A functionalized modified hyperbranched polyethyleneimine and preparation method and application thereof

Abstract

The application discloses a kind of functional modification hyperbranched poly-amine and preparation method and application.The application is synthesized by spermine, the substance providing branched site and N'N-bisacrylamide cystamine to obtain "three-section type" hyperbranched poly-amine, then combined with photosensitizer Ce6, obtained HPSPE-Ce6 is wrapped by hyaluronic acid, obtain functional modification hyperbranched poly-amine.Hyaluronic acid can protect nanoparticle to realize long circulation in blood and target tumor cell, be degraded by hyaluronidase of high expression of tumor microenvironment, so that nanoparticle realizes charge and size reduction, promotes its internalization and deeper tumor tissue penetration.Subsequently, HPSPE-Ce6 enters cell and is degraded into polyamine analogue by GSH, interferes with polyamine metabolism in tumor cell and tumor-associated macrophage, effectively inhibits the proliferation and metastasis of tumor.Therefore, functional modification hyperbranched poly-amine provided by the application can be prepared into antitumor drug.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of materials and biological medicine, and particularly relates to a functionalized hyperbranched polyamine, a preparation method and application thereof. BACKGROUND

[0002] Tumor immunotherapy, as a new cancer treatment method, achieves the purpose of treating tumors by activating the patient's own immune system. It makes up for the deficiency of traditional treatment that cannot be cured, also reduces the recurrence and metastasis of postoperative tumors, and is more accurate in targeting, significantly improves safety, and greatly reduces side effects. However, the response rate of immunotherapy for solid tumors is generally not high, on the one hand, because the immunogenicity of tumor cells is low, which leads to the failure to activate the immune system, on the other hand, because of the existence of tumor inhibitory microenvironment, which makes it transform into "cold tumor".

[0003] At present, in order to successfully cause the immunogenicity of tumor and activate the immune system to re-recognize and kill cancer cells. Small molecule chemical drug treatment (chemotherapy) is the main treatment method in clinical at present, but small molecule drugs achieve their killing effect by means of systemic metabolic circulation, which will produce strong side effects. Ultrasound (US) as a mechanical wave has high tissue penetration ability in soft tissue. In addition, ultrasound can accurately focus on tumor area, target activation of sound sensitive agent, and minimize damage to adjacent normal organ tissues. SDT kills tumor cells by producing ROS, and at the same time causes immunogenic cell death (ICD) of tumor, effectively activates the host immune system, and triggers systemic immune response against cancer.

[0004] Reversing tumor inhibitory microenvironment can effectively inhibit the development and metastasis of tumor, and also can enhance the anti-tumor immune effect. Among them, the presence of tumor associated macrophages (TAM) is significantly related to effective immunosuppressive activity, and the re polarization of macrophages can further enhance the anti-tumor immune response. TAM targeting depletion can achieve TME reprogramming, which can strongly inhibit tumor metastasis and recurrence. Studies have found that immunosuppressive cells have high dependence on polyamines, and they become effective targets for polyamine treatment. Therefore, by interfering with polyamine metabolism to reduce intracellular polyamine levels, it is possible to achieve TME reprogramming, which is expected to improve the efficacy of tumor immunotherapy in the present immunosuppressive "cold" tumor, and provide a new way for anti-tumor treatment.

[0005] Hyperbranched polymers (HBPs) are macromolecules with three-dimensional dendritic structure between traditional linear polymers and dendrimers. Hyperbranched polymers can be prepared by a simple one-pot method, and can retain a large number of groups for functionalization. At present, hyperbranched polymers are mainly used for drug delivery, but the activity of the polymer itself is rarely reported. Previous studies have shown that the polymer can directly exert an anti-tumor effect, so we believe that the products formed after the degradation of the polymer in the body may exert an anti-tumor effect, and therefore it is a promising research direction to use the polymer to interfere with the tumor microenvironment and even reverse the immunosuppressive microenvironment. SUMMARY

[0006] The primary purpose of the present application is to overcome the shortcomings and deficiencies of the prior art, and to provide a preparation method of functionalized modified hyperbranched poly-arginine.

[0007] Another purpose of the present application is to provide a functionalized modified hyperbranched poly-arginine obtained by the above preparation method.

[0008] Still another purpose of the present application is to provide the application of the above functionalized modified hyperbranched poly-arginine.

[0009] The purpose of the present application is achieved by the following technical solution: a preparation method of functionalized modified hyperbranched poly-arginine, comprising the following steps:

[0010] (1) Preparation of hyperbranched polymer HPSPE-Ce6

[0011] 1) Synthesis of N'N-bisacryloyl cystamine (CBA);

[0012] 2) Synthesis of hyperbranched poly-arginine (HPSPE);

[0013] 3) Grafting of the sonosensitizer Ce6 to the hyperbranched poly-arginine to obtain the hyperbranched polymer HPSPE-Ce6;

[0014] (2) Assembly of functionalized modified hyperbranched poly-arginine: vortex mixing of the hyperbranched polymer HPSPE-Ce6 and the shielding component hyaluronic acid in water as a medium, and using the electrostatic attraction between the two to fully mix the hyaluronic acid and the HPSPE-Ce6 to obtain HPSPE-Ce6 / HA composite structure nanoparticles, i.e. functionalized modified hyperbranched poly-arginine.

[0015] The synthesis in step (1) 1) is the synthesis of N'N-bisacryloyl cystamine using cystamine dihydrochloride and acryloyl chloride under the condition of pH value 9-12; specifically comprising the following steps:

[0016] A, dissolve sodium hydroxide in ultrapure water to obtain solution A;

[0017] B, dissolving cystamine dihydrochloride in ultrapure water to obtain solution B;

[0018] C, dissolving acryloyl chloride in an organic solvent under ice bath condition to obtain solution C;

[0019] D, placing solution B under ice bath condition, first adding part of solution A dropwise, first adjusting the initial pH of the reaction system to 9-12, then alternately adding solution A and solution C dropwise, and reacting; extracting the obtained reaction product with an organic solvent, collecting the organic layer, sequentially washing, rotary evaporation, and drying to obtain N', N-bis-acryloyl cystamine.

[0020] The concentration of sodium hydroxide in solution A in step A is preferably 1-20 mol / L; more preferably 10 mol / L.

[0021] The concentration of cystamine dihydrochloride in solution B in step B is preferably 0.01-1 mol / L; preferably 1 mol / L.

[0022] The organic solvent in step C is preferably at least one of dichloromethane, chloroform, ethyl acetate and petroleum ether.

[0023] The concentration of acryloyl chloride in solution C in step C is preferably 1-50 mol / L; more preferably 10 mol / L.

[0024] The part of solution A in step D is preferably 1 / 9-1 / 10 of the total volume of solution A.

[0025] The time of the alternate dropwise addition in step D is not less than 30 min.

[0026] The cystamine dihydrochloride and acryloyl chloride in the reaction system in step D are preferably in a molar ratio of 1:1-8; more preferably in a molar ratio of 1:1-3; most preferably in a molar ratio of 1:2.

[0027] The reaction time in step D is preferably 5-10 h; more preferably 8 h.

[0028] The extraction organic solvent in step D is preferably at least one of dichloromethane, chloroform, ethyl acetate and petroleum ether.

[0029] The sequential washing solvent in step D is preferably saturated sodium bicarbonate solution, saturated sodium chloride solution and ultrapure water.

[0030] The drying in step D is preferably vacuum drying.

[0031] The drying time in step D is preferably 12 h.

[0032] The synthesis described in step (1) 2) is a way of combining a large amount of spermine with a small amount of a branching site-providing substance to form a hyperbranched polyamine by a Michael addition reaction between spermine (SPE), the branching site-providing substance and N'N-diacryl cadaverine (CBA), with the branching site-providing substance as the branching site. The specific steps are preferably as follows:

[0033] E. Dissolve N'N-diacryl cadaverine in an organic solvent to obtain solution D, introduce an inert gas and raise the temperature to react;

[0034] F. Mix and dissolve spermine and a branching site-providing substance in an organic solvent to obtain a mixed solution;

[0035] G. In a dark environment, drop the mixed solution obtained in step F into the product obtained in step E, condense and react;

[0036] H. Add a spermine solution to the product obtained in step G and continue the reaction;

[0037] I. Dilute the reaction solution obtained in step H with ultrapure water, then adjust the pH with an acid solution; dialyze under the condition of 4°C and light avoidance, and dry to obtain a hyperbranched polyamine.

[0038] The concentration of N'N-diacryl cadaverine in solution D described in step E is preferably 1 g / L to 30 g / L; more preferably 10 g / L.

[0039] The organic solvent described in step E is preferably methanol.

[0040] The concentration of the methanol is preferably 40% to 90% by volume; more preferably 70% by volume.

[0041] The inert gas described in step E is preferably nitrogen.

[0042] The temperature of the reaction described in step E is preferably 20 to 70°C; more preferably 40 to 60°C; most preferably 50°C.

[0043] The branching site-providing substance described in step F is preferably at least one of N-aminoethylpiperazine (AEP), spermidine and putrescine.

[0044] The concentration of the branching site-providing substance in the mixed solution described in step F is 1 mmol / L to 2 mmol / L; preferably 2 mmol / L.

[0045] The concentration of spermine in the mixed solution described in step F is 0.5 mmol / L to 6 mmol / L, preferably 4 mmol / L.

[0046] The molar ratio of the spermine and the substance providing branching sites in Step F is 1-16:1; particularly preferably 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1 or 16:1; more preferably 2:1.

[0047] The organic solvent in Step F is preferably methanol.

[0048] The concentration of the methanol is preferably 40%-90% by volume; more preferably 70% by volume.

[0049] The amount of the mixed solution in Step G is in a molar ratio of CBA:SPE:the substance providing branching sites = 0.5-1.5:1-16:1; preferably in a molar ratio of CBA:SPE:the substance providing branching sites = 0.5-1.5:2:1; more preferably in a molar ratio of CBA:SPE:the substance providing branching sites = 1:2:1.

[0050] The temperature of the condensation in Step G is 10-20°C, preferably 15°C.

[0051] The time of the reaction in Step G is preferably 12-60h; more preferably 24h.

[0052] The amount of the spermine added in Step H is preferably more than 40 times the mass of N',N'-bisacryloyl cadaverine; more preferably 40-60 times; most preferably 50 times.

[0053] The concentration of the spermine solution in Step H is preferably 4-6g / mL; more preferably 5g / mL.

[0054] The time of the reaction in Step H is preferably 12-60h; more preferably 24h.

[0055] The acid solution in Step I is preferably hydrochloric acid.

[0056] The pH in Step I is preferably 3-7; more preferably 4-5.

[0057] The dialysis in Step I is preferably dialysis using a dialysis bag with a molecular weight of 1200kDa.

[0058] The drying method in Step I is preferably freeze-drying.

[0059] The hyperbranched polymer in Step (1) 3) is a hyperbranched poly(spermine) grafted with a sonosensitizer Ce6, which is covalently linked to HPSPE through an amide reaction, and the specific steps are preferably as follows:

[0060] J. dissolving Ce6 in an organic solvent to obtain a Ce6 solution;

[0061] K. mixing 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS) and the Ce6 solution in the dark, and reacting;

[0062] L. mixing the hyperbranched polyethylene amine and the reaction product obtained in step K uniformly in the dark to obtain a solution E, and reacting;

[0063] M. dialyzing the product obtained in step L at 4°C in the dark, and drying to obtain HPSPE-Ce6.

[0064] The organic solvent in step J is preferably at least one of dimethyl sulfoxide (DMSO), dimethylformamide (DMF) and acetonitrile.

[0065] The concentration of the Ce6 solution in step J is preferably 0.1 mg / mL to 10 mg / mL; more preferably 1 mg / mL.

[0066] The amount of Ce6 in step K is preferably 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide : N-hydroxysuccinimide : Ce6 = mass ratio 2 to 4 : 2 to 4 : 1, more preferably 2 : 2 : 1.

[0067] The reaction time in step K is preferably 30 min to 4 h; more preferably 2 h.

[0068] The amount of hyperbranched polyethylene amine in step L is preferably calculated according to hyperbranched polyethylene amine : Ce6 = mass ratio 7 to 9 : 1; more preferably according to hyperbranched polyethylene amine : Ce6 = mass ratio 8 : 1.

[0069] The concentration of hyperbranched polyethylene amine in the solution E in step L is 1 mg / mL to 15 mg / mL; more preferably 8 mg / mL.

[0070] The mixing method in step L is preferably stirring.

[0071] The stirring time is preferably 6 to 48 h; more preferably 24 h.

[0072] The dialysis in step M is preferably dialysis using a dialysis bag with a molecular weight of 1000 Da.

