shows the singlet oxygen release amount detection of Ce6, CAR-CS-Ce6, anti CD19-CAR-CS-Ce6 provided in example 12 of the present invention;

FIG. 12 shows the change of cellular uptake of CAR-CS-Ce6, anti CD19-CAR-CS-Ce6 by Raji cells and PBMCs provided in example 13 of the present invention over time;

FIG. 13 shows that MTT provided in example 14 of the present invention detects cytotoxicity of Raji cells and PBMCs of CAR-CS-Ce6, anti CD19-CAR-CS-Ce 6;

fig. 14 is a device for extracorporeal self-blood treatment according to example 14 of the present invention, wherein a is a main body of the device; b is an oxygen injection port; c is a blood transfusion device connecting port; e and D are the light emitting surfaces of the LED light sources.

Detailed Description

In the invention, the photosensitizer compound can form a micelle which is formed by taking a graft polymer as a main body, and the micelle wraps the photosensitizer; the method comprises the following steps of carrying out esterification reaction on a hydrophobic compound and chitosan, grafting through an amido bond to obtain a graft polymer, and wrapping a photosensitizer in the graft polymer through the hydrophobic bond of the hydrophobic compound to form a micelle loaded with the photosensitizer.

In the present invention, the micelle can improve the dispersibility of the photosensitizer compound in an aqueous solution. When the micelle is complete in shape, the photosensitizer has a singlet oxygen quenching effect on a specific excitation wavelength; when the micelle structure is destroyed, the corresponding photodynamic effect is restored for the specific excitation wavelength of the photosensitizer. That is, only when the micelle enters the cell, the micelle structure can be destroyed by the acidic environment of the lysosome due to the need of passing through a lysosome structure, and singlet oxygen can be generated only for the specific excitation wavelength of the photosensitizer, and the generated singlet oxygen can oxidize and destroy the result in the cell; when the micelle is dispersed in blood, the micelle structure is kept intact due to the neutral environment, and no singlet oxygen is generated for the specific excitation wavelength of the photosensitizer.

The photosensitizer compound provided by the invention takes chitosan as a hydrophilic skeleton, and the hydrophilic part outside the chitosan is an important molecule participating in cell recognition and signal conduction; the hydrophobic compound inside chitosan is similar to the long-chain hydrocarbon group of lecithin, and similar to the composition characteristics of cell membranes, and can enter cells in an endocytic form.

In the invention, a hydrophobic compound is coupled inside the chitosan hydrophilic skeleton through an amido bond to form a graft polymer; the amido bond is formed by the esterification reaction of amino inside a chitosan hydrophilic skeleton and a hydrophobic compound; the present invention is not particularly limited with respect to the amount of the hydrophobic compound bonded to the inside of the hydrophilic backbone of the chitosan.

In the present invention, the hydrophobic compound and the photosensitizer interact through a hydrophobic bond to form a coupling, resulting in a core structure.

In the invention, the polyene type hydrophobic compound is preferably a carotenoid compound, more preferably lycopene, β -carotene, lutein or canthaxanthin.

In the present invention, the polyene hydrophobic compound may also be a 2-carboxycarotenoid. The source of the 2-carboxycarotenoid is not particularly limited in the present invention, and a commercially available product of 2-carboxycarotenoid known to those skilled in the art may be used. In the invention, one carboxyl group of two adjacent carboxyl groups of the hydrophobic compound is used for forming an amido bond connection with chitosan of a hydrophilic skeleton, and the other carboxyl group is positioned at the ortho position of the formed amido bond, so that the structure is stable under neutral conditions and can be hydrolyzed under acidic conditions.

In the present invention, the photosensitizer is preferably Ce 6. The Ce6 is a hydrophobic photosensitizer and can be wrapped in a micelle formed by a graft polymer by a hydrophobic compound to form a core structure.

In the invention, the outer part of the hydrophilic skeleton of the chitosan reacts with polar water molecules to form a shell structure. In the present invention, the chitosan hydrophilic backbone interacts with polar water molecules, preferably through hydrogen bonds. In the invention, the shell structure acts like a hydration membrane, can protect the internal hydrophobic core structure and enhance the water solubility of the core structure.

In the invention, the chitosan hydrophilic skeleton is externally modified by a specific antibody. The specific antibody is an anti-CD 19 single-chain antibody, and the gene nucleotide sequence of the coded anti-CD 19 single-chain antibody is shown in SEQ ID NO. 3. In the present invention, the anti-CD 19 single chain antibody enables targeted uptake of the photosensitizer complex by cells expressing CD 19.

In the present invention, the coding sequence of the anti-CD 19 single-chain antibody is obtained by: the VH sequence (shown as SEQ ID NO: 1) and the VL sequence (shown as SEQ ID NO: 2) of the anti-CD 19 antibody (MEDI-551) were obtained from drug bank, and the C-terminal of VL and the N-terminal of VH were connected by a flexible peptide (G4S)3 to obtain the sequence (shown as SEQ ID NO: 3) of the anti-CD 19 single-chain antibody (MEDI-551-scFv). The sequence shown in SEQ ID NO. 3 is specifically:

the method for obtaining the anti-CD 19 single-chain antibody is not particularly limited in the present invention, and protein expression methods well known to those skilled in the art may be used. In the invention, the anti-CD 19 single-chain antibody is obtained by constructing an expression vector and expressing in a host bacterium. The expression vector is preferably used in anti-pET-SUMO, the construction method of the expression vector is not particularly limited, and the construction method of the expression vector, such as an enzyme digestion connection method, which is well known by a person skilled in the art can be adopted; specifically, the double enzyme cutting method preferably adopts BamHI and HinderIII enzyme cutting sites for constructing an expression vector. The choice of the host bacteria is not particularly limited in the present invention, and conventional protein expression strains known to those skilled in the art can be used, and the E.coli strain Origami 2(DE3) is preferably used in the present invention.

