CN113583178B - Branched sugar-containing polymer-based nanoparticle, and preparation method and application thereof - Google Patents
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- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F220/00—Copolymers of compounds having one or more unsaturated aliphatic radicals, each having only one carbon-to-carbon double bond, and only one being terminated by only one carboxyl radical or a salt, anhydride ester, amide, imide or nitrile thereof
- C08F220/02—Monocarboxylic acids having less than ten carbon atoms; Derivatives thereof
- C08F220/52—Amides or imides
- C08F220/54—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide
- C08F220/60—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen
- C08F220/603—Amides, e.g. N,N-dimethylacrylamide or N-isopropylacrylamide containing nitrogen in addition to the carbonamido nitrogen and containing oxygen in addition to the carbonamido oxygen and nitrogen
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
- A61K49/10—Organic compounds
- A61K49/12—Macromolecular compounds
- A61K49/124—Macromolecular compounds dendrimers, dendrons, hyperbranched compounds
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
- A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
- A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y25/00—Nanomagnetism, e.g. magnetoimpedance, anisotropic magnetoresistance, giant magnetoresistance or tunneling magnetoresistance
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/30—Introducing nitrogen atoms or nitrogen-containing groups
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
- C08F8/00—Chemical modification by after-treatment
- C08F8/42—Introducing metal atoms or metal-containing groups
Abstract
The invention provides a preparation method and application of branched sugar-containing polymer nano particles shown in a formula I. The branched sugar-containing polymer prepared by the invention has good self-assembly performance and tumor microenvironment responsiveness. The nanoparticle formed by self-assembly of the branched sugar-containing polymer can remarkably improve the pharmacological properties of the small molecular contrast agent, so that the small molecular contrast agent has longer blood half-life, more accumulation of tumor sites, higher enhancement degree of tumor signals, longer enhancement duration and better tumor enhancement specificity. The branched sugar-containing polymer nanoparticle prepared by the invention can be quickly taken up by cells, releases medicines in tumor microenvironment, has longer in vivo circulation time and targeting property, can obviously improve in vivo distribution of small molecular medicines, ensures that the medicines are effectively accumulated at tumor positions, obviously inhibits tumor growth, has good biological safety, and has great development potential and wide application prospect in the field of tumor diagnosis and treatment integrated medicines. 
Description
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to branched sugar-containing polymer nano particles, a preparation method and application thereof.
Background
The design and preparation of polymer nano-drug delivery systems based on multifunctional and stimulus-responsive are hot spots of current research in the field of tumor diagnosis and therapy. For example, the introduction of specific targeting groups, molecular imaging probes, etc., into the polymer structure can effectively enhance targeting of the delivery system and monitor its pharmacokinetic behavior in vivo. Meanwhile, aiming at the special physiological structure of the tumor part, a plurality of functional groups such as disulfide bond, hydrazone bond, phenylalanyl leucyl glycine (Gly-Phe-Leu-Gly, GFLG) short peptide and the like are also widely used for constructing a polymer drug delivery system with tumor microenvironment responsiveness. The stimulation responsive polymer carrier is used for simultaneously loading diagnostic and therapeutic medicines, so that the loaded medicines can be released in a targeted manner under the tumor-specific microenvironment, and meanwhile, the aggregation condition of the medicines and the change condition of tumors can be displayed on the imaging equipment, and the integration of tumor diagnosis and treatment is realized.
The choice and design of the polymeric carrier is particularly important in order to increase the functionality of the polymeric delivery system. Sugar-containing polymers have been widely used in cell recognition, biosensors, and gene/drug delivery, because of their excellent biocompatibility and specific molecular recognition, which are attracting more and more attention in biotechnology and biomedical applications. However, current drug/gene delivery systems based on sugar-containing polymers are mainly linear polymers with relatively single structure and function. Compared with the linear analogues thereof, the branched polymer has the advantages of relatively simple synthesis steps, high-density functional groups similar to the hyperbranched topology structure of dendrimers, larger nano-size, internal cavities for encapsulating small-molecule drugs and the like, so that the branched sugar-containing polymer has greater application potential. Reversible addition-fragmentation chain transfer free radical polymerization (RAFT polymerization) is an effective method for preparing sugar-containing polymers with precise structures (such as blocks, surface grafts, hyperbranched, dendrimers, etc.), and the prepared sugar-containing polymers have narrow dispersion coefficients and controllable molecular weights within a certain molecular weight range. The patent with application number 201610237966.6 discloses that a RAFT polymerization hyperbranched N- (2-hydroxypropyl) methacrylamide (HPMA) copolymer-DOX conjugate has double-sensitivity characteristics of pH sensitivity and enzyme sensitivity, can be used as an intelligent drug delivery system to rapidly release drugs in a tumor microenvironment, has good anti-tumor effect and biological safety, but lacks certain targeting and tumor diagnosis functionalities.
Due to the multifunction of RAFT polymerization, the sugar-containing monomer containing unsaturated double bond can be copolymerized with monomers containing unsaturated double bond with various functional groups, so that functional molecules such as antitumor drugs, imaging probes and the like can be introduced into the sugar-containing polymer carrier, and the multifunctional modification of the polymer can be realized conveniently. Therefore, the preparation of the multifunctional and stimulus-responsive sugar-containing polymer drug carrier through the selection and the proportion adjustment of the functional comonomer has very important significance for realizing the integration of tumor diagnosis and treatment.
Disclosure of Invention
In order to solve the above problems, the present invention provides a branched sugar-containing polymer nanoparticle.
The present invention first provides a branched sugar-containing polymer of formula I
wherein ,
selected from a cathepsin-sensitive group, a ROS-sensitive group, a glutathione-sensitive group, or a pH-sensitive group;
R 1 is selected from antitumor drugs such as tetracyclic diterpenoid compounds such as curcumin, gemcitabine, paclitaxel, etc., or anthracycline compounds such as doxorubicin, epirubicin, etc.; r is R 2 Is saccharide and its derivative; r is R 3 Is a magnetic resonance imaging developer; r is R 4 Is a fluorescent molecule; r is R 5 Selected from the group consisting of
And dithioesters.
Further, the method comprises the steps of,
is->
Further, the method comprises the steps of,
is->
/>
Further, R 1 Is paclitaxel:
further, R 2 Is that
Further, R 3 Is a metal gadolinium chelate
Further, R 4 Pyropheophorbide a modified with maleimide, or a near infrared fluorescent dye comprising: cyanine dyes, porphyrin dyes or rhodamine dyes, preferably maleimide modified pyropheophorbide a; the structure of the maleimide modified pyropheophorbide A is as follows:
further, R 5 Is that
Further, the structure of the polymer is shown as a formula II
wherein ,
is->
Is that
Further, the branched sugar-containing polymer has an average molecular weight of 200 to 300kDa; 1 to 10 percent of taxol, 1 to 10 percent of metal Gd, 0.5 to 1.5 percent of pyropheophorbide A or fluorescent dye and
and/or +.>
The mass percentage of (2) is 0.5-5%. More preferably, the average molecular weight is 244kDa and the molecular weight distribution is 2.48. The weight percentage of taxol is 6.4 percent, the weight percentage of metal Gd is 4.8 percent, and the weight percentage of pyropheophorbide A is 0.8 percent.
Still further, the branched sugar-containing polymer is prepared from monomers GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA, chain transfer agent MA-GFLG-CTA and cross-linking agent MA-GFLGK-MA and gadolinium ion-containing compound and maleimido-Ppa, wherein the mole ratio of the GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA monomer to the chain transfer agent MA-GFLG-CTA and cross-linking agent MA-GFLGA-MA is as follows: (380-480): (100-200): (30-70): (10-15): (5-10): (5-15), preferably 438:156: 50:13:7.2:9.38;
the mole number of gadolinium ions is the same as MA-DOTA, and the mole number of maleimides-Ppa is the same as PETMA;
wherein the structure of the monomer GAEMA is as follows:
the MA-DOTA structure is as follows:
the MA-GFLG-PTX has the structure:
the structure of PTEMA is:
the chain transfer agent MA-GFLG-CTA has the structure:
the structure of the crosslinking agent MA-GFLGK-MA is as follows:
the maleimid-Ppa has the structure:
the invention also provides a method for preparing the polymer, which comprises the following specific steps:
(1) Preparing a branched sugar-containing polymer matrix coupled with an antitumor drug through RAFT polymerization;
(2) Performing chelation reaction on the branched sugar-containing polymer matrix prepared in the step (1) to prepare a branched sugar-containing polymer containing a magnetic resonance contrast agent;
(3) And (3) taking the branched sugar-containing polymer containing the magnetic resonance contrast agent prepared in the step (2) to perform click reaction with a fluorescent molecular structure, and obtaining the magnetic resonance contrast agent.