[0073] The drying method in step M is preferably freeze-drying.

[0074] The molecular weight of the hyaluronic acid in step (2) is preferably 10 to 120 kDA; more preferably 40 to 80 kDA.

[0075] The HPSPE-Ce6 and the HA in step (2) are preferably mixed at a mass ratio of 1-5:1-5; more preferably, the mass ratio is 1:5, 1:3, 1:2, 1:1, 2:1 or 5:1; most preferably, the mass ratio is 1:3.

[0076] The vortexing time in step (2) is preferably 30 s to 5 min; more preferably, the vortexing time is 1 min.

[0077] The functionalized hyperbranched polyamine is prepared by the preparation method.

[0078] The functionalized hyperbranched polyamine is prepared by the preparation method.

[0079] The present application opens up a new research approach of integrating polyamine metabolic interference and sonodynamic therapy into functionalized polymers and applying them to tumor immunotherapy. The present application constructs a polymer composite structure assembled by a degradable charge shielding component with tumor targeting effect and a polymer component interfering with cell polyamine metabolism in response to intracellular glutathione (GSH). HA can protect the nanoparticles to achieve long circulation in the blood and target tumor cells through CD44. HPSPE-Ce6 is a "three-section" hyperbranched polyamine (HPSPE) formed by Michael addition reaction of spermine, N-aminoethylpiperazine and cystamine, and coupling with Ce6 through amide reaction based on the structure of cystamine. After reaching the tumor tissue, HA is degraded by the high expression of hyaluronidase in the tumor microenvironment to realize the size and charge reduction of the nanoparticles, promote their internalization and deeper tumor tissue penetration. Subsequently, HPSPE-Ce6 enters the cells and is degraded into polyamine analogues (different from the structure of endogenous polyamines) by GSH, interfering with the polyamine metabolism in tumor cells and tumor-associated macrophages. On the one hand, it inhibits tumor cell proliferation and migration, and on the other hand, it promotes the re-polarization of macrophages to the anti-tumor M1 phenotype to reverse the tumor immune microenvironment. At the same time, the sonodynamic therapy (SDT) induces immunogenic death of tumor cells, activates the body's immune response. Subsequently, these damage-associated molecular patterns (DMAP) promote the maturation of dendritic cells (DCs), activate CD8 + T, CD4 + T cells to clear tumor cells. In addition, we found that polyamine metabolic interference can effectively inhibit tumor proliferation and metastasis. This strategy provides a new idea for reversing the tumor immune suppression microenvironment, which may inspire more design and invention of polyamine metabolism applied to tumor immunotherapy, and then be transformed into clinical practice.

[0080] The present application has the following advantages and effects relative to the prior art:

[0081] (1) The present application proposes to interfere with cell polyamine metabolism by using pure polymer (polyamine analogs are produced after polymer degradation, which plays a role in polyamine metabolism interference), which promotes tumor-associated macrophage repolarization while inhibiting tumor proliferation and migration.

[0082] (2) The present application introduces a single functional polymer therapeutic agent to achieve synergistic immunogenic death triggering and microenvironment immune suppression reversal.

[0083] (3) The present application realizes precise tumor targeting by hyperbranched polymer targeting CD44 receptor mediation and enzyme response, and further enhances the body's anti-tumor immune response.

[0084] (3) The composite structure assembly provided by the present application provides a new idea for enhancing tumor immunotherapy. To our knowledge, pure polymer preparations have not been reported for reversing tumor microenvironment by polyamine metabolism interference and combined with sonodynamic therapy for enhancing tumor immunity, so it is innovative. BRIEF DESCRIPTION OF DRAWINGS

[0085] Figure 1 is a characterization result diagram of HPSPE-Ce6 / HA; wherein (A) is the CBA and HPSPE of 1 H NMR spectrum; (B) is the standard curve of Ce6; (C) is the UV-vis-NIR absorption spectrum of HPSPE, HPSPE-Ce6; (D) is the particle size of HPSPE-Ce6 / HA complex under different mass ratios; (E) is the zeta potential of HPSPE-Ce6 / HA complex under different mass ratios; (F) is the morphology and size distribution of HPSPE-Ce6 / HA; (G) is the hydrodynamic particle size and zeta potential of HPSPE-Ce6 / HA in 10% FBS 1640 medium

[0086] Figure 2 is a responsive result diagram of HPSPE-Ce6 / HA composite structure assembly; wherein (A) is the zeta potential of HPSPE-Ce6 / HA after incubation with HAase for different time; (B) is the particle size of HPSPE-Ce6 / HA after incubation with HAase for different time; (C) is the zeta potential of HPSPE-Ce6 after incubation with GSH for different time; (D) is the intracellular GSH content of B16F10 cells after incubation with different concentrations of HPSPE-Ce6 / HA for 12h or 24h; (E) is the fluorescence image of B16F10 cells after incubation with HPSPE-Ce6 / HA for 12h and 24h, wherein the green fluorescence represents GSH; (F) is the relative molecular mass of HPSPE-Ce6 / HA before and after incubation with GSH

[0087] Figure 3Figure for the biocompatibility evaluation results of HPSPE-Ce6 / Ha; wherein, (A) is the hemolysis of red blood cells after incubation with different concentrations of HPSPE-Ce6 solution for different times; (B) is the hemolysis of red blood cells after incubation with different concentrations of HPSPE-Ce6 / HA solution for different times; (C) is the hemolysis of HPSPE-Ce6 and HPSPE-Ce6 / HA after incubation for 6h under different concentrations; (D) is the statistical chart of (C); (E) is the effect of different concentrations of HPSPE-Ce6 on the activity of HUVEC cells; (F) is the effect of different concentrations of HPSPE-Ce6 / HA on the activity of RAW264.7 cells.

[0088] Figure 4 Figure for the results of cell uptake and deep penetration ability of HPSPE-Ce6; wherein, (A) is the endocytosis rate of B16F10 to HPSPE-Ce6 / HA at different times; (B) is the endocytosis rate of RAW264.7 to HPSPE-Ce6 / HA at different times; (C) is the laser confocal imaging of B16F10 after endocytosis of HPSPE-Ce6 / HA at different time points; (D) is the regional fluorescence spectrum of B16F10 3D tumor spheroids after incubation with HPSPE-Ce6 / HA and HPSPE-Ce6 / HA+HAase for 4h; (E) is the CLSM image.

[0089] Figure 5 Figure for the relative expression change results of SMOX, SSAT and PAOX mRNA in B16F10 cells.

[0090] Figure 6 Figure for the results of the influence of polyamine metabolism interference on tumor cells; wherein, (A) is the proliferation curve of B16F10 cells after incubation with different materials; (B) is the relative quantitative result analysis of (C); (C) is the scratch test photo of B16F10 cells after treatment with different materials.

[0091] Figure 7 Figure for the results of tumor-associated macrophage polyamine metabolism interference; wherein, (A) is the representative fluorescence peak chart of CD86-PerCP-Cy5.5 (M1 type macrophages) of RAW264.7 cells after treatment with different experimental groups; (B) is the statistical chart of (A); (C) is the representative fluorescence peak chart of CD206-FITC (M2 type macrophages) of RAW264.7 cells after treatment with different experimental groups; (D) is the statistical chart of (C).

[0092] Figure 8Figures of the ability of the generation of reactive oxygen species after ultrasound stimulation; (A) are fluorescence spectra of HPSPE-Ce6 / HA under different ultrasound powers; (B) are fluorescence spectra of HPSPE-Ce6 / HA under different times; (C) are effects of different ultrasound powers on HUVEC cell activity; (D) are fluorescence images of intracellular ROS generation of B16F10 cells detected by DCFH-DA probe after different treatments; (E) are cell viabilities of B16F10 cells after various treatments (Ce6 concentration is 2 μg / mL); (F) are photographs of live / dead cell fluorescence staining results of B16F10 cells after different experimental group treatments.

[0093] Figure 9 Figures of HPSPE-Ce6 / HA mediated cytotoxicity in vitro; (A) are flow cytometry diagrams of B16F10 cell apoptosis after treatment in different experimental groups; (B) are fluorescence quantitative statistical diagrams.

[0094] Figure 10 Figures of expression of maturation-related molecules of BMDCs after treatment in different experimental groups; (A) are CLSM images of CRT exposed on B16F10 cells after different treatments; (B) are fluorescence intensity distribution diagrams of B16F10 cell CRT-FITC after different treatments; (C) are quantification of CRT expression on B16F10 cells by flow cytometry; (D) are levels of extracellular HMGB1 released by B16F10 cells after treatment in different experimental groups detected by ELISA; (E) are intracellular ATP levels of B16F10 cells after different treatments; (F) are extracellular ATP contents.

[0095] Figure 11 Figures of expression of maturation-related molecules of BMDCs after treatment in different experimental groups; (A) are co-stimulatory molecules CD80; (B) are co-stimulatory molecules CD86.

[0096] Figure 12 Figures of deep tissue penetration ability of ultrasound; (A) are schematic diagrams of effects of different thickness tissue barriers on SDT and PDT; (B) are cell viabilities of B16F10 cells after ultrasound and laser irradiation under different thickness tissue barriers; (C) are fluorescence images of cell live / dead staining.

[0097] Figure 13 Figures of tumor targeting effect of HPSPE-Ce6 / HA; (A) are fluorescence imaging diagrams of Ce6, HPSPE-Ce6 / HA in mice 24 h after tail vein injection and ex vivo fluorescence imaging diagrams of heart, liver, spleen, kidney and tumor; (B) are fluorescence quantification statistics in mice; (C) are fluorescence quantification statistics of ex vivo tumor and main organs of mice.

[0098] Figure 14 Figure 30.4 is a graph of in vivo anti-tumor effect; wherein (A) is a B16F10 tumor model and treatment schematic; (B) is the individual tumor growth of different treated mice; (C) is the image of dissected tumor after treatment.

[0099] Figure 15 Figure 30.8 is a graph of the results of exploring the killing mechanism of HPSPE-Ce6 / HA combined with sonodynamic therapy on tumor at the tissue level; wherein (A) is the curve of tumor volume change of mice after different treatments; (B) is the tumor weight statistics of each group after treatment; (C) is the body weight change of mice in different experimental groups during treatment; (D) is the H&E section and TUNEL immunofluorescence picture of mice after different treatments.

[0100] Figure 16 Figure 30.12 is a graph of the results of immunohistochemical staining to evaluate the induced immunogenic death in vivo; wherein (A) is the immunofluorescence image of CRT of mice after treatment in different treatment groups; (B) is the representative flow fluorescence image of F4 / 80 + CD206 + cells (M2 macrophages) after different treatments; (C) is the representative flow fluorescence image of F4 / 80 + CD86 + cells (M1 macrophages) after different treatments; (D) is the quantitative analysis of F4 / 80 + CD206 + cells in tumor; (E) is the quantitative analysis of F4 / 80 + CD86 + cells.

[0101] Figure 17 Figure 30.16 is a graph of the results of DC activation in vivo; wherein (A) is the representative flow chart of CD11c + CD80 + cells of mice in different treatment groups; (B) is the representative flow chart of CD11c + CD86 + cells; (C) is the percentage of CD80 + expression on CD11c + cells; (D) is the percentage of CD86 + expression on CD11c + cells.

[0102] Figure 18Figure 1 is a diagram of the results of in-depth research on systemic anti-tumor immune mechanisms; wherein (A) is a representative immunofluorescence image of helper T lymphocyte infiltration in tumor tissues after treatment in different treatment groups; (B) is a representative immunofluorescence image of cytotoxic T lymphocyte infiltration in tumor tissues after treatment in different treatment groups; (C) is a representative flow cytometry image of CD3 + CD4 + T cells and CD3 + CD8 + T cells; (D) is a quantitative analysis of CD3 + CD4 + T cells in the spleen; (E) is a quantitative analysis of CD3 + CD8 + T cells.

[0103] Figure 19 Figure 4 is a diagram of serum biochemical test results of mice in different treatment groups.

[0104] Figure 20 Figure 5 is a photograph of organ H&E sections of mice in different treatment groups. DETAILED DESCRIPTION

[0105] The embodiments of the present application will be described in detail below with reference to the embodiments and drawings, but those skilled in the art will understand that the following embodiments and examples are only used to illustrate the present application and should not be regarded as limiting the scope of the present application. If the specific conditions are not specified, the conventional conditions or the conditions recommended by the manufacturer are used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained by market purchase.

