A hydroxyalkyl starch-photosensitizer macromolecular compound encapsulating anthraquinone drugs, a nano-drug delivery system, and its preparation and application.

Abstract

This invention belongs to the interdisciplinary technical field of chemistry, pharmacy, and medicine. More specifically, it relates to a hydroxyalkyl starch-photosensitizer macromolecular compound encapsulating anthraquinone drugs, a nanoparticle drug delivery system, and its preparation and application. The macromolecular compound has a hydrophilic end of hydroxyalkyl starch and a hydrophobic end of a hydrophobic organic cyanine dye molecule. This invention uses a hydrophobic organic cyanine dye molecule photosensitizer with photodynamic and photodetector properties as the hydrophobic end. After coupling with hydroxyalkyl starch, the constructed amphiphilic macromolecule can maintain the stability of the original photosensitizer and improve its water solubility. Furthermore, it can optimize its UV-Vis absorption peak to match the 660nm near-infrared laser irradiation used in photodynamic therapy. The oil-in-water nanosystem constructed through emulsification can also maintain the photodynamic and photodetector properties of the photosensitizer, successfully encapsulating hydrophobic antitumor drugs and achieving tumor-targeted combination therapy while improving the biocompatibility of the photosensitizer.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary technical field of chemistry, pharmacy, medicine and other disciplines. More specifically, it relates to a hydroxyalkyl starch-photosensitizer macromolecular compound encapsulating anthraquinone drugs, a nano-drug delivery system and its preparation and application. Background Technology

[0002] Photodynamic therapy (PDT) is a treatment strategy that uses photosensitizers to transfer energy from laser light to surrounding oxygen molecules, leading to an increase in intracellular reactive oxygen species (ROS) levels. This causes tumor cells to die from oxidative damage that is difficult to repair in time. Compared with traditional therapies, PDT has advantages such as fewer side effects, shorter treatment time, simpler operation, and better killing effect, and therefore its clinical application is increasing.

[0003] Cyanide-based near-infrared fluorescent dyes, such as IR820, possess excellent biocompatibility and good efficacy, making them highly suitable for photodynamic therapy. However, single photosensitizers, being small molecules, exhibit poor solubility, cannot achieve long-term circulation in vivo, lack tumor targeting, and are easily metabolized by the liver and kidneys. Therefore, exploring novel materials loaded with small-molecule photosensitizers is urgently needed.

[0004] Moreover, the accumulation of ROS alone is insufficient to achieve broad and effective killing of tumor cells. This is because the pentose phosphate pathway (PPP), as a branch of cellular glycolysis, is responsible for supplying NADPH (reduced coenzyme II), which helps maintain the antioxidant system of tumor cells.

[0005] Physcion is an anthraquinone compound. Previous studies have shown that it has strong anti-tumor activity, including inducing tumor cell apoptosis, disrupting the cell cycle, and inhibiting tumor metastasis. However, due to its water solubility, physcion has been difficult to apply clinically in the field of cancer treatment.

[0006] Admittedly, single chemotherapy or photodynamic therapy is unlikely to achieve ideal therapeutic effects, but combination therapy can significantly improve tumor treatment outcomes. The strategy of combining photodynamic therapy with chemotherapy drugs can inhibit tumor development and progression from multiple angles. However, the poor water solubility and low biocompatibility of cyanide photosensitizers and anthraquinone drugs limit their clinical application. Therefore, how to achieve co-loading of these two components while improving drug targeting and accumulation is a pressing technical challenge in this field. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a macromolecular compound, a nanoparticle drug delivery system, and its preparation method and applications for encapsulating anthraquinone drugs via a hydroxyalkyl starch-photosensitizer coupling. A photosensitizer with photoresponsive therapeutic properties is used as the hydrophobic end of the macromolecular compound, coupled with hydrophilic hydroxyalkyl starch. The hydrophobic antitumor anthraquinone drug is then encapsulated into nanoparticles through ultrasonic emulsification. This solves a series of problems leading to poor efficacy, such as poor biocompatibility and targeting between cyanine photosensitizers and anthraquinone drugs.

[0008] To achieve the above objectives, the present invention provides an amphiphilic macromolecular compound coupled with a hydroxyalkyl starch-photosensitizer, wherein the macromolecular compound is obtained by chemically coupling hydroxyalkyl starch and a photosensitizer; wherein the hydroxyalkyl starch in the amphiphilic macromolecular compound is the hydrophilic end and the photosensitizer is the hydrophobic end; and the photosensitizer is a hydrophobic organic cyanine dye molecule.

[0009] Preferably, the hydrophobic organic cyanine dye molecule is one or more of IR820, IR780, IR808, IR783 and IR825; the chemical bond is an amide bond or an ester bond.

