CN113209049B

CN113209049B - Tumor weak acid environment mediated construction and deconstruction polymer, preparation method and application

Info

Publication number: CN113209049B
Application number: CN202110445776.4A
Authority: CN
Inventors: 袁友永; 周洁莲; 杨蕊梦; 江新青; 王可伟; 罗诗维; 姚旺
Original assignee: Guangzhou First Peoples Hospital
Current assignee: Guangzhou First Peoples Hospital
Priority date: 2021-04-23
Filing date: 2021-04-23
Publication date: 2022-09-13
Anticipated expiration: 2041-04-23
Also published as: CN113209049A

Abstract

The invention belongs to the technical field of biomedicine and oncology, and particularly relates to a polymer for tumor weak acid environment mediated construction and deconstruction, a preparation method and application thereof, wherein the preparation method of the polymer comprises the following steps: preparing a polymer-drug conjugate PFC by using PAMAM, ferrocenecarboxylic acid and cinnamaldehyde as raw materials; two kinds of nano-particle cPFC are constructed based on the conjugate PFC _DBCO 、cPFC _N3 The nano-particles cPFC _DBCO And nanoparticle cPFC _N3 Constructing a polymer mediated by the tumor acid environment in the acid environment. The polymer has the characteristics of acid-responsive crosslinking and decrosslinking, and in an acid environment, the nanoparticle cPFC _DBCO 、cPFC _N3 The rapid acid response is crosslinked into large particles, which is beneficial to increasing the enrichment of the drug in tumor tissues; nanoparticle cPFC _DBCO 、cPFC _N3 The slow acid response is carried out to release the small-particle-size drug PFC through crosslinking, so that the drug can permeate into a tumor hypoxia area, and the tumor cell killing agent has the advantage of effectively killing tumor cells under both normoxic and hypoxic conditions.

Description

Tumor weak acid environment mediated construction and deconstruction polymer, preparation method and application

Technical Field

The invention belongs to the technical field of biomedicine and oncology, and particularly relates to a polymer for tumor weak acid environment mediated construction and deconstruction, a preparation method and application thereof.

Background

Iron death is a recently discovered non-apoptotic cell death pattern that can be activated by inactivation of glutathione peroxidase 4(GPX4) to break the balance between the production and elimination of reactive oxygen species (ROS, primarily hydroxyl radicals) within the cell. Fenton reaction mediated chemokinetic therapy (CDT) utilizes metal ions to catalyze less active H ₂ O ₂ The generation of highly cytotoxic OH, leading to liposome peroxidation, has become a promising strategy for inducing iron death in tumor cells. Compared to ROS-based treatments such as photodynamic therapy, sonodynamic therapy, etc., the chemodynamic therapy is not limited by oxygen concentration and penetration depth.

The particle size is a key control factor of the processes of effective enrichment of the drug in tumor tissues, tumor permeability, cell internalization and the like in a targeting system. Generally, particles of 10-100 nm are generally considered to have good effects. For the internalization of the cells, the smaller the particle size is, the more beneficial the phagocytosis of the cells is, within the range of 10-100 nm. Thus, a variable-size particle system is likely to be an effective, novel strategy for achieving multiple enhancements in permeability, persistence, and cellular internalization and nuclear phagocytosis simultaneously.

In conclusion, the size of the nanoparticle is variable by response to the tumor acid and crosslinking and decrosslinking so as to obtain a more advanced drug delivery system, and the combination of the size-variable particle system and a stimulation-triggered drug release system has great significance for increasing the effective enrichment and penetration of the drug in tumor tissues and the internalization of cells.

Disclosure of Invention

Aiming at the defects in the prior art, one of the purposes of the invention is to provide a polymer for tumor weak acid environment mediated construction and deconstruction, which has the characteristic of variable size of nano particles and can be used as a more excellent drug carrier, thereby effectively increasing the enrichment and permeation of drugs in tumor tissues and improving the killing effect on tumor cells; the invention also provides a preparation method and application of the tumor weak acid environment mediated constructed and deconstructed polymer.

Based on the purpose, the technical scheme adopted by the invention is as follows:

in a first aspect, the invention provides a method for preparing a tumor weak acid environment mediated constructed and deconstructed polymer, which comprises the following steps:

(1) preparing a polymer-drug conjugate PFC by using PAMAM, ferrocenecarboxylic acid and cinnamaldehyde as raw materials;

(2) coupling Polymer-drug conjugates PFC with CDM-OEG ₄ Carrying out amide reaction on CDM at room temperature according to the molar ratio of 1 (2-4) to prepare a nanoparticle cPFC;

(3) the PAEMA-DBCO after carboxyl activation and the cPFC react at room temperature according to the molar ratio of 1 (4-6) to prepare the nanoparticle cPFC _DBCO ；

(4) Reacting OH-PEG-N3 subjected to acyl chlorination with cPFC according to the molar ratio of 1 (4-6) at room temperature to prepare the nanoparticle cPFC _N3 ；

(5) The nano-particles cPFC _DBCO And nanoparticle cPFC _N3 And (3) constructing a polymer mediated by the tumor acid environment and deconstructing in an acid response way at the pH value of (6.5-6.9).

The invention takes PAMAM, ferrocenecarboxylic acid and cinnamaldehyde as raw materials to prepare a polymer-drug conjugate PFC, and two different nano-particles cPFC are constructed based on the PFC _DBCO And cPFC _N3 The two kinds of nanoparticles have the characteristic of weak acid environment response of tumor, and can be in weak acid environment of tumor tissues, namely cPFCCD _BCO The particles have quick acid response and are converted from hydrophobicity to hydrophilicity to expose pairing groups DBCO and cPFC _DBCO Particles and cPFC _N3 The particles are rapidly cross-linked into particles with larger particle size by click chemistry, thereby realizing the enrichment of the drug in tumor tissues.

