Branched sugar-containing polymer-based nanoparticles and preparation method and application thereof
阅读说明：本技术 一种支化含糖聚合物基纳米粒子及其的制备方法和用途 (Branched sugar-containing polymer-based nanoparticles and preparation method and application thereof ) 是由 罗奎 刘艳辉 蔡豪 向宇凡 于 2021-07-29 设计创作，主要内容包括：本发明提供了式I所示支化含糖聚合物纳米粒子的制备方法和用途。本发明制备得到的支化含糖聚合物具有良好的自组装性能和肿瘤微环境响应性。支化含糖聚合物自组装形成的纳米粒子能够显著改善小分子造影剂的药理学性质,使其具有更长的血液半衰期,更多的肿瘤部位积累量,更高的肿瘤信号强化程度,更长的强化持续时间以及更好的肿瘤强化特异性。本发明制备的支化含糖聚合物纳米粒子能够快速被细胞摄取,并在肿瘤微环境下释放药物,具有较长体内循环时间和靶向性,且能够显著改善小分子药物的体内分布,使药物在肿瘤部位有效积累,显著抑制肿瘤生长,并具有良好的生物安全性,在肿瘤诊疗一体化药物领域具有巨大的发展潜力和广阔的应用前景。(The inventionProvides a preparation method and application of the branched sugar-containing polymer nano-particle shown in the formula I. The branched carbohydrate-containing polymer prepared by the invention has good self-assembly performance and tumor microenvironment responsiveness. The nanoparticles formed by self-assembly of the branched carbohydrate-containing polymer can obviously improve the pharmacological properties of the small-molecule contrast agent, so that the small-molecule contrast agent has longer blood half-life period, more tumor site accumulation amount, higher tumor signal enhancement degree, longer enhancement duration and better tumor enhancement specificity. The branched carbohydrate-containing polymer nanoparticles prepared by the invention can be rapidly taken up by cells, can release the medicine in a tumor microenvironment, has longer in vivo circulation time and targeting property, can obviously improve the in vivo distribution of small molecular medicines, enables the medicine to be effectively accumulated at a tumor part, obviously inhibits the growth of the tumor, has good biological safety, and has huge development potential and wide application prospect in the field of tumor diagnosis and treatment integrated medicines.)
1. A branched saccharide-containing polymer having the structure of formula I:
wherein the content of the first and second substances,selected from a cathepsin-sensitive moiety, a ROS-sensitive moiety, a glutathione-sensitive moiety, or a pH-sensitive moiety;
R1the antitumor drug is a tetracyclic diterpenoid compound or an anthracycline compound, the tetracyclic diterpenoid compound comprises curcumin, gemcitabine and paclitaxel, and the anthracycline compound comprises adriamycin and epiadriamycin; r2Is composed ofR3Is a metal gadolinium chelateR4Is a fluorescent molecule which is maleimide modified pyropheophorbide A
Or a near-infrared fluorescent dye comprising: cyanine dyes, porphyrin-based dyes, or rhodamine-based dyes; r5Are dithioester compounds
2. The branched sugar-containing polymer according to claim 1,is composed of Is composed of
3. The branched saccharide-containing polymer of claim 1 or 2, wherein R is1Is paclitaxel:and/or R4 is maleimide-modified pyropheophorbide A
And/or R5Is composed of
4. The branched sugar-containing polymer of any of claims 1 to 3, having the structure of formula II:
wherein the content of the first and second substances,is composed of
Is composed of
5. The branched sugar-containing polymer of any one of claims 1 to 4, wherein the polymer has an average molecular weight of 200 to 300 kDa; wherein, the weight percentage of the paclitaxel is 1 to 10 percent, the weight percentage of the gadolinium metal is 1 to 10 percent, the weight percentage of the pyropheophorbide A or the fluorescent dye is 0.5 to 1.5 percent, and the content of the pyropheophorbide A or the fluorescent dye isAnd/orThe mass percentage of the component (A) is 0.5-5%.
6. The branched sugar-containing polymer according to any one of claims 1 to 5, which is prepared from monomers GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA, chain transfer agent MA-GFLG-CTA, crosslinking agent MA-GFLGK-MA and gadolinium ion-containing compound, maleimiside-Ppa, wherein the molar ratio of GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA monomer and chain transfer agent MA-GFLG-CTA, crosslinking agent MA-GFLGA-MA is: (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 the gadolinium ions is the same as that of MA-DOTA, and the mole number of the maleimide-Ppa is the same as that of PETMA;
wherein, the structure of the monomer GAEMA is as follows:
the structure of MA-DOTA is:
the structure of MA-GFLG-PTX is as follows:
the structure of PTEMA is:
the chain transfer agent MA-GFLG-CTA has the structure as follows:
the structure of the cross-linking agent MA-GFLGK-MA is as follows:
the structure of maleimide-Ppa is:
7. a process for the preparation of a branched sugar-containing polymer according to any of claims 1 to 6, wherein the process comprises the steps of:
(1) mixing monomers GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA, a chain transfer agent MA-GFLG-CTA and a cross-linking agent MA-GFLGK-MA, adding an initiator dissolved in a solvent for reaction, and precipitating after quenching reaction 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 sulfydryl obtained in the step (3) in deionized water, adding a compound containing gadolinium ions for reaction, and purifying to obtain an intermediate product containing gadolinium;
(5) dissolving the intermediate product containing gadolinium obtained in the step (4) in an organic solvent, adding maleimide-Ppa for reaction, and purifying to obtain the gadolinium intermediate product;
wherein, the structure of the monomer GAEMA is as follows:
the structure of MA-DOTA is:
the structure of MA-GFLG-PTX is as follows:
the structure of PTEMA is:
the chain transfer agent MA-GFLG-CTA has the structure as follows:
the structure of the cross-linking agent MA-GFLGK-MA is as follows:
the structure of maleimide-Ppa is:
preferably, in step (1), the initiator is 2,2' - [ azobis (1-methylethylidene) ] bis [4, 5-dihydro-1H-imidazole ] dihydrochloride, and the molar ratio of the GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA monomer and chain transfer agent MA-GFLG-CTA, cross-linking agent MA-GFLGA-MA to initiator is: (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.
8. a branched sugar-containing polymer nanoparticle which is self-assembled from the branched sugar-containing polymer according to any one of claims 1 to 6.
9. The method of making branched sugar-containing polymer nanoparticles of claim 8, comprising the steps of:
(1) uniformly dispersing the branched sugar-containing polymer in a chromatographic pure solvent, and slowly dropwise adding the branched sugar-containing polymer into vigorously stirred ultrapure water;
(2) dialyzing the stirred solution, and freeze-drying to obtain the final product.
10. Use of the branched sugar-containing polymer nanoparticles according to claim 8 in the preparation of a medicament for tumor diagnosis and treatment.
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a branched sugar-containing polymer nanoparticle and a preparation method and application thereof.