[0106] Example 1: Preparation and characterization of HPSPE-Ce6 / HA

[0107] I. Preparation method of HPSPE-Ce6 / HA, comprising the following steps:

[0108] Spermine, N-aminoethylpiperazine is polymerized with N’N-diacryl cystamine (CBA) by Michael addition to form a “three-section” hyperbranched polyamine (HPSPE), and then Ce6 is coupled by amide reaction to prepare HPSPE-Ce6; finally, HPSPE-Ce6 and HA are mixed in a certain proportion, and a multifunctional composite structure assembly HPSPE-Ce6 / HA is formed by electrostatic self-assembly. The specific steps are as follows:

[0109] (1) Synthesis of N’N-diacryl cystamine (CBA)

[0110] A. 5.8 g of cystamine dihydrochloride (25 mmol) was weighed into a 250 mL three-necked flask, 25 mL of ultrapure water was added, and it was fully dissolved by magnetic stirring to obtain a cystamine dihydrochloride aqueous solution.

[0111] B, weigh 4.0 g of NaOH (0.1 mol) and dissolve in 10 mL of ultrapure water to obtain a sodium hydroxide solution, ready for use; weigh 4.7 g of acryloyl chloride (50 mmol) and dissolve in 5 mL of dichloromethane to obtain an acryloyl chloride solution, ready for use, under ice bath conditions.

[0112] C, place the three-necked flask containing the aqueous solution of cystamine acid salt in an ice bath, and cool the solution to 0-5°C. First, add 1 mL of sodium hydroxide solution to make the pH value of the solution 9-12, and then alternately slowly add the sodium hydroxide solution and the acryloyl chloride solution. This process takes no less than 30 min.

[0113] D, after the addition is complete, remove the ice bath, plug the bottle opening with a glass plug, and continue stirring the reaction at room temperature for 8 h.

[0114] E, collect the reaction mixture obtained in step D, extract with dichloromethane (DCM) multiple times, and collect the organic layer (lower layer)

[0115] F, wash the organic layer with saturated sodium bicarbonate solution, saturated sodium chloride solution, and ultrapure water 2-3 times in sequence.

[0116] G, remove the dichloromethane from the washed solution by rotary evaporation, and then vacuum dry for 12 h to collect the white product.

[0117] (2) Synthesis of hyperbranched polyethylenimine (HPSPE)

[0118] A, dissolve 100 mg of CBA in 10 mL of 70% (v / v) methanol aqueous organic solvent in a flask, introduce nitrogen gas, and raise the temperature to 50°C for reaction.

[0119] B, dissolve SPE and AEP in 5 mL of 70% (v / v) methanol aqueous organic solvent to obtain a mixed solution.

[0120] C, slowly add the mixed solution to the flask containing the reaction product of step A under dark conditions, condense (10-20°C), and react for 24 h; wherein the molar ratio of CBA, SPE, and AEP is 1:2:1.

[0121] D, add 1 mL of excess spermine solution (5 g / mL) and continue to react for 24 h.

[0122] E, dilute the reaction solution with ultrapure water, adjust the pH to 4-5 with hydrochloric acid solution, dialyze (molecular weight: 1200 kDa) under 4°C in the dark, and freeze-dry to obtain hyperbranched polyethylenimine.

[0123] (3) Synthesis of HPSPE-Ce6

[0124] A, 5mg Ce6 was weighed and dissolved in 5mL of dimethyl sulfoxide (DMSO) to obtain a Ce6 solution.

[0125] B, the Ce6 solution was added to the brown reaction bottle, and 10mg EDC and 10mg NHS were added to pre-activate the carboxyl group of Ce6 for 2h.

[0126] C, 40mg of HPSPE prepared in step (2) was weighed and added to the reaction bottle, and stirred at room temperature for 12h.

[0127] D, dialysis (Mw = 1000Da) in pure water at 4°C in the dark for three days to remove unreacted Ce6.

[0128] E, the dialysate was filtered with a 0.8mm filter head, freeze-dried and the product was weighed.

[0129] (4) Synthesis of HPSPE-Ce6 / HA

[0130] A, HPSPE-Ce6 was prepared into a 2mg / mL aqueous solution, and hyaluronic acid was dissolved in the aqueous solution

[0131] B, the two were mixed in different mass ratios (mass ratios were 5:1, 2:1, 1:1, 1:1, 1:2, 1:3 and 1:5, respectively), vortexed for 1min to obtain different specifications of HPSPE-Ce6 nanoparticle solutions, which were detected using a nanolaser particle size instrument to obtain assembly particle size and potential data, which were analyzed by graphpad 8.

[0132] II. Characterization of HPSPE-Ce6 / HA assembly, including the following steps:

[0133] 1) Nuclear magnetic resonance hydrogen spectrum (1H NMR)

[0134] 3mg of the sample to be tested was weighed and dissolved in about 0.6mL of deuterated reagent (heavy water D2O or deuterated dimethyl sulfoxide), and the prepared sample deuterated reagent was loaded into a clean NMR tube. The material structure was determined by nuclear magnetic resonance spectrometer, and the spectrum was analyzed by MestReNova software.

[0135] 2) Ultraviolet-visible spectrophotometry (UV)

[0136] A series of Ce6 solutions with gradient concentrations were prepared and measured using a UV-Vis spectrophotometer. A standard curve was plotted based on the peak area of ​​the characteristic Ce6 absorption peak at 660 nm and its corresponding solution concentration. An appropriate amount of HPSPE-Ce6 was weighed to prepare a 1 mg / mL solution, placed in a UV-Vis dish, and measured using a UV-Vis spectrophotometer, scanning the spectrum in the 300-800 nm range. The grafting rate of Ce6 was calculated using the following formula under the same UV absorbance conditions.

[0137] In the formula, C(Ce6) and C(HPSPE-Ce6) represent the concentrations of Ce6 and HPSPE-Ce6 sample solutions, respectively, under the condition of the same ultraviolet absorption.

[0138] 3) Particle size and potential

[0139] To determine the optimal ratio for assembling the composite structure, HPSPE-Ce6 / HA nanoparticle solutions mixed at mass ratios of 5:1, 2:1, 1:1, 1:2, 1:3, and 1:5 were analyzed using a nanolaser particle size analyzer to obtain the particle size potential data of the assembled structures. The data were then analyzed using GraphPad 8.

[0140] 4) Microstructure of the assembly

[0141] The microstructure of HPSPE-Ce6 / HA nanoparticles was observed using transmission electron microscopy (TEM). An appropriate concentration of the assembly was slowly dropped onto a 200-mesh copper grid and allowed to dry naturally. Subsequently, the microstructure of the product was observed using TEM, and the micromorphology of the HPSPE-Ce6 / HA assemblies obtained at different assembly reaction times was determined.

[0142] III. Characterization results of HPSPE-Ce6 / HA:

[0143] In this invention, HPSPE is mainly prepared via a Michael addition reaction of N'N-bisacryloylcysteine ​​with spermine and N-aminoethylpiperazine (CBA), where N-aminoethylpiperazine serves as the main branching site of the polymer. The sound-sensitizing agent is then prepared by amide grafting after reacting with the terminal amino group of HPSPE. 1 The synthesized product was characterized by ¹H NMR, such as Figure 1 As shown in (A), the peak of 1.5 ppm (-CH2CH2-) ethyl proton and 2.6-2.7 ppm (-NHCH2-) methyl proton, and the 1.6-1.7 ppm ethyl proton peak are attributed to spermine. The peak of 3.4-3.6 ppm (-CONHCH2-) amide proton, as well as the related peaks of piperazine, can all be found in the NMR spectrum, indicating the successful synthesis of HPSPE.

[0144] To further characterize the grafting rate of Ce6, it was detected using a UV spectrophotometer. The Ce6 monomer exhibits two characteristic absorption peaks around 660 nm and 400 nm. First, a concentration-absorbance standard curve was plotted for Ce6 monomer solutions of different concentrations using the 660 nm absorption peak (e.g., ...). Figure 1 (as shown in (B)). HPSPE-Ce6 was then characterized using UV radiation, as shown in... Figure 1 As shown in (C), the material exhibits significant UV absorption peaks at both 400 nm and 660 nm, further confirming the successful grafting of Ce6 onto HPSPE. The calculated drug loading rate of Ce6 is 10.2% (mg / mg).

[0145] HA was coated onto the surface of HPSPE-Ce6 via charge interactions. The size and surface charge properties of the assemblies were optimized by first adjusting the ratio (mass ratio) of HA to HPSPE. When HA and HPSPE-Ce6 were mixed, the particle size decreased significantly. This is due to the compaction of the assemblies caused by the interaction of positive and negative charges. When the HA:HPSPE-Ce6 ratio was 2:1 or smaller, the particle size was less than 200 nm (see...). Figure 1 (D) in the middle). Combined with the zeta potential diagram (see Figure 1 From the perspective of (E), when HA:HPSPE-Ce6 = 3:1, the potential is -20.3 mV. At this point, the charge of cations in the assembly is completely shielded, which allows the nanoparticles to achieve long-term cycling in the bulk. Therefore, the composite structure assembly of HA:HPSPE-Ce6 = 3:1 was selected for subsequent experiments.

[0146] To more intuitively observe the morphology of the assembly, it was directly observed using a transmission electron microscope. For example... Figure 1 As shown in (F), HPSPE-Ce6 / HA exhibits well-dispersed spherical particles with a diameter of 20-40 nm under electron microscopy, indirectly demonstrating good assembly. Furthermore, the hydrodynamic diameter of HPSPE-Ce6 / HA in water was observed to be approximately 126.6 nm, differing from the particle size observed under electron microscopy. This difference is attributed to the chain stretching state of the particles in water, followed by shrinkage after drying. Subsequently, the particle size variation of the HPSPE-Ce6 / HA assembly was measured using a nanolaser particle size analyzer to investigate its stability during in vivo circulation. Two environments were simulated: pure water and 1640 complete culture medium containing 10% fetal bovine serum, mimicking both in vitro and in vivo heterologous circulation. Figure 1As shown in (G) of FIG. 6, the zeta potential of the HPSPE-Ce6 / HA assembly did not change significantly in the two solutions within 48 h, which showed that the assembly had good stability. This indicated that the particle could avoid precipitation after mixing with blood after preparation, which was conducive to achieving long circulation of the particle in the body and increasing effective accumulation and retention of the nanoparticle in tumor tissue.

[0147] Example 2: Responsive effect of HPSPE-Ce6 / HA assembly

[0148] 1 mL of the composite structure assembly was respectively incubated with 5 mL of HAase (hyaluronidase) having a concentration of 10 mmol / L and 5 mL of GSH (glutathione) having a concentration of 10 mmol / L in pure water for 0, 1, 2, 4 and 8 hours to simulate the responsiveness of the assembly in the tumor tissue microenvironment and tumor cells (mouse skin melanoma cells B16-F10). The responsiveness of the material was characterized by particle size and zeta potential, and the change in intracellular GSH level after adding the nanoparticles was detected by a GSH detection kit.

[0149] As shown in (A) and (B) of FIG. 7, with the incubation time, the zeta potential of HPSPE-Ce6 / HA changed significantly, and the charge reversal was achieved after 6 h; at the same time, the particle size of the assembly was gradually reduced. This fully confirmed the enzyme responsiveness of the assembly, that is, the assembly could achieve charge reversal and size shrinkage in the tumor environment. In addition, the degradability of HPSPE in the presence of 10 mM GSH was studied by the change in material potential and the change in intracellular GSH level. As shown in (C) of FIG. 7, the zeta potential of HPSPE was significantly reduced in the presence of 10 mM GSH, which directly proved the degradation of HPSPE. In addition, the intracellular GSH level before and after HPSPE treatment was qualitatively and quantitatively analyzed by a GSH detection kit and GSH-labeled fluorescence. As shown in (E) of FIG. 7, before HPSPE treatment, B16F10 cells showed strong green fluorescence, which indicated that the intracellular GSH level was high, while after 12 h of HPSPE treatment, only weak fluorescence was observed, and after 24 h of treatment, the fluorescence intensity level was lower. This indicated that intracellular HPSPE significantly consumed GSH during intracellular degradation. In (D) of FIG. 7, it can be clearly seen that the intracellular glutathione level decreased with the increase of the concentration of HPSPE and the extension of the treatment time. The degradation of the disulfide bond in HPSPE was indirectly indicated by the change in intracellular GSH level, which confirmed the intracellular responsive degradation of the assembly. In addition, HPSPE-Ce6 / HA was incubated with GSH, and the product after incubation was detected by GPC for relative molecular mass, Figure 2 Figure 2 Figure 2 Figure 2 Figure 2 ​​​​(F) in the figure directly indicates that the relative molecular weight decreases significantly after HPSPE-Ce6 / HA is incubated with GSH.

[0150] Example 3: Evaluation of biocompatibility of HPSPE-Ce6 / HA

[0151] The biocompatibility of HPSPE-Ce6 / HA nanoparticles was evaluated from three aspects of blood compatibility, serum stability and cell compatibility.

