CN115105526B

CN115105526B - Application of modified PAMAM polymer in preparation of antitumor drugs

Info

Publication number: CN115105526B
Application number: CN202110302519.5A
Authority: CN
Inventors: 杜金志; 童其松; 黄华; 缪玮珉; 陈阳; 刘容; 王均
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2021-03-22
Filing date: 2021-03-22
Publication date: 2023-04-07
Anticipated expiration: 2041-03-22
Also published as: WO2022198738A1; CN115105526A

Abstract

The invention relates to an application of a modified PAMAM polymer in preparing an anti-tumor agent. The structural formula of the modified PAMAM polymer is as follows: PAMAM- (NH-C (O) -O-R) _n In the structure-NH-is from amino-NH at the end of PAMAM polymer ₂ (ii) a Wherein R is a C1-C20 alkyl substituted with a tertiary amine group; n is an integer greater than 0. The invention discovers for the first time that the modified PAMAM polymer has the effect of inducing immunogenic cell death, has a stronger anti-tumor effect, and obviously enhances the curative effect of the immune checkpoint inhibitor.

Description

Application of modified PAMAM polymer in preparation of antitumor drugs

Technical Field

The invention relates to the field of medicines, in particular to application of a modified PAMAM polymer in preparation of antitumor drugs.

Background

The traditional treatment means of malignant tumor include surgical excision, radiotherapy, chemotherapy, etc. In recent years, tumor immunotherapy has provided new strategies and hopes for cancer therapy. Among the numerous anti-tumor immunotherapies, immune Checkpoint Blockade (ICB) is one of the most effective strategies. A large number of clinical practices prove that the survival period of responsive tumor patients can be remarkably prolonged by immune checkpoint blockade therapy taking programmed death protein-1 (PD-1), programmed death protein-1 ligand (PD-L1) and the like as targets. However, ICB therapy still faces problems such as low response rates. How to effectively enhance the curative effect of the ICB antibody is a challenge to be solved urgently in both basic research and clinical practice.

Due to the high heterogeneity of tumors, the low exposure rate of tumor cell surface antigens of some cancer types, rendering the tumor microenvironment less immunogenic, so-called "cold" tumors, are one of the major reasons for their poor efficacy on immune checkpoint blockade therapies (such as PD-1 antibodies). Therefore, activation of the immune system by increasing the immunogenicity of tumor cells (i.e. the transition from cold to hot tumors) is a hot point of research to enhance the efficacy of immune checkpoint blockers. Research proves that Immunogenic Cell Death (ICD) can effectively improve the immunogenicity of tumor cells, thereby enhancing the anti-tumor immune response effect. Immunogenic cell death differs from normal death by killing tumor cells while recruiting and activating antigen presenting cells (APCs, such as Dendritic Cells (DCs)) by releasing injury-associated molecules, mainly including Calreticulin (CRT), adenosine Triphosphate (ATP), and high mobility group box 1 (HMGB 1). Ablative cancer treatments of certain chemotherapeutic agents such as (doxorubicin, oxaliplatin, mitoxantrone, etc.), radiation Therapy (RT), photodynamic therapy (PDT) and photothermal therapy (PTT) were found to function to induce ICD effects.

Disclosure of Invention

Based on the situation, the invention aims to provide a novel application of the modified PAMAM polymer in preparing antitumor drugs.

The specific technical scheme is as follows:

the modified PAMAM polymer is used as an anti-tumor active component in the application of preparing anti-tumor drugs, and the structural formula of the modified PAMAM polymer is as follows: PAMAM- (NH-C (O) -O-R) _n In the structural formula, the-NH-is from the-NH at the tail end of the PAMAM polymer ₂ ；

Wherein each R is independently selected from C1-C20 alkyl substituted with a tertiary amine group;

n is an integer greater than 0.

In some of these embodiments, each R is independently selected from C1-C10 alkyl substituted with a tertiary amine group.

In some of these embodiments, each R is independently selected from: - (CH) ₂ ) _m -R ₂ (ii) a Wherein each m is independently selected from: an integer of 1 to 10;

each R ₂ Independently selected from:

* Representing a joint;

the R is ₃ And R ₄ Each independently selected from: unsubstituted or substituted by R ₅ Substituted C1-C20 alkyl, R ₅ Is H, halogen, C1-C5 alkyl, C1-C5 alkoxy or C1-C5 alkylamino;

and x is an integer of 1-10.

In some of these embodiments, the R ₃ And R ₄ Each independently selected from unsubstituted or substituted by R ₅ Substituted C1-C10 alkyl.

In some of these embodiments, the R ₃ And R ₄ Each independently selected from unsubstituted or substituted by R ₅ Substituted C1-C6 alkyl.

In some of these embodiments, x is an integer from 1 to 5.

In some of these embodiments, m is an integer from 1 to 5.

In some of these embodiments, x is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some of these embodiments, m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some of these embodiments, the R ₃ And R ₄ Each independently selected from unsubstituted or substituted by R ₅ Substituted methyl, ethyl, propyl, butyl, pentyl or hexyl.

In some of these embodiments, the R ₅ Is H, fluorine, chlorine, bromine, iodine, methyl, ethyl, propyl, butyl, pentyl or hexyl.

In some of these embodiments, the R ₂ Is composed of

x is 2, 3 or 4.

In some of these embodiments, the R ₂ Comprises the following steps:

in some of these embodiments, n is an integer from 1 to 200.

In some of these embodiments, the PAMAM- (NH-C (O) -O-R) _n The PAMAM in (1) is the 3 rd generation PAMAM, the 4 th generation PAMAM or the 5 th generation PAMAM.

In some of these embodiments, the modified PAMAM polymer is a modified PAMAM polymer nanoparticle.

In some of the embodiments, when the PAMAM is a generation 3 PAMAM polymer, n is an integer of 1 to 32, where n represents the number of PAMAM surface grafted tertiary amine molecules; when the PAMAM is a 4 th generation PAMAM polymer, n is an integer of 1-64, wherein n represents the number of the PAMAM surface grafted tertiary amine molecules; when the PAMAM is the 5 th generation PAMAM polymer, n is an integer of 1-128, wherein n represents the number of the PAMAM surface grafted with tertiary amine molecules.

In some of these embodiments, when the PAMAM is a generation 3 PAMAM polymer, n is an integer from 10 to 30; when the PAMAM is a 4 th generation PAMAM polymer, n is an integer of 20 to 64, more preferably n is an integer of 40 to 50; when the PAMAM is the 5 th generation PAMAM polymer, n is an integer of 60-120.

In some of the embodiments, the PAMAM is a generation 4 PAMAM, and n =20 to 64, preferably 40 to 50.

In some of these embodiments, the modified PAMAM polymer has the formula:

in some of these embodiments, the PAMAM- (NH-C (O) -O-R) of the modified PAMAM polymer _n The medium-NH-C (O) -O-R is from amino-NH at the tail end of PAMAM polymer ₂ And

and reacting to obtain the product.

In some of these embodiments, the modified PAMAM polymer is prepared by a process comprising:

(1) Activating a tertiary amine containing a hydroxyl group with CDI (N, N' -carbonyldiimidazole) to obtain an activated tertiary amine; the tertiary amine containing hydroxyl has the structure of R-OH, wherein R is defined as above; (2) And reacting the activated tertiary amine with the PAMAM polymer, and purifying to obtain the modified PAMAM polymer.

In some of these embodiments, the activated tertiary amine has the structure

R is as defined above.

In some of these embodiments, the hydroxyl containing tertiary amine is selected from

(2- (hexamethyleneimine) ethanol),. Or>

(N- (2-hydroxyethyl) piperidine), N-Dimethylethanolamine (DMEA), N-Diethylethanolamine (DEEA) or N- (2-hydroxyethyl) pyrrolidine.