Background
The design and preparation of polymer nano-drug delivery systems based on multiple functions and stimuli responsiveness are hot spots of research in the field of tumor diagnosis and treatment at present. For example, specific targeting groups, molecular imaging probes and the like are introduced into a polymer structure, so that the targeting property of a delivery system can be effectively improved, and the pharmacokinetic behavior of the delivery system in vivo can be monitored. Meanwhile, aiming at the special physiological structure of the tumor part, a plurality of functional groups, such as disulfide bond, hydrazone bond, phenylalanine 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 loading diagnostic and therapeutic drugs simultaneously, so that the loaded drugs can be released in a targeted manner in a tumor-specific microenvironment, the aggregation condition of the drugs and the change condition of the tumor can be displayed on 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, gene/drug delivery, and the like, because of their excellent biocompatibility and specific molecular recognition, they have attracted increasing attention in the fields of biotechnology and biomedical applications. However, current carriers for drug/gene delivery systems based on sugar-containing polymers are mainly linear polymers, which are structurally and functionally relatively simple. Compared with linear analogues thereof, the branched polymer has the advantages of relatively simple synthetic steps, high-branched topological structure similar to that of a dendrimer, high-density functional groups, larger nano-size, internal cavity 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 grafting, hyperbranched, dendritic molecules, and the like), and the prepared sugar-containing polymers have narrow dispersion coefficients within a certain molecular weight range and controllable molecular weight. The patent with application number 201610237966.6 discloses a hyperbranched N- (2-hydroxypropyl) methacrylamide (HPMA) copolymer-DOX conjugate polymerized by RAFT, which has pH-sensitive and enzyme-sensitive double-sensitive characteristics, 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 property and tumor diagnosis functionality.
Owing to the multifunctionality of RAFT polymerization, the sugar-containing monomer containing unsaturated double bonds can be copolymerized with the monomer containing unsaturated double bonds 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 is facilitated. Therefore, the preparation of the multifunctional sugar-containing polymer drug carrier with stimulation responsiveness by selecting and adjusting the proportion of the functional comonomer has very important significance in 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 invention firstly provides a branched sugar-containing polymer shown as a formula I
Wherein the content of the first and second substances,selected from a cathepsin-sensitive moiety, a ROS-sensitive moiety, a glutathione-sensitive moiety, or a pH-sensitive moiety;
R1selected from antineoplastic drugs, such as tetracyclic diterpenoid compounds like curcumin, gemcitabine and paclitaxel, or anthracycline compounds like adriamycin and epiadriamycin; r2Is a saccharide and a derivative thereof; r3Is a magnetic resonance imaging developer; r4Is a fluorescent molecule; r5Is selected from And the like.
Further, in the above-mentioned case,is composed of
Further, the air conditioner is provided with a fan,is composed of
Further, R1Is paclitaxel:
further, R2Is composed of
Further, R3Is a metal gadolinium chelate
Further, R4Is maleimide modified pyropheophorbide a, or a near infrared fluorescent dye comprising: cyanine dyes, porphyrin-based dyes or rhodamine-based dyes, preferably maleimide-modified pyropheophorbide A; the structure of the maleimide modified pyropheophorbide A is as follows:
further, R5Is composed of
Further, the structure of the polymer is shown as a formula II
Wherein the content of the first and second substances,is composed ofIs composed of
Further, the average molecular weight of the branched sugar-containing polymer is 200-300 kDa; contains taxol in 1-10 wt%, metal Gd in 1-10 wt%, pyropheophorbide A or fluorescent dye in 0.5-1.5 wt% and other componentsAnd/orThe mass percentage of the component (A) is 0.5-5%. More preferably, the average molecular weight is 244kDa and the molecular weight distribution is 2.48. Contains taxol in 6.4 wt%, metal Gd in 4.8 wt% and pyropheophorbide A in 0.8 wt%.
Further, the branched sugar-containing polymer is prepared from monomers GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA, a chain transfer agent MA-GFLG-CTA, a cross-linking agent MA-GFLGK-MA, a gadolinium ion-containing compound and maleimimide-Ppa, wherein the molar ratio of the GAEMA, MA-DOTA, MA-GFLG-PTX, the PTEMA monomer, the chain transfer agent MA-GFLG-CTA and the 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 the gadolinium ions is the same as that of MA-DOTA, and the mole number of the maleimide-Ppa is the same as that of PETMA;
wherein, the structure of the monomer GAEMA is as follows:
the structure of MA-DOTA is:
the structure of MA-GFLG-PTX is as follows:
the structure of PTEMA is:
the chain transfer agent MA-GFLG-CTA has the structure as follows:
the structure of the cross-linking agent MA-GFLGK-MA is as follows:
the structure of maleimide-Ppa is:
the invention also provides a method for preparing the polymer, which comprises the following steps:
(1) preparing a branched carbohydrate-containing polymer matrix of a conjugated antitumor drug by RAFT polymerization;
(2) carrying out chelation reaction on the branched carbohydrate-containing polymer matrix prepared in the step (1) to prepare a branched carbohydrate-containing polymer containing a magnetic resonance contrast agent;
(3) and (3) carrying out click reaction on the branched carbohydrate-containing polymer containing the magnetic resonance contrast agent prepared in the step (2) and a fluorescent molecular structure to obtain the magnetic resonance contrast agent.
Further, in the above preparation method, the following steps are included:
(1) mixing monomers GAEMA, MA-DOTA, MA-GFLG-PTX, PTEMA, a chain transfer agent MA-GFLG-CTA and a cross-linking agent MA-GFLGK-MA, adding an initiator dissolved in a solvent for reaction, and precipitating after quenching reaction 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 sulfydryl obtained in the step (3) in deionized water, adding a compound containing gadolinium ions for reaction, and purifying to obtain an intermediate product containing gadolinium;
(5) dissolving the intermediate product containing gadolinium obtained in the step (4) in an organic solvent, adding maleimide-Ppa for reaction, and purifying to obtain the gadolinium intermediate product;
wherein, the structure of the monomer GAEMA is as follows:
the structure of MA-DOTA is:
the structure of MA-GFLG-PTX is as follows:
the structure of PTEMA is:
the chain transfer agent MA-GFLG-CTA has the structure as follows:
the structure of the cross-linking agent MA-GFLGK-MA is as follows:
the structure of maleimide-Ppa is:
further, in step (1) of the above preparation method, the initiator is 2,2' - [ azobis (1-methylethylidene) ] bis [4, 5-dihydro-1H-imidazole ] dihydrochloride; further, the molar ratio of the monomer 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 of; the solvent is a mixed solvent of water and methanol, and preferably, the ratio of water to methanol is 1: 3; the reaction conditions when the initiator is added in the reaction are ice bath cooling and argon bubbling for 50 min; the reaction temperature is 46 ℃, the reaction time is 20h, and the reaction condition is light-proof; and quenching the quenching reaction by adopting liquid nitrogen, wherein the precipitate is obtained by dropwise adding the solution into acetone when the temperature of the solution is raised to room temperature.
And/or further, the purification method in step (2) is SEC fractionation purification, and lyophilization after dialysis in a 2kDa dialysis bag for 2 days.
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 freeze-drying after dialysis.
And/or further, the step (4) isThe gadolinium ion-containing compound is GdCl3·6H2O, the reaction temperature is room temperature, and the pH value is 5.2-5.4; the purification method comprises 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 condition is light shielding; the purification method is freeze-drying 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 nano particle, which comprises the following specific steps:
(1) uniformly dispersing the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd in a chromatographic pure solvent, and slowly dropwise adding the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd into vigorously stirred ultrapure water;
(2) dialyzing the stirred solution, and freeze-drying to obtain the branched sugar-containing polymer nano-particle pGAEMA-PTX-DOTA-Gd.
Further, the chromatographically pure solvent in the step (1) is chromatographically pure DMSO, pGAEMA-PTX-DOTA-Gd, and the ratio of the DMSO to the ultrapure water is 100 mg: 10 ml: 10ml, and the stirring time is 2 hours; in the step (2), the dialysis conditions are dark and the temperature is 4 ℃.
