CN111040180B - Biological cascade reaction type photodynamic integrated biopolymer and preparation method and application thereof - Google Patents

Biological cascade reaction type photodynamic integrated biopolymer and preparation method and application thereof Download PDF

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CN111040180B
CN111040180B CN202010042976.0A CN202010042976A CN111040180B CN 111040180 B CN111040180 B CN 111040180B CN 202010042976 A CN202010042976 A CN 202010042976A CN 111040180 B CN111040180 B CN 111040180B
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CN111040180A (en
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罗忠
王璇
侯彦华
李孟桓
胡燕
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Chongqing University
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    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
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    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/337—Polymers modified by chemical after-treatment with organic compounds containing other elements

Abstract

The invention relates to a biological cascade reaction type photodynamic integrated biopolymer and a preparation method and application thereof, belonging to the field of medicine preparation. The invention discloses a biological cascade reaction type photodynamic integrated biopolymer (PEG)4000CDM-ZnPc-S-S-HA), the photosensitizer zinc phthalocyanine (ZnPc) being linked to PEG via a pH-acid responsive maleic acid amide linker4000Combining, and then fixing the disulfide linker on a Hyaluronic Acid (HA) chain by using a redox cleavable disulfide linker, wherein a PEG section can enhance the blood circulation of a molecular carrier after intravenous administration and fall off after reaching an acid tumor microenvironment, so that the residual ZnPc-S-S-HA is self-assembled into a large cluster in situ, reverse diffusion is avoided, and the tumor retention rate is improved; while HA is used as a carrier substrate and tumor targeting ligand due to its hydrophilicity, degradability and ability to specifically bind to overexpressed CD44 on tumor cell membranes.
Figure DDA0002368403450000011

Description

Biological cascade reaction type photodynamic integrated biopolymer and preparation method and application thereof
Technical Field
The invention belongs to the field of medicine preparation, and particularly relates to a biological cascade reaction type photodynamic integrated biopolymer and a preparation method and application thereof.
Background
The size of the nano material is a key factor for determining the interaction mode of the nano material with the biological environment, and can fundamentally influence the functional performance of the nano material in clinical application. From a clinical point of view, large-sized nanoparticles (20-200nm) generally exhibit better tumor aggregation effects due to Enhanced Permeation and Retention (EPR) effect, while small-sized nanoparticles (<10nm) tend to have stronger tumor penetration ability due to better diffusion fluidity. The advent of supramolecular nanotechnology offers new opportunities to combine the advantages of large and small nano-biomaterials, most commonly using self-assembly of nanoparticles for size conversion. These resizable nano-assemblies are typically triggered when the tumor microenvironment is specifically stimulated, where they undergo specific biochemical transformations and aggregate/decompose into specific topological nanostructures for the desired purpose, thereby better killing the tumor.
Furthermore, photodynamic therapy (PDT) is a clinically approved method for the treatment of a variety of cancer indications. However, its clinical transformation is hampered by a series of problems associated with the delivery and driving of photoactivatable Photosensitizers (PS), which are generally highly hydrophobic and tend to aggregate in aqueous environments, leading to rapid clearance and self-quenching; an optimal PDT nanopreparative strategy should therefore be able to have a high tumor specificity during systemic redistribution while being able to deliver PS molecules in a single molecule after tumor uptake.
Therefore, a biological cascade reaction type photodynamic integrated biopolymer can be synthesized, can form long-time aggregates in situ in tumor tissues when being used for tumor specific photodynamic therapy, and generates ROS (reactive oxygen species) to kill tumors through PDT under the irradiation of near infrared light.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a bio-cascade reaction type photodynamic integrated biopolymer; the second purpose of the invention is to provide a preparation method of the biological cascade reaction type photodynamic integrated biopolymer; the invention also aims to provide application of the biological cascade reaction type photodynamic integrated biopolymer in preparation of antitumor drugs.
In order to achieve the purpose, the invention provides the following technical scheme:
1. a biological cascade reaction type photodynamic integrated biopolymer has a structural formula as follows:
Figure BDA0002368403430000021
where n is 113.