[0152] I. Experimental process

[0153] (1) Blood compatibility

[0154] Dilute the red blood cells (RBC) to a cell suspension with a body percentage of 16% with PBS (0.01 mol / L, pH 7.4), then configure different concentrations (0.01, 0.1, 0.5, 1 mg / mL) of HPSPE-Ce6 and HPSPE-Ce6 / HA in 1 mL of cell suspension, then incubate at 37°C for 1 h, 3 h, 6 h and 12 h, collect the supernatant by centrifugation at 1000g for 5 min, and take 200 μL of sample and add it to a 96-well plate. Measure the OD value at 540 nm with a microplate reader. RBC with PBS (0% lysis) and water (100% lysis) are used as negative and positive control groups, respectively. Calculate the hemolysis rate according to the following formula:

[0155]

[0156] A is the OD value of the experimental group, A0 is the OD value of the negative control group PBS, A 100 is the OD value of the positive control group pure water.

[0157] (2) Serum stability

[0158] In order to verify that HPSPE-Ce6 / HA can maintain stable size and charge in vitro solution and after injection into the body, the changes in hydrodynamic size and potential of the assembly in different solution environments within 72 h were observed by nanolaser particle size analyzer (DLS), and the specific operation is as follows: weigh an appropriate amount of HPSPE-Ce6 / HA, dissolve the sample in pure water and RPMI-1640 culture medium containing 10% fetal bovine serum (the obtained solution contains HPSPE-Ce6 / HA at 10 μg / mL, 50 μg / mL and 100 μg / mL, respectively), which are used to simulate the assembly in vitro and in vivo circulation environments, respectively. Take 1 mL of sample to be tested in the sample cell, and measure the hydrodynamic size and potential at different time points (1, 2, 4, 8, 12, 24, 48 and 72 h), respectively. The obtained data is analyzed using graphpad 8.

[0159] (3) Cytotoxicity

[0160] The cytotoxicity of HPSPE-Ce6 / HA on B16F10 (Guangzhou Saigun Biotechnology Co., Ltd. (Distributor)), HUVEC (Guangzhou Saigun Biotechnology Co., Ltd. (Distributor)) and RAW264.7 (Guangzhou Saigun Biotechnology Co., Ltd. (Distributor)) was evaluated by CCK-8 kit. First, B16F10 cells were cultured using RPMI 1640 complete medium containing 10% fetal bovine serum by volume, and HUVEC and RAW264.7 cells were cultured using DMEM complete medium. Cells in the logarithmic growth phase were digested and centrifuged, and the three cells were seeded into 96-well plates at a density of 5 x 10 3 After 24 h of continuous incubation, the culture medium was discarded, and residual material was washed off with sterile PBS. CCK-8 reagent was diluted with base medium at a ratio of 1:10, and then 100 μL of staining working solution was added to each well. Three unused wells were selected to add CCK-8 staining solution as a blank control. The cell culture plate was returned to the incubator for 1-4 h, and the OD value at 450 nm was detected by a multifunctional enzyme label instrument. When the OD value of the control group was in the range of 0.8-1.2, the data was available. The activity of cells treated with different materials was calculated by the following formula.

[0161] Where As is the absorbance of the experimental well, Ac is the absorbance of the control well, and Ab is the absorbance of the blank well.

[0162] II. Experimental Results

[0163] (1) Evaluation results of the biocompatibility of HPSPE-Ce6 / HA:

[0164] Generally, a hemolysis rate of less than 5% indicates that the material has good blood compatibility. Figure 3 (A) and (B) in (C) are the hemolysis rates of HPSPE-Ce6 and HPSPE-Ce6 / HA at different incubation times and different concentrations. Figure 3 (C) is a visual view of the hemolysis of HPSPE-Ce6 and HPSPE-Ce6 / HA at different concentrations for 6 h, and the corresponding statistical chart is as follows: Figure 3The results show that HPSPE-Ce6 / HA has a lower hemolysis rate at different concentrations and longer incubation time. In contrast, HPSPE-Ce6 shows a significant concentration and time dependence of hemolysis rate, and the hemolysis rate exceeds 5% when the concentration reaches 20 μg / mL, which indicates poor blood compatibility. This is because HPSPE-Ce6 has a high positive charge, and the electrostatic interaction between it and the negatively charged red blood cell membrane causes damage to the red blood cell membrane. The introduction of HA shields the surface charge of HPSPE-Ce6, significantly improving the biocompatibility.

[0165] Cytotoxicity evaluation is also an important means of evaluating the biocompatibility of materials. Based on the fact that materials are injected intravenously in vivo and are taken up by tumor-associated macrophages and reverse their phenotype, HUVEC (human umbilical vein endothelial cells) and RAW264.7 (mouse monocyte macrophages) were selected to evaluate the in vitro cytotoxicity of HPSPE-Ce6 / HA. As shown in (E) and (F) of Figure 3 As shown in (E) and (F) of, when the concentration of HPSPE-Ce6 / HA is less than or equal to 200 μg / mL, the survival rates of HUVEC and RAW264.7 cells are both more than 90%. This indicates that HPSPE-Ce6 / FA nanoscale assemblies do not have cell damage behavior when the concentration is less than 200 μg / mL, and have good cell compatibility.

[0166] Example 4: Cell uptake and deep penetration ability of HPSPE-Ce6 / HA

[0167] First, the B16F10 tumor cells were collected by centrifugation after digestion, and were configured into a cell suspension at 5 x 10 3 were inoculated in ultra-low adsorption 96-well plates. The plates were placed in a 37°C constant temperature shaker at a speed of 50 rpm for 10 min, at least 5 times a day, and the plates were placed in a cell incubator for the rest of the time. The culture medium was replaced once a day, and when the 3D tumor spheroids grew to the appropriate size, they were transferred to a laser confocal dish.

[0168] Then, the cells were incubated with the basal medium containing HPSPE-Ce6 / HA and HPSPE-Ce6 / HA+HAase (pre-treated with HAase for 4 hours) for 4 hours, respectively. After incubation, the culture medium was discarded, and the residual material was washed with PBS. Finally, observation was carried out by laser confocal.

[0169] The endocytosis of B16F10 and RAW264.7 cells to HPSPE-Ce6 / HA at different times was studied by flow cytometry. Figure 4(A) in the figure is a fluorescence statistical diagram of the internalization of HPSPE-Ce6 / HA by B16F10 cells. This result indicates that after 6 hours of incubation with HPSPE-Ce6 / HA, the internalization rate of B16F10 cells reached over 90%. Figure 4 Similar phenomena were observed in (B) of the study, indicating that both B16F10 and RAW264.7 cells can effectively internalize HPSPE-Ce6 / HA. To more intuitively observe the internalization of the material by cells, laser confocal microscopy was used to observe HPSPE-Ce6 / HA in B16F10 cells. The cell nuclei were stained with DAPI (blue), the cell membrane was stained with Dio membrane dye (green), and Ce6 in the material showed red fluorescence. Figure 4 (C) indicates that the intracellular red fluorescence becomes stronger with prolonged incubation time, which further illustrates the effective internalization of the material.

[0170] The effects of charge reversal and size changes of HPSPE-Ce6 / HA on the permeability of the formulation were evaluated using 3D tumor spheroids (MTS) water. Figure 4 As shown in (E), strong fluorescence was observed in both the HPSPE-Ce6 / HA group and the HAase+HPSPE-Ce6 / HA group when the laser confocal scanning depth was less than 30 μm. Due to the poor penetration of the negatively charged HPSPE-Ce6 / HA, only the outer layer of the tumor sphere was observed with fluorescence signal at 50 to 90 μm, while fluorescence signal penetrating into the interior was observed in the HAase+HPSPE-Ce6 / HA group. This indicates that the degradation of HA by HA enhances the penetration of the nano-formulation. Quantitative fluorescence analysis was performed along the yellow line on the 90 μm crop using ImageJ (e.g., ...). Figure 4 As shown in (D) in the figure, it can be clearly seen that the HAase+HPSPE-Ce6 / HA group has a stronger penetration ability.

[0171] Example 5: In vitro antitumor effect mediated by polyamine metabolism interference

[0172] To verify the interference of the material on intracellular polyamine metabolism, the RNA content of polyamine metabolism-related enzymes was detected by real-time quantitative PCR (qPCR), and then the polyamine level in cell lysates was analyzed by HPLC.

[0173] I. Experimental Procedure

[0174] (1) Cell RNA extraction and qPCR

[0175] First, the expression of polyamine catabolism enzymes SMOX, PAOX, and SSAT in B16F10 cells was quantified by RT-PCR to investigate the polyamine interference of materials on cells. 5 × 10⁶ cells were used. 4B16F10 cells were inoculated into 6-well plates and cultured overnight in a constant temperature incubator at 37°C, 5% CO2. The culture medium was discarded and replaced with 2 mL of HPSPE-Ce6 / HA (10 μg / mL), HPAA / HA (hyperbranched polyamide / hyaluronic acid) (10 μg / mL), and 1640 base medium without other components, and the cells were co-cultured for 24 h. After incubation, the culture medium was aspirated, washed with PBS for 3 times, and then 1 mL of Trizol was added to each well to lyse the cells. The lysed solution was transferred to a new 1.5 mL centrifuge tube and left to stand at room temperature for 2-3 min. Then, 0.2 mL of chloroform was added to the centrifuge tube, which was vortexed for 30 s and then placed in an ice bath environment for 10 min. After ice bath, an ultralow-temperature centrifuge was used to centrifuge at 4°C, 12000 g for 15 min. After centrifugation, the solution was clearly divided into three layers (colorless supernatant aqueous phase, white middle layer, and pink lower organic phase), and the RNA was retained in the upper aqueous phase. The upper aqueous phase was carefully transferred to a new 1.5 mL centrifuge tube, and an equal volume of isopropanol solution was added. After mixing gently, it was ice-bathed for 20 min, and the supernatant was discarded. Then, 1 mL of 75% ethanol was used to resuspend the precipitate, which was again centrifuged at 4°C, 12000 g for 15 min. The supernatant was carefully discarded, and after a short centrifugation, the precipitate was left to dry at room temperature. Finally, a small amount of DEPC-treated water was added to dissolve the RNA, and the RNA purity and concentration were measured by a multifunctional enzyme marker. The samples meeting the standard were sealed and stored in a -80°C refrigerator for later use. According to the manufacturer's protocol, β-action was used as an internal reference, and the SynScriptTM III cDNA Synthesis Mix reverse transcription kit was used to reverse the cDNA. The 2xTSINGKE Master qPCR Mix (SYBR Green I) kit was used for qPCR (the qPCR reaction was performed according to the kit instructions).

[0176] The following primers were used:

[0177]

[0178] Finally, the relative mRNA expression of the enzyme was calculated according to the Ct values of the target gene and the internal reference gene.

[0179] The preparation process of HPAA / HA (hyperbranched polyamide / hyaluronic acid) is as follows: according to "CN201710700712.8 - A glutathione-responsive dual drug carrier and its preparation method and application" Example 5, hyperbranched polyamide amine (PAAs) with amino end groups were prepared; then PAAs were prepared into a 2 mg / mL aqueous solution, hyaluronic acid was dissolved in the aqueous solution, PAAs and hyaluronic acid were mixed at a mass ratio of 1:3, and then HPAA / HA was obtained after vortexing for 1 min.

[0180] (2) Intracellular polyamine content detection

[0181] As usual, first, the B16F10 cells in the logarithmic growth phase were digested, centrifuged and collected, and then inoculated into 60 mm cell culture dishes. When the cells in the dishes grew to 90% density, the old culture medium was discarded and replaced with 1640 basic culture medium containing 20 μg / mL HPSPE-Ce6 / HA, HPAA / HA, respectively, and a blank control group was set up, and incubated for 24 h. After discarding the culture medium, the cells were washed three times with PBS, then trypsinized and centrifuged. The cells were resuspended with 1 mL of 5% pre-cooled perchloric acid, ultrasonically extracted for 1 h in a 4 °C water bath. Then centrifuged at 5000 rpm for 10 min, and the supernatant was taken into a 15 mL centrifuge tube, 1 mL of 1 mmol / mL NaOH solution and 20 μL of 1 mmol / mL benzoyl chloride solution were added and placed in a 37 °C environment for 20 min. After the end, 2 mL of saturated sodium chloride and 2 mL of anhydrous ether were added to the centrifuge tube, vortexed for 30 s, and left at room temperature. After the solution was layered, the supernatant was blown dry with nitrogen, and 500 μL of HPLC-grade methanol was added for dissolution. Finally, 0.22 μm microporous filter membrane was used for filtration, and HPLC machine operation was performed.