The invention also provides application of the branched sugar-containing polymer nano-particle 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 clinical contrast agent Gd-DTPA, the small molecular contrast agent has the advantages that the pharmacological properties of the small molecular contrast agent can be obviously improved, so that the small molecular contrast agent has longer blood half-life period, more tumor part accumulation amount, higher tumor signal strengthening degree, longer strengthening duration and better tumor strengthening specificity. The nano particle can be quickly taken up by cells, releases therapeutic drugs under the stimulation of a tumor microenvironment, has longer in vivo circulation time and targeting property, can obviously improve the in vivo distribution of small molecular drugs so that the small molecular drugs can be effectively accumulated at tumor parts, can obviously inhibit the growth of tumors, has excellent anti-tumor effect in a BALB/c mouse 4T1 xenograft tumor model, has the tumor inhibition rate of over 90 percent, has no obvious system toxicity, has good biological safety, and has huge development potential and application prospect.
In the structural formula of the polymer, m, n, o, p, q, r, x, y and z represent the number of repeating units, and values are reasonably taken within a standard range conforming to the molecular weight of the polymer according to common technical knowledge mastered by a person skilled in the art.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 shows the preparation of the branched polymers pGAEMA-PTX-DOTA and pGAEMA-PTX-DOTA-Gd.
FIG. 2 is a drawing of (A) branched sugar-containing polymer PTX-DOTA-Gd nanoparticles1H NMR. (B) EDX spectra of the prodrug. (C) Fluorescence spectra of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd before and after covalent attachment Ppa. (D) The particle size of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd nanoparticles in water, (E) TEM characterization of the obtained morphology and (F) stability in PBS solution.
FIG. 3 is the Critical micelle concentration (CACs) of the branched polymer pGAEMA-PTX-DOTA-Gd
FIG. 4 is SEC detection results of (A) nanoparticles and their degradation products after 24h incubation with cathepsin B. (B) Enzyme-responsive release profile of PTX. (C) Development effect plots of Gd-DTPA and nanoparticles at different Gd concentration gradients and (D) T1 relaxation efficiency/Gd (III) concentration curves.
FIG. 5 is (A) the 4T1 cellular uptake of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd nanoparticles at different time points observed by CLSM. (B) Flow analysis of nanoparticle uptake by 4T1 cells after different incubation times. (C) Cytotoxicity of nanoparticles and free PTX on 4T1 cells. (D) And (3) infiltrating the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd after being incubated with the 4T1 tumor spheres for 2 and 6 hours. Blue fluorescence is Hoechst33342 stained nuclei and purple fluorescence is Ppa labeled branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd, scale: 50 μm. (E) The distribution of the fluorescence signal of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd along the labeling direction and (F) a 3D topology with a depth of 70 μm. (G) CLSM-photographed multicellular tumor spheres incubated with free PTX and a branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd. Wherein the green fluorescence is Calcein-AM stained living cells, and the red fluorescence is PI stained dead cells. A scale: 50 μm.
FIG. 6 is a multicellular tumor sphering brightfield image of 48 hours co-incubation of free PTX and the branched polymer pGAEMA-PTX-DOTA-Gd. A scale: 200 μm.
FIG. 7 is (A) microtubule aggregation of 4T1 cells treated with free PTX and the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd. Red fluorescence is tubulin-tracker red stained microtubules and blue fluorescence is DAPI stained nuclei. A scale: 10 μm. (B) Semi-quantitative analysis of the cell cycle distribution of 4T1 treated with free PTX and branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd and (C) cycle distribution. (D) Apoptosis of 4T1 cells treated with free PTX and branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd and (E) semi-quantitative analysis of apoptosis. (. P <0.01, vs. control, n-3). Control cells were incubated with fresh medium only.
FIG. 8 is a graph of Gd ion concentration/time in blood of (A) BALB/c mice injected with Gd-DTPA and branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd nanoparticles, respectively. (B) Fluorescence images of major organs and tumors of mice in the nanoparticle and free ppappappa administration groups at different time points after administration. S is normal saline, G is branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd, P is Ppa, H is heart, Li is liver, Sp is spleen, Lu is lung, Ki is kidney and Tu is tumor. (C) Fluorescence semi-quantitative analysis of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd at 1,6,12,24,48 and 72h after administration. (D) Gd content analysis of main organs and tumors after administration of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd.
Figure 9 is a semi-quantitative analysis of fluorescence at the tumor site 1,6,12,24,48,72h after injection of free Ppa and the branched polymer pGAEMA-PTX-DOTA-Gd in tumor-bearing mice (n ═ 3).
FIG. 10 is a semi-quantitative analysis of fluorescence of tissues and organs 1,6,12,24,48,72h after injection of the branched polymer pGAEMA-PTX-DOTA-Gd.
FIG. 11 is an analysis of Gd content of each tissue organ 24h after injection of free Ppa.
Figure 12 is (a) representative MRI imaging pictures of BALB/c mice injected with branched carbohydrate-containing polymers pGAEMA-PTX-DOTA-Gd nanoparticles and Gd-DTPA at different time points at the tumor site and (B) SI% relative signal-to-noise enhancement (p <0.05, n ═ 5). (C) branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd therapeutic strategy. (D) Schematic representation of tumor tissue after treatment for each group. (E) Relative volume of tumors in different groups (. P <0.01, n ═ 7 compared to saline group and free PTX). (F) Tumor tissue weight for each group (. P <0.01,. P <0.001, compared to saline). (G) In-vivo weight change curves 21 days after administration of mice during treatment. (H) Schematic representation of tumor tissue TUNEL and CD31 staining for different treatment groups. (I) Semi-quantitative analysis of CD31 and (J) TUNEL.
FIG. 13 is The tumor growth inhibition index (The tumor growth inhibition, TGI) of free PTX and The carbohydrate-containing branched polymer PTX-DOTA-Gd.
FIG. 14 is histopathological analysis of mice 21 days after treatment with physiological saline, free PTX and the carbohydrate-containing branched polymer PTX-DOTA-Gd (all tissues:. times.100).
Detailed Description
Material
Pyropheophorbide a (ppappapa), gadolinium chloride hexahydrate (GdCl 3.6H 2O), 2,2'- [ azobis (1-methylethylidene) ] bis [4, 5-dihydro-1H-imidazole ] dihydrochloride (VA044), 4-cyanopentanoic acid dithiobenzoic acid (CTA), N' -Dicyclohexylcarbodiimide (DCC), 4-Dimethylaminopyridine (DMAP), 1-hydroxybenzotriazole (HOBt), O-benzotriazol-tetramethyluronium Hexafluorophosphate (HBTU), N-Diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA), cathepsin B was purchased from SIGma-Aldrich and used without further purification. Monomers MA-GFLG-PTX, MA-DOTA, MA-GFLGK-MA were synthesized according to the methods reported previously. All other reagents and solvents were purchased from the corm chemical reagent factory (chengdu, china) and used without further purification.
4T1 mammary tumor cells were purchased from the cell bank of the Chinese academy of sciences type culture Collection (China, Shanghai) and cultured in RPMI 1640 medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin solution, Life technologies, USA) in a constant temperature and humidity cell culture chamber (5% CO)2(ii) a Cultured at 37 ℃). Female BALB/c mice (weight 20. + -. 1.2g and 6-8 weeks old) were purchased from Duoduosho Biotech Inc. All animal experiments were performed according to the rules of the ethical committee of the relevant countries and university of sichuan china.