2. The preparation method of the biological cascade reaction type photodynamic integrated biopolymer comprises the following steps:
(1) preparation of PEG4000-CDM-ZnPc: mixing CDM-PEG4000And tetra-amino zinc phthalocyanine (ZnPc) are fully dissolved in anhydrous tetrahydrofuran, stirred and reacted for 24 hours under the protection of nitrogen, vacuum drying is carried out after the reaction is finished, then the obtained product is dissolved in dichloromethane, and the obtained product is centrifuged to obtain a solid which is dried in vacuum;
(2) preparation of PEG4000-CDM-ZnPc-S-: dissolving carboxyl dithiopyridine in DMF, adding N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), reacting to activate carboxyl for 30min, and adding the PEG in step (1)4000And (3) carrying out stirring reaction on the CDM-ZnPc for 12-24 h at normal temperature under the protection of nitrogen, after the reaction is finished, carrying out vacuum drying to remove a DMF solvent, continuously dissolving the DMF solvent in water, dialyzing to remove excessive EDC, NHS and carboxyl dithiopyridine, and carrying out vacuum freeze drying to obtain a dark green solid, namely the product PEG4000-CDM-ZnPc-S-S-；
(3) Preparation of PEG4000-CDM-ZnPc-S-S-HA: thiolated hyaluronic acid (HA-HS) was dissolved in PBS, PEG4000dissolving-CDM-ZnPc-S-S-in DMF, mixing, stirring under nitrogen protection at 25 deg.C for 48h, dialyzing for 4d after reaction, precipitating with ethanol, and freeze drying to obtain product polymer PEG4000-CDM-ZnPc-S-S-HA。
Preferably, the CDM-PEG in step (1)4000The molar ratio of the ZnPc to the ZnPc is 1: 4; the carboxyl dithiopyridine, NHS, EDC and PEG in the step (2)4000-the molar ratio of CDM to ZnPc is 1:5:5: 2; the HA-HS and PEG in the step (3)4000The molar ratio of-CDM-ZnPc-S-S-is 2: 1.
Preferably, the CDM-PEG in step (1)4000The preparation method comprises the following steps:
(1) dissolving 2-propionyloxy-3-methylbutenedioic anhydride (CDM) in anhydrous dichloromethane, adding oxalyl chloride and a catalyst DMF, reacting on ice for 30min, transferring to room temperature for reaction for 6h to obtain acyl chloride CDM, and drying in vacuum for later use;
(2) dissolving the acyl chloride CDM and the polyethylene glycol monomethyl ether 4000 dried in the step (1) in anhydrous dichloromethane, adding pyridine as a catalyst, reacting for 6 hours at 25 ℃, adding a saturated ammonium chloride solution for extraction after the reaction is finished, collecting an organic phase, and drying in vacuum;
(3) fully dissolving the dried product of the organic phase in the step (2) in dichloromethane, precipitating twice with anhydrous ether, and vacuum drying to obtain a white powder product, i.e. CDM-PEG4000；
Further preferably, the mass-to-volume ratio of CDM, oxalyl chloride, catalyst DMF, polyethylene glycol monomethyl ether 4000 and pyridine is 0.276:0.378:40:0.8:30, g: g: mL: g: mL.
Preferably, the tetraaminozinc phthalocyanine in step (1) is prepared as follows:
(1) mixing 4-nitrophthalimide, urea and ammonium molybdate, heating and melting, adding zinc chloride hexahydrate, heating and reacting at 160 ℃ until no bubbles are generated, cooling, washing with 1mol/L HCL and 1mol/L NaOH solution in sequence, washing with ultrapure water to be neutral, and drying in vacuum to obtain a bluish purple product;
(2) and mixing and heating the bluish violet product and sodium sulfide nonahydrate, adding DMF (dimethyl formamide) for full dissolution, continuously heating, accelerating stirring for 1h when the temperature is 60 ℃, washing with water to be neutral, and drying in vacuum to obtain a dark green solid substance, namely ZnPc.
Further preferably, the mass ratio of the 4-nitrophthalimide to the urea to the ammonium molybdate to the zinc chloride hexahydrate to the sodium sulfide nonahydrate is as follows: 4.1:10:0.05:1.2:2.88.
Preferably, the carboxyl dithiopyridine in the step (2) is prepared according to the following method: dissolving dithiodipyridine in ethanol, adding acetic acid, dropwise adding an ethanol solution of 3-mercaptoacetic acid, reacting at room temperature for 20h, vacuum drying to remove an ethanol solvent, and purifying by column chromatography to obtain the product carboxyl dithiopyridine.