Due to the weak acid environment in the tumor cells, large particles formed by crosslinking are slowly subjected to crosslinking release, and the two types of uncrosslinked nano particles are further subjected to acid response to slowly release PFC with relatively smaller particle size, so that the PFC can be further permeated into a hypoxic region in the deep part of the tumor; when the PFC is taken up by tumor cells, the PAMAM in the PFC realizes lysosome escape through proton sponge effect, simultaneously, the acid response releases medicines of ferrocenecarboxylic acid and cinnamaldehyde, and Fenton reaction occurs in the tumor cells to cause the peroxidation of liposomes and induce the death of tumor cells iron, so that the PFC has huge clinical application potential.

Further, the process for preparing the polymer-drug conjugate PFC by using the PAMAM, the ferrocenecarboxylic acid and the cinnamaldehyde as raw materials in the step (1) comprises the following steps:

I. stirring and reacting carboxyl activated ferrocenecarboxylic acid and PAMAM for 20-30 h at room temperature according to the molar ratio (9-12) of 1 to obtain a PAMAM and ferrocenecarboxylic acid conjugate PF;

II, stirring and reacting the PAMAM and ferrocenecarboxylic acid conjugate PF with cinnamaldehyde for 20-30 h at room temperature in a protective gas atmosphere, and further coupling the cinnamaldehyde on the conjugate PF to obtain the polymer-drug conjugate PFC.

The invention sequentially couples ferrocenecarboxylic acid, cinnamaldehyde and other medicines on macromolecular PAMAM to prepare a polymer PFC (Power factor correction) for tumor acid environment-mediated deconstruction, takes the polymer-medicine conjugate PFC as a medicine carrier, and respectively constructs two nano-particle cPFC (continuous positive current factor correction) containing the medicine carrier _DBCO And cPFC _N3 The two kinds of nano particles can respond to and release PFC in a weak acid environment of tumor tissues, lysosome escape of the released PFC is realized due to PAMAM proton sponge effect, medicines such as ferrocenecarboxylic acid, cinnamaldehyde and the like in the PFC are further released, and the aim of inhibiting the activity of tumor cells is fulfilled.

Furthermore, the molar ratio of the cinnamaldehyde to the PAMAM is (3-5): 1.

Further, the carboxyl activation process of the PAEMA-DBCO in the step (3) comprises the following steps: stirring and reacting PAEMA-DBCO, EDC and NHS for 2-4 h at room temperature in DCM according to the molar ratio of 1 (1-1.5) to (1-1.5), and carrying out carboxyl activation on PAEMA-DBCO.

Further, step (4) is performed on OH-PEG-N ₃ The process of acyl chlorination is as follows: mixing OH-PEG-N ₃ Oxalyl chloride is stirred and reacted for 2 h-4 h in DCM for OH-PEG-N according to the molar ratio of 1 (4-6) under the catalysis of DMF at room temperature ₃ Acyl chlorination is carried out.

Further, the nanoparticle cPFC _DBCO And cPFC _N3 The particle size ranges of the nano particles are 50 nm-200 nm; the particle size range of the PFC is 8-12 nm.

In a second aspect, the invention provides a tumor weak acid environment mediated constructed and deconstructed polymer prepared by the preparation method.

Tumor weak acid environment mediated constructed and deconstructed polymer composed of two nanoparticles of cPFC _DBCO And cPFC _N3 The drug is formed by crosslinking in an acid environment, and the large particles formed by crosslinking the two types of nanoparticles are beneficial to realizing the enrichment of the drug in tumor tissues; in addition, the polymer constructed and deconstructed by the tumor mediated by the weak acid environment can realize decrosslinking in the acid environment, and the nanoparticles released by decrosslinking further release the small molecule drug PFC in an acid response manner, so that the aim of killing tumor cells is fulfilled. Namely, the polymer constructed and deconstructed by the tumor weak acid environment is crosslinked and decrosslinked in the tumor acid environment, so that the enrichment and release of the drug in the tumor environment are realized, and the method has an important clinical application prospect.

Further, the cPFC prepared by the nano-particles _DBCO And cPFC _N3 The particle size range of the polymer constructed by acid response within 30min is 800 nm-1100 nm; the particle size range of a deconstructed product PFC of the polymer after 6 hours of acid response is 8-12 nm.

In a third aspect, the invention provides an application of the polymer which is constructed and deconstructed by the tumor acid environment mediation prepared by the method in preparing an anti-tumor drug.

The polymer for tumor acid environment mediated construction and deconstruction provided by the invention has the characteristics of responding to crosslinking and decrosslinking in a tumor acid environment, the aggregation of a medicament in a tumor tissue is realized through crosslinking, and the penetration of the medicament in the tumor tissue is enhanced through decrosslinking, so that the polymer can be used for preparing an anti-tumor medicament and has the advantage of effectively killing tumor cells under the conditions of normal oxygen and hypoxic.

Compared with the prior art, the invention has the following beneficial effects:

(1) the invention constructs two different nanoparticles with tumor weak acid environment response, and click chemical reaction occurs between the nanoparticles to crosslink into large particles, which is beneficial to increasing the enrichment of the drug in tumor tissues; the acid-response uncrosslinked particles are slowly degraded to release the polymer-drug conjugate PFC with a relatively small particle size, the PFC is further degraded to release smaller-molecular drugs of ferrocenecarboxylic acid and cinnamaldehyde, the drug can be favorably permeated into a tumor hypoxia area, and the PFC-drug conjugate has the advantage of effectively killing tumor cells under the conditions of normal oxygen and hypoxia.