Structural identification of intermediates and final products of various monomers1H NMR (Bruker AV II-400 spectrometer, Bruker, Switzerland), electrospray ionization mass spectrometry (ESI MS, waters, USA) and liquid chromatography mass spectrometry (LC-MS/MS, ABI, USA) were used in combination. Average molecular weight and polydispersity index (PDI) of the polymer Using Rapid protein chromatography (PDI)FPLC system, GE Healthcare, sweden) was measured by Size Exclusion Chromatography (SEC). Sodium acetate buffer (H) was chosen2ACN 70:30, v/v, pH 6.5) as mobile phase. The column was GE Healthcare Superose 6HR10/30 with a flow rate of 0.4 mL/min. By passing1H NMR characterizes the polymer structure. The nanosized particle size and zeta potential (3mg/mL) of the polymers in aqueous solution were measured using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). Each measurement was set up in 3 replicates and the results were processed using DTS software version 3.32. Polymer particle size was measured using Transmission Electron Microscopy (TEM) (GF-20S-TWIN, FEI Tecnai, USA). UV-vis spectra were measured using an ultraviolet-visible spectrophotometer (Cary 400, Varian, USA). Determination of the polymers by means of a fluorescence spectrophotometer (RF-5301-PC, Shimadzu, Japan)Fluorescence spectrum. Statistical analysis was performed using the Student's t test. Results are expressed as mean ± Standard Deviation (SD). p value<0.05 considered 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 (1.34g,4.38mmol), MA-DOTA (803mg,1.56mmol), MA-GFLG-PTX (648mg,0.5mmol), PTEMA (31.8mg,0.13mmol), MA-GFLG-CTA (68.9mg, 72. mu. mol) and MA-GFLGK-MA (61.5mg, 93.8. mu. mol) were added to the polymerization flask and the flask was placed under argon for protection. VAO44 (9.0mg, 28. mu. mol) was dissolved in 15ml H2O/CH31/3, and adding into the polymerization bottle, cooling in ice bath, and bubbling argon for 50 min. The polymerization bottle is heated in an oil bath at 46 ℃ under dark condition for 20 hours, and the reaction is quenched by liquid nitrogen. The reaction solution was warmed to room temperature and then added dropwise to 300ml of acetone solution to obtain a pink precipitate. The crude product was further purified by SEC fractionation. Dialyzing in a 2K dialysis bag for 2 days, and freeze-drying to obtain a pink solid intermediate pGAEMA-DOTA1.63g with the yield of 55%.
pGAEMA-PTX-DOTA (1.5g) was dissolved in 15mL of DMSO under an argon atmosphere, and dithiothreitol (DTT, 300mg) was added to the above solution. Stirring for 10h at room temperature, dialyzing with deionized water in a 2K dialysis bag for 2 days, and freeze-drying to obtain 1.2g of pink solid product. Branched pGAEMA-PTX-DOTA-SH (1.2g) was dissolved in 25mL deionized water and GdCl was added3.6H2O (600mg), and stirring at room temperature to obtain a solution having a pH of 5.2 to 5.4. The product was dialysed for lyophilization and then redissolved in DMSO. maleimide-Ppa (12mg) was dissolved in 2mL of DMSO, and the resulting solution was added to the above reaction system, and the mixture was reacted at room temperature for 6 hours in the dark, dialyzed in RO water in a 2K dialysis bag for 1 day, and lyophilized to obtain 1.07g of a pale green solid. The PTX, Ppa and Gd contents in the final product were 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 (100mg) was uniformly dispersed in 10mL of chromatographically pure DMSO, and slowly added dropwise to 10mL of vigorously stirred ultrapure water under ice bath. After stirring for 2h, the mixture was dialyzed at 4 ℃ against light until the DMSO was removed. And (3) 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, confirmation of chemical Structure and molecular weight
1) Experimental Material
pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
By passing1The H NMR spectrum analyzed the chemical structure of pGAEMA-PTX-DOTA-Gd. As shown in FIG. 2A, a proton peak of the sugar-containing polymer pGAEMA-PTX-DOTA was observed at 7.0 to 8.5ppm, indicating the presence of aromatic groups in the polymer. It may be derived from the benzene ring in PTX, Ppa or GFLG, on the other hand, the polymer does not observe a proton peak at 5.0-6.0ppm, since the peak is observed at 5.60ppm (s, -CH) due to the double bond characteristic of the monomer3-CH(r)-CH2-Ha) And 5.20ppm (s, -CH)3-CH(r)-CH2-Hb) Indicating that the monomer in the reaction has been completely consumed.
Element identification was performed by EDX method. As shown in FIG. 2B, the identification of the elements C, O, S and Gd confirms that Gd is present3+Have been successfully attached to polymers.
Fluorescence spectra were measured by fluorescence spectrophotometer and as shown in FIG. 2C, characteristic peaks of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd and free Ppa were detected at 673nm, indicating that the fluorescence properties of Ppa also remained unchanged during the 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 was 2.57%, 3.26% and 2.54%, respectively, and the molar ratio was about 2:1:1, indicating the presence of GFLG in the polymer.
By rapid protein chromatography (FPLC system, GE Healthcare, sweden) the average molecular weight and polydispersity index (PDI) of the polymer were measured by Size Exclusion Chromatography (SEC). Sodium acetate buffer (H) was chosen2ACN 70:30, v/v, pH 6.5) as mobile phase. The column was GE Healthcare Superose 6HR10/30 with a flow rate of 0.4 mL/min. The results show that the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd has a molecular weight of approximately 244kDa and a PDI of 2.48 (Table 1).
Table 1: characterization parameters of the sugar-containing branched polymer pGAEMA-PTX-DOTA-Gd.
aMolecular Weight (MW) in kDa;
bpercent (%) amino acids, PTX, Gd and Ppa;
cnanoparticle diameter size (nm) by DLS;
dzeta potential (ζ) is in mV.
Test example 2 evaluation of particle size distribution, zeta potential, morphology and stability
1) Experimental Material
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. A nanoparticle solution (1mg/mL) was prepared using deionized water, and then the particle size and zeta potential (n ═ 3) were measured using a malvern nanoparticle size and potential analyzer. The stability of the nanoparticles in PBS was determined in the same manner.
Transmission electron microscopy (TEM Tecnai G2F 20S-TWIN, FEI, Hillsboro, Oregon, USA) was used to detect the surface morphology of the nanoparticles. An aqueous solution of nanoparticles (0.5mg/mL) was added dropwise to the copper mesh, and after excess liquid was removed by suction with filter paper, it was air-dried for TEM detection. As shown in FIG. 2D, the DLS results show that the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd nanoparticles have a hydrodynamic diameter of 95.17nm and a negative surface charge. As shown in FIG. 2E, the TEM test results show that the conjugate can form uniformly distributed nanoparticles of about 70nm, and further prove the existence of self-assembly behavior of the conjugate. As shown in fig. 2F, the stability test result further indicates that the nanoparticles have good stability in PBS, and the particle size and PDI do not change significantly after 48h storage in PBS.
Critical Aggregation Concentration (CAC) of polymer nanoparticles in ultrapure water was measured using pyrene fluorescence. Firstly, accurately weighing and preparing a series of nanoparticle solutions (0.01-500 mu g/mL) with different concentration gradients. Subsequently, 25. mu.L of pyrene in acetone (5X 10) was pipetted-5M) in a series of 10mL sample bottles, after acetone is completely volatilized, respectively adding 2mL of the nanoparticle solutions with the concentration gradients (the final concentration of pyrene is 6.25 multiplied by 10)-7M). Finally, the solution is placed in a constant temperature oscillator at 37 ℃ and incubated for 2h in the dark, and then the fluorescence spectrum (Em:390nm, Ex:300 nm-350 nm) of pyrene is measured by a fluorescence spectrophotometer. As a result, as shown in FIG. 3, the critical aggregation concentration of the formed nanoparticles was 23.7. mu.g/mL, and such a low CAC value indicates that the nanoparticles are expected to have better stability.