Further preferably, the mass-volume ratio of the dithiodipyridine to the acetic acid to the 3-mercaptoacetic acid is 3.75:0.4:0.9, and the mass-volume ratio of the dithiodipyridine to the acetic acid to the 3-mercaptoacetic acid to the g is 3: 2.
Preferably, the molecular weight of the dialysis bag in the dialysis in the step (2) is 1000, and the dialysis time is 3 d.
Preferably, the thiolated hyaluronic acid in step (3) is prepared as follows: adding hyaluronic acid HA into a sextuple dimethyl buffer solution with the pH value of 5 for full dissolution, then adding N-hydroxy thiosuccinimide (EDC) and N-hydroxy succinimide (NHS), reacting and activating for 3-6 h, continuously adding mercaptan ammonia hydrochloride, reacting for 24h under the protection of nitrogen, dialyzing for 2-4 d, and precipitating with ethanol to obtain the product.
Further preferably, the mass ratio of the hyaluronic acid, EDC, N-hydroxysuccinimide (NHS) and mercaptamine hydrochloride is 0.2:0.5:0.3:0.3, and the molecular weight of the dialysis bag used for dialysis is 1000.
Preferably, the molecular weight of the dialysis bag used in the dialysis in the step (3) is 3000.
3. The application of the biological cascade reaction type photodynamic integrated biopolymer in preparing antitumor drugs.
The invention has the beneficial effects that:
1. the invention provides a biological cascade reaction type photodynamic integrated biopolymer PEG4000CDM-ZnPc-S-S-HA, coupling the photosensitizer zinc phthalocyanine (ZnPc) to PEG via a pH-acid responsive maleic acid amide linker4000Combining, and then fixing the disulfide linker on a Hyaluronic Acid (HA) chain by using a redox cleavable disulfide linker, wherein a PEG section can enhance the blood circulation of a molecular carrier after intravenous administration and fall off after reaching an acid tumor microenvironment, so that the residual ZnPc-S-S-HA is self-assembled into a large cluster in situ, reverse diffusion is avoided, and the tumor retention rate is improved; while HA is used as a carrier substrate and tumor targeting ligand due to its hydrophilicity, degradability and ability to specifically bind to overexpressed CD44 on tumor cell membranes. But originally send outThe PEG of the Ming polymer is hydrophilic due to the HA and PEG fragments, and after intravenous administration4000The dispersity of the-CDM-ZnPc-S-S-HA in body fluid is high, and the cycle life is long. When reaching a tumor microenvironment, the pH value of an acidic environment can remove PEG fragments by cracking the maleic acid amide linker, so that the overall hydrophobicity is sharply increased, the steric hindrance is reduced, the remaining ZnPc-S-S-HA is self-assembled into clusters under the driving of hydrophobic interaction among ZnPc molecules, the retention of a PS-carrier in tumor tissues is enhanced, the aggregated ZnPc-S-S-HA can be effectively internalized by tumor cells through hyaluronic acid-mediated endocytosis, and then high-concentration Glutathione (GSH) in the cells can crack disulfide bonds and release ZnPc molecules in a non-aggregated form, so that the curative effect of PDT is optimized. This polymerization-activated sequential tumor targeting is expected to improve the efficiency and specificity of PS delivery to tumor tissue, while further increasing ROS production under mild stimulation to better kill tumor cells.
2. The invention also provides a preparation method of the biological cascade reaction type photodynamic integrated biopolymer, the reactant source is wide, the operation is simple, the mass production can be carried out, and the preparation method is beneficial to the polymer PEG4000Industrial application of CDM-ZnPc-S-S-HA.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention may be realized and attained by the means of the instrumentalities and combinations particularly pointed out hereinafter.