(2) The tumor microenvironment mediated construction and deconstruction polymer prepared by the invention realizes the enrichment and permeation enhancement of the drug in the tumor tissue by combining the weak acid response of the tumor tissue and the quick bioorthogonal crosslinking and slow decrosslinking, and meanwhile, the material has good biocompatibility and degradability.

Drawings

FIG. 1 is a schematic diagram of the synthetic route of the tumor acid environment mediated constructed and deconstructed polymer of the present invention;

FIG. 2 shows PFC ¹ H NMR chart;

FIG. 3 is a graph of cPFC acid response particle size change;

FIG. 4 shows cPFC _DBCO 、cPFC _N3 The particle size distribution map of (a);

FIG. 5 shows cPFC _DBCO A graph of particle size change for acid-responsive crosslinking and decrosslinking;

FIG. 6 shows cPFC _N3 A graph of particle size change for acid-responsive crosslinking and decrosslinking;

FIG. 7 is a graph showing the results of in vitro acid response release test of CA;

FIG. 8 is a graph showing the results of the in vitro acid-responsive release effect test of PAMAM;

FIG. 9 is a graph showing in vitro confirmation of Fenton reaction generation. OH;

FIG. 10 is a graph of the cytotoxicity of drugs on cell lines at different oxygen concentrations for MTT assay;

FIG. 11 is a graph of intracellular ROS levels following drug treatment in confocal assays at different oxygen concentrations;

FIG. 12 shows intracellular H after drug treatment at the usual cost for confocal measurements ₂ O ₂ A horizontal view;

FIG. 13 is a graph showing intracellular OH levels after drug treatment in confocal assays at different oxygen concentrations;

FIG. 14 is a graph of confocal measurements of intracellular GSH levels following drug treatment under normoxic and hypoxic conditions;

figure 15 is a graph of intracellular GPX4 levels following drug treatment under normoxic and hypoxic conditions for confocal assays;

FIG. 16 is a graph of confocal measurements of intracellular LPO levels following drug treatment under normoxic and hypoxic conditions;

FIG. 17 is a confocal detection of acid-responsive cell sphere permeability profile;

FIG. 18 is a graph of tumor volume changes in different experimental groups;

FIG. 19 is a graph showing the change in body weight of mice in different test groups.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments. The raw materials used in the following examples are all commercially available general-purpose products unless otherwise specified.

Example 1 preparation and characterization of Polymer-drug conjugate PFC

Preparation of polymer-drug conjugate PFC

The polymer-drug conjugate PFC, namely PAMAM-Ferr/CA, is synthesized by using Polyaminoamine (G4 PAMAM), ferrocenecarboxylic acid (ferrocenecarboxylic acid) and cinnamaldehyde (cinnamyl aldehyde) as raw materials, wherein the synthetic route of the PFC is shown in figure 1, and the specific preparation process is as follows:

(1) carboxylic activation of ferrocenecarboxylic acid

51.75mg of ferrocenecarboxylic acid (i.e., 0.225mmol of ferrocenecarboxylic acid), 41.85mg of EDC (i.e., 0.27mmol of EDC), 31.05mg of NHS (i.e., 0.27mmol of NHS) were sequentially added to a 50mL round-bottomed flask, and stirred at room temperature for 3 hours with 4mL of DMSO as a reaction solvent to activate the carboxyl group. Wherein EDC is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride; NHS is N-hydroxysuccinimide; DMSO is dimethyl sulfoxide.

(2) Preparation of PAMAM and ferrocenecarboxylic acid conjugate PF

Adding 360mg of PAMAM 4.0 (namely 0.025mmol of PAMAM 4.0) into the ferrocenecarboxylic acid activated by the carboxyl in the step (1), and continuously stirring at normal temperature for reaction for 24 hours; and then transferring the reacted mixture to a 3.5KDa dialysis bag, dialyzing with deionized water for 48h, and performing vacuum freeze drying to obtain the polymer-ferrocenecarboxylic acid conjugate PF.

(3) Preparation of polymer-drug conjugate PFC by coupling PF and cinnamaldehyde

To a 50mL round-bottom flask, PF prepared in step (2), 10mg of cinnamaldehyde (i.e., 0.075mmol of cinnamaldehyde) and 4mL of DMSO as a reaction solvent were sequentially added in N ₂ Stirring and reacting for 24 hours at normal temperature under the condition; transferring the reacted mixture to a 3.5KDa dialysis bag, dialyzing with deionized water for 48h, and performing vacuum freeze drying to obtain the polymer-drug conjugate PFC.

Characterization of intermediates and PFCs

Subjecting the polymer-drug conjugate PFC to hydrogen nuclear magnetic resonance spectroscopy ( ¹ H NMR) analysis, determination of its molecular structure, of PFC ¹ The H NMR is shown in FIG. 2.

As shown in fig. 2, of PFC ¹ The H NMR spectrum letters mark the proton hydrogen ascribed to the PFC. The characteristic peaks of the ferrocenecarboxylic acid appear in 4.189ppm, 4.269ppm and 4.686 ppm. Characteristic peaks of cinnamaldehyde appeared at 6.913ppm, 7.201ppm, 7.423ppm, 7.592ppm, 8.134 ppm. The peak of 8.01ppm belongs to PAMAM, belongs to methine beside amido bond, and the other methylene peaks of PAMAM have chemical shifts of 2.395ppm, 2.596ppm, 2.821ppm, 3.012ppm and 3.305ppm respectively due to different structural environments.