In the present invention, the anti-CD 19 single-chain antibody is obtained by fermentation, and the method of fermentation is not particularly limited in the present invention, and may be any conventional method for fermenting protein with escherichia coli, which is well known to those skilled in the art. The protein obtained by fermentation is preferably purified to obtain the anti-CD 19 single-chain antibody, and the purification method of the protein is not particularly limited, and a protein purification method well known to those skilled in the art, such as a nickel column purification method, can be adopted.

According to the invention, chitosan and a hydrophobic compound are mixed according to a mass ratio of (20-30): 4 for esterification reaction to obtain a graft polymer, and the residual hydrophobic compound is removed through dialysis. In the present invention, the mass ratio of the chitosan to the hydrophobic compound is preferably 25: 4. in the present invention, one of the carboxyl groups of the hydrophobic compound is linked to the 2-amino group of chitosan through an amide bond in the esterification reaction. The method of dialysis in the present invention is not particularly limited, and a conventional dialysis method known to those skilled in the art may be used.

After the graft polymer is obtained, dissolving the graft polymer in a polar solution to obtain a graft polymer solution with the concentration of 0.5-1.5 mg/mL, and dripping a photosensitizer into the graft polymer solution at the speed of 20-40 mu L/min to obtain a micelle loaded with the photosensitizer; in the present invention, the polar solution is preferably PBS, and the PBS is not particularly limited in the present invention, and conventional PBS well known to those skilled in the art may be used. In the present invention, the concentration of the graft polymer in the polar solution is preferably 1mg/mL, and the dropping rate of the photosensitizer is preferably 30. mu.L/min.

After the micelle loaded with the photosensitizer is obtained, the micelle loaded with the photosensitizer and the specific antibody are subjected to esterification reaction to obtain the photosensitizer compound. In the present invention, the specific antibody is preferably linked to the 2-amino group of the chitosan that does not participate in the reaction in the micelle through an amide bond.

Specifically, when the photosensitizer in the photosensitizer compound is Ce6, the nucleotide sequence of a gene encoding anti-CD 19 single-chain antibody is shown as SEQ ID NO:3, and the polyene hydrophobic compound is 2-carboxyl carotenoid, the preparation method of the corresponding photosensitizer compound comprises the following steps:

1) constructing an expression vector (pET-SUMO-MEDI-551-scFv) and an expression strain (Origami 2(DE 3)/pET-SUMO-MEDI-551-scFv);

2) obtaining corresponding anti-CD 19 single-chain antibody protein (MEDI-551-scFv) by using a Ni column purification-enzyme digestion-Ni column repurification mode, and proving that the protein is correctly expressed by using Western Blotting combined SDS-PAGE;

3) carrying out esterification reaction on 2-carboxyl carotenoid and chitosan to form amido bond, and obtaining a graft polymer: 2-carboxycarotenoid-chitosan (CAR-CS);

4) carrying out embedding reaction on the graft polymer and a photosensitizer to form a core structure with the inner part being acted by 2-carboxyl carotenoid and the photosensitizer and a shell structure with the outer part being hydrophilic chitosan, so as to obtain the micelle loaded with the photosensitizer: 2-carboxycarotenoid-chitosan-Ce 6 micelles (CAR-CS-Ce 6);

5) carrying out esterification reaction on the single-chain antibody (MEDI-551-scFv) and the loaded photosensitizer micelle obtained in the step 4) to form an amido bond, and obtaining a final product: anti-CD 19 single chain antibody modified 2-carboxy carotenoid-chitosan-Ce 6 micelles (anti CD19-CAR-CS-Ce 6).

The invention constructs an expression vector (pET-SUMO-MEDI-551-scFv) and an expression bacterium (Origami 2(DE 3)/pET-SUMO-MEDI-551-scFv). In the invention, before constructing an expression vector, the sequence of the antibody is preferably designed, the sequence of the coded antibody is shown as SEQ ID NO. 3, and the design method of the sequence shown as SEQ ID NO. 3 comprises the following steps: the VH sequence (shown as SEQ ID NO: 1) and the VL sequence (shown as SEQ ID NO: 2) of an anti-CD 19 antibody (MEDI-551) are obtained through drug bank, and then a flexible peptide (G) is obtained through an overlap PCR method₄S)₃The C-terminal of VL and the N-terminal of VH were connected to obtain the sequence shown in SEQ ID NO 3. In the present invention, the conditions for the overlap PCR are not particularly limited, and the overlap PCR reaction conditions known to those skilled in the art may be used.

After the expression bacteria are obtained, the corresponding anti-CD 19 single-chain antibody protein (MEDI-551-scFv) is obtained by a Ni column purification-enzyme digestion-Ni column repurification mode, and the protein is proved to be correctly expressed by Western Blotting combined with SDS-PAGE. The method of expressing an antibody protein using an expression strain of the present invention is not particularly limited, and a conventional method for culturing Escherichia coli and a method for inducing expression of a protein, which are well known to those skilled in the art, may be used. The Ni column purification method of the present invention is not particularly limited, and a Ni column purification method known to those skilled in the art may be used. The enzyme digestion method is not specially limited, and a protease digestion method known by a person skilled in the art is adopted, specifically, SUMO enzyme digestion sites are arranged between the protein His label obtained by expression in the invention and the protein, SUMO enzyme is selected for enzyme digestion, the enzyme digestion conditions are not specially limited, and the conventional enzyme digestion conditions of the SUMO enzyme known by the person skilled in the art are adopted.