Drawings
For the purposes of promoting a better understanding of the objects, aspects and advantages of the invention, reference will now be made to the following detailed description taken in conjunction with the accompanying drawings in which:
FIG. 1 is a drawing of the Polymer (PEG) prepared in example 14000-CDM-ZnPc-S-HA), wherein a is a TEM image of the polymer, b is a TEM image of the polymer after treatment under acidic conditions at pH 6.5, c is a SEM image of the polymer after treatment under acidic conditions at pH 6.5;
FIG. 2 is a diagram showing the ultraviolet absorption of the intermediate products ZnPc-S-S-HA and ZnPc;
FIG. 3 is a Polymer (PEG) prepared in example 14000-CDM-ZnPc-S-HA) particle size distribution in solutions at pH 6.5 and pH 7.4, respectively;
FIG. 4 is a graph showing the phagocytic effect of tumor cells, in which a 1-a 4 are the effects of a blank control without any substance added thereto, and b 1-b 4 are the results of adding the Polymer (PEG) prepared in example 14000Effect pattern of CDM-ZnPc-S-S-HA, c 1-c 4 is an additive Polymer (PEG)4000-CDM-ZnPc-S-HA) effect diagram of the material after acid treatment at pH 6.5;
FIG. 5 Polymer (PEG) prepared in example 14000-CDM-ZnPc-S-HA) toxicity effect graph on cells under light and non-light conditions;
FIG. 6 shows the toxic effect of the intermediate (ZnPc-S-S-HA) on cells in both light and non-light conditions.
Detailed Description
The embodiments of the present invention are described below with reference to specific embodiments, and other advantages and effects of the present invention will be easily understood by those skilled in the art from the disclosure of the present specification. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention. It should be noted that the drawings provided in the following embodiments are only for illustrating the basic idea of the present invention in a schematic way, and the features in the following embodiments and examples may be combined with each other without conflict.
Example 1
Preparation of biological cascade reaction type photodynamic integrated biopolymer (PEG)4000-CDM-ZnPc-S-HA) according to the following method:
1. preparation of tetraaminozinc phthalocyanine (ZnPc):
(1) 4.1g of 4-nitrophthalimide, 10g of urea and 0.05g of ammonium molybdate are placed in a three-neck flask, heated and melted, 1.2g of zinc chloride hexahydrate is added, heated to 160 ℃, continuously reacted until no bubbles are generated in the flask, washed by 1mol/L of HCL and 1mol/L of NaOH solution in sequence after being cooled, washed to be neutral by ultrapure water, and dried in vacuum to obtain a bluish purple product;
(2) adding the bluish purple product and 2.88g of sodium sulfide nonahydrate into a three-neck flask, heating and adding DMF15ml to fully dissolve the product, heating to 60 ℃, accelerating the stirring speed, reacting for 1h, washing with water to be neutral, and vacuum drying to remove the solvent to obtain a dark green solid substance, namely the product tetraaminozinc phthalocyanine (ZnPc) to be prepared.
2. Preparation of CDM-PEG4000The synthesis of (2):
(1)0.276g CDM (2-propionyloxy-3-methylbutenedioic anhydride) is dissolved in anhydrous dichloromethane, 0.378g oxalyl chloride and 40 muL DMF are added as catalysts, the mixture is firstly reacted on ice for 30min, then the mixture is transferred to room temperature for reaction for 6h to obtain acyl chloride CDM, and the acyl chloride CDM is prepared for later use after vacuum drying;
(2) adding 800mg of polyethylene glycol monomethyl ether into vacuum-dried acyl chloride CDM, adding anhydrous dichloromethane for dissolving, adding 30 mu L of pyridine as a catalyst, reacting for 6 hours at 25 ℃, adding saturated ammonium chloride solution for extraction after the reaction is finished, collecting an organic phase, drying in vacuum, continuously dissolving in dichloromethane, precipitating twice by using anhydrous ether, and drying the precipitate in vacuum to obtain white powder, namely CDM-PEG4000。
3. Preparation of carboxyl dithiopyridine:
dissolving 3.75g of dithiodipyridine in 10ml of ethanol, adding 0.4ml of acetic acid, then dropwise adding an ethanol solution containing 0.9g of 3-mercaptoacetic acid, reacting for 20 hours at room temperature, removing the ethanol solvent after vacuum drying to obtain a crude product, then performing column chromatography purification by using a mixed solution formed by mixing dichloromethane and ethanol in a volume ratio of 3:2 as an eluent, collecting the product, and performing vacuum drying to obtain the carboxyl dithiopyridine.
4. Preparation of thiolated hyaluronic acid (HA-HS):
adding 200mg of Hyaluronic Acid (HA) into a sextuple dimethyl buffer solution with the pH value of 5, adding 500mg of N-hydroxy thiosuccinimide (EDC) and 300mg of N-hydroxy succinimide (NHS), carrying out activation reaction for 3-6 h, then adding 300mg of thiol ammonia hydrochloride, reacting for 24h under the protection of nitrogen, dialyzing for 2-4 d by using a dialysis bag with the molecular weight of 1000 after the reaction is finished, precipitating by using ethanol, and collecting a product, namely the thiolated hyaluronic acid (HA-HS).