Test example 3 biodegradation and drug Release evaluation
1) Experimental Material
Nanoparticles of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1 and the branched carbohydrate-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 (pH5.4, 50mM citrate, 0.1M phosphate and 2mM EDTA) containing cathepsin B (2.8. mu.M) at a polymer concentration of 6 mg/mL. And (3) incubating the polymer solution in a shaking table at 37 ℃, taking out 1mL of mixed solution at a preset time point (0, 2, 6,12, 20 hours), and determining the molecular weight of the sample by an SEC method, wherein the mobile phase is the mixed solution of sodium acetate buffer solution and methanol, the ratio of the solvent is 7:3, the final pH value of the solvent is 6.5, and the flow rate is 0.4 mL/min. Samples were prepared in triplicate in the experiment.
Drug release experiments with the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd were also performed in McIlvaine buffer (pH 5.4) containing cathepsin B (2.8. mu.M) at a sample concentration of 3 mg/mL. The solution was incubated for 12h at 37 ℃ on a shaker. At a predetermined time point (0, 2, 6,12, 20h), 100. mu.L of the solution was taken out and mixed with equal volume of chromatographically pure methanol, and then detected and analyzed by RP-HPLC high performance liquid chromatography (liquid phase analytical column C8 column 4.6X 150mm), the mobile phase was equal volume of acetonitrile and water, the flow rate was 1.0 mL/min, and the ultraviolet detection wavelength was 227 nm. The drug release properties of the nanoparticles of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd in McIlvaine buffer without cathepsin B (pH5.4 and pH 7.4) 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 significant degradation behavior. In contrast, there was no significant change in polymer molecular weight after 20h incubation in the control group without cathepsin B added. This result indicates that the degradation of the sugar-containing polymer can be attributed to the cleavage of the GFLG short peptide in the polymer structure. FIG. 4A shows the time at which the degradation products peaked after co-incubation of the polymer with cathepsin B. The molecular weight of the degraded polymer (28kDa) is well below the renal threshold, thus facilitating its rapid metabolism out of the body. Meanwhile, the good stability of the polymer under physiological conditions also contributes to prolonging the in vivo circulation time of the polymer.
Table 2: degradation products of 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, almost no PTX release (less than 3%) was observed in PBS solution without cathepsin B (pH5.4, 37 ℃) over 24h, whereas the cumulative amount of PTX released from the sugar-containing polymer was over 90% under the cathepsin B-containing conditions. On the other hand, under simulated physiological conditions (pH 7.4, 37 ℃), PTX is released in about 25% over 24 h. This result is consistent with previous studies, and is attributable to the fact that ester-linked PTX has better stability under weakly acidic conditions than under neutral conditions. The research results show that the glycopolymer can rapidly release the carried PTX drug under the stimulation of a tumor microenvironment after being taken by tumor cells, so as to generate corresponding cytotoxicity.
Test example 4 evaluation of in vitro relaxation Rate
1) Experimental Material
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 concentrations of Gd3+ (0, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4mM) branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd were dissolved in 0.1M PBS and the material was scanned for magnetic resonance signal intensity by T1 SE sequence with the following scanning parameters: TE 8.7ms, TR 20, 30, 50, 70, 90, 125, 150, 175, 200, 300, 400, 500, 700, 850, and 1000ms, Fov 200mm, slice thickness 1.0mm, and matrix dimensions 256 × 256. And acquire the corresponding 1/T1 values by their T1 weighted MR images. By plotting 1/T1 as different Gd3+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 relaxivity of the sample was obtained by the same method as described above.
The results are shown in FIG. 4C, which is related to the relative Gd3+The signal strength of the branched polymer pGAEMA-PTX-DOTA-Gd group is higher than that of the Gd-DTPA group with the same content. FIG. 4D shows the branched polymers pGAEMA-PTX-DOTA-Gd and Gd-DTPA and Gd for clinical use3+Are linearly related, and R of both2Are all equal to about 1.0. Relaxation efficiency r of branched polymers pGAEMA-PTX-DOTA-Gd1Calculated as 7.1L/(mmol. multidot.s) and is approximately Gd-DTPA (r)1＝3.6) of the total weight of the powder. Whether the polyamino polyhydroxy Gd-containing polymer can be mainly used for clinical diagnosisDependent on its rotational correlation time (tau)R) And τ isRIt can be reduced by increasing the molecular mass, thereby increasing the relaxation efficiency. The small molecular Gd-DOTA in pGAEMA-PTX-DOTA-Gd is covalently connected to the branched polymer, so that the relaxation efficiency is greatly improved, and the MRI contrast ratio is improved.
Test example 5 evaluation of in vitro cellular uptake, cytotoxicity and infiltration and growth inhibition
1) Experimental Material
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
Mouse mammary tumor cell (4T1) cell suspension was seeded onto 35X 12mm glass dishes (cell number 1X 10)5) After 24h incubation, the primary medium was removed and pGAEMA-PTX-DOTA-Gd containing a branched carbohydrate-containing polymer (at a concentration corresponding to a PpaPpa concentration of 0.25. mu.g.mL) was added-1) RPMI 1640 medium of (1). Subsequently, the cells were incubated in an incubator for 1, 3, and 5 hours, respectively, the medium was removed, the cells were washed three times with PBS (pH 7.4), and PBS containing Hoechst33342 (10 μ g/mL) was added to the cells and stained in the dark for 15 min. Subsequently, after removing the dye and washing with PBS 3 times, the cell entry was observed with a laser confocal microscope (CLSM) to obtain a fluorescence image of the cell-taken material.
Mouse breast tumor cells (4T1) were selected to evaluate the relative cytotoxicity of 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 plated in 96-well plates at a cell density of 5X 10 per well3After 24h of adherent growth in the incubator, the RPMI 1640 medium was removed and the free PTX and the branched glycopolymer pGAEMA-PTX-DOTA-Gd media containing different concentration gradients (concentrations corresponding to PTX concentrations from 0.039 to 40. mu.M) were added, respectively. After another 48h incubation, the medium was removed and washed three times with PBS. A cytotoxicity evaluation kit CCK-8(Dojindo, Japan) was added to each well. After incubation for 2h in a cell incubator, absorbance was measured by a multifunctional enzyme labeling apparatus (Thermo Fisher SCIENTIFIC), and absorbance of each sample was measured by an enzyme labeling apparatus, as statedCell viability was calculated as the instructions.
Multicellular Tumor Spheres (MTS) of 4T1 cells were prepared and used to study the tumor penetration and tumor cell growth inhibition of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd. First 2mL of a molten 2% agarose solution was added to a glass dish 5cm in diameter. After the agarose solution had solidified, 4mL of 4T1 cell suspension (5 mL. times.10) containing fresh RMPI 1640 medium was added6cells/mL) was added to agarose. After about 5 days of incubation MTS was formed and the cell pellet diameter was about 200 μm. 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, both viable and dead 4T1 cells were stained. First, after the MTS diameter reached about 200 μm, fresh RPMI 1640 medium was added, wherein the branched glycopolymers pGAEMA-PTX-DOTA-Gd containing 2 μ g/mL PTX and free PTX were added to the medium in both groups, while the control group contained no PTX related therapeutic. After 48h of continued incubation of the MTS, the medium was removed and the MTS was transferred to a 1mL EP tube. Thereafter, the surviving and dead 4T1 cells were differentiated by double staining with fluorescein calcein AM/propidium iodide (CAM/PI), and MTS was fixed and transferred to glass dishes, and the stained tumor cell spheres were observed under CLSM. The experimental data obtained were further processed using NIS-Elements AR software.