Example 2 preparation of nanoparticle cPFC

The synthesis route of the nanoparticle cPFC is shown in figure 1, and the specific preparation process is as follows:

(1) 30mg of the PFC (i.e., 0.002mmol of PFC) obtained in example 1 and 8mL of DMSO were sequentially added to a 50mL round-bottomed flask, and 100. mu.L of 0.1mol/L PBS (phosphate buffered saline) was added to adjust the reaction solution to a weakly alkaline state, thereby obtaining a PFC reaction solution.

(2) 3.2mg of CDM-OEG ₄ CDM (i.e. 0.006mmol of CDM-OEG) ₄ CDM) in DMSO to give 1mg/mL CDM-OEG ₄ CDM solution, wherein CDM-OEG ₄ The CDM is a double-arm cross-linking agent synthesized by the inventor, and the specific structure and the synthesis path are as follows:

(3) mixing CDM-OEG ₄ Dropping the CDM solution into the PFC reaction solution at the speed of 1mL/h by using a syringe pump, and continuing stirring and reacting for 8h at normal temperature after the dropping is finished. And transferring the reacted mixture to a 100KDa dialysis bag, and dialyzing with deionized water for 48 hours to obtain a cross-linked product, namely the nanoparticle cPFC.

Example 3 nanoparticle cPFC _DBCO Preparation of

Nanoparticle cPFC _DBCO The synthesis route is shown in figure 1, and the specific preparation process is as follows:

(1) carboxyl activation of PAEMA-DBCO

In a 50mL round bottom flask were added 12mg of PAEMA-DBCO (i.e., 0.002mmol of PAEMA-DBCO), 0.372mg of EDC (i.e., 0.0024mmol of EDC), 0.276mg of NHS (i.e., 0.0024mmol of NHS) in sequence, and 2mL of DCM was used as a reaction solvent, and the mixture was stirred at room temperature for 3h to activate the carboxyl group. Wherein PAEMA-DBCO is poly (2-azepane ethyl methacrylate) -Dibenzo cyclooctanesAn alkyne; EDC is 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride; NHS is N-hydroxysuccinimide; DCM is dichloromethane.

(2) Nanoparticle cPFC _DBCO Synthesis of (2)

The PAEMA-DBCO activated by carboxyl is slowly dripped into the cPFC solution of DMSO phase, 150mg, namely 0.01mmol of cPFC is contained in the cPFC solution of DMSO phase, and the stirring reaction is continued at normal temperature for 24 h. Transferring the reacted mixture to a 100KDa dialysis bag, and dialyzing with deionized water for 48h to obtain a cross-linked product, namely the nanoparticle cPFC _DBCO 。

Example 4 nanoparticle cPFC _N3 Preparation of

Nanoparticle cPFC _N3 The synthesis route of (2) is shown in figure 1, and the specific preparation process is as follows:

(1)OH-PEG-N ₃ by acid chlorination of

4mg of OH-PEG-N was added to a 50mL round bottom flask in sequence ₃ (i.e., 0.002mmol of OH-PEG-N ₃ ) 1.27mg oxalyl chloride (0.01 mmol oxalyl chloride), 50 μ L DMF (N, N-dimethylformamide) as catalyst, 4mL DCM as reaction solvent, stirring at room temperature for 3h, and vacuum drying the mixture after reaction to obtain acyl-chlorinated OH-PEG-N ₃ 。

(2) Nanoparticle cPFC _N3 Preparation of

To the acyl-chlorinated OH-PEG-N prepared in step (1) ₃ 2ml of EDC was added to the reaction solution, followed by addition of cPFC dissolved in DMSO, which contained 150mg of cPFC (i.e., 0.01mmol of cPFC), and the reaction was continued at room temperature with stirring for 24 hours. Transferring the reacted mixture to a 100KDa dialysis bag and dialyzing the mixture for 48 hours by using deionized water to obtain a cross-linked product, namely the nano-particle cPFC _N3 。

Example 5 detection of Properties of nanoparticles

1. Particle size of nanoparticles

The nanoparticles cPFC and cPFC prepared in examples 2-4 were prepared by Dynamic Light Scattering (DLS) respectively _DBCO 、cPFC _N3 The particle size of the nanoparticle cPFC before the acid response and the change of the particle size of the nanoparticle cPFC after the acid response are detected, and a graph showing the change of the particle size of the nanoparticle cPFC before the acid response is shown in FIG. 3, it can be seen that the particle size range of the nanoparticle cPFC before the acid response is 50nm to 200nm, and the particle size range of the nanoparticle cPFC after the acid response is 8nm to 12 nm.

Nanoparticle cPFC _DBCO And cPFC _N3 The particle size distribution before acid response is shown in figure 4, the nanoparticle cPFC _DBCO And cPFC _N3 The particle size of the nano particle is 50 nm-200 nm, which is similar to the particle size range of the nano particle cPFC before acid response, and the influence of PAEMA-DBCO or OH-PEG-N3 grafted on the nano particle cPFC on the particle size of the nano particle cPFC is small.

2. Construction and deconstruction properties of acid-responsive nanoparticles

Monitoring cPFC with dynamic light scattering instrument _N3 And cPFC _DBCO Acid-responsive crosslinking and decrosslinking between particles. Deionized water is used as a solvent to prepare 10mg/mL of nanoparticle cPFC _N3 And cPFC _DBCO . Subsequently, cPFC _N3 And cPFC _DBCO Mixing 50 μ L of the above solutions, adding buffer solution of 1mL, pH 7.4 or pH 6.5, respectively, mixing, and detecting the cPFC by dynamic light scattering instrument for 15min and 24h _N3 And cPFC _DBCO The particle size varied.