5. Preparation of PEG4000-CDM-ZnPc：
Mixing CDM-PEG4000Adding the mixture and tetra-amino zinc phthalocyanine (ZnPc) into anhydrous tetrahydrofuran according to the molar ratio of 1:4 for full dissolution, adjusting the stirring speed of a rotor to 400rpm, reacting for 24 hours under the protection of nitrogen, vacuum drying to obtain a crude product, dissolving the crude product into dichloromethane, centrifuging to collect solids, removing excessive ZnPc in a supernatant solution, and vacuum drying to obtain a PEG product4000-CDM-ZnPc。
6. Preparation of CDM-PEG4000-ZnPc-S-S-：
Dissolving carboxyl dithiopyridine in DMF, adding N-hydroxysuccinimide (NHS) and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC), reacting to activate carboxyl for 30min, and adding PEG4000Carrying out normal temperature reaction on the CDM-ZnPc for 12-24 h under the protection of nitrogen, adjusting the stirring speed of a rotor to 400rpm, carrying out vacuum drying after the reaction is finished to remove a DMF solvent in a reaction system, continuously dissolving the DMF solvent in water, carrying out dialysis treatment for 3d by using a dialysis bag with the molecular weight of 1000 to remove excessive EDC, NHS and carboxyl dipyridyl disulfide, and finally carrying out vacuum freeze drying to obtain a dark green solid, namely a PEG product4000-CDM-ZnPc -S-S-；
Carboxylic dithiopyridine, NHS, EDC and PEG during the reaction4000The molar ratio of-CDM-ZnPc is 1:5:5: 2.
7. Preparation of PEG4000-CDM-ZnPc-S-S-HA
The prepared thiolated hyaluronic acid (HA-HS) was dissolved in 10ml of PBS solution, PEG4000Dissolving CDM-ZnPc-S-S-in DMF, mixing, stirring under nitrogen protection at 25 deg.C for 48h, dialyzing with dialysis bag with molecular weight of 3000 for 4d, precipitating with ethanol, and freeze drying to obtain target product biological cascade reaction type photodynamic integrated biopolymer (PEG)4000-CDM-ZnPc-S-HA), the structure of which is as follows:
Figure BDA0002368403430000061
where n is 113.
Example 2
For the bio-cascade reaction type photodynamic integration biopolymer (PEG) prepared in example 14000CDM-ZnPc-S-HA) was carried out in vitro simulation experiments under acidic conditions:
the bio-cascade reaction type photodynamic integrated biopolymer (PEG) prepared in example 14000TEM image of-CDM-ZnPc-S-S-HA) is shown in a in FIG. 1, and it can be seen that the Polymer (PEG)4000-CDM-ZnPc-S-HA) is an irregular macromolecule, without forming nanoparticles. Next, the bio-cascade reaction type photodynamic integration biopolymer (PEG) prepared in example 1 will be described4000-CDM-ZnPc-S-HA) under acidic condition of pH 6.5, TEM and SEM images of the treated product are shown as b and c in fig. 1, respectively, and it can be seen that the polymer HAs formed nanoparticles after being treated under acidic condition of pH 6.5, illustrating the bio-cascade reaction type photodynamic integrated biopolymer (PEG) prepared by the present invention4000-CDM-ZnPc-S-HA) is capable of self-assembly to form nanoparticles under acidic conditions at pH 6.5.
The intermediate products ZnPc-S-S-HA and ZnPc of the present invention were tested for their UV absorption, and the results are shown in FIG. 2, in which the UV absorption of tetraamino zinc phthalocyanine (ZnPc) is 650cm-1And 720-1The corresponding characteristic peak disappears to form a broad peak in the intermediate ZnPc-S-S-HA formed after S-S and HA are connected on ZnPc; shows that the disulfide bond (S-S) and the hyaluronic acid molecule (HA) are actually connected on the tetra-amino zinc phthalocyanine in the reaction process of the invention, so that the red shift phenomenon can be generated on the ultraviolet absorption peak, and the PEG can be subsequently performed4000Modified ligation is performed.