[0010] More preferably, the photosensitizer is IR820, and the IR820 is coupled to the hydroxyalkyl starch via an amide bond.

[0011] According to another aspect of the present invention, a method for preparing the amphiphilic macromolecular compound is provided, comprising the following steps:

[0012] (1) The hydroxyl groups in hydroxyalkyl starch are replaced by chlorine atoms in a compound containing at least one acyl chloride and a nitro group to obtain hydroxyalkyl starch containing ketone carbonyl and nitro groups;

[0013] (2) The hydroxyalkyl starch containing ketone carbonyl and nitro groups is subjected to a substitution reaction with a primary amine compound containing at least two amino groups, so that the ketone carbonyl group is replaced with an amide bond, and hydroxyalkyl starch containing amino groups is obtained.

[0014] (3) The amino-containing hydroxyalkyl starch is subjected to a substitution reaction with the chlorine atom contained in the photosensitizer to obtain the amphiphilic macromolecular compound coupled with the hydroxyalkyl starch-photosensitizer.

[0015] Preferably, the compound containing at least one acyl chloride and one nitro group is p-nitrophenyl chloroformate.

[0016] Preferably, the primary amine compound containing at least two amino groups is one or more of 1,4-cyclohexanediamine, 1,6-hexanediamine, and 1,2-cyclohexanediamine.

[0017] According to another aspect of the present invention, a nano-drug delivery system based on the amphiphilic macromolecular compound is provided, comprising the amphiphilic macromolecular compound and further comprising anthraquinone antitumor drugs.

[0018] Preferably, the anthraquinone antitumor drug and the amphiphilic macromolecular compound are constructed into oil-in-water nanoparticles by ultrasonic emulsification, wherein the anthraquinone antitumor drug is the oil phase and the amphiphilic macromolecular compound is the aqueous phase.

[0019] Preferably, the anthraquinone antitumor drug includes one or more of emodin methyl ether, rhein, chrysophanol, emodin and aloe-emodin.

[0020] According to another aspect of the invention, an antitumor drug is provided, comprising the aforementioned nano-drug delivery system and pharmaceutically acceptable additives.

[0021] In summary, the technical solutions conceived by this invention have the following beneficial effects compared with the prior art:

[0022] (1) This invention provides an amphiphilic macromolecular compound of hydroxyalkyl starch-photosensitizer, wherein the hydrophilic end is hydroxyalkyl starch and the hydrophobic end is a hydrophobic organic cyanine dye molecule, thereby improving the stability and water solubility of the photosensitizer. This invention uses a hydrophobic organic cyanine dye molecule photosensitizer with photoresponsive therapeutic and photodetector properties as the hydrophobic end. After coupling with hydroxyalkyl starch, it was found that this molecule can optimize the photosensitizer's photoresponsiveness while maintaining its good photoresponsive therapeutic and photodetector properties and reducing its toxicity.

[0023] (2) The organic cyanine dye molecules used in this invention, such as IR820, have poor water solubility, are small molecules, and do not have retention at the tumor site. In addition, their UV-Vis absorption peak is located at 690 nm, which means they do not have photoresponsive properties. This invention prepares the hydroxyalkyl starch-photosensitizer IR820 conjugate by successively nitrifying and amino-modifying hydroxyalkyl starch, and then further substituting the amino-modified hydroxyalkyl starch with the chlorine atom in the photosensitizer IR820. This conjugate is an amphiphilic macromolecular compound. Experiments have shown that this modification and grafting method not only maintains the good photothermal response and photodevelopment properties of the photosensitizer and reduces the toxicity of the photosensitizer IR820, but also adjusts the position of the ultraviolet absorption peak of the photosensitizer IR820 to match the position of the near-infrared laser (adjusted to 655nm, which can match the 660nm near-infrared laser), giving it photodynamic therapy properties. Furthermore, the conjugate has good solubility in deionized water. More unexpectedly, this specific modification and grafting method can maintain the good photostability of the photosensitizer.

[0024] (3) The oil-in-water nanoparticle drug delivery system based on anthraquinone drugs provided in this embodiment of the invention has tumor-targeting and pH-responsive drug release capabilities. In vivo tumor-targeting evaluation of this nanoparticle drug delivery system revealed that it can accumulate more at the tumor site and significantly prolong the drug's half-life in vivo. Furthermore, experiments suggest that anthraquinone drugs may inhibit NADPH production via the pentose phosphate pathway. The anthraquinone-loaded nanoparticles can achieve synergistic effects with HES-IR820 on redox-damaged tumor cells, exhibiting a more significant tumor-killing effect compared to nanoparticles without anthraquinone drugs. Moreover, this nanoparticle drug delivery system itself has good biocompatibility.