After mixing, the particles cPFC is mixed for 15min and 24h _N3 And cPFC _DBCO The results of the particle size variation test are shown in FIG. 5 and FIG. 6, respectively, under the condition of pH 6.5, cPFC _N3 And cPFC _DBCO The particle size of the nanoparticles is rapidly increased by rapid click crosslinking. cPFC with extended mixing time _N3 And cPFC _DBCO The PFC internally crosslinked is slowly crosslinked, and the particle size of the nano particles is reduced.

3. In vitro drug Release test

To investigate whether cpfcs can release Cinnamaldehyde (CA) in an in vitro acid response, the following experiment was performed:

taking 2mL of the cPFC nanoparticle solution prepared in example 2, placing the solution into a dialysis bag (MWCO 3.5KD), adding 2mL of buffer solutions with pH 7.4, pH 6.5 and pH 5.0, respectively, placing the dialysis bag into a 50mL centrifuge tube, adding 10mL of buffer solution with corresponding pH into the centrifuge tube as dialysis external liquid, placing the centrifuge tube in a constant-temperature shaking water tank at 37 ℃, sampling 1mL of dialysis external liquid into an EP tube at specific time intervals, supplementing fresh dialysis external liquid with the same volume, testing absorbance of the sampled liquid by an ultraviolet spectrophotometer, drawing a standard curve by an external standard method, calculating drug release content, cumulative release rate and drug release ratio at corresponding time, setting 3 experiments in each group in parallel, and testing results are shown in FIG. 7.

4. In vitro cPFC _RhB Release test

To verifycPFC _RhB Acid response is adopted to perform crosslinking decomposition, and rhodamine B is adopted to mark PFC to obtain PFC _RhB Then by PFC _RhB With crosslinking agent CDM-OEG ₄ CDM preparation of cPFC _RhB The following experiments were carried out:

2mL of cPFC were taken _RhB The particle solution is filled into a dialysis bag (MWCO 3500), 2mL of buffer solution with pH 7.4 and pH 6.5 is respectively added into the dialysis bag, the dialysis bag is filled into a 50mL centrifuge tube, 10mL of buffer solution with corresponding pH is added into the centrifuge tube to be used as dialysis external liquid, the centrifuge tube is placed in a constant-temperature oscillation water tank with the temperature of 37 ℃, 500 mu L of dialysis external liquid is sampled into an EP tube at intervals of specific time, fresh dialysis external liquid with the same volume is supplemented, the absorbance of the sampled liquid is tested by a fluorescence spectrophotometer, a standard curve is drawn by an external standard method, the drug release content, the cumulative release rate of corresponding time and the drug release ratio are calculated, 3 parallels are set in each group of experiment, the test result is shown in figure 8, under the acidic condition of pH 6.5, the cPFC of the nanoparticles is used for realizing the parallel reaction, and the pH value of the nanoparticles is controlled by the pH value of the PCR _RhB PFC capable of being slowly released by acid-response uncrosslinking _RhB Is favorable for promoting the penetration of the medicine in tumor tissues.

5. In vitro nanoparticle Fenton reaction hydroxyl radical generation test

In order to investigate whether the nanoparticles prepared according to the present invention can generate hydroxyl radicals through fenton reaction, the following experiment was performed:

disodium terephthalate is taken as a trapping agent of hydroxyl free radicals, and finally, the solution is subjected to fluorescence tracing at the excitation wavelength of 310nm and the fluorescence tracing at the wavelength of 425nm by a fluorescence spectrophotometer.

4 5mL centrifuge tubes were added with 50. mu.L of H ₂ O ₂ (100. mu.M) and 100. mu.L of a disodium terephthalate solution (50.0mmol/L), and finally 100. mu.L of PBS buffer, 100. mu.L of PC (8mg/mL), 100. mu.L of PF (40mg/mL) and 100. mu.L of PFC (40mg/mL) were added to each of the 4 centrifuge tubes, and each of the centrifuge tubes was placed in a shaking bath at a constant temperature of 37 ℃ for 12 hours, and then the generation of hydroxyl radicals was measured by a fluorescence spectrophotometer. Wherein, PC is a graft copolymer formed by PAMAM and cinnamaldehyde; PF is a graft copolymer formed by PAMAM and ferrocenecarboxylic acid.

As shown in fig. 9, it can be seen that PF and PFC can effectively catalyze the conversion of hydrogen peroxide into hydroxyl radicals due to the grafting of ferrocenecarboxylic acid on PAMAM, which contains iron ions, while PC and PBS buffer without iron ions can not catalyze the conversion of hydrogen peroxide into hydroxyl radicals.

Example 6 tumor weak acid environment mediated constructed and deconstructed nanoparticles killing effect test on tumor cells

This example will analyze the killing effect of the nanoparticles prepared by the present invention on tumor cells from seven aspects as follows.