[0182] (3) Cell proliferation experiment

[0183] The effect of polyamine metabolism interference on cell growth and proliferation was verified by cell proliferation experiment. First, B16F10 was prepared into a cell suspension by trypsin digestion, and the cells were counted by a hemocytometer. The cells were evenly inoculated into 60 mm cell culture dishes and placed in a 37 °C, 5% CO2 incubator. After the cells were completely adherent, the supernatant was discarded, 6 mL of RPMI 1640 complete medium containing 10 μg / mL of HPSPE / HA and HPAA / HA was added, respectively, and a blank group was set as a negative control, with 3 parallel groups in each group. After incubation for 24 h, 48 h and 72 h, respectively, the cells in different treatment groups were digested and centrifuged. Finally, the cells in different treatment groups were counted using a hemocytometer, and the data were processed and analyzed by graphpad.

[0184] (4) Cell scratch experiment

[0185] The effect of HPSPE-induced polyamine metabolism interference on the migration ability of B16F10 cells was verified by cell scratch experiment. A six-well plate was prepared in advance, and a straight line was drawn along the long edge of the well plate with a black marker pen, so that each well was evenly crossed by three lines for subsequent marking. The digested B16F10 cells were inoculated into the six-well plate at 1 × 10 6Cells were evenly seeded into 6-well plates. When the cells reached 90% confluence, a pipette tip was held perpendicular to the plate and used to make three scratches along the short side of each well, perpendicular to the line drawn on the back of the plate the previous day. After scratching, the cells were washed with PBS to remove any floating cells. The plates were then observed under a microscope, and the scratches at the designated points (the intersection of the drawn line and the scratches on the back of the plate) were photographed. Cells were co-incubated with 2 mL of serum-free medium containing 10 μg / mL HPSPE, 10 μg / mL SPE, and RPMI 1640 (containing no other media), and the plates were placed in a 37°C, 5% CO2 incubator. The scratches at the designated points were photographed after 12 h and 24 h of culture. Finally, the images were processed using ImageJ to quantify the area of ​​the cell scratches, and the cell migration rate was calculated using a formula:

[0186]

[0187] (5) Macrophage polarization

[0188] RAW264.7 cells were spaced at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 g / mL in 24-well plates and incubated overnight at 37°C with 5% CO2. The old medium was discarded and replaced with medium containing 20 ng / mL IL-4 (interleukin-4), and incubated for 12 h to stimulate macrophage polarization towards the M2 morphology. The original medium was aspirated, and the cells were carefully washed twice with PBS. 600 μL of medium containing the material was added, with HPSPE concentration of 10 μg / mL and 1 μg / mL LPS (lipopolysaccharide) as a positive control. Wells containing only cells and 1640 complete medium were set up as negative controls, with three replicates for each group. After incubation, the cells were washed with PBS, and cells from different wells were collected in 1.5 mL centrifuge tubes. Under light-protected conditions, FITC-anti-CD206 and PerCP-Cy5.5-anti-CD86 were diluted at a volume ratio of 1:200, and 200 μL of antibody dilution was added to each well. The plates were incubated at 4°C for 30 min. Finally, the cells were centrifuged and the supernatant was discarded. Unbound antibodies were washed away with PBS, and the cells were resuspended in 200 μL PBS for flow cytometry analysis.

[0189] II. Experimental Results

[0190] Polyamine analogues can be used as effective inducers of the elevated expression of catabolic enzymes of polyamines such as SMOX (spermine oxidase), SSAT (spermine / spermidine N1-acetyltransferase) and PAOX (polyamine oxidase) by interfering with intracellular polyamine metabolism in various ways, thus causing rapid depletion of polyamines and leading to tumor-selective cytotoxicity. The present application uses q-PCR to study the effect of HPSPE-Ce6 / HA treatment on the expression of SMOX, SSAT and PAOX. In order to exclude the influence of disulfide bonds and other degradation products, hyperbranched polyamides obtained by Michael addition of N-aminoethylpiperazine and N'N-diallyl cystamine are selected as a control group (which has a disulfide bond structure as HPSPE and can be degraded in cells) to explore the effect of cationic polymers or disulfide bonds on polyamine metabolism. The results are shown in Figure 5 , which show that the mRNA total level of SSAT, SMOX and PAOX is significantly up-regulated after HPSPE-Ce6 treatment, while the Control group and HPAA group have no effect on the content of intracellular polyamine catabolic enzymes. The experimental results of the HPAA group show that the breaking of polymer disulfide bonds and other small molecules of structure in cells do not affect intracellular polyamine metabolism.

[0191] Studies have shown that polyamines are important molecules for maintaining continuous cell proliferation, and depletion of polyamines and overexpression of SSAT will lead to reduced cell growth, migration and invasion. In order to explore the effect of polyamine metabolism interference on tumor cells, the present application verifies the effect of the decrease of polyamine concentration and the increase of polyamine catabolic enzyme expression on cell growth and migration by cell proliferation experiment and cell scratch experiment, respectively. Figure 6 (A) is the cell proliferation curve after treatment with different materials. It can be seen that there is little difference in cell proliferation rate after incubation with different materials for one day, which is presumably due to the low basic cell density and the influence of cell activity after trypsin digestion and re-plating. On the second and third days, it can be seen that the proliferation rate of the SPE group with additional polyamines is higher than that of the negative control group, while the cell proliferation of the HPSPE group is significantly inhibited, which shows that polyamine metabolism interference can seriously affect the proliferation of B16F10 tumor cells. The migration rate quantitative analysis and scratch healing pictures of B16F10 cells treated with different materials for 12h and 24h are shown in Figure 6 (B) and (C). Figure 6 As can be seen from (C) in Figure 6 , the scratch healing of the SPE positive control group is the fastest, and the cells on both sides of the scratch will converge after 24h; while the scratch healing rate of the HPSPE group is the slowest, and is significantly lower than that of the PBS group. This phenomenon shows that polyamines promote cell migration, and polyamine metabolism interference can effectively inhibit the migration of tumor cells.

[0192] Macrophages are an important component of innate immune cells, which play an important role in the regulation of tumor microenvironment. Studies have found that the activation of M1 macrophages is regulated by polyamines, and ornithine decarboxylase (ODC) and its product putrescine play a key role in inhibiting the activation of M1 macrophages. The absence of putrescine in macrophages will lead to chromatin remodeling and enhanced expression of M1 genes. In order to explore the effect of HPSPE-Ce6 / HA-mediated polyamine metabolism interference on tumor-associated macrophages, the present application uses 50 ng / mL IL-4 to co-incubate with macrophages for 12 h to induce M2 phenotype. After being treated with 10 μg / mL HPSPE-Ce6 / HA and 20 μg / mL LPS, the phenotype of RAW264.7 in two polarization states, i.e. CD86 (M1 type macrophage marker) and CD206 (M2 type macrophage marker) are analyzed by flow cytometry. + (A) and (C) in FIG. 1 are the flow cytometry fluorescence peak diagrams of CD86 Figure 7 + and CD206 + , respectively, Figure 7 (B) and (D) in FIG. 1 are the corresponding statistical diagrams. From the results, it can be seen that after IL-4 stimulation, the expression levels of CD86 + and CD206 + are 0.44% and 15.63%, respectively, and the macrophages are in an anti-inflammatory (M2) state, which indicates that the M2 immune-inhibitory macrophage model is successfully established. Then HPSPE-Ce6 / FA is co-incubated with M2 type RAW264.7 for 24 h, and LPS is used as a positive control for M1 type induction. Figure 7 In FIG. 2, the expression of CD86 + and CD206 + on the surface of cells in the HPSPE-Ce6 / HA group is 25.57% and 5.39%, respectively, although the expression of CD86 + is not as high as 56.23% of LPS, but it also significantly increases the expression level of M1 type macrophages. In general, these results show that HPSPE-Ce6 / HA can promote the polarization of macrophages to M1 type, and can effectively reverse the phenotype of tumor-associated macrophages and remodel the tumor immunosuppressive microenvironment.

[0193] Example 6: Production of ROS and HPSPE-Ce6 / HA-mediated in vitro cytotoxicity

[0194] (1) Detection of reactive oxygen species in solution

[0195] ​To verify the active oxygen yield of HPSPE-Ce6, the present application detects it by SOSG probe. First, HPSPE-Ce6 is configured into a solution with a concentration of 1 μM under dark conditions. Then the HPSPE-Ce6 solution is subjected to ultrasonic treatment at 1 MHz, 50% duty cycle, 0.8 W / cm 2 After 0 min, 2 min, 4 min, 8 min, and 10 min of treatment, respectively, the appropriate amount of SOSG probe working solution is added according to the kit operation, and it is left to stand for 5 min in a room temperature light-proof environment. Finally, the fluorescence of the SOSG probe is determined by a fluorescence spectrophotometer with an excitation light of 480 nm.

[0196] In addition, in order to explore the effect of ultrasonic power on the yield of active oxygen, the following experiment is set: the HPSPE-Ce6 solution is subjected to ultrasonic treatment at a power of 0 W / cm 2 , 0.16 W / cm 2 , 0.32 W / cm 2 , 0.48 W / cm 2 , 0.6 W / cm 2 , and 0.8 W / cm 2 , 50% duty cycle for 4 min. Then the detection is carried out according to the above same steps.

[0197] (2) Intracellular active oxygen detection

[0198] First, B16F10 cells are configured into a cell suspension with a concentration of 5×10 4 cells / mL, and the cells are uniformly inoculated into 24-well plates (1 mL of cell suspension is added to each well, i.e. 5×10 4 cells are plated per well), and cultured overnight in a 37°C cell incubator. The next day, 1 mL of material (HPSPE / HA, Ce6, HPSPE-Ce6 / HA, wherein the equivalent of Ce6 is 2 μM, and the concentration of HPSPE / HA and HPSPE-Ce6 / HA is 10 μg / mL) is added to the cells for 6 h of incubation. DCFH-DA is diluted with serum-free culture medium at a volume ratio of 1:1000 to make the probe detection working solution with a concentration of 10 μmol / L. The culture medium containing the material is discarded and washed with sterile PBS, and an appropriate volume of diluted DCFH-DA is added. The volume added is appropriate to cover the cells completely, and the cells are incubated in a 37°C cell incubator for 20 min. The cells are washed with serum-free cell culture medium three times to thoroughly remove the DCFH-DA that has not entered the cells. The material groups with Ce6 and HPSPE-Ce6 / HA are subjected to ultrasonic treatment, and the PBS group is set as a blank control, and the PBS+US group is set as a negative control (ultrasonic treatment is 1 MHz, 50% duty cycle, 0.64 W / cm 2Finally, the cells were directly observed and photographed by an inverted fluorescence microscope, and the cells were collected to detect the fluorescence intensity of the cells after stimulation by a fluorescence spectrophotometer with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0199] (3) Intracellular GSH concentration detection

[0200] In order to verify the GSH-responsive degradation of the disulfide bond in the HPSPE polymer in cells, the present application quantitatively detects the glutathione in cells in different treatment groups. Since the GSH detection requires a large amount of cells, the cells are expanded into a 60 mm cell culture dish, and when the cells grow to 90% density in the culture dish, the old culture medium is discarded, and 5 μg / mL, 10 μg / mL, and 20 μg / mL of HPSPE-Ce6 / HA are added to the culture dish, and the solvent is 1640 basic culture medium; after incubation for 12 h and 24 h, the GSH content in the cells is detected by using a GSH detection kit. The present application also stains the cells incubated with the material for different time by using a GSH staining kit, and the cells are observed by an inverted fluorescence microscope.

[0201] (4) Cytotoxicity experiment

[0202] In order to explore the effect of the HPSPE-Ce6-mediated sonodynamic combination with polyamine metabolism interference on the anti-tumor effect in vitro, the present application detects by using a CCK-8 method. Five groups, i.e., a control group, a US group, an HPSPE / HA group, a Ce6+US group, and an HPSPE-Ce6 / HA+US group, are set, and three parallel controls are set in each group. First, B16F10 is uniformly inoculated into a 96-well plate at a density of 1×10 4 cells / well, and is incubated in a cell culture box overnight. After the culture medium containing HPSPE / HA, Ce6, and HPSPE-Ce6 / HA (wherein the Ce6 equivalent is 2 μM, and the concentration of HPSPE / HA and HPSPE-Ce6 / HA is 10 μg / mL) is replaced, the incubation is continued for 12 h. The culture medium is discarded, and the residual material is washed away with PBS, and the sample wells are subjected to ultrasonic treatment, and the ultrasonic parameters are 1 MHz, 50% duty cycle, and 0.64 W / cm 2 , 2 min. The PBS is discarded, and a proper amount of CCK-8 staining working solution diluted in advance is added to the 96-well plate, three staining working solution groups are set as blank controls, the reagent is gently shaken to make it fully contact with the cells. The plate is placed in a 37°C incubator, and is incubated in the dark, and the absorbance value at 450 nm is detected by using an enzyme marker until the OD value of the control group reaches the required range, and the detection is terminated. The cell survival rate is calculated according to the following formula:

[0203] wherein As is the absorbance of the experimental well, Ac is the absorbance of the control well, and Ab is the absorbance of the blank well.