The invention carries out esterification reaction on 2-carboxyl carotenoid and chitosan to form amido bond, and obtains a graft polymer: 2-carboxycarotenoid-chitosan (CAR-CS). The source of the 2-carboxycarotenoid and chitosan is not particularly limited in the present invention, and conventional commercial products of 2-carboxycarotenoid and chitosan known to those skilled in the art may be used. In the invention, the mass ratio of the chitosan to the 2-carboxyl carotenoid is preferably (20-30) to 4, more preferably 25: 4. the esterification reaction conditions in the present invention are not particularly limited, and the esterification reaction conditions known to those skilled in the art may be used.

After 2-carboxyl carotenoid-chitosan (CAR-CS) is obtained, the graft polymer and a photosensitizer are subjected to embedding reaction to form a core structure with the inner part being the action of the 2-carboxyl carotenoid and the photosensitizer and a shell structure with the outer part being hydrophilic chitosan, so that the photosensitizer-loaded micelle is obtained: 2-carboxycarotenoid-chitosan-Ce 6 micelles (CAR-CS-Ce 6). In the invention, the mass ratio of the 2-carboxyl carotenoid-chitosan to the photosensitizer is preferably (10-20): 1, more preferably 15: 1. in the present invention, the photosensitizer is preferably Ce 6. The conditions for the embedding reaction are not particularly limited in the present invention, and a conventional method for embedding reaction known to those skilled in the art may be used.

After obtaining the target single-chain antibody and the loaded photosensitizer micelle after the excision tag expression is correctly purified, the invention carries out esterification reaction on the single-chain antibody (MEDI-551-scFv) and the loaded photosensitizer micelle to form amido bond, and obtains the final product: anti-CD 19 single chain antibody modified 2-carboxy carotenoid-chitosan-Ce 6 micelles (anti CD19-CAR-CS-Ce 6). The esterification reaction conditions in the present invention are not particularly limited, and the esterification reaction conditions known to those skilled in the art may be used.

In the present invention, the photosensitizer complex is stable at neutral pH, undergoes about 30% dissociation at pH6.5, and gradually increases in dissociation to about 80% at pH below 6.0.

Specifically, when the photosensitizer complex of the present invention is anti cd19-CAR-CS-Ce6, the photosensitizer complex has pH sensitivity, the hydrophobic compound forming micelles is a 2-carboxyl carotenoid analog, one end of the hydrophobic compound participating in the reaction has 2 carboxyl groups, the hydrophobic compound is covalently bound to the main chain chitosan through amide bond formation of one of the carboxyl groups, and the other carboxyl group remains in a free state, and the micelle photosensitizer complex is easily hydrolyzed under acidic conditions (pH6.0) due to the amide bond having the carboxyl group at the ortho position, so that the original core-shell structure is lost due to dissociation of the hydrophobic compound at pH below 6.0, and the photosensitizer encapsulated therein is released.

When the photosensitizer compound anti CD19-CAR-CS-Ce6 is structurally stable, an active oxygen quenching effect is generated. Because the hydrophobic compound 2-carboxyl carotenoid analogue is a conjugated polyene hydrophobic compound, when the hydrophobic compound is in close contact with Ce6, a conjugated double bond can be well overlapped with a pyrrole ring in Ce6, and the energy generated by the absorption of a specific wavelength by Ce6 is transferred through a FRET effect, so that the active oxygen yield of Ce6 is quenched.

The dosage form of the drug is not particularly limited in the present invention, and a conventional dosage form of the drug known to those skilled in the art may be used. In the invention, the dosage form of the medicine is preferably freeze-dried powder, the medicine is preferably dissolved by adopting physiological saline under the condition of keeping out of the sun, and the concentration of the medicine is preferably 5-10 mg/mL.

The process of entry of the photosensitizer complex into the cell is: the photosensitizer complex is decomposed in an acidic environment of 2 organelles by targeting to the surface of a cell expressing CD19 through a surface modified anti-CD 19 single-chain antibody, then entering the cell through endocytosis and being sequentially encapsulated in 2 organelles including an endocytosis body (pH6.0) and a lysosome (pH5.0), and releasing a free photosensitizer Ce 6. In blood, the photosensitizer compound anti CD19-CAR-CS-Ce6 has the characteristic of cell dependence in stability, only enters cells to release free photosensitizer Ce6, and the corresponding singlet oxygen yield also has the characteristic of cell dependence, namely only the photosensitizer compound (anti CD19-CAR-CS-Ce6) taken by the cells can generate singlet oxygen under a specific excitation wavelength.

Specifically, in order to verify the treatment process and effect of the photosensitizer compound, the photosensitizer compound (anti CD19-CAR-CS-Ce6) is administered to a patient at a dose of 5-10 mg/mL, a part of blood of the patient is extracted after 24 hours, and the patient is irradiated with 671nm wavelength in vitro, so that singlet oxygen can be generated only by the photosensitizer compound entering target cells expressing CD19, and singlet oxygen cannot be generated by the photosensitizer compound (anti CD19-CAR-CS-Ce6) which is not taken in the blood, and thus, the target cells are damaged, and normal cells (normal white blood cells, red blood cells and the like) and other non-cellular components (platelets, plasma albumin and the like) in the blood are not damaged. Therefore, safety is provided. Because the photosensitizer compound (anti CD19-CAR-CS-Ce6) can circulate in blood for a long time, after one extracorporeal autoblood treatment, the residual photosensitizer compound (anti CD19-CAR-CS-Ce6) can be continuously taken up by target cells in the subsequent blood circulation, so that the effects of one-time administration and multiple treatments are achieved.