1. Experiment of killing effect of acid-response drug-loaded nanoparticles on 4T1 cells

The specific method of the killing effect experiment of the acid response drug-loaded nanoparticles on the 4T1 cells is as follows:

a) cell plating: the 96-well plate was inoculated with 10000 cell number of 4T1 cells per well at 37 ℃ with 5% CO ₂ And different oxygen concentrations (1% O) ₂ 、5％O ₂ 、10％O ₂ 、21％O ₂ ) Culturing for 24h by using a DMEM medium containing 10% FBS under the condition;

b) adding medicine: diluting the drug solution with serum-free RPMI 1640 medium to obtain a medium containing 8mg/mL PC drug, a medium containing 40mg/mL PF drug and a medium containing 40mg/mL PFC drug, replacing the original medium of a 96-well plate with the medium containing the drug, and incubating the drug for 24 h; each group is provided with 3 multiple holes, and a drug-free group is arranged as a blank control; wherein PF is a PAMAM and ferrocenecarboxylic acid graft copolymer; PC is a PAMAM and cinnamaldehyde graft copolymer; the PFC is a double-drug polymer containing ferrocenecarboxylic acid and cinnamaldehyde.

c) MTT detection: dissolving an MTT reagent in PBS to the concentration of 0.5mg/mL, replacing a drug-containing culture medium in a 96-well plate with a PBS solution containing MTT, incubating for 4 hours in a cell incubator, absorbing the PBS containing the MTT reagent, adding 0.1mL of DMSO into each hole to dissolve formazan crystals, oscillating for 30min, and detecting the 570nm absorption value by a micropore detector; and calculating the cell activity of the experimental group according to the ratio of the absorption values of the experimental group and the non-medicated control group.

The test result is shown in fig. 10, and it can be seen that under different oxygen concentrations, the PFC double drug group can kill tumor cells more effectively than the single drug group (PC and PF).

2. Intracellular ROS levels of acid-responsive drug-loaded nanoparticles at different oxygen concentrations

And observing the ROS level of the acid response drug-loaded nanoparticles under different oxygen concentrations by using a laser confocal scanning microscope. 4 experimental groups of PBS, PC, PF and PFC are respectively arranged, wherein the drug-loaded nanoparticles in the PC experimental group are nanoparticles cPC formed by graft copolymerization of PAMAM and cinnamaldehyde, the drug-loaded nanoparticles in the PF experimental group are nanoparticles cPF formed by graft copolymerization of PAMAM and ferrocenecarboxylic acid, and the drug-loaded nanoparticles in the PFC experimental group are nanoparticles cPFC formed by graft copolymerization of PAMAM, ferrocenecarboxylic acid and cinnamaldehyde. Controlling the concentrations of the single medicine group PC and PF and the concentrations of the cinnamaldehyde and the ferrocenecarboxylic acid in the double medicine groups to be consistent, and incubating the nanoparticles and the 4T1 cells for 4 h. Intracellular ROS levels (green) and nuclei (blue) were observed by confocal microscopy, stained with ROS probe (DCFH-DA; 2',7' -dichlorodihydrofluorescin diacetate) and 4', 6-diamidino-2-phenylindole (DAPI). As shown in fig. 11A, the PC and PFC groups showed strong green fluorescence compared to the PBS group at different oxygen concentrations, and the PFC group fluorescence was significantly stronger than the PC group, mainly because the active oxygen content was increased due to the stimulation of hydrogen peroxide by CA, the generated hydrogen peroxide was further catalytically decomposed into hydroxyl radicals by iron ions, and the ROS probe (DCFH-DA) was more sensitive to hydroxyl radicals, so the PFC group fluorescence was significantly stronger than the PC group. The intracellular active oxygen content after cell administration was quantified by blackboard fluorescence, and the quantification result was consistent with the confocal imaging result as shown in fig. 11B.

In order to further verify that cinnamaldehyde can stimulate cells to produce hydrogen peroxide, intracellular hydrogen peroxide is quantified by a titanyl sulfate colorimetric method. 4 experimental groups of PBS, PC, PF and PFC are respectively arranged, and the concentrations of the PC and PF of the single medicine group and the cinnamaldehyde and the ferrocenecarboxylic acid in the double medicine group are controlled to be consistent. The nanoparticles were incubated with 4T1 cells for 6h, the cells were collected and resuspended in acetone in an ice bath. 1ml of acetone supernatant was collected and centrifuged at 8000rpm/min for 10min to remove suspended cells. Then 100. mu.L of 0.03mol/L TiOSO was added to each sample ₄ 、200μLNH ₃ ·H ₂ O, a yellow precipitate appeared. After centrifugation at 5000rpm/min for 10 minutes, the precipitate was collected and dissolved in 2mL of 1mol/L sulfuric acid solution, and the solution was measured at λ 405nm using an ultraviolet-visible spectrophotometer. Finally, determining the intracellular H according to the drawn standard curve ₂ O ₂ The concentration of (2). The results are shown in FIG. 12, where the intracellular hydrogen peroxide was significantly elevated in the PC-administered group under normoxic and hypoxic conditions. Indicating that cinnamaldehyde can effectively stimulate the production of hydrogen peroxide by cell number.

3. Intracellular OH levels of acid-responsive drug-loaded nanoparticles at different oxygen concentrations

And (3) observing the OH levels of the acid response drug-loaded nanoparticles under different oxygen concentrations by using a laser confocal scanning microscope. 4 experimental groups of PBS, PC, PF and PFC are respectively arranged, the concentrations of the PC and PF of the single medicine group and the cinnamaldehyde and the ferrocenecarboxylic acid in the double medicine group are controlled to be consistent, and the nano particles and 4T1 cells are incubated for 4 hours. OH probes (APF; Aminophenyl fluorochein) and 4', 6-diamidino-2-phenylindole (DAPI) were stained, and intracellular OH levels (green) and nuclei (blue) were observed by confocal microscopy. The results are shown in FIG. 13A. The PFC group showed the strongest green fluorescence, indicating that cinnamaldehyde stimulates cells to produce hydrogen peroxide, which is further catalytically decomposed by iron ions into hydroxyl radicals. As shown by the results, the PF group also shows weak green fluorescence compared with the PBS group, mainly the tumor cells highly express hydrogen peroxide, and the iron ions in the PF group can catalyze and convert the hydrogen peroxide inherent in the cells into hydroxyl radicals. Intracellular hydroxyl radical content was quantified by blackboard fluorescence after cell administration, as shown in fig. 13B, the quantification results were consistent with confocal imaging results.