Further, in the above preparation method, the method comprises the steps of:
(1) Mixing monomers GAEMA, MA-DOTA, MA-GFLG-PTX, PTMA, chain transfer agent MA-GFLG-CTA and cross-linking agent MA-GFLGK-MA, adding an initiator dissolved in a solvent for reaction, quenching the reaction, and precipitating to obtain a crude product;
(2) Purifying the crude product obtained in the step (1) to obtain an intermediate product pGAEMA-PTX-DOTA;
(3) Dissolving the intermediate product pGAEMA-PTX-DOTA obtained in the step (2) in an organic solvent, adding a reducing agent for reaction, and purifying to obtain a sulfhydryl-containing intermediate product;
(4) Dissolving the intermediate product containing the sulfhydryl group obtained in the step (3) in deionized water, adding a compound containing gadolinium ions for reaction, and purifying to obtain a gadolinium-containing intermediate product;
(5) Dissolving the gadolinium-containing intermediate product obtained in the step (4) in an organic solvent, adding maleimide-Ppa for reaction, and purifying to obtain the gadolinium-containing intermediate product;
wherein the structure of the monomer GAEMA is as follows:
The MA-GFLG-PTX has the structure:
the structure of PTEMA is:
the chain transfer agent MA-GFLG-CTA has the structure:
the structure of the crosslinking agent MA-GFLGK-MA is as follows:
The maleimid-Ppa has the structure:
further, in the step (1) of the above preparation method, the initiator is 2,2' - [ azobis (1-methylethylene) ] bis [4, 5-dihydro-1H-imidazole ] dihydrochloride; further, the molar ratio of the monomers GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA, chain transfer agent MA-GFLG-CTA cross-linking agent MA-GFLGK-MA to the initiator is as follows: (380-480): (100-200): (30-70): (10-15): (5-10): (5-15): (1-5), preferably 438:156:50:13:7.2:9.38:2.8; the solvent is a mixed solvent of water and methanol, preferably, the ratio of water to methanol is 1:3; the reaction condition when the initiator is added in the reaction is ice bath cooling, argon bubbling is carried out for 50min; the reaction temperature of the reaction is 46 ℃, the reaction time is 20 hours, and the reaction conditions are light-proof; the quenching reaction is quenched by liquid nitrogen, and the precipitation is carried out by dripping the solution into acetone after the solution temperature is raised to room temperature.
And/or further, the purification method described in step (2) is SEC fractionation purification, dialysis in a 2kDa dialysis bag for 2 days followed by lyophilization.
And/or further, the reducing agent in step (3) is dithiothreitol; the reaction temperature is room temperature, the reaction time is 10 hours, and the reaction condition is argon atmosphere; the purification method is that freeze-drying is carried out after dialysis.
And/or further, the gadolinium ion-containing compound in the step (4) is GdCl 3 ·6H 2 O, the reaction temperature is room temperature, and the pH is 5.2-5.4; the purification method is dialysis and freeze-drying.
And/or further, the organic solvent in the step (5) is DMSO; the reaction temperature is room temperature, the reaction time is 6 hours, and the reaction conditions are light-shielding; the purification method is that freeze-drying is carried out after dialysis.
The invention also provides a branched sugar-containing polymer nanoparticle, which is formed by self-assembly of the branched sugar-containing polymer.
The invention also provides a preparation method of the branched sugar-containing polymer nanoparticle, which comprises the following specific steps:
(1) The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd is uniformly dispersed in a chromatographic pure solvent and slowly added into vigorously stirred ultrapure water in a dropwise manner;
(2) Dialyzing the stirred solution, and freeze-drying to obtain the branched sugar-containing polymer nanoparticle pGAEMA-PTX-DOTA-Gd.
Further, the chromatographic pure solvent in the step (1) is chromatographic pure DMSO, and the ratio of pGAEMA-PTX-DOTA-Gd, DMSO and ultrapure water is 100mg:10ml: 10ml, stirring for 2h; the dialysis conditions in the step (2) are light-shielding, and the temperature is 4 ℃.
The invention also provides the application of the branched sugar-containing polymer nanoparticle in tumor diagnosis and treatment medicines.
Experimental results show that the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd nano particle prepared by the invention has good self-assembly performance, stability and tumor microenvironment responsiveness. Compared with a clinical contrast agent Gd-DTPA, the pharmacological property of the small molecular contrast agent can be obviously improved, so that the small molecular contrast agent has longer blood half-life, more accumulation of tumor parts, higher enhancement degree of tumor signals, longer enhancement duration and better tumor enhancement specificity. The nanoparticle can be rapidly taken up by cells and stimulated by tumor microenvironment to release therapeutic drugs, has longer in vivo circulation time and targeting property, can obviously improve in vivo distribution of small molecular drugs to effectively accumulate at tumor sites, can obviously inhibit tumor growth, has excellent anti-tumor effect and tumor inhibition rate of more than 90 percent in a 4T1 xenograft tumor model of a BALB/c mouse, has no obvious systemic toxicity, has good biological safety, and has great development potential and application prospect.
M, n, o, p, q, r, x, y, z in the structural formula of the polymer of the invention represents the number of the repeating units and is reasonably valued in a standard range conforming to the molecular weight of the polymer according to the common technical knowledge of the person skilled in the art.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
FIG. 1 is a scheme for the preparation of branched polymers pGAEMA-PTX-DOTA and pGAEMA-PTX-DOTA-Gd.
FIG. 2 is a schematic diagram of (A) branched sugar-containing polymer PTX-DOTA-Gd nanoparticles 1 H NMR. EDX spectra of prodrugs (B). (C) Fluorescence spectra of branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd before and after covalent attachment Ppa. (D) Particle size of branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd nanoparticle in water, (E) TEM characterization of the obtained morphology and (F) stability in PBS solution.
FIG. 3 is the critical micelle concentration (Critical aggregation concentrations, CACs) of the branched polymer pGAEMA-PTX-DOTA-Gd
FIG. 4 is a SEC assay of (A) nanoparticles and their degradation products after 24h incubation with cathepsin B. (B) enzyme-responsive Release profile of PTX. (C) A graph of the development effect of Gd-DTPA and nanoparticles and (D) T1 relaxation efficiency/Gd (III) concentration profile at different Gd concentration gradients.
FIG. 5 is (A) the observation by CLSM of 4T1 cell uptake of branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd nanoparticles at various time points. (B) Flow analysis of nanoparticle uptake by 4T1 cells after incubation for different times. (C) Cytotoxicity of nanoparticles and free PTX on 4T1 cells. (D) The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was co-incubated with 4T1 tumor balls for 2 and 6h for penetration. Blue fluorescence is the nucleus stained by Hoechst33342, purple fluorescence is the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd marked by Ppa, and the scale is: 50 μm. (E) Distribution of fluorescence signal of branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd along the labeling direction and (F) 3D topology map with depth of 70 μm. (G) CLSM photographed multicellular tumor spheres incubated with free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd. Wherein green fluorescence is living cells stained with Calcein-AM, and red fluorescence is dead cells stained with PI. Ruler: 50 μm.
Fig. 6 is a bright field image of a multicellular tumor sphere co-incubated with free PTX and branched polymer pGAEMA-PTX-DOTA-Gd for 48 hours. Ruler: 200 μm.