The Polymer (PEG) prepared in example 1 was tested4000-CDM-ZnPc-S-HA) in pH 6.5 and pH 7.4 respectively, the results of which are shown in fig. 3, illustrating the particle size distribution of the present inventionPolymer (PEG) prepared by invention4000-CDM-ZnPc-S-HA) is capable of self-assembling to form nanoparticles in solutions at pH 6.5 and pH 7.4, while different acidic environments have different effects on the products formed by its self-assembly.
The phagocytic effect of tumor cells formed by adding different substances into tumor cells is shown in FIG. 4, wherein a 1-a 4 is not added with any substance and belongs to blank control group, and b 1-b 4 are added with the Polymer (PEG) prepared by the invention4000CDM-ZnPc-S-S-HA), c 1-c 4 is added with the Polymer (PEG) prepared by the invention4000-CDM-ZnPc-S-HA) by acid treatment at pH 6.5. From the comparison of the phagocytic effect of tumor cells shown in fig. 4, it can be seen that the self-assembled nanoparticles of the polymer prepared by the present invention after acid treatment at pH 6.5 are more easily phagocytized by tumor cells.
To Polymers (PEG) in light and non-light conditions4000-CDM-ZnPc-S-HA) and intermediate ZnPc-S-HA, and the resulting MTT maps are shown in fig. 5 and 6. It can be seen that the Polymer (PEG) is present in both the light and non-light conditions4000-CDM-ZnPc-S-HA) is non-toxic to cells; while the intermediate product ZnPc-S-S-HA is due to a Polymer (PEG)4000-CDM-ZnPc-S-S-HA) is treated by acid, so that PEG in the CDM-ZnPc-S-HA is broken, the formed hydrophilic and hydrophobic product ZnPc-S-HA can be self-assembled to form nanoparticles, targets tumor cells under the action of HA (hyaluronic acid) and is more phagocytized by the tumor cells, under the action of high GSH of the tumor cells, disulfide bonds are broken, photosensitizer is released, and under the irradiation of a laser, the photosensitizer generates ROS to kill the tumor cells.
The test results show that the biological cascade reaction type photodynamic integrated biopolymer PEG can be obtained by the preparation method4000CDM-ZnPc-S-S-HA, coupling the photosensitizer zinc phthalocyanine (ZnPc) to PEG via a pH-acid responsive maleic acid amide linker4000Conjugated and then immobilized to Hyaluronic Acid (HA) chains using redox cleavable disulfide linkers, wherein the PEG moiety enhances blood circulation of the molecular carrier after intravenous administrationThe residual ZnPc-S-S-HA falls off after reaching an acid tumor microenvironment so that the residual ZnPc-S-S-HA is self-assembled into a large cluster in situ, reverse diffusion is avoided, and the tumor retention rate is improved; while HA is used as a carrier substrate and tumor targeting ligand due to its hydrophilicity, degradability and ability to specifically bind to overexpressed CD44 on tumor cell membranes. The polymer of the invention HAs hydrophilic effect of HA and PEG segment, and PEG is administered intravenously4000The dispersity of the-CDM-ZnPc-S-S-HA in body fluid is high, and the cycle life is long. When reaching a tumor microenvironment, the pH value of an acidic environment can remove PEG fragments by cracking the maleic acid amide linker, so that the overall hydrophobicity is sharply increased, the steric hindrance is reduced, the remaining ZnPc-S-S-HA is self-assembled into clusters under the driving of hydrophobic interaction among ZnPc molecules, the retention of a PS-carrier in tumor tissues is enhanced, the aggregated ZnPc-S-S-HA can be effectively internalized by tumor cells through hyaluronic acid-mediated endocytosis, and then high-concentration Glutathione (GSH) in the cells can crack disulfide bonds and release ZnPc molecules in a non-aggregated form, so that the curative effect of PDT is optimized. This polymerization-activated sequential tumor targeting is expected to improve the efficiency and specificity of PS delivery to tumor tissue, while further increasing ROS production under mild stimulation, resulting in better killing of tumor cells.
Finally, the above embodiments are only intended to illustrate the technical solutions of the present invention and not to limit the present invention, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions, and all of them should be covered by the claims of the present invention.

Claims (10)

1. A biological cascade reaction type photodynamic integrated biopolymer is characterized in that the structural formula of the biopolymer is as follows:
Figure FDA0003321822930000011
where n is 113.