4. Intracellular GSH levels of acid-responsive drug-loaded nanoparticles under normoxic and hypoxic conditions

In order to verify that cinnamaldehyde can effectively consume intracellular glutathione GSH, a laser confocal scanning microscope is utilized to observe GSH levels of the acid response drug-loaded nanoparticles under different oxygen concentrations. PBS, PC, PF, PFC + DFO are respectively arranged in 5 experimental groups, the concentrations of the PC and PF of the single medicine group and the cinnamaldehyde and the ferrocenecarboxylic acid in the double medicine group are controlled to be consistent, and the nanoparticles and 4T1 cells are incubated for 4 hours. GSH probe (ThiolTracker) ^TM Violet) dyeing, copolymerizationIntracellular GSH levels (green) were observed under a focal microscope. The results are shown in FIG. 14. The green fluorescence of the PC and PFC groups is obviously weakened, which indicates that the cinnamaldehyde can effectively consume the GSH. The PFC group showed a stronger GSH consumption capability. After the iron ion chelating agent Deferoxamine (DFO) is added into the PFC, the iron ions in the PFC are chelated, so that hydrogen peroxide cannot be further catalyzed and converted into hydroxyl radicals, the oxidizing capability is reduced, and the GSH consumption capability is reduced.

5. Intracellular GPX4 levels of acid-responsive drug-loaded nanoparticles under normoxic and hypoxic conditions

GSH consumption can lead to GPX4 inactivation and induce tumor cell iron death, and the expression level of GPX4 protein is observed through immunofluorescence labeling. PBS, PC, PF, PFC + DFO are respectively arranged in 5 experimental groups, the concentrations of the PC and PF of the single medicine group and the cinnamaldehyde and the ferrocenecarboxylic acid in the double medicine group are controlled to be consistent, and the nanoparticles and 4T1 cells are incubated for 4 hours. The test result is shown in fig. 15, the PFC can effectively down-regulate the expression of GPX4 protein to induce the death of tumor cells, and after the PFC is added with the iron ion chelator, Deferoxamine (DFO), since the iron ions in the PFC are chelated, the PFC cannot further catalyze the conversion of hydrogen peroxide into hydroxyl radicals, the oxidizing ability is reduced, the GSH consumption ability is reduced, and the activity of the GPX4 protein is increased.

6. Intracellular Liposome Peroxidation (LPO) levels of acid-responsive drug-loaded nanoparticles under normoxic and hypoxic conditions

And (3) observing the LPO level of the acid response drug-loaded nanoparticles under different oxygen concentrations by using a laser confocal scanning microscope. Respectively setting 5 experimental groups of PBS, PC, PF, PFC + DFO, controlling the concentrations of the PC and PF of the single medicine group and the cinnamaldehyde and ferrocenecarboxylic acid of the double medicine group to be consistent, and incubating the nanoparticles and 4T1 cells for 4 h. LPO Probe (BODIPY) ^665/676 ) And 4', 6-diamidino-2-phenylindole (DAPI) staining, confocal microscopy to observe intracellular LPO levels (red) and nuclei (blue). As a result, as shown in fig. 16A, the PFC group generated hydroxyl radicals intracellularly, so that the liposome was more oxidized and red fluorescence was stronger than the PC group. After the addition of the iron chelator to the PFC, the fluorescence decreases. Intracellular liposome peroxidation levels after cell administration were quantified by flow fluorescence, as shown in figures 16B and 16C,the quantitative results are consistent with the confocal imaging results.

7. Acid-responsive cell sphere penetration

To verify that the crosslinked PFCs can achieve acid-responsive release, enhancing permeation. Marking PFC with rhodamine B, and obtaining cPFC after crosslinking _RhB And a cross-linking product pPFC formed by acid-insensitive pPAMAM, ferrocenecarboxylic acid and cinnamaldehyde _RhB Each set of nanoparticles was incubated with 4T1 cells for 8h at Ph 6.5 and Ph 7.4, respectively. And (3) observing the acid response decrosslinking and permeation enhancement of the particles by using a laser confocal scanning microscope. The results are shown in FIG. 17. Acid-responsive decrosslinked cPFCs _RhB Can maximally permeate into the cell ball under the acidic condition.

Example 7 animal test for antitumor efficacy of tumor-weak acid environment-mediated constructed and deconstructed nanoparticles

35 BALB/C mice, which were implanted with the 4T1 subcutaneous tumor model, were randomly divided into 7 groups of 5 mice each. 100 μ L of the following seven solutions were injected separately into the tail vein:

(1) PBS group: PBS buffer solution;

(2) cP group: cP buffer, namely PAMAM acid is crosslinked to form nanoparticles, and the injection concentration of the cP buffer is 1mg/kg of mouse body weight calculated by the PAMAM;

(3) cPF group: cPF buffer solution, cPF is nanoparticles formed by acid response crosslinking after graft copolymerization of PAMAM and ferrocenecarboxylic acid, and the injection concentration of cPF buffer solution is 50mg/kg mouse weight based on ferrocenecarboxylic acid;

(4) cPC group: cPC buffer solution, cPC is nanoparticles formed by acid response crosslinking after PAMAM and cinnamaldehyde graft copolymerization, and the injection concentration of cPC buffer solution is 10mg/kg mouse weight based on cinnamaldehyde;

(5) and (c) cPFC group: cPFC buffer solution, cPFC is nanometer particles formed by responding PFC acid to crosslinking, and the injection concentration is 10mg/kg mouse body weight calculated by cinnamaldehyde;

(6) pPFC group: pPFC buffer solution, wherein pPFC is an acid-insensitive nanoparticle formed by PFC non-acid-responsive crosslinking, and the injection concentration of the pPFC buffer solution is 10mg/kg of the body weight of a mouse in terms of cinnamaldehyde; (ii) a

(7)cPF _CDBCO +cPFC _N3 Group (2): 50 μ L of cPF _CDBCO +50μL cPFC _N3 The injection concentration is 10mg/kg of mouse body weight calculated by cinnamaldehyde.