The anti-CD 33 single-chain antibody and photosensitizer complex and the preparation method thereof provided by the present invention are further described in detail with reference to the following embodiments, but the technical solutions of the present invention include, but are not limited to, the following embodiments.

Example 1

Construction of MEDI-551-scFv expression vector:

VH sequence (shown as SEQ ID NO: 1) and VL sequence of anti-CD 19 antibody (MEDI-551) were obtained by drug bank: (As shown in SEQ ID NO: 2), followed by a flexible peptide (G) by the overlap PCR method₄S)₃Connecting the C end of VL with the N end of VH to obtain a sequence (shown as SEQ ID NO: 3) of a single-chain antibody (MEDI-551-scFv) for resisting CD19, adding BamHI and HinderIII enzyme cutting sites at the 2 end of the sequence, inserting the sequence into an expression vector pET-SUMO in an enzyme cutting enzyme connection mode, and screening out a correct recombinant expression vector through colony PCR verification and sequencing: the result of pET-SUMO-MEDI-551-scFv electrophoresis is shown in FIG. 1, in which Lane1 is a single chain antibody (MEDI-551-scFv); lane2 is a recombinant expression vector (pET-SUMO-MEDI-551-scFv).

The recombinant expression vector was transformed into Origami 2(DE3) and the correct expression strain was selected using 50ug/mL kanamycin: origami 2(DE 3)/pET-SUMO-MEDI-551-scFv.

Example 2

Obtaining MEDI-551-scFv protein:

carrying out large-scale fermentation on an expression strain (Origami 2(DE3)/pET-SUMO-MEDI-551-scFv) for 2L, extracting cytoplasmic protein, and obtaining the corresponding single-chain antibody (MEDI-551-scFv) protein by adopting a Ni column purification-enzyme digestion-Ni column repurification mode because the 5' end of the target protein obtained by the expression vector is provided with a his label and then contains a SUMO enzyme digestion site.

The method comprises the following specific steps:

1) the cultured E.coli was collected and centrifuged at 8000rpm for 30min at 4 ℃ to obtain a bacterial pellet.

2) The pellet was resuspended in 20mL of cell lysate containing 0.1mg/mL lysozyme (50mM Tris-HCl,0.5M NaCl, 1% Triton X-100, pH 8.0) and sonicated for 10 min.

3) The mixture was magnetically stirred at 4 ℃ for 30min, then centrifuged at 14000rpm for 30min, and the supernatant was collected and filtered through a 0.22um filter.

4) The filtered supernatant was loaded onto a Ni column equilibrated with PBS in advance, and after the loading was finished, the column was washed with 5 column volumes of PBS.

5) Gradient elution was carried out with eluents having imidazole concentrations of 10mM, 20mM, 50mM, 100mM, 200mM, and 500mM in this order, and 10 tubes, 1 mL/tube were collected for each elution concentration.

6) And (3) determining elution components containing the target protein after SDS-PAGE verification of elution products of each tube, and combining the elution components to obtain a fused form of the target protein with a his tag at the 5 end and a subsequent SUMO enzyme cutting site.

7) The obtained solution was packed in a dialysis belt and dialyzed with 500ml PBS 3 times for 12 hours each time.

8) SUMO protease (the enzyme also contains His tag) was added to the resulting dialyzed product at 100IU/mL, and reacted at 37 ℃ for 10 hours, the reaction product was again adsorbed by Ni column, and the effluent was collected and purified by using an ultrafiltration tube (MWCO: 50kDa), ultrafiltration to obtain a concentrated solution, and WB verification of the concentrated product (results shown in FIG. 2: 2a is his tag in the product detected with anti-his antibody; 2b is the detection of the SUMO tag in the product with an anti-SUMO antibody; lane 1: after enzyme digestion, the effluent liquid of the product purified by a Ni column; lane 2: ni column purified product before cleavage), verified by SDS-PAGE (results see fig. 3: m: marker; lane 1: after enzyme digestion, the effluent liquid of the product purified by a Ni column; lane 2: ni column purification product before cleavage). The purification tag of the correctly expressed target protein is proved to be effectively cut off, and the final sequence of the obtained protein is shown as SEQ ID NO. 4.

Example 3

Synthesis of 2-carboxycarotenoid-chitosan (CAR-CS)

1) Chitosan 50mg dissolved in 20mL ddH was weighed out₂In O, 8mg of 2-carboxycarotenoid anhydride was weighed and dissolved in 20mL of 45 ℃ ethanol.

2) The above 2 solutions were mixed at 80 ℃ and stirred overnight with a magnetic stirrer at 250 rpm.

3) 250mL of cold ether was added to the reaction mixture to produce a precipitate, which was filtered and washed with 100mL of cold ether several times.

4) With 50ml ddH₂And (3) redissolving the precipitate, adjusting the pH of the reaction mixture to 10 by using 20% NaOH in an ice water bath, and performing suction filtration.

5) The filtrate is filled in a dialysis bag (MWCO is 10000), the dialysate is 10% ethanol solution, and the dialysis is carried out for 3 times to remove impurities in the reaction.

6) Subsequent dialysate was changed to ddH₂And O, dialyzing for 2 times again to remove residual ethanol.

7) The dialyzed product was lyophilized. The dark yellow product 2-carboxycarotenoid-chitosan (CAR-CS) was obtained.