In some of these embodiments, the molar ratio of N, N' -carbonyldiimidazole CDI to hydroxyl containing tertiary amine is (1.5 to 5): 1.

in some of these embodiments, the activating comprises the steps of: mixing tertiary amine containing hydroxyl with N, N' -carbonyl diimidazole at room temperature in organic solvent, activating for 10-72h, washing the obtained product with water, and drying. Further, the organic solvent is at least one of dichloromethane, chloroform and ethyl acetate. Further, the tertiary amine containing a hydroxyl group is added dropwise to an organic solution of N, N' -carbonyldiimidazole.

In some of these embodiments, the molar ratio of terminal amino groups to activated tertiary amine in the PAMAM polymer is 1: (0.01-5).

In some of the embodiments, the solvent of the reaction of step (2) is an organic solvent, further at least one of DMSO and methanol.

In some of these embodiments, the mass to volume ratio of PAMAM polymer to solvent in step (2) is (10 to 30) mg: (1-5) mL.

In some of the embodiments, the reaction temperature in the step (2) is 30-50 ℃, and the reaction time is 15-168h. The further reaction time is 20-30 h.

In some embodiments, the purification in step (2) refers to separation and purification by using a Sephadex chromatographic column, and further separation and purification by using a Sephadex LH20 gel column.

In some of these embodiments, the modified PAMAM polymer nanoparticles are prepared by a process comprising:

and dissolving the modified PAMAM polymer by using an organic solvent, adding water, and stirring to obtain the modified PAMAM polymer nano-particles.

In some of these embodiments, the organic solvent for preparing the modified PAMAM polymer nanoparticles is at least one of chloroform, acetone, or dimethylformamide; the volume ratio of the organic solvent to the water is (1-1.5): (6-8); the volume-mass ratio of the organic solvent to the modified PAMAM polymer is (1-1.5) mL: (20-35) mg.

In some embodiments, the rotation speed of the stirring is 2000-3000rpm, the stirring is performed for 8-18 h based on complete volatilization of the organic solvent.

In some of these embodiments, the tumor is colorectal cancer, breast cancer, melanoma, liver cancer.

In some of these embodiments, the anti-tumor drug is an anti-tumor drug that induces immunogenic cell death.

It is another object of the present invention to provide the use of a modified PAMAM polymer, as defined above, in the preparation of an immunogenic cell death inducing agent.

It is another object of the present invention to provide an immunogenic cell death inducer or anti-tumor drug comprising an active ingredient comprising a modified PAMAM polymer as defined above and pharmaceutically acceptable excipients.

Another objective of the invention is to provide an immunogenic cell death inducer or an antitumor drug, which comprises a modified PAMAM polymer and a modified PAMAM polymer, and comprises a PD-1 antibody or a PD-L1 antibody; the modified PAMAM polymer is as defined above.

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

the invention provides application of a modified PAMAM polymer in preparation of an anti-tumor drug or an immunogenic cell death inducer. The invention discovers for the first time that the modified PAMAM polymer has the effect of inducing immunogenic cell death, has a strong anti-tumor effect, and remarkably enhances the curative effect of the immune checkpoint blocker.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum of CDI activated 2- (hexamethyleneimine) ethanol (C7A-CDI);

FIG. 2 shows D47 (D47) ₄₄ ) Nuclear magnetic hydrogen spectrum of (a);

FIG. 3 shows D47 (D47) ₄₄ ) Carbon spectrum of (a);

FIG. 4 is an electron micrograph of D47 nanoparticles;

FIG. 5 is a D47 nanoparticle size plot;

FIG. 6 shows D47 ₁₀ Nuclear magnetic hydrogen spectrum of (a);

FIG. 7 shows D47 ₂₀ Nuclear magnetic hydrogen spectrum of (a);

FIG. 8 shows D47 ₃₀ Nuclear magnetic hydrogen spectrum of (a);

FIG. 9 is a nuclear magnetic hydrogen spectrum of D37;

FIG. 10 is a nuclear magnetic hydrogen spectrum of D57;

FIG. 11: (a) Is nuclear magnetic hydrogen spectrum diagram of CDI activated N- (2-ethoxyl) piperidine (C6A-CDI), and (b) is nuclear magnetic hydrogen spectrum diagram of D47-D46;

FIG. 12 is a nuclear magnetic hydrogen spectrum of G4-DMEA;

FIG. 13 is a nuclear magnetic hydrogen spectrum of G4-DEEA;

FIG. 14 is a nuclear magnetic hydrogen spectrum of D45;

FIG. 15 is a nuclear magnetic hydrogen spectrum of D46;

FIG. 16 is a graph showing that D47 induces calreticulin eversion in CT26 cells at a cellular level;

FIG. 17 shows that D47 induces calreticulin eversion in 4T1 cells at the cellular level;

FIG. 18 is a graph demonstrating at the cellular level that D47 induces calreticulin eversion in MC38 cells;

FIG. 19 is a graph demonstrating at the cellular level that D47 induces calreticulin eversion in HCT116 cells;

FIG. 20 is a graph demonstrating at the cellular level that D47 induces calreticulin eversion in B16F10 cells;

FIG. 21 is a graph demonstrating at the cell level that D47 induces adenosine 5' -triphosphate (ATP) secretion in CT26 and 4T1 cells;

FIG. 22 is a cellular level demonstration that D47 induces secretion of adenosine 5' -triphosphate (ATP) by HCT116 cells;

FIG. 23 shows that D47 induces the release of high mobility group B1 protein (HMGB 1)) from CT26 cells and 4T1 cells at the cellular level;

FIG. 24 is a graph demonstrating at the cellular level that D47 induces the release of high mobility group box protein B1 (HMGB 1)) from HCT116 cells;

FIG. 25 is a graph demonstrating at the cellular level that different graft numbers D47 (D47 n) induce calreticulin eversion in CT26 cells;

FIG. 26 is a graph demonstrating at the cellular level that different numbers of grafts, D47 (D47 n), induce calreticulin eversion in MC38 cells;

FIG. 27 shows the cellular level of validation that different graft numbers D47 (D47 n) induce calreticulin evasion in HCT116 cells;

FIG. 28 shows cell level assays D45, D46, D47 ₄₄ Inducing calreticulin evagination in CT26 cells;

FIG. 29 shows cell level assays D46 and D47 ₄₄ Inducing the eversion of the calreticulin of the MC38 cells;

FIG. 30 shows cell level assays D37, D46, D47 ₄₄ Inducing calreticulin eversion in HCT116 cells;

FIG. 31 shows that D45, D46, and D47 induce calreticulin evagination in CT26 cells at the cellular level;

FIG. 32 shows cell level assays D47-D46, D57, D47 ₄₄ Inducing calreticulin evagination in CT26 cells;

FIG. 33 is a D47 "vaccine" experiment demonstrating that D47 induces immunogenic death;

FIG. 34 shows D47 at different concentrations ₄₄ Treatment experiments on immunodeficient BALB/cnude mice and immunocompromised BALB/c mice;

FIG. 35 shows D47 ₄₄ Therapeutic experiments on the CT26 model in combination with PD-1 antibody;

FIG. 36 is D47 ₄₄ Therapeutic experiments in combination with PD-1 antibodies on the 4T1 model.

Detailed Description

Experimental procedures according to the invention, in which no particular conditions are specified in the following examples, are generally carried out under conventional conditions, or under conditions recommended by the manufacturer. The various chemicals used in the examples are commercially available.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, article, or device that comprises a list of steps is not limited to only those steps or modules listed, but may alternatively include other steps not listed or inherent to such process, method, article, or device.

The "plurality" referred to in the present invention means two or more. "and/or" describes the association relationship of the associated objects, meaning that there may be three relationships, e.g., a and/or B, which may mean: a exists alone, A and B exist simultaneously, and B exists alone. The character "/" generally indicates that the former and latter associated objects are in an "or" relationship.

The present invention will be described in further detail with reference to specific examples.