As shown in fig. 5A, laser confocal microscopy (CLSM) showed Ppa with red fluorescence mainly distributed in the cytoplasm. After incubation, the Ppa fluorescence enhancement of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd overlaps well with lysosomal fluorescence staining of lysosomes, suggesting that the polymer is likely to be taken up by cells via the endocytosomal pathway. The fluorescence intensity gradually increased with time, and showed a clear time dependence. As shown in fig. 5B, flow quantification further revealed time-dependent cellular uptake of nanoparticles, consistent with the results obtained from laser confocal microscopy (CLSM) experiments.
As shown in FIG. 5C, both the free PTX group and the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd group showed significant concentration-dependent cytotoxicity. Wherein the IC of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd50The value was 1.99. mu.M, which is about 1.7 times (1.17. mu.M) the free PTX. This result may be due to the free PTX diffusing into the cell by free diffusion, while the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd employs a different endocytosis pathway and drug release process.
Furthermore, two-dimensional cytotoxicity studies on 4T1 cells showed that the antitumor effect of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd is comparable to that of free PTX. Its antitumor effect was further confirmed by applying it to three-dimensional MTS. From the topological 3D images and the CLSM images (FIGS. 5D-F) it can be seen that 2h after application of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd, the polymer is concentrated to a large extent in the periphery of the MTS. After the incubation time was prolonged to 6h, a significant enhancement of the signal on MTS could be observed, demonstrating the deep penetration of the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd into the three-dimensional MTS. At the same time, there were a large number of morphologic abnormalities and isolated cells in MTS incubated with the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd, compared to MTS treated with free PTX. After 48h incubation, MTS incubated with both free PTX and branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd showed an increase in PI-positive cells (dead cells), whereas MTS treated with branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd produced more heterotypic 4T1 cells, whereas the whole tumor sphere was relatively smaller. Thus, these experimental results demonstrate that the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd is able to penetrate deeply into three-dimensional solid tumor tissues and enhance the anti-tumor effect of PTX.
The inhibitory effect of branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd on 4T1 MTS is shown in FIG. 5G. The growth of MTS is shown in FIG. 6. Both morphological abnormalities and cell death were observed in large pieces of cells in MTS treated with free PTX and branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd. After 48h incubation, a significant increase in PI positive area was observed for both the free PTX group and the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd group MTS. Cellular morphological abnormalities may be caused by prolonged incubation and cell necrosis. Thus, the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd has a deep penetrating power for 3D solid tumors and an equivalent anti-tumor power as free PTX.
Test example 6 in vitro antitumor mechanism study
1) Experimental Material
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
And (3) detecting cell tubulin: the 4T1 cell line was seeded on a 20mm diameter glass dish (cell number 5X 10)3) In (1). After 24h incubation, the original medium was removed, fresh RPMI 1640 medium containing free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd (both at 0.5. mu.g/mL) was added separately, and a set of fresh RPMI 1640 medium added without free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was set as a control. The 4T1 cells were then incubated for 24 h. The medium was removed and cell microtubules were assayed by a Tubulin-Tracker Red solution (C1050, Beyotime, chinese achievements) according to the protocol provided by the manufacturer. After staining of the cell microtubes, the cells were rinsed twice with PBS and then incubated in Hoechst33342 (10. mu.g/mL) PBS for 10 minutes. The dye was removed, the cells were washed twice with PBS and observed under CLSM. The data from the experiment 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) of 5cm diameter4) In (1). The original medium was removed and fresh RPMI 1640 medium containing free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd (both at a concentration of 0.5. mu.g/mL) was added separately and a set of fresh RPMI 1640 medium without free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was added as a control. The cells were then incubated for 24 h. The medium was removed and the cells were trypsinized and centrifuged. Subsequently, the proliferation cycle was determined by PI/RNase staining solution according to the protocol provided by the manufacturer. After the cells were fixed and stained, the cell proliferation cycle was examined using a flow cytometer. The data obtained from the experiment are further processed by ModFit LT 3.1 softwareAnd (5) physical and analysis.
And (3) detecting cell apoptosis: 4T1 cell suspensions were seeded into 6-well plates (cell number 1.5X 10)5Each well) were incubated for 24h, the original medium was removed, media containing free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd (concentrations of 0.8 μ g/mL PTX and 1.8 μ g/mL PTX, respectively) were added, and a set of fresh RPMI 1640 media without free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd was set as a control. After further incubation for 24h, the medium was removed and the cells were trypsinized and centrifuged. And then, staining the cells according to the operation steps in the Annexin V-FITC apoptosis test box, quantitatively detecting the apoptosis condition by using a flow cytometer, and further processing and analyzing the data obtained by the experiment by using WinMDI 2.9 software.
Fig. 7A clearly shows the microtubule structure of 4T1 cells after staining with tubulin. The normal microtubule structure labeled by Tubulin-Tracker Red has been latticed in 4T1 cells, and after the treatment with branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd and free PTX, microtubule aggregation and multinuclear structure are observed in single cells, which proves that the microtubule stability is enhanced during cell division. Two groups of cell results show that compared with free PTX, the branched carbohydrate-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 the G1 phase (37.84%) was significantly higher than the number in the G2/M phase (9.39%). While the percentage of the cells in the G1 phase and the G2/M phase in the 4T1 cells treated by the free PTX is 14.06 percent and 23.34 percent respectively, and the percentage is 18.33 percent and 22.98 percent respectively in the group of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd, which proves that the addition of the free PTX and the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd can effectively inhibit the division of tumor cells and obviously increase the blocking rate of the G2/M phase.
As shown in fig. 7D and 7E, both free PTX and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd were able to induce apoptosis and necrosis of cells efficiently after drug treatment, wherein free PTX induced apoptosis and necrosis in 37.2% of cells and branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd induced 31.2%, compared to the control group. The results show that the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd based on the nano structure can be effectively taken up by cells, specifically releases chemotherapeutic drugs in the microenvironment of the cells and efficiently kills tumor cells.
Test example 7, pharmacokinetics and evaluation of drug Dispersion in vivo
1) Experimental Material
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
Metabolic analysis of the branched carbohydrate-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 carbohydrate-containing polymers pGAEMA-PTX-DOTA-Gd (0.08mmol/kg Gd) were injected separately via the tail vein3+) After the dosage, 20 μ L of blood was collected by fundus vein blood collection at time points of 0min, 5min, 15min, 30min, 1h, 2h, 4h, 8h, 12h, 24h and 48h, and the blood was subjected to HNO3And H2O2(1: 3) digesting, and measuring the content of Gd (III) by Inductively coupled plasma mass spectrometry (ICP-MS). The pharmacokinetic parameters were calculated by PKSolver software.
And (3) detecting the biodistribution condition of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd in the main organs of the mouse in the mouse body at different time points by adopting fluorescence imaging. Growing the tumor to about 150mm3The mice were randomly divided into two groups, each injected via the tail vein with 1.5mg/kg of a branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd, and free Ppa, relative to Ppa. Live fluorescence pictures were taken before and after injection at 1,6,12,24,48 and 72h, and the drug distribution at the tumor sites of the mice was detected and recorded.