FIG. 7 is a graph showing the aggregation of 4T1 cell microtubules treated with (A) free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd. Red fluorescence is tubulin-tracker red stained microtubules and blue fluorescence is DAPI stained nuclei. Ruler: 10 μm. (B) Semi-quantitative analysis of 4T1 cell cycle distribution treated with free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd (C) cycle distribution. (D) Semi-quantitative analysis of apoptosis in 4T1 cells treated with free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd. P <0.01, n=3 compared to control group). Control cells were incubated with fresh medium only.
FIG. 8 is a graph showing Gd ion concentration/time in blood of (A) BALB/c mice after injection of Gd-DTPA and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd nanoparticles, respectively. (B) Fluorescent images of the major organs and tumors of the mice in the group dosed with nanoparticles and free ppapppa at different time points post-dosing. S is normal saline, G is branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd, P is Ppa, H is heart, li is liver, sp is spleen, lu is lung, ki is kidney, tu is tumor. (C) Fluorescent semi-quantitative analysis of branched sugar-containing polymers pGAEMA-PTX-DOTA-Gd for 1,6,12,24,48 and 72h after dosing. (D) Gd content analysis of branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd after administration in major organs and tumors.
Fig. 9 is a semi-quantitative analysis of fluorescence of 1,6,12,24,48,72h tumor sites after tumor-bearing mice were injected with free Ppa and branched polymer pGAEMA-PTX-DOTA-Gd (n=3).
FIG. 10 is a semi-quantitative analysis of fluorescence of tissues and organs of 1,6,12,24,48,72h after injection of branched polymer pGAEMA-PTX-DOTA-Gd.
FIG. 11 is a Gd content analysis of each tissue organ 24h after injection of free Ppa.
Fig. 12 is a representative MRI image of (a) BALB/c mice injected with branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd nanoparticles and Gd-DTPA at different time points of tumor sites and (B) relative signal to noise ratio enhancement SI% (< 0.05 p, n=5). (C) Branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd treatment strategy. Schematic of tumor tissue after treatment of each group (D). (E) Relative volumes of different groups of tumors (< 0.01P, n=7 compared to saline group and free PTX). (F) Tumor tissue weights of each group (< 0.01 for P, <0.001 for P, and saline group comparison). (G) Body weight change profile in 21 days after dosing of mice during treatment. (H) Schematic representation of tumor tissue TUNEL and CD31 staining of different treatment groups. Semi-quantitative analysis of (I) CD31 and (J) TUNEL.
FIG. 13 is a tumor growth inhibition index (The tumor growth inhibitions, TGI) of free PTX and sugar-containing branched polymer PTX-DOTA-Gd.
FIG. 14 is a histopathological analysis (all tissues:. Times.100) of mice 21 days after treatment with physiological saline, free PTX and the sugar-containing branched polymer PTX-DOTA-Gd.
Detailed Description
Material
Pyropheophorbide A (PpaPpa), gadolinium chloride hexahydrate (GdCl 3. 6H 2O), 2'- [ azobis (1-methylethylidene) ] bis [4, 5-dihydro-1H-imidazole ] dihydrochloride (VA 044), 4-cyanovaleric acid dithiobenzoic acid (CTA), N, N' -Dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), O-benzotriazol-tetramethylurea Hexafluorophosphate (HBTU), N, N-Diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), cathepsin B purchased from SIGma-Aldrich company, can be used without further purification. The monomers MA-GFLG-PTX, MA-DOTA, MA-GFLGK-MA were synthesized as previously reported. All other reagents and solvents were purchased from the Kelong chemical reagent plant (Chengdu, china) and used without further purification.
4T1 mammary tumor cells were purchased from the China academy of sciences' typical culture Collection cell Bank (China, shanghai) and cultured in a constant temperature and humidity cell incubator (5% CO) with RPMI 1640 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin solution, america Life technologies Co.) 2 The method comprises the steps of carrying out a first treatment on the surface of the 37 ℃) culture. Female BALB/c mice (weight 20.+ -. 1.2g and 6-8 weeks old) were purchased from Chengdu Biotech Co. All animal experiments were performed according to the regulations of the ethical committee of the relevant China and university of Sichuan.
Structural identification of intermediates and end products of various monomers by 1 H NMR (Bruker AV II-400 spectrometer, bruker, switzerland) electrospray ionization Mass Spectrometry (ESI MS, waters, USA)Department of nuda) and liquid chromatography mass spectrometry (LC-MS/MS, ABI, usa). Average molecular weight and polydispersity index (PDI) of the polymer using flash protein chromatography
FPLC system, GE Healthcare, sweden) by Size Exclusion Chromatography (SEC). Sodium acetate buffer (H) 2 Acn=70:30, v/v, ph=6.5) as mobile phase. The chromatographic column adopts GE Healthcare Superose HR 10/30 with the flow rate of 0.4mL/min. By passing through 1 H NMR characterizes the polymer structure. The nanoparticle size and zeta potential (3 mg/mL) of the polymer in aqueous solution were measured using Zetasizer Nano ZS (Malvern Instruments, worcestershire, UK). 3 replicates were set for each measurement and the results were processed with DTS software version 3.32. The polymer particle size was measured using a Transmission Electron Microscope (TEM) (GF-20S-TWIN, FEI Tecnai, USA). UV-vis spectra were tested using an ultraviolet visible spectrophotometer (Cary 400, varian, usa). The fluorescence spectrum of the polymer was measured by a fluorescence spectrophotometer (RF-5301-PC, shimadzu, japan). Statistical analysis was performed using the Student's t test. Results are expressed as mean ± Standard Deviation (SD). p value <0.05 is considered to have a statistically significant difference, p-value<0.01 is considered to have a highly significant difference.
EXAMPLE 1 Synthesis of branched sugar-containing Polymer pGAEMA-PTX-DOTA-Gd
The synthesis procedure is shown in FIG. 1.
GAEMA (1.34 g,4.38 mmol), MA-DOTA (803 mg,1.56 mmol), MA-GFLG-PTX (640 mg,0.5 mmol), PTEMA (31.8 mg,0.13 mmol), MA-GFLG-CTA (68.9 mg, 72. Mu. Mol) and MA-GFLGK-MA (61.5 mg, 93.8. Mu. Mol) were added to the polymerization flask and the flask was protected under argon. VAO44 (9.0 mg, 28. Mu. Mol) was dissolved in 15ml H 2 O/CH 3 1/3 of OH, and adding into the polymerization bottle, cooling in ice bath, and bubbling argon for 50min. The polymerization bottle was heated in an oil bath at 46℃under dark conditions for 20h, and the reaction was quenched with liquid nitrogen. The reaction mixture was warmed to room temperature and then added dropwise to 300ml of acetone solution to give a pink precipitate. The crude product was further purified by SEC fractionation. And permeate in a 2K dialysis bagAfter 2 days of separation, the pink solid intermediate pGAEMA-DOTA1.63g is obtained by freeze-drying, and the yield is 55%.
pGAEMA-PTX-DOTA (1.5 g) was dissolved in 15mL DMSO under argon and dithiothreitol (DTT, 300 mg) was added to the solution. Stirring at room temperature for 10 h, dialyzing with deionized water in a 2K dialysis bag for 2 days, and lyophilizing to obtain 1.2g of pink solid product. Branched pGAEMA-PTX-DOTA-SH (1.2 g) was dissolved in 25mL deionized water and GdCl was added 3 .6H 2 O (600 mg), stirring at room temperature, wherein the pH value of the solution is 5.2-5.4. The product was dialyzed and lyophilized before being dissolved again in DMSO. maleimide-Ppa (12 mg) was dissolved in 2mL of DMSO and added to the above reaction system, reacted at room temperature in the absence of light for 6 hours, dialyzed in RO water in a 2K dialysis bag for 1 day, and lyophilized to give 1.07g of pale green solid. The final product had PTX, ppa and Gd contents of 6.4%,0.8% and 4.9%, respectively.
EXAMPLE 2 preparation of nanoparticles of branched sugar-containing Polymer pGAEMA-PTX-DOTA-Gd
Polymer branched sugar-containing Polymer pGAEMA-PTX-DOTA-Gd (100 mg) was uniformly dispersed in 10mL of chromatographically pure DMSO, and slowly added dropwise to vigorously stirred 10mL of ultra-pure water under ice bath. After stirring for 2h, the mixture was dialyzed against light at 4℃until DMSO was removed. And freeze-drying the dialyzed solution to obtain the drug-loaded nanoparticle branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd NPs, and storing at 4 ℃ for later use.