Example 4

Synthesis of 2-carboxycarotenoid-chitosan-Ce 6 micelle (CAR-CS-Ce 6):

1) weighing 15mg of 2-carboxyl carotenoid-chitosan, dissolving in 15ml PBS solution, and magnetically stirring at 37 ℃ overnight to completely dissolve the 2-carboxyl carotenoid-chitosan; ce61mg was weighed and dissolved in 600. mu. LTHF solution to obtain Ce6 solution.

2) Slowly dripping the Ce6 solution into the 2-carboxyl carotenoid-chitosan solution, controlling the dripping speed at 30 mu L/min, and stirring and mixing by using a magnetic stirrer in the dripping process.

3) After the dropwise addition, stirring was continued at 37 ℃ for 24 hours to obtain 2-carboxycarotenoid-chitosan-Ce 6 micelle (CAR-CS-Ce 6).

Example 5

Synthesis of anti-CD 19 single chain antibody modified 2-carboxy carotenoid-chitosan-Ce 6 micelle (anti CD19-CAR-CS-Ce 6):

1) to the micelle solution prepared above, 0.15mL each of EDC solution (50mg/mL) and NHS solution (50mg/mL) was added, and after incubation at room temperature for 20min, 0.15mL of MEDI-551-scFv solution (0.16mg/mL) was added, and the mixture was gently stirred at room temperature for 1 hour,

2) the reaction product was passed through a Sephadex G200 Sephadex column, using 0.01M PBS (pH7.4) as an elution phase, and the eluate was collected at 2 mL/tube.

3) The absorption value of 280nm in each tube of eluent is detected, an elution curve is drawn, the result is shown in figure 4,

4) as can be seen, the front and back 2 elution peaks in the figure correspond to the micelles of the single chain antibody (MEDI-551-scFv) conjugated to anti-CD 19, and the free MEDI-551-scFv that did not participate in the reaction, respectively. And collecting the eluent in the tube corresponding to the mixed and first elution peak.

5) And (3) carrying out ultrafiltration and concentration on the combined phase to obtain the 2-carboxyl carotenoid-chitosan-Ce 6 micelle with the surface modified by MEDI-551-scFv.

The whole preparation process and the pH stability are shown in FIG. 5.

Example 6

UV-vis spectroscopic analysis:

a PBS solution of anti CD19-CAR-CS-Ce6 (concentration of 0.01mg/mL) was placed in a quartz dish, and the absorbance at a wavelength of 250-700nm was measured to plot a UV-vis curve, the results of which are shown in FIG. 6.

As can be seen, there are 5 absorption peaks, of which 280nm is a protein, i.e., a characteristic absorption peak of MEDI-551-scFv; the absorption peaks at 414 and 664nm are peculiar to Ce 6; the 472 nm and the 501nm are absorption peaks special for carotenoid; in the range of 260-320nm, there is a broad absorption band, and the protein absorption peak at 280 has some overlap, which is the absorption band of chitosan derivative.

Example 7

TEM inspection

Dissolve the sample in ddH₂O, and 3% phosphotungstic acid in a volume ratio of 50: 1, after mixed dyeing, dripping 3 mu L of mixed liquid on a copper net covered with a carbon film, and naturally drying; the morphology and size of the particles were observed by transmission electron microscopy. The results are shown in FIG. 7.

It can be seen that the prepared colloidal particles have smooth surfaces and are in a sphere-like shape.

Example 8

Determination of drug loading and encapsulation efficiency

The following Ce6 dilutions were diluted with PBS: 0.8, 1, 2, 4 and 5 mu g/mL, exciting with 667nm wavelength, detecting the fluorescence emission intensity at 715nm, drawing a Ce6 concentration-fluorescence intensity standard curve (the result is shown in FIG. 8) by taking the group as an abscissa and the detected corresponding fluorescence emission intensity as an ordinate, and obtaining a regression equation of Ce6 concentration-fluorescence intensity as follows: 0.464C3+1.736 (R)²＝0.9988)

And (3) centrifuging 500 mu L of nanoparticle suspension in a Millipore ultrafiltration centrifugal tube (MWCO is 10kDa) at 8000rpm for 40min, collecting filtrate, diluting the filtrate by 10 times with PBS, exciting the filtrate by using a 671nm wavelength, detecting the fluorescence emission intensity at 715nm, and converting the fluorescence emission intensity into the content of Ce6 to obtain the free dosage of Ce 6.

The encapsulation rate is (Ce6 total dosage-Ce 6 free dosage)/Ce 6 total dosage × 100, and the drug loading rate is (Ce6 total dosage-Ce 6 free dosage)/nanoparticle mass × 100.

The results show that the encapsulation efficiency is: (64.2 ± 1.6)%; the drug loading rate is as follows: (6.5. + -. 1.6)%.

Example 9

Measurement of average particle diameter and Zeta potential

The synthesized anti CD19-CAR-CS-Ce6 was dissolved in PBS to obtain a solution with a concentration of 1 mg/mL. The average hydrated particle size of the compound was measured using a dynamic light scattering instrument (DLS).

As a result, the average particle diameter was (35.4. + -. 2.4) nm, and the average Zeta potential was (23.5. + -. 1.7) mV.