The drug was given every two days and the mice were subjected to the 14d course of treatment. The tumor volume was measured with a vernier caliper every two days throughout the treatment and the weight change of the mice in each experimental group was examined. The formula for tumor volume is as follows: volume (mm) ³ ) 0.5 x length x width ² 。

The results of the experiment are shown in FIG. 18, in PBS, cP and cPF, the tumors grew rapidly. cPC group has certain effect in inhibiting tumor growth, because Cinnamaldehyde (CA) enters tumor site to increase intracellular ROS, and has killing effect on tumor.

Compared with an acid-insensitive pPFC group, the cPFC group has a more obvious tumor growth inhibition effect. The PAMAM is released by acid response to decrosslinking, and carried medicaments of cinnamaldehyde and iron ions are deeply penetrated into the deep part of a tumor to induce intracellular Fenton reaction to generate more active oxygen, so that a better treatment effect is obtained.

cPFC _DBCO +cPFC _N3 The nano-particles are subjected to double acid response crosslinking and decrosslinking, so that the cinnamaldehyde and iron ions serving as the medicaments can be greatly enriched in tumor tissues, the medicaments can be continuously released, and the anti-tumor effect is achieved.

In addition, the body weight of each group of mice is monitored in the whole treatment process, as shown in fig. 19, it can be seen that the body weight of each group of mice does not change obviously in the whole treatment process, and the acid-responsive drug-loaded nanomaterial disclosed by the invention is proved to have good biocompatibility.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A preparation method of a polymer for tumor acid environment mediated construction and deconstruction is characterized by comprising the following steps:

(2) coupling Polymer-drug conjugates PFC with CDM-OEG ₄ Carrying out amide reaction on CDM at room temperature according to the molar ratio of 1 (2-4) to prepare nano-particle cPFC; the CDM-OEG ₄ The structure of CDM is as follows:

(3) the PAEMA-DBCO after carboxyl activation and the cPFC react at room temperature according to the molar ratio of 1 (4-6) to prepare the nanoparticle cPFC _DBCO (ii) a The PAEMA-DBCO is poly (2-azepane ethyl methacrylate) -dibenzocyclooctyne;

(4) reacting OH-PEG-N3 subjected to acyl chlorination with cPFC according to the molar ratio of 1 (4-6) at room temperature to prepare the nanoparticle cPFC _N3 (ii) a The structure of the OH-PEG-N3 is as follows:

(5) the nano-particles cPFC _DBCO And nanoparticle cPFC _N3 Constructing a polymer mediated by a tumor acid environment and deconstructed in an acid response way at the pH value of (6.5-6.9); the nanoparticle cPFC _DBCO And cPFC _N3 The particle size range of the polymer constructed by acid response within 30min is 800 nm-1100 nm;

the process for preparing the polymer-drug conjugate PFC by using the PAMAM, the ferrocenecarboxylic acid and the cinnamaldehyde as raw materials in the step (1) comprises the following steps:

II, stirring and reacting the PAMAM and ferrocenecarboxylic acid conjugate PF with cinnamaldehyde for 20-30 h at room temperature in a protective gas atmosphere, and further coupling the cinnamaldehyde on the conjugate PF to obtain a polymer-drug conjugate PFC;

the molar ratio of the cinnamaldehyde to the PAMAM is (3-5) to 1.

2. The method for preparing a tumor weak acid environment mediated constructed and deconstructed polymer according to claim 1, wherein the step (3) of carboxyl activation of PAEMA-DBCO comprises: stirring PAEMA-DBCO, EDC and NHS according to the molar ratio of 1 (1-1.5) to 1-1.5 in DCM for 2-4 h, and activating carboxyl of PAEMA-DBCO.

3. The method for preparing a tumor weak acid environment mediated constructing and deconstructing polymer according to claim 1, wherein the step (4) is performed on OH-PEG-N ₃ The process of acyl chlorination is as follows: mixing OH-PEG-N ₃ Oxalyl chloride is stirred and reacted for 2 h-4 h in DCM for OH-PEG-N according to the molar ratio of 1 (4-6) under the catalysis of DMF at room temperature ₃ Acyl chlorination is carried out.

4. The method for preparing a weak acid environment mediated constructed and deconstructed polymer for tumor according to claim 1, wherein the nanoparticle cPFC _DBCO And cPFC _N3 The particle size ranges of the nano particles are 50 nm-200 nm; the particle size range of the PFC is 8-12 nm.

5. The preparation method of any one of claims 1 to 4, wherein the polymer for the environment-mediated construction and deconstruction of the tumor acid is prepared.

6. The tumor acid environment mediated constructed and deconstructed polymer of claim 5, wherein the nanoparticle cPFC _DBCO And cPFC _N3 The particle size range of the polymer constructed by acid response within 30min is 800 nm-1100 nm; the particle size range of a deconstructed product PFC of the polymer after 6 hours of acid response is 8-12 nm.

7. The use of the polymer for the environmental-mediated construction and deconstruction of tumor acids prepared by the preparation method of any one of claims 1 to 4 in the preparation of anti-tumor drugs.