Test example 1, chemical Structure and molecular weight confirmation
1) Experimental materials
pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
By passing through 1 The H NMR spectrum analysis pGAEMA-PTX-DOTA-Gd chemical structure. As shown in FIG. 2A, proton peaks of the sugar-containing polymer pGAEMA-PTX-DOTA can be observed at 7.0-8.5ppm, indicating the presence of aromatic groups in the polymer. It may be derived from benzene rings in PTX, ppa or GFLG, on the other hand, the polymer does not observe proton peaks at 5.0 to 6.0ppm, and the peak occurs at 5.60ppm (s, -CH) due to double bond characteristics in the monomer 3 -CH(r)-CH 2 -H a ) And 5.20ppm(s,-CH 3 -CH(r)-CH 2 -H b ) Indicating that the monomer in the reaction has been completely consumed.
Element recognition was performed by EDX method. As shown in FIG. 2B, recognition of the elements C, O, S and Gd may confirm Gd 3+ Has been successfully attached to the polymer.
The fluorescence spectra were measured by fluorescence spectrophotometry, as shown in FIG. 2C, and characteristic peaks of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd and free Ppa were detected at 673nm, indicating that the fluorescence properties of Ppa remained unchanged during covalent coupling.
The specific gravity of the amino acids was verified by amino acid analysis, and the results showed that the specific gravity of the three amino acids glycine, phenylalanine and leucine in the polymer were 2.57%,3.26% and 2.54%, respectively, with a molar ratio of about 2:1:1, indicating the presence of GFLG in the polymer.
Through a rapid protein chromatograph
The average molecular weight and polydispersity index (PDI) of the polymer were measured by Size Exclusion Chromatography (SEC) by the FPLC system, GE Healthcare, sweden). Sodium acetate buffer (H) 2 Acn=70:30, v/v, ph=6.5) as mobile phase. The chromatographic column adopts GE Healthcare Superose HR10/30 with the flow rate of 0.4 mL/min. The results show that the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd has a molecular weight of about 244kDa and a PDI of 2.48 (Table 1).
Table 1: characterization parameters of the sugar-containing branched polymer pGAEMA-PTX-DOTA-Gd.
a Molecular Weight (MW) units are kDa;
b amino acid, PTX, gd and Ppa percent (%);
c nanoparticle diameter size (nm) measured by DLS;
d zeta potential (ζ) units are mV.
Test example 2 evaluation of particle size distribution, zeta potential, morphology and stability
1) Experimental materials
Nanoparticles of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 2.
2) Experimental methods and results
The particle size and zeta potential of the nanoparticles were characterized by DLS. Nanoparticle solutions (1 mg/mL) were prepared using deionized water, followed by particle size and zeta potential (n=3) measurements using a malvern nanoparticle size and potential analyzer. The stability of the nanoparticles in PBS was determined in the same way.
Transmission electron microscopy (TEM Tecnai G2F 20S-TWIN, FEI, hillsboro, oregon, USA) was used to detect the surface morphology of the nanoparticles. The aqueous solution of the nanoparticles (0.5 mg/mL) was added dropwise to the copper mesh, and after the excess liquid was sucked away with filter paper, the sample was air-dried for TEM detection. As shown in fig. 2D, DLS results showed that the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd nanoparticle had a hydrodynamic diameter of 95.17nm and had a negative surface charge. As shown in fig. 2E, the test results of TEM show that the conjugate can form nanoparticles with a uniform distribution of about 70nm, further demonstrating the existence of self-assembly behavior of the conjugate. As shown in fig. 2F, the stability test results further indicate that the nanoparticles have good stability in PBS, and no significant changes in particle size and PDI occurred after 48h storage in PBS.
The Critical Aggregation Concentration (CAC) of polymer nanoparticles in ultrapure water was measured using pyrene fluorescence. Firstly, accurately weighing and configuring a series of nanoparticle solutions (0.01 mug/mL-500 mug/mL) with different concentration gradients. Subsequently, 25. Mu.L of pyrene in acetone (5X 10) -5 M) in a series of 10mL sample bottles, after the acetone was completely volatilized, 2mL of nanoparticle solutions of the above concentration gradients (final concentration of pyrene 6.25X10 were added respectively -7 M). Finally, the solution was incubated in a constant temperature shaker at 37℃for 2 hours in the absence of light, and the fluorescence spectrum of pyrene was measured by a fluorescence spectrophotometer (Em: 390nm, ex:300nm to 350 n)m). As a result, as shown in FIG. 3, the critical aggregation concentration of the formed nanoparticle was 23.7. Mu.g/mL, and such a low CAC value suggests that the nanoparticle is expected to have good stability.
Test example 3, evaluation of biodegradation and drug Release
1) Experimental materials
Nanoparticles of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1 and the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 2.
2) Experimental methods and results
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was dissolved in McIlvaine buffer (pH 5.4, 50mM citrate, 0.1M phosphate and 2mM EDTA) containing cathepsin B (2.8. Mu.M) at a polymer concentration of 6mg/mL. The polymer solution was incubated in a shaker at 37℃at predetermined time points (0,2,6, 12, 20 h), 1mL of the mixed solution was taken out and the molecular weight of the sample was determined by SEC, the mobile phase was a mixed solution of sodium acetate buffer and methanol, the solvent ratio was 7:3, the final pH of the solvent was 6.5, and the flow rate was 0.4mL/min. Three replicates were prepared for the samples in the experiment.
The drug release experiment of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was also performed in a buffer (pH 5.4) containing McIlvaine of cathepsin B (2.8. Mu.M) at a sample concentration of 3mg/mL. The solution was incubated in a shaker at 37℃for 12h. At predetermined time points (0,2,6, 12, 20 h), 100. Mu.L of the solution was taken out and mixed with an equal volume of chromatographically pure methanol, and then subjected to detection analysis (liquid phase analysis column C8 column 4.6X150 mm) by RP-HPLC, the mobile phase was equal volumes of acetonitrile and water, the flow rate was 1.0 mL/min, and the ultraviolet detection wavelength was 227nm. The drug release properties of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd in McIlvaine buffer (pH 5.4 and pH 7.4) without cathepsin B were also determined as described above.
As shown in table 2, the molecular weight of the sugar-containing polymer gradually decreased with increasing incubation time, showing a remarkable degradation behavior. In contrast, there was no significant change in polymer molecular weight after incubation for 20h in the control group without cathepsin B added. This result indicates that the degradation of the carbohydrate-containing polymer can be attributed to cleavage of the GFLG oligopeptide in the polymer structure. Figure 4A shows the time at which degradation products peak after co-incubation of the polymer with cathepsin B. The molecular weight of the degraded polymer (28 kDa) is well below the renal threshold, thus facilitating its rapid metabolism out of the body. At the same time, the good stability of the polymer under physiological conditions also helps to prolong its in vivo circulation time.
Table 2: degradation products of the sugar-containing branched polymer pGAEMA-PTX-DOTA-Gd after co-incubation with cathepsin B (2.8. Mu. Mol/L, pH=5.4) in McIlvaine buffer solution at 37 ℃.
As shown in fig. 4B, little PTX release (less than 3%) was observed in PBS solution without cathepsin B (ph=5.4, 37 ℃) over 24h, whereas the cumulative PTX release of the sugar-containing polymer exceeded 90% in the case of cathepsin B. On the other hand, under simulated physiological conditions (pH 7.4, 37 ℃), the amount of PTX released within 24h was about 25%. This result is consistent with previous studies, and can be attributed to the fact that ester-linked PTX has better stability under weakly acidic conditions than under neutral conditions. The research results show that the sugar-containing polymer can quickly release the carried PTX drug under the stimulation of tumor microenvironment after being taken up by tumor cells, thereby generating corresponding cytotoxicity.