The modified dendritic PAMAM (PAMAM is polyamidoamine dendrimer) polymer is obtained by reacting dendritic PAMAM polymer with tertiary amine molecules activated by CDI (N, N' -carbonyldiimidazole). CDI activated tertiary amine molecules refer to tertiary amine molecules activated with CDI.

The tertiary amine molecule may be

(2- (hexamethyleneimine) ethanol, C7A),

(N- (2-hydroxyethyl) piperidine, C6A), N-Dimethylethanolamine (DMEA), N-Diethylethanolamine (DEEA), N- (2-hydroxyethyl) pyrrolidine (C5A), and the like.

In the embodiment of the invention: the 3 rd generation PAMAM is the 3 rd generation polyamidoamine dendrimer, the 4 th generation PAMAM is the 4 th generation polyamidoamine dendrimer, and the 5 th generation PAMAM is the 5 th generation polyamidoamine dendrimer. The molecular weights of the generation 3, the generation 4 and the generation 5 PAMAM are 6909, 14215 and 28826g/mol respectively; the 1mol of the third, fourth and fifth PAMAM respectively contains 32, 64 and 128mol of amino groups. Examples of CDI-activated tertiary amine molecules of the present invention include C7A-CDI and C6A-CDI, C5A-CDI, DMEA-CDI, DEEA-CDI, and the like.

Example 1: dendrimer D47 (D47) ₄₄ ) Synthesis of (2)

Dendrimer D47 (D47) ₄₄ ) Is prepared by the reaction of the surface amino group of G4-PAMAM and 2- (hexamethylene imine) ethanol (C7A-CDI) activated by CDI.

Preparation of CDI-activated C7A (C7A-CDI):

dissolving C7A (2- (hexamethylene imine) ethanol) (0.02 mol) in 20mL dichloromethane to obtain a C7A solution; dissolving CDI (0.05 mol) in 40mL of dichloromethane to obtain a CDI solution; dripping the C7A solution into the CDI solution (the dripping is completed within 30 minutes), and reacting for 24 hours at room temperature after the dripping is completed; after the reaction was completed, the product was washed three times with deionized water (lower layer product was collected), a small amount of water was removed with anhydrous magnesium sulfate, the solvent dichloromethane was removed by rotary evaporation, and a small amount of the dichloromethane solvent which was not removed was removed by vacuum pumping to obtain CDI-activated C7A, i.e., C7A-CDI, in a yield of 68%, and nuclear magnetic (1H NMR chart) results of C7A-CDI are shown in fig. 1.

D47(D47 ₄₄ ) The preparation of (1):

(1) In 1.5ml of anhydrous DMSO, 25mg of a fourth generation PAMAM (G4-PAMAM) was reacted with 80mg of C7A-CDI in an oil bath pan at 40 ℃ for 24 hours, the molar ratio of C7A-CDI to the terminal amino groups of the G4-PAMAM was 192:64, obtaining a crude product.

(2) Purifying the crude product with Sephadex LH20 column, collecting the product with methanol as mobile phase, removing solvent to obtain purified modified dendritic PAMAM polymer D47 ₄₄ . The overall yield was 85% (from reaction through purification).

The nuclear magnetic hydrogen spectrum result of the modified dendritic PAMAM polymer D47 is shown in FIG. 2, the C7A grafting number is 44 (G4-PAMAM has 996 methylene H, and the chemical shifts of H are all between 2-4ppm, as shown in FIG. 2, the hydrogen on the first carbon (G) close to oxygen is a characteristic peak, and the chemical shift is between 4.2-4.5, while the chemical shift of the hydrogen of three carbons (H + i) adjacent to the tertiary amine molecule N is between 2-4ppm, and is coincided with PAMAM, assuming that x tertiary amine molecules are grafted, 2 x/(996 x) =0.07/1, and x =44 is calculated). The nuclear magnetic carbon spectrum result of the modified dendritic PAMAM polymer D47 is shown in FIG. 3.

D47(D47 ₄₄ ) Preparing nano particles:

the modified dendritic PAMAM polymer D47 was dissolved in a small amount of chloroform (1-1.5 ml) ₄₄ The product (30 mg) was added (6-8 ml) with deionized water, stirred overnight until chloroform was completely removed, concentrated to about 1.5-2ml on a rotary evaporator, and stored in a refrigerator at 4 ℃. D47 (D47) ₄₄ ) The electron micrograph of the nanoparticles of (A) is shown in FIG. 4, D47 (D47) ₄₄ ) The particle size diagram of the nanoparticles of (a) is shown in fig. 5.

Example 2: dendrimer D47 ₁₀ The preparation of (1):

dendrimer D47 ₁₀ Is prepared by the reaction of CDI activated tertiary amine small molecules (C7A-CDI) and PAMAM surface amino.

The conditions and procedures for preparing CDI-activated 2- (hexamethyleneimine) ethanol (C7A-CDI) were the same as in example 1.

D47 ₁₀ Preparation of

(1) In 1.5ml of anhydrous DMSO, 25mg of the fourth generation PAMAM (G4-PAMAM) was reacted with 6mg of C7A-CDI in an oil bath pan at 40 ℃ for 24 hours, the molar ratio of C7A-CDI to the terminal amino group of G4-PAMAM was 15:64, obtaining a crude product;

(2) Product D47 ₁₀ The purification procedure was as in example 1 to obtain a purified modified dendritic PAMAM polymer, D47 ₁₀ The total yield was 87%.

Modified dendritic PAMAM polymers namely D47 ₁₀ The nuclear magnetic results of (A) are shown in FIG. 6, the number of grafts was about 10, and the nuclear magnetic calculation method was the same as that of example 1.

D47 ₁₀ The nanoparticles were prepared as in example 1.

Example 3: dendrimer D47 ₂₀ The preparation of (1):

dendrimer D47 ₂₀ Is prepared by the reaction of CDI activated tertiary amine small molecules (C7A-CDI) and PAMAM surface amino.

D47 ₂₀ The preparation of (1):

(1) In 1.5ml of anhydrous DMSO, 25mg of the fourth generation PAMAM (G4-PAMAM) was reacted with 12mg of C7A-CDI in an oil bath pan at 40 ℃ for 24 hours, the molar ratio of C7A-CDI to the terminal amino group of G4-PAMAM was 30:64, obtaining a crude product;

(2) Product D47 ₂₀ The purification procedure was as in example 1 to obtain a purified modified dendritic PAMAM polymer, D47 ₂₀ The yield was 70%.

Modified dendritic PAMAM polymers namely D47 ₂₀ The nuclear magnetic results of (A) are shown in FIG. 7, the number of grafts was about 20, and the nuclear magnetic calculation method was the same as that of example 1.

D47 ₂₀ The nanoparticles were prepared as in example 1.

Example 4: dendrimer D47 ₃₀ The preparation of (1):

dendrimer D47 ₃₀ Is prepared by the reaction of CDI activated tertiary amine small molecules (C7A-CDI) and PAMAM surface amino.

D47 ₂₀ The preparation of (1):

(1) In 1.5ml of anhydrous DMSO, 25mg of the fourth generation PAMAM (G4-PAMAM) was reacted with 27mg of C7A-CDI in an oil bath pan at 40 ℃ for 24 hours, the molar ratio of C7A-CDI to the terminal amino group of G4-PAMAM was 64:64, obtaining a crude product;

(2) Product D47 ₃₀ The purification procedure was as in example 1 to obtain a purified modified dendritic PAMAM polymer, D47 ₃₀ The overall yield was 77%.

Modified dendritic PAMAM polymers namely D47 ₃₀ The nuclear magnetic results of (A) are shown in FIG. 8, the number of grafts was about 20, and the nuclear magnetic calculation method was the same as that of example 1.

D47 ₃₀ The nanoparticles were prepared as in example 1.

Example 5: preparation of dendrimer D37:

dendrimer D37 was prepared by reacting CDI-activated tertiary amine small molecules (C7A-CDI) with PAMAM surface amino groups.