Example 10

Critical Micelle Concentration (CMC) determination

Pyrene fluorescence was used. Adding 30 mu mol/L pyrene acetone solution into a series of 15mL centrifuge tubes, 100 mu L/tube, completely volatilizing the acetone at 37 ℃, adding 5mL of anti CD19-CAR-CS-Ce6 solutions with different mass concentrations (0.01, 0.02, 0.04, 0.06, 0.08, 0.1, 0.2, 0.4, 0.6, 0.8 and 1mg/mL), carrying out ultrasonic treatment at room temperature for 40min, then carrying out constant temperature in a water bath at 65 ℃ for 3h, cooling at room temperature, standing for 12h, exciting by using a wavelength of 334nm, detecting the fluorescence intensity ratio of the solutions at 373nm and 384nm, drawing a curve by taking the ratio as an ordinate and taking the logarithm of the mass concentration of the corresponding micelle in each solution as an abscissa, and determining the CMC value of the carrier to be 2.4 mu g/L as shown in FIG. 9.

Example 11

The amount of Ce6 released varied with pH:

1) 6.0mL of anti CD19-CAR-CS-Ce6 (containing 1.5mg of Ce6) is put into 6 dialysis bags and respectively put into 6 dialysis solutions of 150mL with different pH values, the dialysis medium is PBS buffer solution, and the pH values are sequentially; 5.0, 5.5, 6.0, 6.5, 7.0, 7.5.

2) 1.5mL of the dialysate was removed at l, 2, 3, 4, and 5 hours, and the same volume of dialysate corresponding to pH was added back.

3) The dialysate taken out at different time points was excited with light having an excitation wavelength of 671nm, and the emission intensity at 715nm was detected. And calculating the corresponding Ce6 content according to a Ce6 concentration-fluorescence intensity standard curve.

4) The measured ratio of the Ce6 content to the total Ce6 was used as the release amount, and the release amount of anti cd19-CAR-CS-Ce6 in different pH environments was plotted using different time points as abscissa and corresponding Ce6 release amount as ordinate, with the results shown in fig. 10.

It can be seen that the release was very low, not substantially exceeding 10%, and not significant over time at pH 7.0 and 7.5; at 6.5, the amount released increases with time, but eventually does not exceed 30%; the release of the drug increased significantly after pH6.0, reaching 60% after 1h at pH 5.5 and 80% after 1h at pH 5.0.

Example 12

Singlet oxygen release detection

The method for detecting the release amount of singlet oxygen by using RNO (ribonucleic acid) decoloration reaction comprises the following specific steps:

1) dissolving RNO in heavy water (D)₂O), a solution A was prepared at a final concentration of 250. mu.M.

2) Dissolving histidine in heavy water (D)₂O), a solution B was prepared to a final concentration of 0.03M.

3) Mixing the solution A and the solution B according to a volume ratio of 1: 3 to obtain a solution C.

4) 6.67. mu.M free Ce6, CAR-CS-Ce6 and anti CD19-CAR-CS-Ce6, respectively, dissolved in 700. mu.L of heavy water containing 1% DMSO, followed by addition of 400. mu.L of solution C to the corresponding test group, placing the mixed solution in a quartz dish and immediately detecting the luminescence intensity at 440nm, recorded as the initial luminescence intensity.

5) The wavelength is 671nm and the intensity is 6J/cm²The solution in the quartz cell was irradiated for 30min and during this time, the luminescence intensity at 440nm was detected every 2min, the ratio of the singlet oxygen production at each time point was (initial luminescence intensity-luminescence intensity at that time point)/initial luminescence intensity × 100%.

6) And taking the time as an abscissa and the singlet oxygen yield ratio of the time point as an ordinate to obtain a singlet oxygen yield curve. The results are shown in FIG. 11.

It can be seen that the singlet oxygen release amount of the simple photosensitizer Ce6 is remarkably increased along with time, and the release amount at 30min reaches about 85%.

The release amount of the singlet oxygen of CAR-CS-Ce6 is very low, and the maximum is not more than 20%; the singlet oxygen release amount of the anti CD19-CAR-CS-Ce6 is lower, the maximum singlet oxygen release amount is not more than 15%, and the time dependence is not existed,

probably, because the carotenoid in the CAR-CS-Ce6 colloid has good photobleaching effect on the encapsulated Ce6, the energy generated by the absorption of corresponding wavelengths by the Ce6 is mostly transferred to the carotenoid instead of surrounding oxygen atoms through FRET effect, and the yield of singlet oxygen is greatly reduced. In the anti CD19-CAR-CS-Ce6 colloid, the outer surface is modified by hydrophilic anti CD19, so that the structure of the micelle is more compact, the photobleaching effect of carotenoid on Ce6 is stronger, and the yield of singlet oxygen is lower.

Example 13

Measurement of cellular uptake

Since Raji cells are B-ALL cells that have been reported to express CD19 molecules at high surface levels, PBMC has little expression of CD19 molecules at the surface.

1) The 5 × 107 cells of Raji in logarithmic growth phase after PBS washing were collected, centrifuged at 3000rpm for 5min to obtain a cell pellet, and 1mL of cell lysate (0.1M NaOH, 1% SDS) was added to obtain a cell lysate.

2) Adding 10 μ L of CAR-CS-Ce6 and anti CD19-CAR-CS-Ce6 with different dosages into 100 μ L of the cell lysate to sequentially obtain lysate mixtures with CAR-CS-Ce6 final concentrations of 0.8, 1, 2, 4 and 5 μ g/mL,

3) the mixture was sonicated for 40min, then centrifuged at 12000rpm for 30min and the supernatant was taken.

4) And (3) detecting the absorbance value of the corresponding concentration at 715nm of the supernatant under the excitation wavelength of 671nm to obtain the relation between the micelle concentration and the absorbance value in the lysate.