In addition, to further explore the in vivo distribution of the compounds, the tumor-bearing mice injected with the above drugs were euthanized at 1,6,12,24,48 and 72h after administration, 3 mice per each time point. Tumors and major organs (heart, liver, spleen, lung, kidney) were denuded, weighed, and fluorescence images of these tissues and organs were semi-quantitatively analyzed using a Maestro In-Vivo imaging system. Saline injected mice were used as a control group for analysis.
Simultaneously adopting ICP-MS to detect Gd in main visceral organs of mice3+Content to investigate the distribution of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd in the body of mice at different time points. 4T1 tumor-bearing mice (tumor volume about 150mm)3) Randomly divided into 4 groups (n-5). Clinical contrast agent Gd-DTPA and branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd are injected through tail veins respectively. Each reagent is given to two groups, which are marked respectively, and the administration dosage is 0.08mmol Gd3+Mice/kg. One group of mice in the Gd-DTPA and branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd dosing groups were euthanized 24h and 96h after dosing, respectively. Tumors and major organs (heart, liver, spleen, lung, kidney) were denuded, 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 it. Each of the completely digested sample solutions was diluted to the same volume with ultrapure water, and Gd in the sample was detected using ICP-MS3+The concentration of (c). The distribution of Gd in each organ is calculated as: gd (Gd)3+Relative content (ng Gd/g tissue) Gd in each sample3+Quality of the corresponding organ.
As shown in fig. 8A, the clinical agent Gd-DTPA showed a very rapid blood clearance rate, and the presence of Gd was barely detectable in plasma 1h after administration. In contrast, the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd decreases in concentration in blood significantly slower, showing a significantly prolonged blood circulation time, with Gd concentrations up to 10. mu.g/mL being detectable in plasma after 4h of administration, and Gd concentrations of 7. mu.g/mL remaining detectable after 12 h. As shown in table 3, the results of the two-compartment model analysis showed that Gd-DTPA showed a very short blood half-life (14.2min), in contrast to nanoparticles showing a significantly prolonged blood half-life time of 1255.4 min. In addition, the nanoparticles also have a significantly reduced mean systemic clearance rate and an extended mean residence time compared to Gd-DTPA.
Table 3: the two-compartment model pharmacokinetic related parameters of the PKSolver 2.0 software.
As shown in FIG. 9, the signal distribution of Ppa at the tumor site of tumor-bearing mice is shown. In free Ppa group, only very weak Ppa signal was detected at 12 h. Whereas the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd group detected a higher Ppa fluorescence signal at all time points, with the fluorescence signal increasing continuously before 24h and then beginning to decrease. Furthermore, as shown in fig. 8B, free Ppa and Ppa labeled nanoparticles exhibited significantly different organ tissue distributions after administration. Among them, in the free Ppa-administered group, a strong fluorescence signal was observed at the liver site 1h after the administration, indicating that it was accumulated in a large amount at the liver site, and a fluorescence signal was hardly observed at the tumor site. The fluorescence intensity of each organ decreased significantly with time, and almost no fluorescence signal was observed after 12 hours. In contrast, Ppa-labeled nanoparticles showed a gradual and persistent fluorescence distribution, and a course of significant fluorescence signal enhancement was observed at the tumor site. The results are further revealed by semiquantitative fluorescence analysis, as shown in fig. 8C, wherein the fluorescence signal at the tumor site reached a maximum and then slowly decreased after 24h of administration in the group of Ppa-labeled nanoparticles. The fluorescence signals of the ppappappa group were gradually reduced in each organ and tumor after administration (fig. 10). These results indicate that the Ppa-labeled nanoparticles have longer in vivo circulation time and can be targeted to tumor sites through the EPR effect, so that the in vivo distribution of small molecule drugs is expected to be improved, and the anti-tumor efficacy of the small molecule drugs is improved.
As shown in FIG. 11, trace amounts of Gd were detectable in the organs and tumors of the mice 24h after Gd-DTPA injection3+Wherein 0.1% ID/g Gd is detectable in the liver3+Is total Gd3+0.2% of the injected amount, 0.1% ID/g detectable in tumors, Gd in other organs3+The concentration is less than 0.1% ID/g. This result and fluorescenceThe imaging results were consistent. However, after 24h injection of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd, large amounts of Gd could be detected in the liver (30.9% ID/g) and spleen (18.4% ID/g) of mice3+Whereas in the kidney this value is 4.8% ID/g. At the same time, about 4.3% ID/g Gd is detectable in tumor tissue3+. And Gd in liver and spleen 96h after injection3+The concentration was significantly reduced, 11.9% ID/g for the former and 6.1% ID/g for the latter. And Gd detected in tumors and other organs3+The concentration was extremely low (fig. 8D). Despite Gd3+The biodistribution results are similar to those of Ppa fluorescence imaging, but when Gd is present3+After dissociation from the polymer, Gd3+Has a relatively high rate of clearance, while Ppa-bonded carbohydrate-containing polymer backbones and their degradation products clear relatively slowly from the body. The results of these in vivo distribution experiments show that the biodegradable branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd can be rapidly excreted out of the body through metabolism after exerting its biological function in vivo, and Gd is avoided3+Potential toxicity caused by long-term retention in the body.
Test example 8 evaluation of in vivo magnetic resonance imaging
1) Experimental Material
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
Xenografted 4T1 tumor-bearing mice (tumor volume approximately 150mm)3) The samples were randomized into two groups (n ═ 5) and injected separately via the tail vein with the clinical contrast agent Gd-DTPA and the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd (the dose administered corresponds to 0.08mmol Gd)3+In kg mice). Mice were anesthetized with isoflurane gas and then fixed in custom coils. Contrast enhanced imaging scans were performed using a T1 SE sequence as follows: TE 20ms, TR 500ms, FOV 40ms, thickness 1.0mm, Flip angle 90 °. MRI images were taken for each experimental group before injection, 10min, 30min, 1h, 4h and 24h post injection, respectively. The relative enhancement of MRI signal intensity (SI%) is defined as SI (post-injection)/SI (pre-injection) x 100%. By plotting SI% vs. timeTo assess signal changes at the tumor site using semi-quantitative analysis.
Changes in tumor MRI signals from mice in the branched carbohydrate-containing polymers pGAEMA-PTX-DOTA-Gd and Gd-DTPA groups at various time points before and after injection are shown in FIG. 12A. For the group of branched carbohydrate-containing polymers pGAEMA-PTX-DOTA-Gd, the contrast between the tumor site and the surrounding tissues of the mice is gradually enhanced with the time after the injection of the contrast agent, the outline of the tumor is clearly visible, and the contrast is always kept high within 24 h. 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 substantially returned to the level before injection after 4 h. Semi-quantitative analysis of The relative enhanced intensity Signal (SI) at The tumor site is shown in fig. 12B. The branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd group SI was continuously enhanced within 24h, while the Gd-DTPA group reached a peak 30min after injection and then began to decline. This result indicates that the small molecule Gd-DTPA contrast agent has no tumor targeting in vivo and can be rapidly cleared from the body. Notably, at each time point after dosing, the relative enhanced signal intensity was higher for the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd group than for the Gd-DTPA group. These results demonstrate that the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd has a better in vivo imaging effect than the small molecule Gd-DTPA contrast agent. This may be due on the one hand to its better relaxation efficiency and on the other hand to its longer circulation time in vivo due to its larger molecular weight, thus enabling more accumulation at the tumor site.