Preparation of D37:

(1) In 1.5ml of anhydrous DMSO, 25mg of a tertiary PAMAM (G3-PAMAM) was reacted with 79mg of C7A-CDI in an oil bath pan at 40 ℃ for 24 hours, the molar ratio of C7A-CDI to the terminal amino group of the G3-PAMAM was 96:32, obtaining a crude product;

(2) The purification method of the product D37 was the same as that of example 1, to obtain a purified modified dendritic PAMAM polymer D37 with an overall yield of 75%.

The nuclear magnetic results of D37, which is a modified dendritic PAMAM polymer, are shown in fig. 9, where the number of grafts was about 20 and the nuclear magnetic calculation method was the same as in example 1.

The preparation of D37 nanoparticles was performed as in example 1.

Example 6: preparation of dendrimer D57:

the dendrimer D57 is prepared by reacting CDI-activated tertiary amine small molecules (C7A-CDI) with PAMAM surface amino groups.

Preparation of D57:

(1) In 1.5ml of anhydrous DMSO, 25mg of the pentabasic PAMAM (G5-PAMAM) was reacted with 81.5mg of C7A-CDI in an oil bath at 40 ℃ for 24 hours, with a molar ratio of C7A-CDI to the terminal amino group of the G5-PAMAM of 384:128, obtaining a crude product;

(2) The purification method of the product D57 was the same as that of example 1, to obtain a purified modified dendritic PAMAM polymer D57 with an overall yield of 87%.

The nuclear magnetic results of the modified dendritic PAMAM polymer, D57, are shown in fig. 10, with the number of grafts being about 90, and the nuclear magnetic calculation method being the same as in example 1.

The preparation of D57 nanoparticles was performed as in example 1.

Example 7: preparation of dendrimers D47-D46:

dendrimers D47-D46 were prepared from the reaction of the surface amino groups of G4-PAMAM with CDI-activated N- (2-hydroxyethyl) piperidine (C6A-CDI) and CDI-activated 2- (hexamethyleneimine) ethanol (C7A-CDI).

Preparation of CDI-activated C6A (C6A-CDI):

dissolving C6A (1- (2-hydroxyethyl) piperidine) (0.02 mol) in 20mL dichloromethane to obtain a C6A solution; dissolving CDI (0.05 mol) in 40mL of dichloromethane to obtain a CDI solution; dripping the C6A solution into the CDI solution (the dripping is completed within 30 minutes), and reacting for 24 hours at room temperature after the dripping is completed; after the reaction was completed, the product was washed three times with deionized water (lower layer product was collected), a small amount of water was removed with anhydrous magnesium sulfate, the solvent dichloromethane was removed by rotary evaporation, and a small amount of the dichloromethane solvent that was not removed was removed by vacuum pumping to obtain CDI-activated C6A, i.e., C6A-CDI, with a yield of 70%. FIG. 11 (a) is a nuclear magnetic hydrogen spectrum of CDI-activated N- (2-hydroxyethyl) piperidine (C6A-CDI).

Preparation of D47-D46

(1) In 1.5ml of anhydrous DMSO, 25mg of the fourth generation PAMAM (G4-PAMAM) was reacted with 5.9mg of C6A-CDI in an oil bath at 40 ℃ for 12 hours, the molar ratio of C6A-CDI to the terminal amino group of G4-PAMAM was 20: and 64, adding 80.0mg of C7A-CDI, and reacting in an oil bath kettle at the temperature of 40 ℃ for 24 hours, wherein the molar ratio of the C7A-CDI to the terminal amino group of the G4-PAMAM is 192:64, obtaining a crude product;

(2) The purification method of the product D47-D46 is the same as that of example 1, and the purified modified dendritic PAMAM polymer D47-D46 is obtained, and the total yield is 81%.

The nuclear magnetic results of D47-D46, which are modified dendritic PAMAM polymers, are shown in FIG. 11 (b), in which the number of C6A grafts was about 14 and the number of C7A grafts was about 30.

The preparation of D47-D46 nanoparticles was performed as in example 1.

Example 8: synthesis of dendrimer G4-DMEA

The dendritic macromolecule G4-DMEA is prepared by reacting surface amino groups of G4-PAMAM with CDI activated N, N-dimethylethanolamine (DMEA-CDI).

Preparation of CDI-activated DMEA (DMEA-CDI):

DMEA (N, N-dimethylethanolamine) (0.02 mol) is dissolved in 20mL of dichloromethane to obtain a DMEA solution; dissolving CDI (0.05 mol) in 40mL of dichloromethane to obtain a CDI solution; dripping the DMEA solution into the CDI solution (dripping is completed within 30 minutes), and reacting for 24 hours at room temperature after finishing dripping; after the reaction was completed, the product was washed three times with deionized water (lower layer product was collected), a small amount of water was removed with anhydrous magnesium sulfate, the solvent dichloromethane was removed by rotary evaporation, and a small amount of the dichloromethane solvent that was not removed was removed by vacuum pump, to obtain DMEA activated by CDI, i.e., DMEA-CDI.

Preparation of G4-DMEA:

(1) In 1.5ml of anhydrous DMSO, 25mg of quadruplicate PAMAM (G4-PAMAM) was reacted with 62mg of DMEA-CDI in an oil bath pan at 40 ℃ for 24 hours, with a molar ratio of DMEA-CDI to terminal amino groups of G4-PAMAM of 3:1, obtaining a product;

(2) The purification method of the product G4-DMEA is the same as that of example 1, and the purified modified dendritic PAMAM polymer, namely G4-DMEA, is obtained, and the total yield is 85%.

The nuclear magnetism result of the modified dendritic PAMAM polymer, namely G4-DMEA, is shown in FIG. 12, and the grafting number of DMEA is 44.

The preparation process of the G4-DMEA nanoparticles is the same as that of example 1.

Example 9: synthesis of dendrimer G4-DEEA

The dendrimer G4-DEEA is prepared by reacting a CDI activated tertiary amine small molecule (DEEA-CDI) with PAMAM surface amino.

Preparation of CDI-activated DEEA, DEEA-CDI: DEEA (N, N-diethylethanolamine) was used in place of DMEA (N, N-dimethylethanolamine) in example 9, and the other conditions and procedures were the same as in example 9.

Preparation of G4-DEEA:

(1) In 1.5ml of anhydrous DMSO, 25mg of the fourth generation PAMAM (G4-PAMAM) was reacted with 71mg of DEEA-CDI in an oil bath pan at 40 ℃ for 24 hours, the molar ratio of DEEA-CDI to the terminal amino group of G4-PAMAM being 3:1, obtaining a crude product;

(2) The purification method of the product G4-DEEA is the same as that of the example 1, and the purified modified dendritic PAMAM polymer, namely G4-DEEA is obtained, and the total yield is 90%.

The nuclear magnetic results of the modified dendritic PAMAM polymer, G4-DEEA, are shown in FIG. 13, with a DMEA graft number of 44.

The preparation of G4-DEEA nanoparticles was performed as in example 1.

Example 10: synthesis of dendrimer D45

Dendrimer D45 was prepared by reacting CDI-activated tertiary amine molecule N- (2-hydroxyethyl) pyrrolidine (C5A-CDI) with PAMAM surface amino groups.

Preparation of CDI-activated N- (2-hydroxyethyl) pyrrolidine, C5A-CDI: n- (2-hydroxyethyl) pyrrolidine was substituted for DMEA (N, N-dimethylethanolamine) in example 9, and the other conditions and procedures were the same as in example 9.

Preparation of D45:

(1) Reacting 25mg of G4-PAMAM with 71mg of C5A-CDI in 1.5mL of anhydrous DMSO at 40 ℃ for 24 hours, wherein the molar ratio of the C5A-CDI to amino on the surface of the PAMAM is 3:1 to obtain a crude product;

(2) The purification method of the product D45 is the same as that of example 1, and the purified modified dendritic PAMAM polymer D45 is obtained, and the total yield is 88%.