Test example 4, in vitro relaxation rate evaluation
1) Experimental materials
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
The 1H water relaxation rate of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd in PBS was measured by a clinical Siemens 3.0T MRI scanner. Different Gd3+ concentrations (0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4 mM) of branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd were dissolved in 0.1M PBS and scanned by T1 SE sequence The magnetic resonance signal intensity of the material, the scan parameters are as follows: te=8.7 ms, tr=20, 30, 50, 70, 90, 125, 150, 175, 200, 300, 400, 500, 700, 850 and 1000ms, fov= 200mm,slice thickness = 1.0mm,matrix dimensions =256×256. And obtain corresponding 1/T1 values from their T1 weighted MR images. By plotting 1/T1 as different Gd 3+ The relaxation rate value r1 is calculated as a function of the concentration. In addition, the same concentration of clinical DTPA-Gd was used as a control, and then the longitudinal relaxation rate of the sample was obtained by the same method as described above.
The results are shown in FIG. 4C, vs. Gd 3+ The branched polymer pGAEMA-PTX-DOTA-Gd has higher signal intensity than Gd-DTPA with the same content. FIG. 4D shows the branched polymer pGAEMA-PTX-DOTA-Gd and the clinically used Gd-DTPA and Gd 3+ And R of both are linearly related 2 Are each approximately equal to 1.0. Relaxation efficiency r of branched Polymer pGAEMA-PTX-DOTA-Gd 1 Calculated as 7.1L/(mmol.s), was about Gd-DTPA (r) 1＝ 3.6 1.97 times more than before). Whether the polyaminopolyhydroxy Gd-containing polymer can be used for clinical diagnosis is largely dependent on its rotation-dependent time (τ R ) Whereas τ R The relaxation efficiency can be improved by increasing the molecular mass. The small molecule Gd-DOTA of pGAEMA-PTX-DOTA-Gd is connected to the branched polymer through covalent bond, so that the relaxation efficiency is greatly improved, and the contrast ratio of MRI is improved.
Test example 5 in vitro cell uptake, cytotoxicity, and penetration and growth inhibition evaluation
1) Experimental materials
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
A35X 12mm glass dish (cell number: 1X 10) was inoculated with a cell suspension of mouse mammary tumor cells (4T 1) 5 ) After 24h incubation, the original medium was removed and a solution containing the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd (corresponding to a concentration of PpaPpa of 0.25. Mu.g.mL was added -1 ) Is a medium of RPMI 1640. Next, after incubating the cells in an incubator for 1,3,5 hours, respectively, the medium was removed and the cells were washed three times with PBS (pH=7.4)PBS containing Hoechst 33342 (10. Mu.g/mL) was added and stained for 15min under dark conditions. Subsequently, after removing the dye and washing 3 times with PBS, the cell entry was observed with a confocal laser microscope (CLSM), and a fluorescent image of the cell uptake material was obtained.
Mouse mammary tumor cells (4T 1) were selected to evaluate the relative cytotoxicity of the comparative free paclitaxel (free PTX) and the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd on 4T1 cells to verify the safety of the material. 4T1 cells were seeded in 96-well plates with a cell density of 5X 10 per well 3 After 24h of adherent growth in an incubator, the RPMI 1640 medium was removed and free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd medium were added, respectively, with different concentration gradients (concentrations corresponding to PTX concentrations 0.039 to 40. Mu.M). After 48h of incubation again, the medium was removed and washed three times with PBS. A cytotoxicity assessment kit CCK-8 (Dojindo, japan) was then added to each well. After incubation for 2h in the cell incubator, absorbance was detected by a multifunctional enzyme-labelling instrument (Thermo Fisher SCIENTIFIC), absorbance of each sample was detected with an enzyme-labelling instrument, and cell viability was calculated according to instructions.
Multicellular Tumor Spheres (MTS) of 4T1 cells were prepared and used to study the tumor penetrating power and the growth inhibitory capacity of tumor cells of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd. First 2mL of a molten 2% agarose solution was added to a glass dish having a diameter of 5 cm. After solidification of the agarose solution, 4mL of 4T1 cell suspension (5 mL. Times.10) containing fresh RMPI 1640 medium 6 cells/mL) was added to agarose. MTS was formed after about 5 days of incubation and the cell sphere diameter was about 200. Mu.m. To investigate the penetration of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd, MTS was incubated with the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd for 2h and 6h and the cell pellet was observed under CLSM. The data were further processed by Image J software. To investigate the growth inhibitory capacity of tumor cells, surviving and dying 4T1 cells were stained. Firstly, after MTS diameter reaches about 200 μm, fresh RPMI 1640 medium is added, wherein branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd and free PTX containing 2 μg/mL PTX are respectively added into two groups of medium The control group did not contain PTX-related therapeutic agents. After 48h of continued incubation of MTS, the medium was removed and MTS was transferred to a 1mL EP tube. Thereafter, surviving and dying 4T1 cells were differentiated using dual staining with fluorescein calcein AM/propidium iodide (CAM/PI), and MTS was fixed and transferred to glass dishes, and stained tumor cell spheres were observed under CLSM. The resulting experimental data were further processed using NIS-Elements AR software.
As shown in fig. 5A, laser confocal microscopy (CLSM) showed that the red fluorescence of Ppa was mainly distributed in the cytoplasm. After incubation, ppa fluorescence enhancement of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was well coincident with lysosomal fluorescence of lysosomal fluorescent staining, suggesting that the polymer may be taken up by cells via the endocytic lysosomal pathway. Over time, the fluorescence intensity gradually increased, exhibiting a pronounced time dependence. As shown in fig. 5B, flow quantitative analysis further revealed a time-dependent cellular uptake of nanoparticles, consistent with the results obtained from the confocal laser microscopy (CLSM) experiments.
As shown in fig. 5C, both the free PTX group and the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd group showed significant concentration-dependent cytotoxicity. Wherein the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd IC 50 The value was 1.99. Mu.M, approximately 1.7 times (1.17. Mu.M) that of the free PTX. This result may be due to free PTX being free to diffuse into the cells, while the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd adopts a different endocytosis cell route and drug release process.
Furthermore, two-dimensional cytotoxicity studies on 4T1 cells revealed that the antitumor effect of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was comparable to that of free PTX. Its antitumor effect was also confirmed by applying it to three-dimensional MTS. From the topological 3D image and the CLSM image (FIGS. 5D-F), it can be seen that 2h after application of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd, the polymer is largely concentrated at the periphery of the MTS. After the incubation time is prolonged to 6 hours, a significant increase in the signal on MTS can be observed, demonstrating that the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd penetrates deeply into the three-dimensional MTS. Meanwhile, compared to MTS treated with free PTX, there are a large number of morphologically abnormal and isolated cells in the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd incubated MTS. After 48h incubation, the MTS incubated with both free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd showed an increase in PI-positive cells (dead cells), while the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd treated MTS produced more atypical 4T1 cells, while the whole tumor sphere was relatively smaller. Thus, these experimental results demonstrate that the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd is able to penetrate into three-dimensional solid tumor tissue and enhance the antitumor effect of PTX.
The inhibitory effect of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd on 4T1 MTS is shown in FIG. 5G. The growth of MTS is shown in FIG. 6. In MTS treated with both free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd, both large cell morphology abnormalities and cell death were observed. After 48h incubation, a significantly increased PI positive region was observed for both the free PTX group and the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd group MTS. Cell morphology abnormalities may be caused by prolonged incubation and cell necrosis. Thus, the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd has deep penetration ability to 3D solid tumors and equivalent antitumor ability to free PTX.
Test example 6 in vitro anti-tumor mechanism study
1) Experimental materials
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
Cell tubulin detection: the 4T1 cell line was seeded on a glass dish (cell number: 5X 10) 3 ) Is a kind of medium. After 24h incubation, the original medium was removed, fresh RPMI 1640 medium containing free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd (at a concentration of 0.5. Mu.g/mL) was added, respectively, and a set of fresh RPMI 1640 medium containing no free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was set as a control. The 4T1 cells were then incubated for 24h. The medium was removed and the cell micro-was determined by a Tubulin-Tracker Red solution (C1050, beyotime, chinese adults) according to the manufacturer's protocol A tube. After cell microtube staining, cells were rinsed twice with PBS and then incubated in Hoechst 33342 (10. Mu.g/mL) PBS for 10 min. The dye was removed, the cells were washed twice with PBS and observed under CLSM. The experimental data were further processed and analyzed by NIS-Elements AR software.