2. The method for preparing the biological cascade reaction type photodynamic integrated biopolymer of claim 1, wherein the method comprises the following steps:
(1) preparation of PEG4000-CDM-ZnPc: modifying 2-propionyloxy-3-methylbutenedioic anhydride with PEG4000The resulting CDM-PEG4000Fully dissolving tetra-amino zinc phthalocyanine in anhydrous tetrahydrofuran, stirring and reacting for 24 hours under the protection of nitrogen, drying in vacuum after the reaction is finished, then dissolving in dichloromethane, centrifuging to obtain a solid, and drying in vacuum;
(2) preparation of PEG4000-CDM-ZnPc-S-: dissolving carboxyl dithiopyridine in DMF, adding N-hydroxysuccinimide and 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, reacting to activate carboxyl for 30min, and adding PEG in step (1)4000Carrying out normal-temperature stirring reaction on the CDM-ZnPc for 12-24 h under the protection of nitrogen, carrying out vacuum drying to remove a DMF solvent after the reaction is finished, continuously dissolving the DMF solvent in water, dialyzing to remove excessive 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and carboxyl dithiopyridine, and carrying out vacuum freeze drying to obtain dark green solid, namely the product PEG4000-CDM-ZnPc-S-S-；
(3) Preparation of PEG4000-CDM-ZnPc-S-S-HA: dissolving thiolated hyaluronic acid in PBS, PEG4000dissolving-CDM-ZnPc-S-S-in DMF, mixing, stirring under nitrogen protection at 25 deg.C for 48h, dialyzing for 4d after reaction, precipitating with ethanol, and freeze drying to obtain product polymer PEG4000-CDM-ZnPc-S-S-HA。
3. The method of claim 2, wherein the CDM-PEG is used in step (1)4000The molar ratio of the quaternary ammonium zinc phthalocyanine to the quaternary ammonium zinc phthalocyanine is 1: 4; the carboxyl dithiopyridine, the N-hydroxysuccinimide, the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and the PEG in the step (2)4000-the molar ratio of CDM to ZnPc is 1:5:5: 2; mercapto group in step (3)Dissolving hyaluronic acid and PEG4000The molar ratio of-CDM-ZnPc-S-S-is 2: 1.
4. The method of claim 2, wherein the CDM-PEG is used in step (1)4000The preparation method comprises the following steps:
(1) dissolving 2-propionyloxy-3-methylbutenedioic anhydride in anhydrous dichloromethane, adding oxalyl chloride and DMF, reacting on ice for 30min, transferring to room temperature, reacting for 6h to obtain acylchlorinated 2-propionyloxy-3-methylbutenedioic anhydride, and drying in vacuum for later use;
(2) dissolving the acyl-chlorinated 2-propionic-3-methylbutenedioic anhydride and polyethylene glycol monomethyl ether 4000 dried in the step (1) in anhydrous dichloromethane, adding pyridine as a catalyst, reacting at 25 ℃ for 6 hours, adding a saturated ammonium chloride solution after the reaction is finished, extracting, collecting an organic phase, and drying in vacuum;
(3) fully dissolving the dried product in the step (2) in dichloromethane, precipitating twice with anhydrous ether, and vacuum drying to obtain white powder product, i.e. CDM-PEG4000。
5. The method for preparing the bio-cascade reaction type photodynamic integrated biopolymer according to claim 4, wherein the mass-to-volume ratio of the 2-propionyloxy-3-methylbutenedioic anhydride, the oxalyl chloride, the DMF, the polyethylene glycol monomethyl ether 4000 and the pyridine is 0.276:0.378:40:0.8:30, g: mL: g: mL.
6. The method for preparing a bio-cascade reaction type photodynamic integrated biopolymer according to claim 2, wherein the tetraaminozinc phthalocyanine in the step (1) is prepared by the following method:
(1) mixing 4-nitrophthalimide, urea and ammonium molybdate, heating and melting, adding zinc chloride hexahydrate, heating and reacting at 160 ℃ until no bubbles are generated, cooling, washing with 1mol/L HCL and 1mol/L NaOH solution in sequence, washing with ultrapure water to be neutral, and drying in vacuum to obtain a bluish purple product;
(2) and mixing and heating the bluish violet product and sodium sulfide nonahydrate, adding DMF (dimethyl formamide) for full dissolution, continuously heating, accelerating stirring for 1h when the temperature is 60 ℃, washing with water to be neutral, and drying in vacuum to obtain a dark green solid substance, namely the tetraamino zinc phthalocyanine.