5) Raji cells in logarithmic growth phase were plated in 6 wells of 6-well plates, 1 × 106 cells/well.

6) After 24h, the fresh medium was replaced, CAR-CS-Ce6 diluted with PBS was added and incubated for additional 0h, 1h, 2h, 4h, 8h, 16h, 24 h.

7) When the sampling time point is reached, transferring the cell suspension in the corresponding hole to a clean EP tube, centrifuging at 3000rpm for 5min to obtain cell sediment, washing for 2 times by PBS, and then obtaining the corresponding micelle content according to the steps 1), 3) and 4).

8) And (3) obtaining a change curve of Raji cell uptake of the 2 micelles along with time by taking different time points as abscissa and corresponding micelle content as ordinate.

9) In the same manner, the time-dependent intake of PBMC of 2 kinds of micelles was obtained. The results are shown in FIG. 12.

As can be seen, the leukemia Raji cell has high intake of anti CD19-CAR-CS-Ce6, about 40% in 4h and strong time dependence, and the intake of 24h is about 82%; in contrast, the intake of CAR-CS-Ce6 is low, the increase of the intake with time is gentle, and the intake of 24h is only about 30%; the uptake of these 2 micelles by normal cells was not high and varied only slightly over time.

The anti CD19-CAR-CS-Ce6 micelle is modified by a specific antibody, so that the anti CD19-CAR-CS-Ce6 micelle has a good leukemia cell targeting function, and meanwhile, the aggregation in normal cells is reduced.

Example 14

Cytotoxicity assays

The cytotoxicity of CAR-CS-Ce6 and anti CD19-CAR-CS-Ce6 on Raji cells and healthy human PBMC was examined,

1) laying Raji cells in logarithmic growth phase in 96-well plate, 5000 cells/well, culturing for 24 hr,

2) centrifuging at 3000rpm for 5min, removing supernatant, replacing with serum-free 1640 medium, dividing each cell into 3 groups, adding PBS, equimolar amounts of CAR-CS-Ce6 and anti CD19-CAR-CS-Ce6 to each group of 3 wells. So that the final concentration of Ce6 is 5ug/mL,

3) After incubation in the incubator for 1h in the absence of light, the medium was replaced with fresh 1640+ 10% FBS medium at 100. mu.L/well, and incubated in the incubator for 24h in the absence of light.

4) Irradiating with 671nm wavelength light for 5min at an irradiation energy of 9J/cm 2. After that, the cells were incubated in an incubator for 2 hours without light.

5) Adding 11 mu of LMTT solution (5mg/mL) into each hole, incubating for 4h, centrifuging at 3000rpm for 5min, discarding the supernatant, adding 200 mu of DMSO into each hole to dissolve the formazan, and detecting the absorbance at 570nm of an microplate reader, wherein the cell inhibition ratio (%) -1-absorbance value of the experimental group/absorbance value of the control group is × 100%.

6) The cytotoxicity of the above 2 kinds of micelles to PBMC of healthy people was examined in the same manner,

the results are shown in FIG. 13. As can be seen, the anti CD19-CAR-CS-Ce6 has strong toxicity to Raji cells, and the inhibition rate is as high as 90%. The inhibition rate of CAR-CS-Ce6 on Raji cells is relatively low, and is about 50%; the 2 micelles all showed low toxicity to PBMC, with an inhibition rate of about 10%. The anti CD19-CAR-CS-Ce6 has certain cytotoxicity to the partial cells because the normal human blood also contains a very small amount of pre-B cells expressing CD19, but has little influence on normal components in the human blood because the normal hematopoietic stem cells and mature B cells are not influenced.

The cytotoxicity exhibited by these 2 micelles was consistent with the cellular uptake rate results. The colloid modified by the antibody has targeting property, thereby showing more efficient and specific killing power.

Example 15

Extracorporeal circulation photodynamic therapy:

with one treatment device, as shown in fig. 14, 400mL of blood can be contained, and the basic material is polydimethylsiloxane, which has good optical transparency.

The body A of the device, which has a flat structure with an internal dimension of 40cm × 40cm × 0.25cm, i.e. a length and a width of 40cm, and a thickness of 0.25 cm., the upper and lower 2 faces being irradiation faces, has 2 ports (B and C) on its side for injecting oxygen, and connecting to a blood transfusion set, respectively.

The light source of the therapeutic device is an LED light emitting array with the same irradiation area (40cm × 40cm), and the light source density is that per cm²There are 16 LED micro-LEDs with an emission wavelength of 671 nm. When in use, the utility model is used,the light emitting surfaces (D and E) of the 2 light sources were closely attached to the upper and lower 2 irradiation surfaces of the treatment main body a.

The specific treatment process is as follows:

1) a patient with B-ALL in which the expression of CD19 was detected was intravenously infused with 10mg/Kg of anti CD19-CAR-CS-Ce6 after 0.22 μm filtration, and after administration, the patient was protected from light for 24 hours.

2) 350mL of blood was drawn from the patient and C-terminally perfused into the above-described treatment device containing 50mL of anticoagulant.

3) Injecting 1mL of pure oxygen filtered by 0.22 μm into the B end, and mixing the mixture 3-4 times clockwise.

4) The treatment device is placed in a dark room, and the light source is used for irradiating for 3 times, each time for 3min, and the interval between the two times is 1 min. The irradiated autologous blood is then returned to the patient as a treatment.