Cell cycle detection: the 4T1 cell line was seeded on a glass dish (cell number: 2X 10) with a diameter of 5cm 4 ) Is a kind of medium. The original medium was removed, fresh RPMI 1640 medium containing free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd (each at a concentration of 0.5. Mu.g/mL) was added separately, and a set of fresh RPMI 1640 medium containing no free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was set as a control. The cells were then incubated for 24h. The medium was removed and the cells were trypsinized and centrifuged. Subsequently, proliferation cycles were determined by PI/RNase staining solutions according to the protocol provided by the manufacturer. After fixing and staining the cells, the cell proliferation cycle was examined using a flow cytometer. The experimental data were further processed and analyzed by ModFit LT 3.1 software.
Apoptosis detection: 4T1 cell suspensions were seeded in 6-well plates (cell number 1.5X10) 5 Per well), the original medium was removed after 24 hours of incubation, media containing free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd (at concentrations of 0.8 μg/mL PTX and 1.8 μg/mL PTX, respectively) were added, respectively, and a set of fresh RPMI 1640 media containing no free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was set as a control. After an additional 24h incubation, the medium was removed and the cells were trypsinized and centrifuged. Subsequently, cells were stained according to the procedure in the Annexin V-FITC apoptosis test kit, the apoptosis was quantitatively detected using a flow cytometer, and the experimental data were further processed and analyzed by WinMDI 2.9 software.
The microtubule structure of 4T1 cells after staining by tubulin can be clearly seen in FIG. 7A. The normal microtubule structure marked by Tubulin-Tracker Red has been distributed in 4T1 cells in a grid, whereas after treatment with the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd and free PTX, both aggregation of microtubules and the presence of polynuclear structures in single cells and the like can be observed, demonstrating that an enhanced visualization of microtubule stability occurs during cell division. The two sets of cell results show that compared with free PTX, the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd has the same anti-tumor mechanism and anti-tumor effect.
As shown in fig. 7B and 7C, the number of untreated 4T1 cells in G1 phase (37.84%) was significantly higher than the number in G2/M phase (9.39%). In the 4T1 cells treated by the free PTX, the percentages of the cells in the G1 phase and the G2/M phase are 14.06% and 23.34% respectively, and in the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd group, the percentages are 18.33% and 22.98% respectively, which proves that the addition of the free PTX and the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd can effectively inhibit the division of tumor cells, so that the G2/M phase blocking rate of the tumor cells is obviously increased.
As shown in fig. 7D and 7E, both free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd were able to effectively induce apoptosis and necrosis of cells after drug treatment, wherein free PTX induced apoptosis and necrosis of 37.2% of cells and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd induced 31.2% compared to control group. These results indicate that the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd based on the nanostructure can be effectively taken up by cells, and can specifically release chemotherapeutic drugs in the cell microenvironment to kill tumor cells with high efficiency.
Test example 7, pharmacokinetic and in vivo drug spread evaluation
1) Experimental materials
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
Metabolic analysis of branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd in normal mice: 10 female healthy BALB/c mice (20.+ -.2 g,8-10 weeks) were arbitrarily divided into two groups (n=5). Branched sugar-containing Polymer pGAEMA-PTX-DOTA-Gd (0.08 mmol/kg Gd) by Tail vein separate injection 3+ ) After dosing, the ocular fundus vein harvest was performed at time points of 0min, 5min, 15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h and 48hBlood collection of 20. Mu.L of blood, blood passed through HNO 3 and H2 O 2 (1: 3) digestion, gd (III) content was measured by inductively coupled plasma mass spectrometry (Inductively coupled plasma mass spectrometry, ICP-MS). Pharmacokinetic parameters were calculated by PKSolver software.
The biological distribution of branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd in the main organ of the mouse in the mouse at different time points is detected by adopting fluorescence imaging. Tumors were grown to about 150mm 3 The mice of (2) were randomly divided into two groups, the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd and free Ppa, each having a relative Ppa content of 1.5mg/kg, by tail vein injection. Living body fluorescence pictures are taken before injection and after injection for 1,6,12, 24,48 and 72 hours, and the distribution of the medicine at the tumor part of the mice is detected and recorded.
Furthermore, to further investigate the in vivo distribution of the branched, tumor-bearing mice injected with the above drugs were additionally euthanized for 3 mice per time point per group, respectively at 1,6, 12, 24, 48 and 72h after administration. Tumors and major organs (heart, liver, spleen, lung, kidney) were stripped, weighed, and fluorescence images of these tissues and organs were semi-quantitatively analyzed using a Maestro In-Vivo imaging system. Normal saline injected mice were used as a control group analysis.
Simultaneously, ICP-MS is adopted to detect Gd in main organs of mice 3+ The distribution of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd in mice at different time points was investigated. 4T1 tumor-bearing mice (tumor volume about 150 mm) 3 ) The randomization was divided into 4 groups (n=5). The clinical contrast agent Gd-DTPA and the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd are injected through tail veins respectively. Two groups of each reagent are respectively marked, and the dosage of each reagent is 0.08mmol Gd 3+ Mice/kg. One group of mice in the Gd-DTPA and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd dosing group were euthanized 24h and 96h after dosing, respectively. Tumors and major organs (heart, liver, spleen, lung, kidney) were stripped, weighed, and digested in a mixed solution of concentrated hydrochloric acid and concentrated nitric acid (3:1, v/v). Each sample was then heated at 120 ℃ to completely dissolve. By using ultrapure water The completely digested sample solutions were diluted to the same volume and the samples were examined for Gd using ICP-MS 3+ Is a concentration of (3). The calculation formula of the distribution of Gd in each organ is as follows: gd (Gd) 3+ Relative content (ng Gd/g tissue) =gd in each sample 3+ Mass of the corresponding organ.
As shown in fig. 8A, the clinical reagent Gd-DTPA showed a very rapid blood clearance rate, and the presence of Gd was hardly detected in plasma after 1h of administration. In contrast, the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd showed a significantly slower decrease in blood concentration, a significantly prolonged blood circulation time, and a Gd concentration of up to 10. Mu.g/mL was detectable in plasma after 4 hours of administration, while a Gd concentration of 7. Mu.g/mL was still detectable after 12 hours. As shown in table 3, the results of the two-chamber model analysis showed that Gd-DTPA showed a very short blood half-life (14.2 min), whereas the nanoparticles showed a significantly prolonged blood half-life time of 1255.4min. Furthermore, the nanoparticles also have significantly reduced average system clearance rates and prolonged average residence times compared to Gd-DTPA.
Table 3: two-compartment model pharmacokinetic-related parameters for PKSolver 2.0 software.
As shown in fig. 9, the signal distribution of tumor sites Ppa of tumor-bearing mice is shown. In the free Ppa group, only a very weak Ppa signal was detected at 12 h. While the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd detected a higher Ppa fluorescence signal at all time points, which increased continuously until 24h before it began to decrease. Furthermore, as shown in fig. 8B, after administration, free Ppa and Ppa labeled nanoparticles exhibited significantly different organ tissue distribution. Among them, in the free Ppa administration group, a strong fluorescent signal was observed at the liver site 1h after administration, indicating that it was largely aggregated at the liver site, whereas a fluorescent signal was hardly observed at the tumor site. Over time, the fluorescence intensity in each organ was significantly reduced, and almost no fluorescence signal was observed after 12 hours. In contrast, ppa labeled nanoparticles showed a gradual and sustained fluorescence distribution, and a clear process of fluorescence signal enhancement was observed at the tumor site. The semi-quantitative analysis of fluorescence further reveals this result, as shown in fig. 8C, in the Ppa labeled nanoparticle group, the fluorescence signal at the tumor site reached a maximum 24h after administration, followed by a slow decrease. The fluorescence signal in each organ and tumor gradually decreased after administration of the PpaPpa group (fig. 10). These results show that the Ppa labeled nanoparticle has longer in vivo circulation time and can be targeted to tumor sites through EPR effect, so that the in vivo distribution of the small molecular medicine is hopeful to be improved, and the anti-tumor curative effect of the small molecular medicine is improved.