[0204] (5) Cell viability assay

[0205] To evaluate the tumor killing effect of the treatment in vitro more intuitively, the present application carried out cell viability assay experiment. Set up control group, US group, HPSPE / HA group, Ce6+US group and HPSPE-Ce6 / HA+US a total of five groups, each group set three parallel controls. First, B16F10 was inoculated into 24-well plates at a density of 5x10 5 cells / well and incubated in a cell incubator overnight. After replacing with 1 mL of culture medium containing HPSPE / HA, Ce6, HPSPE-Ce6 / HA (wherein the equivalent of Ce6 is 2 μM, and the concentration of the rest of the substances is 10 μg / mL), continue to incubate for 12 h. Discard the culture medium, wash the residual material with PBS, and sonicate the sample wells with the following parameters: 1 MHz, 50% duty cycle, 0.64 W / cm 2 , 2 min. Dilute the Live Dye and Nuclei Dye 1:1 to prepare the staining working solution. Collect the cells in each well into a 1.5 mL centrifuge tube, wash with PBS, centrifuge, discard the supernatant, and add 0.5 mL of the staining working solution to resuspend the cells. Incubate the cell suspension at 37°C for 30 min in the dark, wash the cells with PBS twice, resuspend the cells with a small amount of PBS, and drop them onto a glass slide, cover with a cover glass, and observe the cells using an inverted fluorescence microscope.

[0206] (6) Cell apoptosis assay

[0207] To determine the level of cell apoptosis after ultrasonic treatment of different formulations, the cells were subjected to Annexin V-APC and PI double staining experiment. B16F10 cells were plated and incubated with the material and then treated with ultrasound. The specific operation was the same as the cell viability assay experiment, which is not repeated here. The treated cells were collected in 1.5 mL centrifuge tubes, resuspended with 200 μL of binding buffer, added with 5 μL of Annexin V-APC dye, mixed evenly, and incubated at room temperature for 15 min in the dark. Then, 2 μL of PI dye was added and mixed evenly, and incubated for another 10 min in the dark. Finally, the cells were detected by flow cytometry, and the test results were analyzed by Flow Jo software.

[0208] First, the yield of reactive oxygen species under different ultrasonic times and powers was detected by SOSG probe. The results are as follows Figure 8As shown in (A) and (B), both increasing the ultrasound time and ultrasound power can effectively increase the reactive oxygen species (ROS) production. To avoid damage to normal tissue cells, the toxic effects of different ultrasound powers on HUVEC cells were tested, such as... Figure 8 As shown in (C), it was found that when the ultrasonic power is less than or equal to 0.64 W / cm², 2 At that time, the survival rate of HIUVEC cells was higher than 90%. Through the above experiments, 1 MHz, 50% duty cycle, and 0.64 W / cm² were selected. 2 The sonication conditions for subsequent experiments were 2 min. Furthermore, the reactive oxygen species (ROS) levels in B16F10 cells were detected using the DCFH-DA probe. Four groups were established: PBS, HPSPE-Ce6 / HA, US, Ce6+US, and HPSPE-Ce6 / HA+US. Intracellular ROS levels were characterized using fluorescence microscopy. Figure 8 Image (D) shows the fluorescence microscopy image of the ROS probe. The HPSPE-Ce6 / HA+US group exhibits the strongest fluorescence intensity, while the Ce6+US group shows significantly weaker fluorescence. Neither the PBS nor the US group shows significant fluorescence. This indicates that the sonosensitive agent Ce6 can generate ROS intracellularly. The HPSPE-Ce6 / HA+US group, due to its disulfide bonds, consumes intracellular GSH, altering the redox homeostasis within tumor cells and effectively increasing intracellular ROS production. Surprisingly, the HPSPE-Ce6 / HA group showed a lower level of ROS, which may be due to the synergistic effect of disulfide bond-induced GSH consumption and polyamine metabolism interference-induced increases in polyamine catabolism enzyme levels. SMOX, SSAT, and PAOX generate ROS while regulating intracellular polyamine levels through catabolism. These results demonstrate that HPSPE-Ce6 / HA can induce high levels of ROS through ultrasound, and amplify ROS generation during sonodynamic processes through polyamine metabolism interference and GSH consumption by disulfide bonds.

[0209] Subsequently, this invention tested the in vitro cytotoxicity of SDT on tumor cells. First, cell viability after treatment with different materials was assessed using CCK-8 assay. For example... Figure 8(E) shows that HPSPE-Ce6 / HA+US group has stronger anti-tumor toxicity in vitro than free Ce6+US group, which corresponds to the intracellular reactive oxygen species level above, and the consumption of intracellular glutathione by HPSPE disulfide bond achieves the sonodynamic sensitization, resulting in the strong tumor killing ability of HPSPE-Ce6 / HA+US. Further, the in vitro toxicity of SDT was more intuitively detected by the live and dead staining kit, in which dead cells were labeled with propidium iodide (PI) and showed red color, and live cells were labeled with Calcein AM and showed green color. The experimental results are as shown in Figure 8 (F) shows that the proportion of red fluorescent cells in the HPSPE-Ce6 / HA+US group is the largest, and there are also more red fluorescent cells in the Ce6+US group, while most of the cells in the other three groups show green fluorescence. This result is consistent with the cell toxicity results, and HPSPE-Ce6 / HA mediated SDT can cause a large number of tumor cell death.

[0210] During SDT, one of the main mechanisms of action is to activate the sonosensitizer by low-intensity US and produce ROS to induce apoptosis. The present application evaluates the apoptosis by Annexin V-APC / PI apoptosis detection kit. Early apoptotic cells specifically bind Annexin V-APC due to phosphatidylserine exposure, and necrotic cells can bind PI. On the other hand, late apoptotic cells lose membrane integrity and nuclear shrinkage and / or fragmentation can bind PI well, so late apoptotic cells show double positive signals. As shown in Figure 9 As shown in (E), HPSPE-Ce6 / HA+US group has stronger anti-tumor toxicity in vitro than free Ce6+US group, which corresponds to the intracellular reactive oxygen species level above, and the consumption of intracellular glutathione by HPSPE disulfide bond achieves the sonodynamic sensitization, resulting in the strong tumor killing ability of HPSPE-Ce6 / HA+US. Further, the in vitro toxicity of SDT was more intuitively detected by the live and dead staining kit, in which dead cells were labeled with propidium iodide (PI) and showed red color, and live cells were labeled with Calcein AM and showed green color. The experimental results are as shown in

[0211] Example 7: SDT mediated immunogenic death and in vitro DC activation

[0212] (1) Calreticulin (CRT) detection

[0213] CRT was detected by cell immunofluorescence. First, 20-30 million B16F10 cells were evenly plated in a 12-well plate and incubated overnight, and then treated with 2 mL of different materials, and then ultrasonicated (1 MHz, 50% duty cycle, 0.64 W / cm 2After 2 min, cells were collected. Cells were incubated with 4% paraformaldehyde in PBS at room temperature for 10 min, and then centrifuged to pellet the cells, and washed twice with PBS. Subsequently, cells were permeabilized with 0.1% Triton X-100 (diluted in PBS) for 5 min, and washed three times with PBS. Cells were incubated with PBST (PBS + 0.1% Tween 20) containing 1% BSA and 22.52 mg / mL glycine for 30 min to block non-specific binding of antibodies. Subsequently, cells were incubated with diluted Anti-Calreticulin antibody at 4°C overnight, and centrifuged to pellet the cells, and the primary antibody solution was aspirated, and the cells were washed twice with PBS. Then, goat anti-rabbit IgG secondary antibody was added and incubated at room temperature for 1 h (2 pg / mL Alexa Fluor® 488), and the secondary antibody solution was aspirated, and the cells were washed with PBS, and then detected by flow cytometry (three replicates per group). For laser confocal imaging, the cell slides were sterilized and placed in a 12-well plate, and the cells were allowed to grow on the slides, and the subsequent processing steps were similar to the flow detection method. After the cells were fixed, permeabilized, blocked, and stained, the cells were stained with DAPI at room temperature for 10 min, and the slides were gently transferred to a glass slide using tweezers, and a drop of mounting medium was used to cover the coverslip, and the coverslip was sealed to prevent the sample from drying and moving under the microscope. The processed samples were stored at 4°C in the dark, and laser confocal imaging was performed as soon as possible. 488), and the secondary antibody solution was aspirated, and the cells were washed with PBS, and then detected by flow cytometry (three replicates per group). For laser confocal imaging, the cell slides were sterilized and placed in a 12-well plate, and the cells were allowed to grow on the slides, and the subsequent processing steps were similar to the flow detection method. After the cells were fixed, permeabilized, blocked, and stained, the cells were stained with DAPI at room temperature for 10 min, and the slides were gently transferred to a glass slide using tweezers, and a drop of mounting medium was used to cover the coverslip, and the coverslip was sealed to prevent the sample from drying and moving under the microscope. The processed samples were stored at 4°C in the dark, and laser confocal imaging was performed as soon as possible.

[0214] (2) High mobility group box 1 (HMGB1) detection

[0215] HMGB1 released by cells was detected by enzyme-linked immunoassay (ELISA). After B16F10 cells were incubated with different materials and ultrasonicated, the cell supernatant was collected. First, the HMGB1 standard solution in the kit was diluted according to the appropriate concentration gradient, and 50 pL of the standard diluent was added to the HMGB1 antibody-coated well plate. At the same time, the supernatant of different treatment samples was added to the same amount of well plate, and a blank control well (no sample and enzyme-labeled reagent, the same operation in the following steps) was set, and three replicates were set for each group. Subsequently, 50 pL of enzyme-labeled reagent in the kit was added to each well, and the plate was sealed with a sealing film and incubated at 37°C for 30 min. The sealing film was carefully removed, and the liquid in the well plate was discarded, and the well plate was shaken dry, and the well plate was washed with washing solution for 5 times. Protein color developing solution was added, and the plate was incubated at 37°C for 10 min, and the reaction was terminated by adding a termination solution. Finally, the blank well was zeroed, and the absorbance of each well at 450 nm was measured by a multifunctional enzyme-labeled instrument.

[0216] (3) Adenosine triphosphate (ATP) detection

[0217] The ATP content inside and outside the cells was detected by chemiluminescence method described by ATP assay kit. After different experimental groups of B16F10 cells were treated, the supernatant was collected by centrifugation. In addition, cell lysis solution was added to the cell pellet and mixed well by blowing. After lysis, centrifugation was performed at 4°C, 12000g for 5min, and the supernatant was aspirated for subsequent determination. ATP detection working solution was added to a black 96-well plate, which was placed at room temperature for 3-5min to consume background ATP, then the sample to be tested was added and mixed quickly (three parallel groups for each group). Multifunctional enzyme labeler was used to detect its chemiluminescence.

[0218] (4) In vitro DC activation

[0219] To verify the effect of sonodynamic-mediated tumor cell immunogenic cell death (ICD) on the activation of dendritic cells, the stimulation and activation response of ICD on dendritic cells was explored by extracting and culturing mouse bone marrow-derived dendritic cells (BMDCs). First, balb / c mice (Guangdong Medical Laboratory Animal Center) were euthanized, and the bone marrow in the bilateral tibia and fibula of the mice was extracted in a sterile environment. The cells in the bone marrow were gently blown out using a syringe, and mouse bone marrow cells were obtained after treatment with red blood cell lysis solution. The bone marrow cells were cultured with RPMI-1640 complete medium containing granulocyte-macrophage colony-stimulating factor (GM-CSF, 20ng / mL) and interleukin-4 (IL-4, 10ng / mL) to direct the induction of DC cells, which generally takes about one week. During the culture process, the culture medium needs to be replaced in time according to the actual situation. When the cells grow to the 6th day, in vitro stimulation and activation experiment can be carried out.