After each administration, the patients need to receive a course of treatment, including 5 treatments, with 30min intervals between the two treatments.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

SEQUENCE LISTING

<110> Lissvin

<120> photosensitizer compound and preparation method and application thereof

<130>2017

<160>4

<170>PatentIn version 3.3

<210>1

<211>363

<212>DNA

<213> Artificial sequence

<400>1

gaagtgcagc tggtggaaag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60

agctgcgcgg cgagcggctt tacctttagc agcagctgga tgaactgggt gcgccaggcg 120

ccgggcaaag gcctggaatg ggtgggccgc atttatccgg gcgatggcga taccaactat 180

aacgtgaaat ttaaaggccg ctttaccatt agccgcgatg atagcaaaaa cagcctgtat 240

ctgcagatga acagcctgaa aaccgaagat accgcggtgt attattgcgc gcgcagcggc 300

tttattacca ccgtgcgcga ttttgattat tggggccagg gcaccctggt gaccgtgagc 360

agc 363

<210>2

<211>333

<212>DNA

<213> Artificial sequence

<400>2

gaaattgtgc tgacccagag cccggatttt cagagcgtga ccccgaaaga aaaagtgacc 60

attacctgcc gcgcgagcga aagcgtggat acctttggca ttagctttat gaactggttt 120

cagcagaaac cggatcagag cccgaaactg ctgattcatg aagcgagcaa ccagggcagc 180

ggcgtgccga gccgctttag cggcagcggc agcggcaccg attttaccct gaccattaac 240

agcctggaag cggaagatgc ggcgacctat tattgccagc agagcaaaga agtgccgttt 300

acctttggcg gcggcaccaa agtggaaatt aaa 333

<210>3

<211>757

<212>DNA

<213> Artificial sequence

<400>3

ggatccatgg aaattgtgct gacccagagc ccggattttc agagcgtgac cccgaaagaa 60

aaagtgacca ttacctgccg cgcgagcgaa agcgtggata cctttggcat tagctttatg 120

aactggtttc agcagaaacc ggatcagagc ccgaaactgc tgattcatga agcgagcaac 180

cagggcagcg gcgtgccgag ccgctttagc ggcagcggca gcggcaccga ttttaccctg 240

accattaaca gcctggaagc ggaagatgcg gcgacctatt attgccagca gagcaaagaa 300

gtgccgttta cctttggcgg cggcaccaaa gtggaaatta aaggcggcgg cggcagcggc 360

ggcggcggca gcggcggcgg cggcagcgaa gtgcagctgg tggaaagcgg cggcggcctg 420

gtgcagccgg gcggcagcct gcgcctgagc tgcgcggcga gcggctttac ctttagcagc 480

agctggatga actgggtgcg ccaggcgccg ggcaaaggcc tggaatgggt gggccgcatt 540

tatccgggcg atggcgatac caactataac gtgaaattta aaggccgctt taccattagc 600

cgcgatgata gcaaaaacag cctgtatctg cagatgaaca gcctgaaaac cgaagatacc 660

gcggtgtatt attgcgcgcg cagcggcttt attaccaccg tgcgcgattt tgattattgg 720

ggccagggca ccctggtgac cgtgagcagc taagctt 757

<210>4

<211>247

<212>PRT

<213> Artificial sequence

<400>4

Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Asp Thr Phe

20 25 30

Gly Ile Ser Phe Met Asn Trp Phe Gln Gln Lys Pro Asp Gln Ser Pro

35 40 45

Lys Leu Leu Ile His Glu Ala Ser Asn Gln Gly Ser Gly Val Pro Ser

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn

6570 75 80

Ser Leu Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Lys

85 90 95

Glu Val Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Gly

100 105 110

Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val

115 120 125

Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu

130 135 140

Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Ser Trp Met

145 150 155 160

Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Gly Arg

165 170 175

Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Val Lys Phe Lys Gly

180 185 190

Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser Leu Tyr Leu Gln

195 200 205

Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg

210 215 220

Ser Gly Phe Ile Thr Thr Val Arg Asp Phe Asp Tyr Trp Gly Gln Gly

225 230235 240

Thr Leu Val Thr Val Ser Ser

245

Claims

1. A photosensitizer compound is of a shell-core structure, chitosan is used as a hydrophilic framework, a hydrophobic compound is coupled inside the hydrophilic framework of the chitosan through an amido bond, and the hydrophobic compound is coupled with a photosensitizer to form a core structure; the outer part of the chitosan hydrophilic skeleton acts with polar water molecules, and is modified by a specific antibody to form a shell structure;

the hydrophobic compound is a polyene hydrophobic compound and has two adjacent carboxyl groups;

the photosensitizer is Ce 6.

2. The photosensitizer complex according to claim 1, wherein the polyene-based hydrophobic compound contains conjugated double bonds.

3. The photosensitizer complex according to claim 2, wherein the polyene hydrophobic compound is a carotenoid compound.

4. The photosensitizer complex according to claim 3, wherein the polyene hydrophobic compound is a 2-carboxycarotenoid.

5. The photosensitizer complex of claim 1, wherein the specific antibody is an anti-CD 19 single-chain antibody, and the nucleotide sequence of the gene encoding the anti-CD 19 single-chain antibody is shown in SEQ ID NO:3, respectively.

6. A process for preparing a photosensitizer complex according to any one of claims 1 to 5, comprising the steps of:

1) mixing chitosan and a hydrophobic compound according to the mass ratio of (20-30) to 4 for esterification reaction to obtain a graft polymer, and removing the residual hydrophobic compound through dialysis;

7. Use of the photosensitizer compound according to any one of claims 1 to 5 or the photosensitizer compound obtained by the preparation method according to claim 6 in the preparation of a medicament for preventing and/or treating acute B-lymphocyte leukemia.