7. The method for preparing the biological cascade reaction type photodynamic integrated biopolymer according to claim 6, wherein the mass ratio of the 4-nitrophthalimide to the urea to the ammonium molybdate to the zinc chloride hexahydrate to the sodium sulfide nonahydrate is as follows: 4.1:10:0.05:1.2:2.88.
8. The method for preparing a bio-cascade reaction type photodynamic integrated biopolymer according to claim 2, wherein the carboxyl dipyridyl disulfide in the step (2) is prepared by the following method: dissolving dithiodipyridine in ethanol, adding acetic acid, dropwise adding an ethanol solution of 3-mercaptoacetic acid, reacting at room temperature for 20h, vacuum drying to remove an ethanol solvent, and purifying by column chromatography to obtain a product, namely carboxyl dithiopyridine;
the mass-volume ratio of the dithiodipyridine to the acetic acid to the 3-mercaptoacetic acid is 3.75:0.4:0.9, the mass-volume ratio of the dithiodipyridine to the acetic acid to the 3-mercaptoacetic acid is 3: 0.4:0.9, and the mass-volume ratio of the dithiodipyridine to the acetic acid to the 3-mercaptoacetic acid to the g: mL: g, and the eluent in the column chromatography is a mixed solution of dichloromethane and ethanol with the volume ratio of 3: 2.
9. The method for preparing a bio-cascade reaction type photodynamic integrated biopolymer according to claim 2, wherein the thiolated hyaluronic acid in the step (3) is prepared as follows: adding hyaluronic acid HA into a sextuple dimethyl buffer solution with pH being 5 for full dissolution, then adding N-hydroxy thiosuccinimide and N-hydroxy succinimide, reacting and activating for 3-6 h, continuously adding mercaptan ammonia hydrochloride, reacting for 24h under the protection of nitrogen, dialyzing for 2-4 d, and precipitating with ethanol to obtain a product;
the mass ratio of the hyaluronic acid to the N-hydroxythiosuccinimide to the N-hydroxysuccinimide to the thiol ammonia hydrochloride is 0.2:0.5:0.3:0.3, and the molecular weight of the dialysis bag used in dialysis is 1000.
10. The use of the bio-cascade reaction type photodynamic integration biopolymer as set forth in claim 1 in the preparation of an antitumor drug.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008041610A1 (en) * 2006-10-03 2008-04-10 Nippon Kayaku Kabushiki Kaisha Compound of resorcinol derivative with polymer
CN102949728A (en) * 2012-12-12 2013-03-06 重庆大学 Meso-porous silicon nano-drug carrier with both reduction responsiveness and targeting ability and preparation method thereof
CN104013965A (en) * 2014-05-06 2014-09-03 重庆大学 Method for preparing meso-porous silicon nano medicine carrier with cell specificity target, reduction responsiveness and triple anticancer treatment effects
CN108456298A (en) * 2018-05-15 2018-08-28 长春理工大学 A kind of amphipathic nature polyalcohol and synthetic method of the modification of solubility unsymmetrical phthalocyanine
CN108653288A (en) * 2018-05-29 2018-10-16 福建医科大学孟超肝胆医院 A kind of weary oxygen responsive polymer nanoparticle and its application

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008041610A1 (en) * 2006-10-03 2008-04-10 Nippon Kayaku Kabushiki Kaisha Compound of resorcinol derivative with polymer
CN102949728A (en) * 2012-12-12 2013-03-06 重庆大学 Meso-porous silicon nano-drug carrier with both reduction responsiveness and targeting ability and preparation method thereof
CN104013965A (en) * 2014-05-06 2014-09-03 重庆大学 Method for preparing meso-porous silicon nano medicine carrier with cell specificity target, reduction responsiveness and triple anticancer treatment effects
CN108456298A (en) * 2018-05-15 2018-08-28 长春理工大学 A kind of amphipathic nature polyalcohol and synthetic method of the modification of solubility unsymmetrical phthalocyanine
CN108653288A (en) * 2018-05-29 2018-10-16 福建医科大学孟超肝胆医院 A kind of weary oxygen responsive polymer nanoparticle and its application

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