Test example 9 evaluation of in vivo antitumor Activity
1) Experimental Material
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 carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd was studied by establishing a 4T1 tumor model for subcutaneous xenografts. 4T1 cells were suspended in 70. mu.L PBS (1.4X 10)5Individual cells) and injected subcutaneouslyRight hind leg of female BALB/c mice. Tumor volumes were calculated as follows: v ═ lxw2X 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 60-80mm3When mice were randomly divided into saline, PTX combination and branched glycopolymer-containing PTX-DOTA-Gd (n ═ 7) groups. Mice were treated with the above formulations by tail vein injection every 3 days at a dose of 10mg PTX/kg mice for 4 total doses. At the same time, body weight and tumor volume of each mouse were recorded every 2 days. All mice were euthanized on day 21, and tumors and major organs (heart, liver, spleen, lung, kidney) were dissected out, respectively. Tumors from each group were weighed and tumor inhibition rate (TGI) was calculated using the formula: TGI ═ 100% (1-W1/W2) where W1 and W2 represent the mean tumor weights of the treated and control groups, respectively.
The tumor growth inhibition of the mice by different administration groups was observed in a model of xenografted 4T1 breast tumor as shown in FIG. 12C, the change of the tumor volume of the mice in each administration group is shown in FIGS. 12D and 12E, after the first administration, the tumor growth of the normal saline group showed a rapid growth trend, the tumor growth rate of the PTX group was similar to that of the normal saline group, and the tumor growth of the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd group was significantly inhibited. From the second dose, the growth rate of tumors began to slow in PTX group mice compared to saline group, but tumor growth inhibition was more pronounced in mice treated with branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd and tumor growth was consistently inhibited during the subsequent treatment without a tendency to regrow. After day 21 of administration, the group of branched carbohydrate-containing polymers pGAEMA-PTX-DOTA-Gd showed a very significant anti-tumor effect compared to the group of physiological saline (approximately 1631% relative to the tumor volume), and some of the mice had completely disappeared tumors, and the tumor size of the mice, from which the remaining tumors had not disappeared, was also maintained at a very small level. While free PTX exhibits a relatively weak anti-tumor effect (approximately 1049% relative to tumor volume), this is probably due to the fact that small molecules of PTX do not have the ability to target and circulate long, and the drug is rapidly cleared from the body after entering the body, making it difficult to achieve sufficient drug concentration at the tumor site.
As shown in fig. 12F, the tumor weight of the material group was much 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) compared to the saline group, showing a very significant tumor inhibition (TGI of 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 we prepared the nano-scale branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd material has long in vivo circulation time, can be passively targeted to the tumor area, thus 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, thus improving its tumor killing effect. In addition, the body weight change curves of tumor-bearing mice throughout the treatment period are shown in FIG. 12G. The figure shows that the body weight of each group of mice does not change obviously in the whole treatment period, which indicates that the branched carbohydrate-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 evaluation of TUNEL analysis
1) Experimental Material
The branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd prepared in example 1.
2) Experimental methods and results
The H & E staining method was as follows: washing the fixed mouse viscera with tap water overnight, dehydrating with ethanol of each grade by using a full-automatic dehydrator, performing xylene transparency, and performing wax dipping twice. Routine paraffin embedding was performed using an embedding machine, and paraffin blocks were cut into 5 μm paraffin sections for hematoxylin-eosin (H & E) staining using a rotary microtome. And finally performing microscopic examination and photographing the used specimen.
Immunohistochemical analysis was performed using a streptavidin-peroxidase method: the deparaffinized and rehydrated tumor sections were first incubated overnight at 4 ℃ with monoclonal anti-CD 31 antibody (1:200) (Beijing Biotechnology Co., Ltd.) and anti-Ki-67 monoclonal antibody (1:200) (Beijing Biotechnology Co., Ltd.), respectively. Then adding a biotinylated goat anti-mouse/rabbit IgG secondary antibody (1:200) dropwise for reaction at room temperature for 20 min; immunohistochemical Images were finally acquired by Motic Images Advanced software (Motic China Group CO., LTD.) and the positive stain Integrated Optical Density (IOD) of CD31 was measured 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.
Deoxynucleotide terminal transferase mediated dUTP nick end labeling (TUNEL) assay method as follows: TUNEL analysis was performed using the in situ apoptosis detection kit (Roche Molecular Biochemicals, Laval, Quebec, Canada) according to the protocol provided by the manufacturer. Light microscopy was used to observe TUNEL staining positive cells (i.e. apoptotic cells) and the ratio of the number of apoptotic cells to the total number of tumor cells in each microscopic field was calculated as the 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 more severe infiltration of inflammatory cells in liver tissue sections from both the saline and PTX groups compared to normal liver tissue from the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd. In all other groups of organs, no significant organic toxicity was observed in any of the three experimental groups.
CD31 antigen staining is used for evaluating angiogenesis during tumor cell proliferation, and the result of detecting apoptosis by using a TUNEL method is shown in FIG. 12H and FIG. 12I. Compared with the normal saline group, the count of the mouse tumor tissue MVD-CD31 in the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd group is obviously reduced, and the statistical significance is very remarkable (p is less than 0.05). The free drug PTX group has no obvious difference in MVD-CD31 count compared with the physiological saline group, and has no statistical significance (p > 0.05). The results show that the branched carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd has very good effect of inhibiting the angiogenesis of tumors. As shown by TUNEL staining results in fig. 12J, the apoptosis rate of tumor cells in 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 carbohydrate-containing polymer pGAEMA-PTX-DOTA-Gd treated group (37.7%). These results indicate that the branched carbohydrate-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 the apoptosis necrosis of tumor cells.
In conclusion, the multifunctional biodegradable branched polymer drug delivery system (branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd) based on the sugar-containing polymer is prepared by RAFT polymerization to realize the integration of diagnosis and treatment of tumors. The branched polymer selects a branched sugar-containing polymer which has higher functional group density and larger hydrodynamic size and has a topological structure similar to that of a dendrimer as a skeleton, and loads fluorescent dyes pyropheophorbide A (Ppa) and metal gadolinium ion chelate (Gd-DOTA) as imaging groups and Paclitaxel (PTX) as a therapeutic group. In addition, in order to ensure the biodegradability and biosafety of the branched polymer, GFLG tetrapeptide linker, which is degradable in tumor microenvironments overexpressing cathepsin B, was used to make the branched structure. Compared with a clinical contrast agent Gd-DTPA, the branched sugar-containing polymer pGAEMA-PTX-DOTA-Gd nano particle is prepared by designing the proportion of each functional monomer and the cross-linking agent, and compared with the clinical contrast agent Gd-DTPA, the nano particle prepared by the invention can obviously improve the pharmacological property of a small molecular contrast agent, so that the small molecular contrast agent has longer blood half-life period, more tumor part accumulation amount, higher tumor signal strengthening degree, longer strengthening duration, better tumor strengthening specificity and the like. Meanwhile, in vitro experiments show that the nano particle can be rapidly taken up by cells, and cathepsin B in tumor cells is used for responsively degrading and releasing chemotherapeutic drug PTX, so that cytotoxicity close to that of free drugs is induced. In vivo experiments show that the nanoparticles can obviously improve the in vivo distribution of small molecular drugs, can be effectively accumulated at tumor sites through EPR effect, can obviously inhibit the growth of 4T1 xenograft tumor by reducing the generation of new vessels, inducing the apoptosis of tumor cells and the like, and have good biological safety. Therefore, the multifunctional nano-drug delivery system based on the branched carbohydrate-containing polymer carrier has great development potential in the field of tumor diagnosis and treatment integrated research.