The nuclear magnetic results of the modified dendritic PAMAM polymer, D45, are shown in fig. 14, with a C5A graft number of 44.

The preparation of D45 nanoparticles was performed as in example 1.

Example 11: synthesis of dendrimer D46

Dendrimer D46 was prepared by reacting CDI-activated tertiary amine molecule N-hydroxyethylpiperidine (C6A-CDI) with PAMAM surface amino groups.

The conditions and procedures for preparing CDI-activated N-hydroxyethylpiperidine, C6A-CDI, were the same as in example 8.

Preparation of D46:

(1) Reacting 25mg of G4-PAMAM and 75mg of C6A-CDI in 1.5mL of anhydrous DMSO at 40 ℃ for 24 hours, wherein the molar ratio of the C6A-CDI to amino on the surface of the PAMAM is 3:1 to obtain a crude product;

(2) The purification method of the product D46 was the same as that of example 1, to obtain a purified modified dendritic PAMAM polymer, D45, with an overall yield of 88%.

The nuclear magnetic results of the modified dendritic PAMAM polymer, D46, are shown in fig. 15, with a C6A graft number of 44.

The preparation of D46 nanoparticles was performed as in example 1.

Example 12: cell level validation D47 (D47) ₄₄ ) Induction of calreticulin evagination in CT26 cells

Detection of FACs (flow cytometry) modified dendrimer Polymer D47 prepared in example 1 ₄₄ Nanoparticles, induced calreticulin eversion in CT26 cells (purchased from shanghai cell bank, chinese academy of sciences). The specific method comprises the following steps: firstly, 1X 10 seeds are planted in each hole on a 12-hole plate ⁵ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ 5%) was added, and after 12 hours of culture, the medium was changed to a medium containing 5. Mu. M D47 ₄₄ The complete medium of (4) was co-cultured, and the complete medium containing 200. Mu.M OXA (oxaliplatin) was used as a positive control group. Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The results of the experiment are shown in FIG. 16 (the ordinate represents the relative expression compared with the PBS group).

The results in FIG. 16 show that the modified dendrimer polymer D47 prepared according to the present invention ₄₄ The nano-particles can induce the expression of calreticulin of CT26 cells, and the inducing effect of the nano-particles is superior to that of oxaliplatin.

Example 13: cell level validation D47 (D47) ₄₄ ) Induction of calreticulin eversion in 4T1 cells

FACs (streaming)Cytometry) detection of the modified dendrimer Polymer D47 prepared in example 1 ₄₄ Calreticulin was induced in 4T1 cells (professor Wang Jun, university of southern china) to evaginate. The specific method comprises the following steps: firstly, 1X 10 seeds are planted in each hole on a 12-hole plate ⁵ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ 5%) was added, and after 12 hours of culture, the medium was changed to a medium containing 5. Mu. M D47 ₄₄ The complete medium of (4) was co-cultured, and complete medium containing 200 μ M OXA (oxaliplatin) was a positive control group. Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The results of the experiment are shown in FIG. 17 (plotted on the ordinate) is the relative expression compared to the PBS group).

The results in FIG. 17 show that the modified dendrimer polymer D47 prepared according to the present invention ₄₄ The nanoparticle can induce the expression of 4T1 cell calreticulin, and the inducing effect of the nanoparticle is obviously superior to that of oxaliplatin.

Example 14: cell level validation of D47 (D47) ₄₄ ) Induction of MC38 cell calreticulin eversion

Detection of FACs (flow cytometry) modified dendrimer Polymer D47 prepared in example 1 ₄₄ Induces calreticulin eversion in MC38 cells (professor Wang Jun, professor laboratories, university of southern China). The specific method comprises the following steps: firstly, 1X 10 seeds are planted in each hole on a 12-hole plate ⁵ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ 5%) was added, and after 12 hours of culture, the medium was changed to a medium containing 5. Mu. M D47 ₄₄ The complete medium of (4) was co-cultured, and complete medium containing 200 μ M OXA (oxaliplatin) was a positive control group. Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The results of the experiment are shown in FIG. 18 (the ordinate represents the relative expression compared to the PBS group).

The results in FIG. 18 show that the modified dendrimer polymer D47 prepared according to the present invention ₄₄ The nanoparticle can induce the expression of the MC38 cell calreticulin, and the inducing effect of the nanoparticle is obviously superior to that of oxaliplatin.

Example 15: cell level validation of D47 (D47) ₄₄ ) Induction of HCalreticulin evagination of CT116 cells

Detection of FACs (flow cytometry) modified dendrimer Polymer D47 prepared in example 1 ₄₄ HCT116 cells (donated by the university of southern china philosophy male professor laboratory) were induced to calreticulin eversion. The specific method comprises the following steps: firstly, 1X 10 seeds are planted in each hole on a 12-hole plate ⁵ Cells in DMEM (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ 5%) was added, and after 12 hours of culture, the medium was changed to a medium containing 5. Mu. M D47 ₄₄ The complete medium of (4) was co-cultured, and complete medium containing 200 μ M OXA (oxaliplatin) was a positive control group. Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The results of the experiment are shown in FIG. 19 (the ordinate represents the relative expression amount compared with the PBS group).

The results in FIG. 19 show that the modified dendrimer polymer D47 prepared according to the present invention ₄₄ The nanoparticle can induce the expression of HCT116 cell calreticulin, and the inducing effect of the nanoparticle is obviously superior to that of oxaliplatin.

Example 16: cell level validation of D47 (D47) ₄₄ ) Induction of calreticulin eversion in B16F10 cells

Detection of FACs (flow cytometry) modified dendrimer Polymer D47 prepared in example 1 ₄₄ Calreticulin eversion was induced in B16F10 cells (purchased from shanghai cell bank, chinese academy of sciences). The specific method comprises the following steps: firstly, 1X 10 seeds are planted in each hole on a 12-hole plate ⁵ Cells in DMEM (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ 5%) was added, and after 12 hours of culture, the medium was changed to a medium containing 5. Mu. M D47 ₄₄ The complete medium of (4) was co-cultured, and complete medium containing 200 μ M OXA (oxaliplatin) was a positive control group. Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The results of the experiment are shown in FIG. 20 (the ordinate represents the relative expression amount compared with the PBS group).

The results in FIG. 20 show that the modified dendrimer polymer D47 prepared according to the present invention ₄₄ The nanoparticle can induce the expression of B16F10 cell calreticulin, and the inducing effect of the nanoparticle is obviously superior to that of oxaliplatin.

Example 17: cell level validation of D47 (D47) ₄₄ ) Induction of adenosine 5' -triphosphate (ATP) secretion by CT26 cells

Kit detection of modified dendrimer polymer D47 prepared in example 1 ₄₄ Inducing release of 5' -Adenosine Triphosphate (ATP) from CT26 cells. The specific method comprises the following steps: firstly, 5X 10 of each pore is planted on a 24-pore plate ⁴ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ Concentration of 5%), after overnight adherent culture, the cells were replaced with cells containing 50. Mu.g/mL and 150. Mu.g/mL of D47 ₄₄ Co-culturing with the complete medium (D47) ₄₄ Mixed with PBS solution, and then co-cultured with medium added) for 24 hours, complete medium containing PBS, 200 μ M OXA (oxaliplatin) was a positive control group. After 24 hours, the cell supernatant was collected, impurities and cell debris were removed, and the release of 5' -Adenosine Triphosphate (ATP) from the cell culture supernatant was examined. The experimental results are shown in fig. 21.

The left panel of FIG. 21 shows the results of the modified dendrimer polymer D47 prepared according to the present invention ₄₄ The nano-particles have the function of remarkably inducing the secretion of 5' -Adenosine Triphosphate (ATP) of CT26 cells.