As shown in FIG. 11, trace amounts of Gd can be detected in organs and tumors of mice 24h after Gd-DTPA injection 3+ Wherein 0.1% ID/g Gd is detectable in the liver 3+ Is total Gd 3+ 0.2% of the injected amount, 0.1% ID/g was detected in the tumor, and Gd in other organs 3+ The concentration is less than 0.1% ID/g. This result is consistent with that of fluorescence imaging. However, after 24h injection of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd, a large amount of Gd was detected in the liver (30.9% ID/g) and spleen (18.4% ID/g) of the mice 3+ While in the kidney this value was 4.8% ID/g. At the same time, about 4.3% ID/g Gd was also detectable in tumor tissue 3+ . And Gd in liver and spleen 96h after injection 3+ The concentration was significantly reduced, the former 11.9% ID/g and the latter 6.1% ID/g. Whereas Gd detected in tumors and other organs 3+ The concentration was very low (fig. 8D). Although Gd 3+ Is similar to the result of Ppa fluorescence imaging, but when Gd 3+ Gd after dissociation from the polymer 3+ Has a relatively high clearance rate, while Ppa bonded carbohydrate-containing polymer backbones and degradation products thereof are cleared from the body at a relatively slow rate. The results of in vivo distribution experiments show that after the biodegradable branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd plays the biological function in vivo, the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd can be rapidly discharged out of the body through metabolism, so that Gd is avoided 3+ Potential for long-term retention in vivoToxicity.
Test example 8 in vivo magnetic resonance imaging evaluation
1) Experimental materials
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
Xenograft 4T1 tumor-bearing mice (tumor volume approximately 150 mm) 3 ) Randomly divided into two groups (n=5), and the clinical contrast agent Gd-DTPA and the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd were injected by tail vein respectively (the administration dose is equivalent to 0.08mmol Gd) 3+ /kg mice). Scanning mice were anesthetized with isoflurane gas and then fixed in custom coils. Contrast enhanced imaging scans were performed using the T1 SE sequence, which was as follows: te=20 ms, tr=500 ms, fov=40 ms, thiokness=1.0 mm, flip angle=90°. MRI images of each experimental group were acquired before injection, 10min,30min,1h,4h and 24h after injection, respectively. The relative enhancement of MRI signal intensity (SI%) was defined as SI (post injection)/SI (pre injection) ×100%. Semi-quantitative analysis was applied to assess signal changes at tumor sites by plotting SI% versus time.
Tumor MRI signal changes in mice with branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd and Gd-DTPA groups at various time points before and after injection are shown in fig. 12A. For the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd group, after the mice are injected with the contrast agent, the contrast between the tumor part and surrounding tissues is gradually enhanced along with the time extension, the outline of the tumor is clearly visible, and the contrast is always kept high within 24 hours. In contrast, the Gd-DTPA group gradually increased in brightness within 30 minutes of administration, followed by a rapid decrease in tumor signal values, became very weak already 1h after administration, and had been restored to a level substantially before injection after 4 h. Semi-quantitative analysis of the relative enhancement signal (The relative enhanced intensity signal, SI) at the tumor site is shown in fig. 12B. The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd group SI continued to increase for 24h, while the Gd-DTPA group reached peak 30min after injection and then began to decrease. This result indicates that small molecule Gd-DTPA contrast agent has no tumor targeting in vivo and can be rapidly cleared from the body. Notably, the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd group had a higher relative enhancement signal intensity than the Gd-DTPA group at various time points after administration. These results demonstrate that the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd has better in vivo imaging effect compared to the small molecule Gd-DTPA contrast agent. This may be due on the one hand to its better relaxation efficacy and on the other hand to its longer in vivo circulation time due to its larger molecular weight, thus being able to accumulate more at the tumor site.
Test example 9 in vivo antitumor evaluation
1) Experimental materials
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
The in vivo antitumor effect of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was studied by establishing a 4T1 tumor model of subcutaneous xenograft. 4T1 cells were suspended in 70. Mu.L PBS (1.4X10) 5 Individual cells) and injected subcutaneously into the right hind leg position of female BALB/c mice. Tumor volumes were calculated as follows: v=l×w 2 X 0.5, where L refers to the longest diameter of the tumor and W refers to the shortest diameter of the tumor). When the tumor volume reaches about 60-80mm 3 At this time, the mice were randomly divided into saline group, PTX combination and branched sugar-containing polymer PTX-DOTA-Gd group (n=7). Mice were treated with the above formulation by tail vein injection every 3 days at a dose of 10mg PTX/kg mice for a total of 4 administrations. At the same time, body weight and tumor volume of each mouse were recorded every 2 days. On day 21, all mice were euthanized and tumors and major organs (heart, liver, spleen, lung, kidney) were dissected out separately. Tumors of each group were weighed and tumor inhibition rate (TGI) was calculated using the formula: tgi= (1-W1/W2) ×100%, where W1 and W2 represent average tumor weights of the treatment group and control group, respectively.
The inhibition of tumor growth in mice was observed in different dosing groups using xenograft 4T1 breast tumors as shown in fig. 12C, the tumor volume change in mice in each dosing group was shown in fig. 12D and 12E, the tumors in the saline group showed a rapid growth trend after the first dosing, the tumor growth rate in the PTX group was similar to that in the saline group, and the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd group tumor growth was significantly inhibited. From the second dose, the growth rate of the tumors in the PTX group mice began to slow down compared to the saline group, but the tumor growth inhibition was more pronounced in mice treated with the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd, and the tumor growth was in an inhibited state all the time during the following treatment without a trend of regrowth. After day 21 of administration, the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd group showed very remarkable antitumor effect compared to the physiological saline group (about 1631% relative to tumor volume), and the tumor size of the mice in which the remaining tumor had not disappeared was also kept at a very small level. The anti-tumor effect exhibited by free PTX is relatively weak (about 1049% relative to tumor volume), probably because small-molecule PTX does not have tumor targeting and long circulation capabilities, and the drug can be rapidly cleared from the body after entering the body, so that it is difficult to achieve a sufficient drug concentration at the tumor site.
As shown in fig. 12F, the tumor weight of the material group was far lower than that of the PTX group and the saline group (114.0±49.1mg,873.8±178.1mg and 1214.1 ±133.1mg, respectively), showing a very significant tumor suppression effect (TGI 90.6%). In contrast, the TGI of the free PTX group administered at the same PTX concentration was only 28.0% (fig. 13). The reason for this result is probably because the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd material prepared by us has long in vivo circulation time, can be passively targeted to the tumor region, thereby improving the enrichment of the drug at the tumor site, and can trigger the rapid release of the therapeutic drug in the tumor-specific microenvironment, thereby improving the tumor killing effect thereof. In addition, the body weight change profile of tumor-bearing mice throughout the treatment period is shown in fig. 12G. The figure shows that the body weight of each group of mice does not change obviously during the whole treatment period, which indicates that the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd material has excellent anti-tumor curative effect and better in-vivo safety.
Test example 10, H & E staining, immunohistochemical analysis and TUNEL analysis evaluation
1) Experimental materials
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
The H & E staining method is as follows: washing fixed mouse viscera with tap water overnight, dehydrating with ethanol of all levels of full-automatic dehydrator, and waxing twice. Conventional paraffin embedding was performed using an embedding machine, and paraffin blocks were cut into 5 μm paraffin pieces for hematoxylin-eosin (H & E) staining by a rotary microtome. Finally, the used specimens are photographed by microscopic examination.
Immunohistochemical analysis employed streptavidin-peroxidase method: the dewaxed and rehydrated tumor sections were first incubated overnight at 4℃with monoclonal anti-CD 31 antibody (1:200) (Beijing biosynthesized Biotechnology Co., ltd.) and anti-Ki-67 monoclonal antibody (1:200) (Beijing biosynthesized Biotechnology Co., ltd.). Then, the secondary anti-mouse/rabbit IgG antibody (1:200) of the biotinylated goat is dripped to react for 20min at room temperature; immunohistochemical images were finally obtained by Motic Images Advanced software (Motic China Group co., ltd.) and positive Integrated Optical Density (IOD) measurements of CD31 were made using Image-Pro Plus 6.0 software (Media Cybernetics, bethesda, MD). Tumor microvascular density (MVD) was measured by calculating the ratio of CD31 to the total area of each photograph.