[0220] First, the cultured DC cells were gently blown up in the well plate with a pipette gun, and then collected by centrifugation. The cells were counted by a hemocytometer, and the cells were configured into 1×10 6 / mL suspension, 500 μL cell suspension was added into each well of 24-well plate. Subsequently, the PBS, HPSPE, US, Ce6+US, HPSPE-Ce6 / HA+US treated B16F10 cells were collected, and the cells were resuspended with 100 μL corresponding supernatant, and co-cultured with DC cells for 24 h using Transwell chamber. The DC cells (i.e. BMDCs) obtained after co-culture were collected into 1.5 mL EP tube, centrifuged, washed with PBS twice, and prepared for staining. PerCP-Cy5.5-anti-CD86 and FITC-anti-CD80 were diluted with PBS at a ratio of 1:100 by volume, and two kinds of staining working solutions were prepared. The staining working solution was added to the EP tube containing BMDCs, and the cells were mixed with the staining solution by blowing with a gun head. The cells were incubated in a dark environment at 4°C for 30 min. Then the unbound antibody dye was removed by centrifugation, and the cells were washed twice with PBS, and then resuspended with 200 mL PBS. Finally, the percentage of B16F10 stimulated and activated BMDCs under different treatment conditions was obtained by flow cytometry detection, and the data were analyzed by Flowjo X software.

[0221] SDT shows great potential to induce ICD of tumor cells, by releasing molecular damage associated patterns (DMAP) and by activating dendritic cells (DCs) to trigger anti-tumor immune response. Now, nanoscale particles loaded with small molecule sonodynamic agents have been widely used to enhance the efficacy of SDT on ICD generation. The present application evaluates the SDT-induced ICD by CRT evertion, HIMGB1 and ATP release. First, the CRT expressed on the membrane surface of B16F10 cells in different treatment groups was visualized by laser confocal microscopy. The results are shown in (A) of FIG. 6, only the HPSPE-Ce6 / HA+US and Ce6+US groups have CRT expression on the cell membrane surface, and the fluorescence intensity of HPSPE-Ce6 / HA+US is higher. In order to quantify the CRT expression level on the cell membrane surface, the present application further analyzes by flow cytometry. Figure 10 Figure 10 (B) and (C) of FIG. 6 represent the representative fluorescence peak graph and statistical graph of CRT labeled flow cytometry analysis, respectively. The results show that the CRT expression level of the HPSPE-Ce6 / HA+US group is significantly higher than that of the Ce6+US group, which is due to the fact that the HPSPE-Ce6 / HA+US group can produce higher ROS, and the ROS level and ER (endoplasmic reticulum) stress are important factors for regulating ICD. The low level of CRT expression of HPSPE-Ce6 / HA is due to the ROS produced by polyamine metabolism. Interestingly, we found very low level of CRT expression in the US group, which may be due to slight physical damage to the cells caused by shear stress in the process of acoustic cavitation.

[0222] ​HMGB1 is another hallmark molecule of ICD, and the extracellular HMGB1 levels of different treatment groups were detected by ELISA. As shown in (D) of FIG. 6, Figure 10 The trend of extracellular HMGB1 levels released by different groups was basically consistent with the expression of CRT, as shown in (D) of FIG. 6. Figure 10 The intracellular and extracellular ATP levels were detected by ATP detection kit. The detection results are shown in (E) and (F) of FIG. 6. After treatment by Ce6+US group and HPSPE-Ce6 / HA+US group, the intracellular ATP level was significantly reduced to 24.91% and 14.11% of the initial level, respectively, and the trend of extracellular ATP level change showed a good corresponding relationship with the decrease of intracellular ATP.

[0223] To further study the effect of DMAP released by ICD on the maturation of DCs, the present application extracts and induces BMDCs cells in vitro and incubates them with B16F10 cells treated by different experimental groups, and then uses flow cytometry to analyze the expression of CD80 and CD86 on BMDCS. Figure 11 As shown in (A) and (B) of FIG. 7, HPSPE-Ce6 / HA+US and Ce6-US groups significantly increased the expression of two kinds of costimulatory factors CD80 and CD86 on DCs, and the degree of activation of BMDCs was closely related to the level of DMAP release. These results prove that SDT-mediated ICD can effectively promote the maturation of BMDCs, and the HPSPE-Ce6 / HA+US treatment group has the best promoting effect.

[0224] Example 8: Study on deep tissue penetration of ultrasound

[0225] To study deep tissue SDT, the present application uses pork to simulate tissue barriers and studies the effect of tissue thickness on cell viability by CCK-8 and cell live and dead staining. The pork is cut into 0.1 cm, 0.5 cm and 1 cm thick to simulate different depth tissue barriers. After 12-well plates with 80% volume of B16F10 cells were incubated with 1 mL HPSPE-Ce6 / HA (Ce6 equivalent to 2 μM) for 6 h, different thickness of pork tissue was used to cover the bottom of the well plate, and ultrasound (US) and 660 laser treatment were used as photodynamic control group, and different treatment groups were done in triplicate. The cell viability of different treatment groups was evaluated by CCK-8 and cell live and dead staining method.

[0226] Ultrasound (US) -induced SDT is considered as an alternative therapeutic technique to overcome the limited penetration depth of phototherapy such as photothermal therapy and photodynamic therapy. In this study, we used pork tissue to mimic the tissue barrier and compared the penetration effect of SDT with photodynamic therapy (PDT) (see Figure 12 Figure 1). The effect of SDT and PDT on tumor cell viability under different tissue thickness was investigated by CCK-8 and live-dead staining. Figure 12 Figure 1(B) shows that both therapies have a strong cytotoxic effect (cell survival rate less than 20%) without tissue barrier. Once the tissue barrier is added, the cytotoxicity of PDT decreases rapidly, with a cell survival rate of more than 60% under a 0.1 cm thick tissue barrier, and almost no killing effect under 0.5 cm and 1 cm thick tissue barriers. SDT still has a certain cytotoxicity even when there is a 1 cm thick tissue barrier. Figure 12 Figure 1(C) is the co-staining of different treated cells with calcein-AM and PI. Without tissue barrier, both SDT and PDT treated cells show strong red fluorescence, representing dead cells. As the tissue barrier thickens, the green fluorescence gradually becomes stronger. This trend is consistent with the cytotoxicity data measured by CCK-8. In general, the cytotoxicity of SDT is significantly higher than that of PDT when facing deep tissue. The results of this experiment show that US can at least penetrate a 0.5 cm thick tissue barrier to exert the effect of SDT.

[0227] Example 9: Tumor targeting effect of HPSPE-Ce6 / HA

[0228] The targeting effect of HPSPE-Ce6 / HA on tumors and the effective accumulation at tumors were evaluated by a small animal live imaging system. First, a tumor-bearing mouse model was constructed, and 5 x 10 6 When the tumor size reached the appropriate size, the mice were anesthetized with isoflurane, and 100 μL of HPSPE-Ce6 / HA or free Ce6 was injected into the mice through the tail vein. The fluorescence intensity of Ce6 in the mice was observed by a small animal live imaging system at 0 h, 3 h, 6 h, 12 h, and 24 h after tail vein injection. Finally, the mice were euthanized, and the mouse heart, liver, spleen, lung, kidney, and tumor were collected for ex vivo imaging.

[0229] As Figure 13As shown in (A), after drug injection, HPSPE-Ce6 / HA exhibited a strong fluorescence signal in tumor tissue, and some material remained within the tumor tissue even after 24 hours. In contrast, free Ce6 only showed weak fluorescence signals at 3 and 6 hours, with virtually no significant fluorescence signal observed thereafter. This indicates that HPSPE-Ce6 / HA nanoparticles have a long in vivo circulation time and can effectively target the tumor site. Subsequently, tumor tissues and major organs from both groups of mice were collected for in vivo imaging. In the HPSPE-Ce6 / FIA group, inevitable liver accumulation was observed, and significant fluorescence signals remained in the tumor tissue, consistent with in vivo fluorescence imaging results. The fluorescence intensity of the tumor and the final excised tissue in both groups was quantified using Living Image software, and the results are shown below. Figure 13 As shown in (B) and (C), this result demonstrates that HPSPE-Ce6 assembly with HA enables long-term in vivo circulation and enhances the tumor targeting and accumulation capabilities of nanoparticles.

[0230] Example 10: In vivo anti-tumor effect

[0231] To investigate the antitumor effects of polyamine metabolism interference and sonodynamic therapy in vivo, B16F10 tumor-bearing mice were treated with different methods, and the tumor size was observed for evaluation. First, 100 μL of a 1×10⁻⁶ m² solution was added... 7 A cell suspension of B16F10 cells / mL was injected subcutaneously into the left hind limb of C57BL / 6J mice to establish a subcutaneous melanoma model. Approximately 6 days later, the subcutaneous tumor grew to about 80 mm. 3 B16F10 tumor-bearing mice were randomly divided into five groups: PBS, US, HPSPE / HA, Ce6+US, and HPSPE-Ce6 / HA+US, with eight mice in each group. Subsequently, 100 μL of PBS, HPSPE, Ce6, and HPSPE-Ce6 / HA were administered via tail vein injection, respectively. Three hours after administration, the mouse tumors were subjected to ultrasound treatment, as described below. Figure 14 As shown in (A) above. The ultrasonic treatment conditions were 1 MHz, 50% duty cycle, and 0.64 W / cm². 2 Treatment lasted 5 minutes. Treatment was administered every two days for a total of three treatments. The mouse's body weight and the long and short diameters of the tumor were recorded, and the tumor volume was calculated using the following formula:

[0232]

[0233] When the tumor volume in mice exceeds 2000 mm 3Alternatively, euthanasia is performed on mice when they exhibit pre-defined humane endpoint characteristics. Tumor tissue is collected after dissection, weighed, and photographed. Finally, tumor tissue from different treatment groups is collected for H&E staining and TUNEL immunofluorescence staining to further assess tumor tissue damage.

[0234] like Figure 14 As shown in (B) and (C), there was almost no difference in tumor size between the PBS group and the PBS+US group, indicating that the current power of US had no effect on tumor treatment, which also reflects the low tissue toxicity of US. The HPSPE group showed a certain tumor-suppressive effect, which we speculate is due to the effective consumption of intracellular polyamine levels by polyamine metabolism interference, inhibiting cell proliferation. The HPSPE-Ce6 / HA+US group, by consuming intracellular GSH in tumor cells through disulfide bonds, achieved a certain tumor-killing effect through sonodynamic sensitization. Subsequently, sonodynamically induced immunogenic death activated the body's anti-tumor immune response to clear tumor cells. Furthermore, polyamine metabolism interference effectively inhibited tumor cell growth and reversed the tumor-associated macrophage phenotype, further inhibiting tumor growth. It exhibited the most effective tumor suppression among all groups.

[0235] The volume and mass of tumor tissues in different groups of mice were statistically analyzed, and the results are as follows: Figure 15 As shown in (A) and (B) in the figure, this viewpoint is further confirmed. Analysis of the body weight of different groups of mice (…) Figure 15 In (C) of the study, it was found that there was no difference in body weight among the groups during the observation period, indicating that HPSPE-Ce6 / HA had good biocompatibility during treatment. Subsequently, the tumor-killing mechanism of HPSPE-Ce6 / HA combined with sonodynamics was examined at the tissue level. TUNEL staining (to detect tumor cell apoptosis) and H&E staining were performed on B16F10 melanoma tissues from mice, respectively. Figure 15 As shown in (D), TUNEL staining results indicated that the number of apoptotic tumor cells in the HPSPE-Ce6 / HA+US group was significantly higher than that in the Ce6+US group, while almost no TUNEL fluorescence signal was observed in the PBS, PBS+US, and HPSPE groups. H&E staining of tumor tissue sections is shown in Figure [Figure number missing]. Figure 15 As shown in (D), extensive tumor necrosis was observed in the HPSPE-Ce6 / HA+US treatment group. This result indicates that the synergistic effect of sonodynamics and elicited immune response can effectively kill tumor cells, thereby inhibiting tumor growth. It is evident that HPSPE-Ce6 / HA-mediated sonodynamics and polyamine metabolism interference enhance tumor immunotherapy, effectively killing tumor cells in vivo and significantly inhibiting the proliferation of B16F10 tumor cells.

[0236] Example 11: Immunogenic cell death and reversal of immunosuppressive microenvironment

[0237] To verify the ICD induced by SDT in vivo, the CRT egress was evaluated by immunofluorescence staining of tumor tissues after different treatments. The specific experimental steps are as follows: the tumor tissues of different experimental groups were soaked in tissue fixative, embedded with paraffin, and cut into 4 pm-thick tissue sections at the maximum cross-section of the tumor tissue using a microtome. Then the tissue sections were fixed on glass slides for later use. The tumor tissue sections were washed twice with xylene, then gradient dehydrated with ethanol, and then incubated with proteinase K working solution for 30 min, followed by PBS washing and CRT staining. After staining, the sections were observed, photographed and analyzed under an inverted microscope for the egress of immunogenic death marker-calreticulin in tumor tissues of different treatment groups.