Example 18: cell level validation of D47 (D47) ₄₄ ) Induction of 5' -Adenosine Triphosphate (ATP) secretion by 4T1 cells

Kit for detecting modified dendrimer polymer D47 prepared in example 1 ₄₄ Induces the release of 5' -Adenosine Triphosphate (ATP) from 4T1 cells. The specific method comprises the following steps: firstly, 5X 10 of each pore is planted on a 24-pore plate ⁴ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ Concentration of 5%), after overnight adherent culture, the cells were replaced with cells containing 50. Mu.g/mL and 150. Mu.g/mL of D47 ₄₄ Co-culturing the cells in the complete medium for 24 hours (D47 is ₄₄ Mixed with PBS solution, and then co-cultured with the addition of culture medium), culture medium containing PBS, 200 μ M OXA (oxaliplatin) was a positive control group. After 24 hours, the cell supernatant was collected, impurities and cell debris were removed, and the release of 5' -Adenosine Triphosphate (ATP) from the cell culture supernatant was examined. The experimental results are shown in fig. 21.

FIG. 21, the results of the right graph show that the modified dendritic macromolecular polymer D47 prepared by the invention ₄₄ The nano-particles have the function of remarkably inducing the secretion of 5' -Adenosine Triphosphate (ATP) of 4T1 cells.

Example 19: cell level validation of D47 (D47) ₄₄ ) Induction of adenosine 5' -triphosphate (ATP) secretion by HCT116 cells

Kit for detecting modified dendrimer polymer D47 prepared in example 1 ₄₄ Induces 5' -Adenosine Triphosphate (ATP) release from HCT116 cells. The specific method comprises the following steps: firstly, 5X 10 of each pore is planted on a 24-pore plate ⁴ Cells in DMEM (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ Concentration of 5%), after overnight adherent culture, the cells were replaced with cells containing 50. Mu.g/mL and 150. Mu.g/mL of D47 ₄₄ Co-culturing the cells in the complete medium for 24 hours (D47 is ₄₄ Mixed with PBS solution and then co-cultured with media added), complete media containing PBS, 200 μ M OXA (oxaliplatin) was a positive control group. After 24 hours, the cell supernatant was collected, impurities and cell debris were removed, and the release of 5' -Adenosine Triphosphate (ATP) from the cell culture supernatant was examined. The results of the experiment are shown in FIG. 22.

The results in FIG. 22 show that the modified dendrimer polymer D47 prepared according to the present invention ₄₄ The nanoparticles have the function of inducing the secretion of adenosine 5' -triphosphate (ATP) by HCT116 cells at a concentration of 150 mug/mL.

Example 20: cell level validation of D47 (D47) ₄₄ ) Inducing the release of high mobility group B1 (HMGB 1)) of CT26 cells

ELISA detection (from arigo, cat. ARG 81351) of the modified dendrimer Polymer D47 prepared in example 1 ₄₄ Induces HMGB1 release from CT26 cells. The specific method comprises the following steps: firstly, 5X 10 of each pore is planted on a 24-pore plate ⁴ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ Concentration of 5%), and after 24 hours of adherent culture, the cells were replaced with D47 containing 50. Mu.g/mL and 150. Mu.g/mL ₄₄ Co-culturing the cells in the complete medium for 24 hours (D47 is ₄₄ Mixed with PBS solution, then co-cultured with medium added), containing PBS, 200 μ M OXA (oxaliplatin)The medium of (3) is a positive control group. Cell supernatants were harvested after 24 hours, freed of impurities and cell debris, and cell culture supernatants were examined for release of high mobility group box 1 protein (HMGB 1)). The results of the experiment are shown in FIG. 23.

The results in the left panel of FIG. 23 show that the modified dendrimer polymer D47 prepared according to the present invention ₄₄ The nanoparticle has the function of remarkably inducing the release of HMGB1 of CT26 cells, and the inducing effect of the nanoparticle is remarkably superior to that of oxaliplatin.

Example 21: cell level validation of D47 (D47) ₄₄ ) Induction of 4T1 cell high mobility group protein B1 (HMGB 1) release

ELISA detection of the modified dendrimer Polymer D47 prepared in example 1 ₄₄ Induces HMGB1 release from 4T1 cells. The specific method comprises the following steps: firstly, 5X 10 of each pore type is arranged on a 24-pore plate ⁴ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ Concentration of 5%), and after 24 hours of adherent culture, the cells were replaced with D47 containing 50. Mu.g/mL and 150. Mu.g/mL ₄₄ Co-culturing for 24 hours (D47 is used) ₄₄ Mixed with PBS solution and then co-cultured with media added), complete media containing PBS, 200 μ M OXA (oxaliplatin) was a positive control group. Cell supernatants were harvested after 24 hours, freed of impurities and cell debris, and cell culture supernatants were examined for release of high mobility group box 1 protein (HMGB 1)). The results of the experiment are shown in FIG. 23.

The results in the right panel of FIG. 23 show that modified dendrimer polymer D47 according to the present invention was prepared ₄₄ The nanoparticle has the function of remarkably inducing the release of HMGB1 of 4T1 cells, and the inducing effect of the nanoparticle is remarkably superior to that of oxaliplatin.

Example 22: cell level validation of D47 (D47) ₄₄ ) Inducing HCT116 cell high mobility group protein B1 (HMGB 1)) release

ELISA detection of the modified dendrimer Polymer D47 prepared in example 1 ₄₄ Induces HMGB1 release from HCT116 cells. The specific method comprises the following steps: firstly, 5X 10 of each pore is planted on a 24-pore plate ⁴ Cells in DMEM (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ Concentration of 5%), and after 24 hours of adherent culture, the cells were replaced with D47 containing 50. Mu.g/mL and 150. Mu.g/mL ₄₄ Co-culturing the cells in the complete medium for 24 hours (D47 is ₄₄ Mixed with PBS solution and then co-cultured with media added), complete media containing PBS, 200 μ M OXA (oxaliplatin) was a positive control group. Cell supernatants were harvested after 24 hours, freed of impurities and cell debris, and cell culture supernatants were examined for release of high mobility group box 1 protein (HMGB 1)). The results of the experiment are shown in FIG. 24.

The results in FIG. 24 show that the modified dendrimer polymer D47 prepared according to the present invention ₄₄ The nano-particles have the function of remarkably inducing HMGB1 release of HCT116 cells.

Example 23: verification of the different grafting numbers D47 (D47) at the cellular level ₂₀ 、D47 ₃₀ 、D47 ₄₄ ) Induction of calreticulin evagination in CT26 cells

FACs (flow cytometry) detection of different graft numbers D47 (D47) ₂₀ 、D47 ₃₀ 、D47 ₄₄ ) Induce calreticulin eversion in CT26 cells. The specific method comprises the following steps: firstly, 1X 10 seeds are planted in each hole on a 12-hole plate ⁵ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ Concentration of 5%), after 12 hours of cultivation, the cells were replaced with cells containing 5. Mu.M, 7.5. Mu.M different graft numbers D47 (D47) ₂₀ 、D47 ₃₀ 、D47 ₄₄ ) Co-culturing with the complete medium (D47) ₄₄ Mixed with PBS solution and then co-cultured with media added), complete media containing PBS, 200 μ M OXA (oxaliplatin) was a positive control group. Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The results of the experiment are shown in FIG. 25.

The results in FIG. 25 show that the modified dendrimer polymer D47 prepared according to the present invention ₂₀ 、D47 ₃₀ 、D47 ₄₄ The nano-particles have the function of remarkably inducing the calreticulin expression of CT26 cells under the concentration of 5 mu M (low) and 7.5 mu M (high).

Example 24: verification of the different grafting numbers D47 (D47) at the cellular level ₂₀ 、D47 ₃₀ 、D47 ₄₄ ) Induction of MC38 cell calreticulin eversion

Different numbers of grafts D47 (D47) prepared in the examples for the detection of FACs (flow cytometry) ₂₀ 、D47 ₃₀ 、D47 ₄₄ ) Induces the evagination of calreticulin of MC38 cells. The specific method comprises the following steps: firstly, 1X 10 seeds are planted in each hole on a 12-hole plate ⁵ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ Concentration of 5%), after 12 hours of cultivation, the cells were replaced with cells containing a different number of grafts D47 (D47) of 5. Mu.M ₂₀ 、D47 ₃₀ 、D47 ₄₄ ) The complete medium of (1) was co-cultured, and the complete medium containing 200. Mu.M OXA (oxaliplatin) was a positive control group, and the complete medium containing 200. Mu.M CDDP (cisplatin) was a negative control group. Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The results of the experiment are shown in FIG. 26.