The deoxynucleotide terminal transferase mediated dUTP notch end-labeling (TUNEL) assay was as follows: TUNEL analysis was performed using an in situ apoptosis detection kit (Roche Molecular Biochemicals, laval, quebec, canada) according to the manufacturer's protocol. Optical microscopy was used to observe TUNEL staining positive cells (i.e. apoptotic cells) and to calculate the ratio of the number of apoptotic cells to the total tumor cells in each microscopic field as an apoptosis index.
As shown in fig. 14, histological examination of the major organs of mice in each group after the end of treatment by H & E staining revealed a more severe inflammatory cell infiltration than that of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd group in the liver tissue sections of both the saline group and the PTX group. In the other groups of organs, no significant organic toxicity was found in all three experimental groups.
The angiogenesis during the proliferation of tumor cells was evaluated by staining with CD31 antigen, and the results of detecting apoptosis by TUNEL method are shown in fig. 12H and fig. 12I, angiogenesis is critical for the growth and metastasis of most solid tumors, and inhibition of angiogenesis is an important method for inhibiting tumor growth. Compared with the physiological saline group, the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd group has obviously reduced MVD-CD31 count of mouse tumor tissue, and has very obvious statistical significance (p < 0.05). The free drug PTX group showed no significant difference in MVD-CD31 counts compared with the physiological saline group, and had no statistical significance (p > 0.05). The result shows that the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd has very good effect of inhibiting tumor angiogenesis. As shown by TUNEL staining results in fig. 12J, the apoptosis rate of the tumor cells in the mice in the free drug PTX-treated group was about 33.6%, higher than that in the saline group (15.7%), but lower than that in the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd-treated group (37.7%). These results indicate that the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd can effectively inhibit the growth of 4T1 tumor by inhibiting the generation and development of new blood vessels at the tumor site and efficiently inducing apoptosis necrosis of tumor cells.
In conclusion, the invention prepares the multifunctional biodegradable branched polymer drug delivery system (branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd) based on the sugar-containing polymer through RAFT polymerization to realize the diagnosis and treatment integration of tumors. The branched polymer adopts a branched sugar-containing polymer with higher functional group density and larger hydrodynamic size and similar topological structure to that of a dendrimer as a framework, and is loaded with fluorescent dye pyropheophorbide A (Ppa) and metal gadolinium ion chelate (Gd-DOTA) as imaging groups, and simultaneously loaded with Paclitaxel (PTX) as a therapeutic group. In addition, in order to ensure biodegradability and biosafety of the branched polymer, a GFLG tetrapeptide linker degradable in tumor microenvironment over-expressing cathepsin B was used to make a branched structure. The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd nano particles are prepared by designing the proportion of each functional monomer to the cross-linking agent, and compared with a clinical contrast agent Gd-DTPA, the nano particles prepared by the invention can obviously improve the pharmacological properties of a small molecular contrast agent, so that the small molecular contrast agent has longer blood half-life, more accumulation amount of tumor parts, higher enhancement degree of tumor signals, longer enhancement duration, better tumor enhancement specificity and the like. Meanwhile, in vitro experiments show that the nanoparticle can be quickly taken up by cells, and cathepsin B in tumor cells is responsively degraded and releases chemotherapeutic drugs PTX to induce cytotoxicity similar to that of free drugs. In vivo experiments show that the nanoparticle can remarkably improve in vivo distribution of small molecular drugs, can effectively accumulate at tumor sites through EPR effect, remarkably inhibit growth of 4T1 xenograft tumors by reducing angiogenesis, inducing apoptosis of tumor cells and the like, and has good biological safety. Therefore, the multifunctional nano-drug delivery system based on the branched sugar-containing polymer carrier, which is developed by the invention, has great development potential in the field of tumor diagnosis and treatment integrated research.
Claims (8)
1. A branched sugar-containing polymer characterized in that it has the structure of formula II:
wherein ,
is->
The average molecular weight of the polymer is 200-300 kDa; wherein the weight percentage of the taxol-containing material is 6.4 percent, the weight percentage of the gadolinium-containing material is 4.9 percent, the weight percentage of the pyropheophorbide A-containing material is 0.8 percent, and the weight percentage of the pyropheophorbide A-containing material is
And/or +.>
The mass percentage of (2) is 0.5-5%.
2. The branched sugar-containing polymer of claim 1, prepared from monomers GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA, chain transfer agent MA-GFLG-CTA, cross-linker MA-GFLGK-MA and gadolinium ion containing compound, maleimido-Ppa, in a molar ratio of GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA monomer and chain transfer agent MA-GFLG-CTA, cross-linker MA-GFLGA-MA of: (380-480): (100-200): (30-70): (10-15): (5-10): (5-15);
the mole number of gadolinium ions is the same as MA-DOTA, and the mole number of maleimides-Ppa is the same as PTEMA;
wherein the structure of the monomer GAEMA is as follows:
the MA-DOTA structure is as follows:
the MA-GFLG-PTX has the structure:
the structure of PTEMA is:
the chain transfer agent MA-GFLG-CTA has the structure:
the structure of the crosslinking agent MA-GFLGK-MA is as follows:
The maleimid-Ppa has the structure:
3. the branched sugar-containing polymer of claim 2, wherein the molar ratios of GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA monomer and chain transfer agent MA-GFLG-CTA, crosslinker MA-GFLGA-MA are: 438:156:50:13:7.2:9.38.
4. a process for the preparation of a branched sugar-containing polymer according to any one of claims 1 to 3, characterized in that it comprises the steps of:
(1) Mixing monomers GAEMA, MA-DOTA, MA-GFLG-PTX, PTMA, chain transfer agent MA-GFLG-CTA and cross-linking agent MA-GFLGK-MA, adding an initiator dissolved in a solvent for reaction, quenching the reaction, and precipitating to obtain a crude product;
(2) Purifying the crude product obtained in the step (1) to obtain an intermediate product pGAEMA-PTX-DOTA;
(3) Dissolving the intermediate product pGAEMA-PTX-DOTA obtained in the step (2) in an organic solvent, adding a reducing agent for reaction, and purifying to obtain a sulfhydryl-containing intermediate product;
(4) Dissolving the intermediate product containing the sulfhydryl group obtained in the step (3) in deionized water, adding a compound containing gadolinium ions for reaction, and purifying to obtain a gadolinium-containing intermediate product;
(5) Dissolving the gadolinium-containing intermediate product obtained in the step (4) in an organic solvent, adding maleimide-Ppa for reaction, and purifying to obtain the gadolinium-containing intermediate product;
Wherein the structure of the monomer GAEMA is as follows:
the MA-DOTA structure is as follows:
the MA-GFLG-PTX has the structure:
the structure of PTEMA is:
the chain transfer agent MA-GFLG-CTA has the structure:
the structure of the crosslinking agent MA-GFLGK-MA is as follows:
the maleimid-Ppa has the structure:
5. the process of claim 4 wherein in step (1) the initiator is 2,2' - [ azobis (1-methylethylene) ] bis [4, 5-dihydro-1H-imidazole ] dihydrochloride and the molar ratio of GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA monomer and chain transfer agent MA-GFLG-CTA, crosslinker MA-GFLGA-MA to initiator is: 438:156:50:13:7.2:9.38:2.8.
6. a branched sugar-containing polymer nanoparticle, characterized by being self-assembled from a branched sugar-containing polymer according to any one of claims 1 to 3.
7. A method of preparing branched sugar-containing polymer nanoparticles according to claim 6, comprising the steps of: (1) Uniformly dispersing the branched sugar-containing polymer in a chromatographic pure solvent, and slowly dripping the solution into vigorously stirred ultrapure water; and (2) dialyzing the stirred solution, and freeze-drying to obtain the product.
8. Use of the branched sugar-containing polymer nanoparticle of claim 6 in the preparation of a medicament for tumor diagnosis and treatment.
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