[0238] As shown in (A) of Figure 16 , PBS, PBS+US and HPSPE-Ce6 / HA groups rarely or did not expose CRT, but the B16F10 melanoma treated by HPSPE-Ce6 / HA+US group obviously exposed more CRT. In addition, there was also a small amount of CRT exposure with Ce6+US, which was similar to the cell experiment. This result showed that the HPSPE-Ce6 / HA+US group could effectively generate ROS in the tumor tissue and significantly promote the immunogenic death of tumor cells, and release a large amount of DMAP, which potentially promoted the maturation of DC cells.

[0239] Tumor-associated macrophages (TAMs) are one of the main culprits in the tumor immunosuppressive microenvironment, which exerts its immunosuppressive effect through various ways. Polyamine metabolism interference can effectively reverse the macrophage phenotype and reverse the tumor microenvironment. We further analyzed the ratio of M1 and M2 macrophages in the tumor after treatment by flow cytometry. As shown in (B) and (C) of Figure 16 , the ratio of M1 (F4 / 80 + and CD86 + ) macrophages was higher than that of PBS and PBS+US groups in the HPSPE-Ce6 group, while the ratio of M2 (F4 / 80 + and CD206 +) were lower than these two groups. This phenomenon indicated that HPSPE-Ce6 could interfere with the macrophage phenotype in vivo by polyamine metabolism, thus reprogramming the immunosuppressive microenvironment. Surprisingly, we found a similar phenomenon in the Ce6+US group, which we speculate is due to the induction of subsequent tumor immune enhancement by sonodynamic-induced immunogenic cell death. While HPSPE-Ce6 / HA+US activated the body's anti-tumor immune effect by sonodynamic-induced immunogenic death, it also maximized the reversal of the macrophage phenotype by interfering with polyamine metabolism. The corresponding quantitative analysis of (D) and (E) was obtained by graphpad Figure 16

[0240] Example 12: DC activation in vivo

[0241] To further study the effect of ICD-induced release of damage-associated molecular patterns (DAMP) on dendritic cell maturation, the B16F10 cells from the normal tumor-bearing mouse near the tumor end of the draining lymph node were collected for flow cytometry analysis, as follows: the collected draining lymph nodes were ground into a single cell suspension, and after centrifugation, stained with APC-anti-CD11c, FITC-anti-CD80 and Percp / Cy5.5-anti-CD86, and the percentage of mature DC cells was detected by flow cytometry.

[0242] As shown in (A) and (B) of Figure 17 , compared with the PBS group, the proportion of mature DC cells (CD80 + CD86 + in CD11c + ) in the draining lymph nodes of mice in the PBS+US and HPSPE-Ce6 / HA groups did not change significantly. While the proportion of mature dendritic cells in the HPSPE-Ce6 / HA+US and Ce6+US groups increased, and the proportion of DC mature dendritic cells in the HPSPE-Ce6 / HA+US group was the highest. This indicates that sonodynamic-induced immunogenic cell death can promote the maturation of dendritic cells in vivo, and has the potential to activate the body's anti-tumor immune response. The corresponding expression percentage of (C) and (D) was obtained by graphpad Figure 17

[0243] Example 13: Cytotoxic T cell (CTL) infiltration

[0244] The reversal of the tumor microenvironment and the induction of systemic immune response by DC maturation were studied. CD4 + T, CD8 + T cells in the tumor tissue and spleen tissue of Example 10 were stained by immunofluorescence and flow cytometry.​The T ratio was analyzed. For flow cytometry, staining was performed using APC-anti-CD3, FITC-anti-CD4, and Percp / Cy5.5-anti-CD8 antibodies, followed by flow cytometry analysis.

[0245] An in-depth study was conducted on the systemic antitumor immune mechanism induced by HPSPE-Ce6 / HA+US-mediated sonodynamics and polyamine metabolism interference. Immunofluorescence staining was used to analyze the infiltrating CD4+ cells in the tumor tissue of Example 10. + T and CD8 + The proportion of T cells. For example... Figure 18 As shown in (A) and (B), the PBS+US group compared to the PBS group showed that almost no CD4 was observed in the tumor tissue of either group. + T and CD8 + T fluorescence, while a small amount of CD4 could be observed in tumors of the HPSPE-Ce6 / HA group and the Ce6+US group. + T and CD8 + The fluorescence signal of T indicates that simple sonodynamics and polyamine metabolism interference promote anti-tumor immune responses to some extent, and the highest CD4 count was observed in tumor tissues of the HPSPE-Ce6 / HA+US group. + T and CD8 + The proportion of T cell infiltration. This indicates that immunogenic death and macrophage phenotype reversal can effectively activate the tumor immune system and recruit cytotoxic T cells to infiltrate the tumor.

[0246] In anti-tumor immunotherapy, CTLs that kill tumor cells originate from CD8. + T cells, their differentiation process is influenced by CD4 + T cell regulation. Therefore, CD8 in peripheral lymphoid organs + T cells and CD4 + The number of T cells is an important indicator of CTLs, and it is one of the main drivers of cellular immunity. Further detection of CD8+ in mouse spleen was performed. + T cells and CD4 + The percentage of T cells, such as Figure 18 As shown in (C), (D), and (E), the HPSPE-Ce6 / HA+US group mice exhibited a higher percentage of CD3 in the spleen compared to other groups. + CD8 + and CD3 + CD4 + T cells indicate that sonodynamically induced immunogenic death combined with macrophage phenotype reversal can induce a stronger immune response to kill tumors.

[0247] Example 14: In vivo biocompatibility

[0248] The biological compatibility of the material was verified by serum biochemical indicators and main tissue H&E pathological sections. After the anti-tumor experiment of Example 10 was completed, about 500 μL of mouse whole blood was collected by orbital plexus venous blood, and after standing at room temperature for 4 h, mouse serum was collected by centrifugation, and the levels of blood urea nitrogen (BUN), creatinine (CREA), uric acid (UA), glucose (GLU), lactate dehydrogenase (LDH), cholesterol (CHO), alanine aminotransferase (ALT), albumin (ALB) and aspartate aminotransferase (AST) were detected to evaluate the toxicity of the material. The results are shown in Table 1. Figure 19 As shown in Table 1, the indicators of the HPSPE-Ce6 / HA group had no significant difference compared with the PBS group and other control groups, further confirming that HPSPE-Ce6 / HA had good in vivo biocompatibility. Subsequently, we collected mouse heart, liver, spleen, lung and kidney tissues, and used hematoxylin-eosin (H&E) staining, and observed the tissue damage by digital slide scanning system. The main organs of mice in different treatment groups were observed, and the results are shown in FIG. 6. Figure 20 As shown in FIG. 6, no obvious pathological damage and changes were found in all groups.

[0249] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, and are all included in the protection scope of the present application.

Claims

1. A process for the preparation of a functionally modified hyperbranched poly- ethylenediamine characterized in that The method comprises the following steps: (1) preparing hyperbranched polymer HPSPE-Ce6 1) synthesis of N'N-bisacryloyl cystamine; 2) synthesis of hyperbranched polyamine; 3) grafting of the sonosensitizer Ce6 to the hyperbranched polyamine to obtain the hyperbranched polymer HPSPE-Ce6; (2) assembly of the functionalized modified hyperbranched polyamine: mixing the hyperbranched polymer HPSPE-Ce6 and the shielding component hyaluronic acid in water as a medium, and stirring them to fully mix the hyaluronic acid and the HPSPE-Ce6 by electrostatic attraction, to obtain HPSPE-Ce6 / HA composite structure nanoparticles, i.e. the functionalized modified hyperbranched polyamine; The synthesis in step (1) 1) comprises the following steps: A. Dissolve sodium hydroxide in ultrapure water to obtain solution A; B. Dissolve cystamine dihydrochloride in ultrapure water to obtain solution B; C. Dissolve acryloyl chloride in an organic solvent under ice bath conditions to obtain solution C; D. Place solution B in an ice bath, first add part of solution A dropwise, and first adjust the initial pH of the reaction system to 9-12; then alternately add solution A and solution C, and react; extract the obtained reaction product with an organic solvent, collect the organic layer, sequentially wash, rotary evaporate, dry, and obtain N'N-bisacryloyl cystamine; The specific steps of the synthesis in step (1) 2) are as follows: E. Dissolve N'N-bisacryloyl cystamine in an organic solvent, introduce inert gas, and increase the temperature to react; F. Dissolve the hyperbranched site-providing substance and spermine in an organic solvent to obtain a mixed solution; G. In a dark environment, drop the mixed solution obtained in step F into the product obtained in step E, condense, and react; H. Add a spermine solution to the product obtained in step G, and continue to react; I. Dilute the reaction liquid obtained in step H with ultrapure water, and then adjust the pH with an acid solution; dialyze at 4°C in the dark, and dry to obtain the hyperbranched polyamine; The hyperbranched polymer in step (1) 3) is prepared by the following steps: J. Dissolve Ce6 in an organic solvent to obtain a Ce6 solution; K. Mix 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, N-hydroxysuccinimide, and the Ce6 solution in the dark, and react; L. Mix the hyperbranched polyamine and the reaction product obtained in step K uniformly in the dark to obtain solution E, and react; M. Dialyze the product obtained in step L at 4°C in the dark, and dry to obtain HPSPE-Ce6; The hyperbranched site-providing substance in step F is at least one of N-aminoethylpiperazine, spermidine, and putrescine.

2. The method for preparing the functionalized modified hyperbranched polyamine according to claim 1, characterized in that: The organic solvent in step C is at least one of dichloromethane, chloroform, ethyl acetate, and petroleum ether; The organic solvent for extraction in step D is at least one of dichloromethane, chloroform, ethyl acetate, and petroleum ether; The solvents for sequentially washing in step D are saturated sodium bicarbonate solution, saturated sodium chloride solution, and ultrapure water; The organic solvent in step E is methanol; The inert gas in step E is nitrogen; The organic solvent in step F is methanol; The acid solution in step I is hydrochloric acid; The organic solvent in step J is at least one of dimethyl sulfoxide, dimethylformamide and acetonitrile.

3. The method of claim 1, wherein: the molar ratio of cystamine dihydrochloride to acryloyl chloride in the reaction system in step D is 1:1-8; the molar ratio of CBA to SPE to the substance providing branching sites in the mixed solution in step G is 0.5-1.5:1-16:1; the amount of spermine added in step H is more than 40 times the mass of N', N'-bisacryloyl cystamine; the molar ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide to N-hydroxysuccinimide to Ce6 in step K is 2-4:2-4:1; and the mass ratio of hyperbranched poly(spermine) to Ce6 in step L is 7-9:

1.

4. The method of claim 1, wherein: the concentration of sodium hydroxide in solution A in step A is 1-20 mol / L; the concentration of cystamine dihydrochloride in solution B in step B is 0.01-1 mol / L; the concentration of acryloyl chloride in solution C in step C is 1-50 mol / L; the volume of solution A used in step D is 1 / 9-1 / 10 of the total volume of solution A; the concentration of N', N'-bisacryloyl cystamine in solution D in step E is 1-30 g / L; the concentration of the substance providing branching sites in the mixed solution in step F is 1-2 mmol / L; the concentration of spermine in the mixed solution in step F is 0.5-2 mmol / L; the concentration of the spermine solution in step H is 4-6 g / mL; the concentration of the Ce6 solution in step J is 0.1-10 mg / mL; and the concentration of hyperbranched poly(spermine) in solution E in step L is 1-15 mg / mL.

5. The method of claim 1, wherein: the time for the alternate dropwise addition in step D is no less than 30 min; the reaction time in step D is 5-10 h; the drying in step D is vacuum drying; the reaction temperature in step E is 20-70 °C; the condensation temperature in step G is 10-20 °C; the reaction time in step G is 12-60 h; the reaction time in step H is 12-60 h; the pH in step I is 3-7; the dialysis in step I is dialysis using a dialysis bag with a molecular weight of 1200 kDa; the drying method in step I is freeze drying; the reaction time in step K is 30 min-4 h; and the mixing method in step L is stirring. ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ The dialysis in step M is dialysis using a dialysis bag with a molecular weight of 1000 Da; The drying method in step M is freeze-drying.

6. The method for preparing the functionalized modified hyperbranched poly-precine amine according to claim 1, characterized in that: The molecular weight of the hyaluronic acid in step (2) is 10-120 kDA; The HPSPE-Ce6 and the HA in step (2) are mixed in a mass ratio of 1-5:1-5; The vortexing time in step (2) is 30 s-5 min.

7. A functionally modified hyperbranched polyethyleneimine, characterized in that: The functionalized modified hyperbranched poly-precine amine is prepared by the method according to any one of claims 1-6.

8. The use of the functionalized modified hyperbranched poly-precine amine according to claim 7 in the preparation of an anti-tumor drug.

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