The results in FIG. 26 show that the modified dendrimer polymer D47 prepared according to the present invention ₂₀ 、D47 ₃₀ 、D47 ₄₄ The nanoparticle has the function of remarkably inducing the expression of the calreticulin of the MC38 cells, and the inducing effect of the nanoparticle is remarkably superior to that of oxaliplatin.

Example 25: verification of the different grafting numbers D47 (D47) at the cellular level ₂₀ 、D47 ₃₀ 、D47 ₄₄ ) Induction of calreticulin eversion in HCT116 cells

Different numbers of grafts D47 (D47) prepared in the examples for the detection of FACs (flow cytometry) ₂₀ 、D47 ₃₀ 、D47 ₄₄ ) Induces calreticulin eversion in HCT116 cells. The specific method comprises the following steps: firstly, 1X 10 seeds are planted in each hole on a 12-hole plate ⁵ Cells in DMEM (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ Concentration of 5%), after 12 hours of cultivation, the cells were replaced with cells containing a different number of grafts D47 (D47) of 5. Mu.M ₂₀ 、D47 ₃₀ 、D47 ₄₄ ) The complete medium of (1) was co-cultured, and the complete medium containing 200. Mu.M OXA (oxaliplatin) was a positive control group, and the complete medium containing 200. Mu.M CDDP (cisplatin) was a negative control group. Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The results of the experiment are shown in FIG. 27.

FIG. 27 shows the results of modified dendrons prepared according to the present inventionDendrimer polymer D47 ₂₀ 、D47 ₃₀ 、D47 ₄₄ The nano-particles have the function of remarkably inducing the expression of calreticulin of HCT116 cells, and the inducing effect of the nano-particles is remarkably superior to that of oxaliplatin. And also found D47 ₂₀ 、D47 ₃₀ 、D47 ₄₄ The induction effect of the compound is better than that of D47 ₁₀ 。

Example 26: cell level validation of D45, D46, D47 ₄₄ Induction of calreticulin evagination in CT26 cells

Modified dendrimers D45, D46, D47 prepared in the detection examples of FACs (flow cytometry) ₄₄ Calreticulin eversion of CT26 cells was induced. The specific method comprises the following steps: firstly, 1X 10 seeds are planted in each hole on a 12-hole plate ⁵ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ 5%) and 12 hours after the culture, the medium was changed to a medium containing D45, D46 and D47 at concentrations of 2.5. Mu.M and 5. Mu.M, respectively ₄₄ The complete medium of (1) was co-cultured, and the complete medium containing 200. Mu.M OXA (oxaliplatin) was a positive control group, and the complete medium containing 200. Mu.M CDDP (cisplatin) was a negative control group. Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The results of the experiment are shown in FIG. 28.

The results in FIG. 28 show that the modified dendrimers D45, D46, D47 prepared according to the invention ₄₄ The nano-particles have the function of remarkably inducing the calreticulin expression of CT26 cells.

Example 27: cell level validation of D46, D47 ₄₄ Induction of MC38 cell calreticulin eversion

Detection of modified dendrimers D46, D47 by FACs (flow cytometry) ₄₄ Induces the evagination of calreticulin of MC38 cells. The specific method comprises the following steps: firstly, 1X 10 of each pore type is arranged on a 12-pore plate ⁵ Cells in RMPI1640 (10% primary bovine serum) complete Medium, CO ₂ Incubator (37 ℃, CO) ₂ 5%) and 12 hours after the cultivation, the cells were replaced with cells containing 5. Mu. M D, D47 ₄₄ The complete medium of (1) was cocultured, the complete medium containing 200. Mu.M OXA (oxaliplatin) was used as a positive control group, and the complete medium containing 200. Mu.M CDDP (cisplatin) was used as a negative control groupAnd (4) a sexual control group. Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The results of the experiment are shown in FIG. 29.

The results in FIG. 29 show that the modified dendritic macromolecular polymers D46, D47 prepared according to the present invention ₄₄ The nanoparticle has the function of remarkably inducing the expression of the calreticulin of the MC38 cells, and the inducing effect of the nanoparticle is remarkably superior to that of oxaliplatin.

Example 28: validation of D37, D46, D47 at cellular level ₄₄ Induction of calreticulin eversion in HCT116 cells

FACs (flow cytometry) detection modified dendrimers D37, D46, D4744 induced calreticulin eversion in HCT116 cells. The specific method comprises the following steps: firstly, 1X 10 seeds are planted in each hole on a 12-hole plate ⁵ Cells in DMEM (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ 5%) and cultured for 12 hours, and then replaced with a culture medium containing 5. Mu. M D, D46 and D47 ₄₄ The complete medium of (4) was co-cultured, and the complete medium containing 200. Mu.M OXA (oxaliplatin) was used as a positive control group, and the complete medium containing 200. Mu.M CDDP (cisplatin) was used as a negative control group. Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The results of the experiment are shown in FIG. 30.

The results in FIG. 30 show that the modified dendrimers D37, D46, D47 prepared according to the invention ₄₄ The nanoparticle has the function of remarkably inducing the expression of calreticulin of HCT116 cells, and the inducing effect of the nanoparticle is remarkably superior to that of oxaliplatin.

Example 29: cell level validation of D45, D46, D47 ₄₄ Inducing calreticulin eversion of CT26 cells

Detection of modified dendrimers D45, D46, D47 by FACs (flow cytometry) ₄₄ Induce calreticulin eversion in CT26 cells. The specific method comprises the following steps: firstly, 1X 10 seeds are planted in each hole on a 12-hole plate ⁵ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ 5%) and 12 hours after the culture, the concentration was changed to D45, D46 and D47 at 2.5. Mu.M and 5. Mu.M, respectively ₄₄ Co-culturing in complete medium. Detection of Calreticulin (CR) on the cell membrane after 4hT) expression. The experimental results are shown in fig. 31.

The results in FIG. 31 show that modified dendrimers D45, D46, D47 prepared according to the present invention ₄₄ The nano-particles have the function of obviously inducing the calreticulin expression of CT26 cells.

Example 30: cell level validation of D47-D46, D57, D47 ₄₄ Induction of calreticulin evagination in CT26 cells

Detection of FACs (flow cytometry) modified dendrimers D47-D46, D57, D47 prepared in the examples ₄₄ Calreticulin eversion of CT26 cells was induced. The specific method comprises the following steps: firstly, 1X 10 of each pore type is arranged on a 12-pore plate ⁵ Cells in RMPI1640 (10% primary bovine serum) complete medium, CO ₂ Incubator (37 ℃, CO) ₂ Concentration of 5%), after 12 hours of culture, the medium was changed to 5. Mu.M medium containing D47-D46, D57 and D47 ₄₄ Co-culturing with the complete medium of (3). Expression of Calreticulin (CRT) on the cell membrane was detected after 4 h. The experimental results are shown in fig. 32.

The results in FIG. 32 show that the modified dendrimers D47-D46, D57, D47 according to the invention were prepared ₄₄ The nano-particles have the function of obviously inducing the calreticulin expression of CT26 cells.

Example 31: two experimental protocols verify D47 ₄₄ Induction of immunogenic death:

scheme (1): verification of the "vaccine" test D47 ₄₄ Inducing immunogenic death. The specific method comprises the following steps: addition of the solution containing D47 prepared in example one to CT26 cells ₄₄ PBS (50. Mu.g/mL) was used as the culture medium, and a PBS solution containing OXA (200. Mu.M, oxaliplatin) was used as the positive control group. After 24h of co-culture, cells were harvested and injected in 100 ten thousand/100. Mu.L 1 XPBS on the left dorsal side of BALB/c mice (Hunan Slek Jing Da). After 7 days, 50 ten thousand/100. Mu.L of 1 XPBS of CT26 cells without any treatment were re-inoculated on the right dorsal side of the mice. Tumor growth was observed and the results are shown in figure 33. FIG. 33 shows the ratios of tumor-producing mice treated with different drugs, from which D47 is shown ₄₄ The treatment group produced a stronger vaccine effect.

Scheme (2): getImmunodeficient BALB/c nude mice (Hunan Slek Jing Da) and immunocompromised BALB/c mice (Hunan Slek Jing Da) (6 weeks, male) were injected with PBS suspension of 100 ten thousand CT26 cells without any treatment on the right back, and after 8 days, different concentrations D47 were administered ₄₄ (D47 prepared in example 1) ₄₄ 1/2/3.3/5 mg/kg) administered intraperitoneally once every three days for a total of three times. BALB/c nude mice (FIG. 34, left panel) and BALB/c mice (FIG. 34, right panel) were observed for tumor growth. The experimental results of FIG. 34 show that the modified dendrimer polymer D47 ₄₄ The compound has the effect of inhibiting tumor growth in immune-competent mice (BALB/c) and has no effect of inhibiting tumor growth in immune-deficient mice (BALB/c nude), which proves that the compound plays an anti-tumor role through an immune system.

Example 32: animal level verification D47 ₄₄ Therapeutic experiments on CT26 model in combination with PD-1 antibody

The specific method comprises the following steps:

BALB/c mice (purchased from sllakedak laboratory animals ltd, 6 weeks, male, in the south of the hu) were injected subcutaneously with 100 ten thousand CT26 cell suspensions, and on day 7 (day 0 on the day of tumor implantation), all mice were randomly and equally divided into four groups: isotype control antibody (100. Mu.g/mouse) (clone 2A3, bioXcell), D47 ₄₄ (3.3 mg/kg, solvent 1 XPBS) + Isotype (100. Mu.g/mouse), PD-1 group (100. Mu.g/mouse), D47 ₄₄ (3.3 mg/kg) + PD-1 (100. Mu.g/mouse). Each mouse was intraperitoneally injected with 100. Mu. L D47 ₄₄ The drug was administered once every three days for 2 times, starting with 100 μ L of PD-1 antibody or isotype control antibody per mouse intraperitoneally 13 days, once every two days for 3 times. BALB/c mice (FIG. 35) were observed for tumor growth. The experimental result shows that D47 is used in the treatment experiment of the CT26 model ₄₄ The combination with PD-1 has strong effect of inhibiting tumor growth.

Example 33: animal level verification D47 ₄₄ Therapeutic experiments in combination with PD-1 antibodies on the 4T1 model

The specific method comprises the following steps:

BALB/c mice (purchased from St. Ex. Sciada, hu., 6 weeks, female) injected subcutaneously with 50 ten thousand 4T1 thin stripsCell suspension, day 6 (day 0 on day of tumor implantation), all mice were randomly and equally divided into four groups: isotype control antibody (100. Mu.g/mouse) (clone 2A3, bioXcell), D47 ₄₄ (3.3 mg/kg, solvent 1 XPBS) + Isotype (100. Mu.g/mouse), PD-1 group (100. Mu.g/mouse), D47 ₄₄ (3.3 mg/kg) + PD-1 (100. Mu.g/mouse). Each mouse was injected intraperitoneally with 100. Mu. L D47 ₄₄ Every other day, 2 times, starting with 100. Mu.L of PD-1 or _ isotype control antibody _ antibody per mouse, every 10 days, once every three days, 3 times. BALB/c mice (FIG. 36) were observed for tumor growth. The experimental result shows that D47 is used in the treatment experiment of the 4T1 model ₄₄ The combination with PD-1 has strong effect of inhibiting tumor growth.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is specific and detailed, but not to be understood as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. The application of the modified PAMAM polymer as an anti-tumor active ingredient in preparing anti-tumor drugs is characterized in that the structural formula of the modified PAMAM polymer is as follows: PAMAM- (NH-C (O) -O-R) _n In the formula, the-NH-is derived from the-NH at the end of the PAMAM polymer ₂ ；

Wherein, when the PAMAM is the 3 rd generation PAMAM, n is an integer of 1-32; when the PAMAM is the 4 th generation PAMAM, n is an integer of 1-64; when the PAMAM is the 5 th generation PAMAM, n is an integer of 1-128;

and each R is independentlySelected from: - (CH) ₂ ) _m -R ₂ (ii) a Wherein each m is independently selected from: an integer of 1 to 10;

each R ₂ Independently selected from:

* Representing a joint;

and x is an integer of 1 to 10.

2. Use according to claim 1, wherein R is ₃ And R ₄ Each independently selected from unsubstituted or substituted by R ₅ Substituted C1-C10 alkyl.

3. Use according to claim 2, wherein R is ₃ And R ₄ Each independently selected from unsubstituted or substituted by R ₅ Substituted C1-C6 alkyl; and/or, x is an integer of 1 to 5; and/or m is an integer of 1 to 5.

4. Use according to claim 3, wherein R is ₂ Is composed of

x is 2, 3 or 4.

5. Use according to claim 3, wherein each R is ₂ Independently selected from:

6. the use according to any one of claims 1 to 5, wherein the modified PAMAM polymer is a modified PAMAM polymer nanoparticle.

7. The use of claim 1, wherein the PAMAM is a generation 4 PAMAM and n is an integer from 20 to 64.

8. Use according to claim 7, wherein n is an integer from 40 to 50.

9. The use of claim 1, wherein the modified PAMAM polymer has the formula:

10. the use according to claim 1, wherein said PAMAM- (NH-C (O) -O-R) _n The medium-NH-C (O) -O-R is from amino-NH at the tail end of PAMAM polymer ₂ And

and reacting to obtain the product.

11. The use according to any one of claims 1 to 7, wherein the modified PAMAM polymer is prepared by a process comprising the steps of:

(1) Activating tertiary amine containing hydroxyl by adopting N, N' -carbonyl diimidazole to obtain activated tertiary amine; the tertiary amine containing a hydroxyl group has the structure R-OH, wherein R is defined in any one of claims 1 to 9;

(2) And (2) reacting the activated tertiary amine obtained in the step (1) with the PAMAM polymer, and purifying to obtain the modified PAMAM polymer.

12. Use according to claim 11, characterized in that the tertiary amine containing a hydroxyl group is selected from 2- (hexamethyleneimine) ethanol, N- (2-hydroxyethyl) piperidine, N-dimethylethanolamine, N-diethylethanolamine or N- (2-hydroxyethyl) pyrrolidine;

and/or the molar ratio of the N, N' -carbonyldiimidazole to the hydroxyl-containing tertiary amine is (1.5-5): 1;

and/or, the activation of the step (1) comprises the following steps: mixing tertiary amine containing hydroxyl with N, N' -carbonyl diimidazole at room temperature in an organic solvent, activating for 10-72h, washing the obtained product with water, and drying;

and/or the molar ratio of the terminal amino group to the activated tertiary amine in the PAMAM polymer is 1: (0.01-5).

13. The use according to any one of claims 1 to 9, wherein the tumour is colorectal cancer, breast cancer, melanoma or liver cancer; and/or the anti-tumor drug is an anti-tumor drug for inducing immunogenic cell death.

14. Use of a modified PAMAM polymer as defined in any one of claims 1 to 12 in the preparation of an immunogenic cell death inducing agent.

15. The modified PAMAM polymer and the application of the PD-1 antibody or the PD-L1 antibody as an anti-tumor active ingredient in the preparation of an immunogenic cell death inducer or an anti-tumor medicament; the modified PAMAM polymer is as defined in any one of claims 1 to 12.