[0025] (4) The nano-drug delivery system provided in this embodiment of the invention exhibits excellent photodynamic performance under 660nm laser irradiation, significantly increasing the reactive oxygen species level in the target area after irradiation both in vivo and in vitro. Short-duration laser irradiation can significantly enhance the antitumor effect of hydrophobic anthraquinone chemotherapy drugs; compared with the PBS group, the tumor inhibition rate of this novel nano-drug delivery system reaches 86.1%. Attached Figure Description

[0026] Figure 1 The UV-Vis absorption spectra are those of free IR820, amino-substituted IR820, and IR820 coupled with hydroxyethyl starch.

[0027] Figure 2A This is a flowchart illustrating the preparation process of amino-substituted IR820 (IR820-NH2) and hydroxyethyl starch-IR820 (HES-IR820) from Example 1 of the present invention. Figure 2B , Figure 2C The 1H NMR spectra of hydroxyethyl starch and hydroxyethyl starch-IR820 (HES-IR820), and amino-substituted IR820 (IR820-NH2), respectively. Figure 2D Fourier transform infrared spectra of HES and HES-IR820. Figure 2E To determine the UV-Vis spectrum of the IR820 concentration standard curve using IR820-NH2 standard.

[0028] Figure 3 Content A describes the hydrated particle size of the Phy@HES-IR820 nanoparticles prepared in Example 2 of this invention, as detected by dynamic light scattering (DLS). Figure 3 Content B describes the seven-day stability of the Phy@HES-IR820 nanoparticles prepared in Example 2; Figure 3 Content C shows the zeta potential of the Phy@HES-IR820 nanoparticles prepared in Example 2 in ultrapure water; Figure 3Content D shows the morphology of Phy@HES-IR820 nanoparticles as detected by transmission electron microscopy; Figure 3 Content E shows the ultraviolet-visible absorption spectra of free Phy, free IR820, IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles in Example 3 of this invention. Figure 3 Content F is the Fourier transform infrared spectrum of free Phy, free IR820, IR820-NH2, HES-IR820 and Phy@HES-IR820 nanoparticles in Example 3 of the present invention; Figure 3 Contents G and H are the IR820 fluorescence emission spectra of IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles in DMSO under different excitation light in Example 4 of the present invention; Figure 3 Content I is the drug release curve of Phy@HES-IR820 nanoparticles in Example 5 of the present invention under the conditions of pH=7.4, pH=6.8 and pH=5.0.

[0029] Figure 4 Content A describes the photostability of IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles under 660nm near-infrared laser irradiation in Example 6 of this invention. Figure 4 Content B describes the detection of reactive oxygen species (ROS) generation of IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles under 660nm near-infrared laser irradiation using DPBF scavenger in Example 7 of this invention. Figure 4 Content C describes the singlet oxygen generation of IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles under 660nm near-infrared laser irradiation, as detected by ESR in Example 7 of this invention.

[0030] Figure 5 Content A describes the use of confocal microscopy (CLSM) in Example 8 of this invention to examine the uptake of different drugs by 4T1 tumor cells; Figure 5 Content B and Figure 5 Content C describes the use of flow cytometry in Example 9 of this invention to investigate the uptake and relative quantification of different drugs by 4T1 tumor cells.

[0031] Figure 6 Contents A and B show the survival of 4T1 cells in Example 10 that were treated with Phy, IR820-NH2, HES-IR820 and Phy@HES-IR820 nanoparticles for different times and cultured in strict darkness. Figure 6Contents C and D describe the survival of 4T1 cells after being treated with the same drug group as in Example 10 for different durations, followed by near-infrared laser irradiation.

[0032] Figure 7 Content A and Figure 7 Content B describes the use of confocal microscopy in Example 12 to examine and quantify intracellular ROS production and apoptosis in 4T1 cells treated with different drugs. Figure 7 Content C and Figure 7 Content D describes the use of flow cytometry in Example 12 to investigate and quantify intracellular ROS production and apoptosis in 4T1 cells after treatment with different drugs.

[0033] Figure 8 Content A is Example 13, which uses a kit to determine the effect of different concentrations of Phy and Phy@HES-IR820 nanoparticles on the activity of G6PD enzyme in the pentose phosphate pathway in 4T1 tumor cells; Figure 8 Contents B, C, and D represent the effects of different concentrations of Phy and Phy@HES-IR820 nanoparticles, as determined by the corresponding kits, on 6PGD activity, NADPH / NADP+ ratio, and GSH / GSSG ratio in 4T1 tumor cells.

[0034] Figure 9 Content A contains in vivo imaging images of different drugs at different time points on the 4T1 subcutaneous tumor sites in mice in Example 14; Figure 9 Content B and Figure 9 Content C presents the enrichment and relative quantification of different nanoparticles in various organs and tumor regions of mice;

[0035] Figure 10 The changes in tumor volume in mice after different drug treatments are shown in Example 15.

[0036] Figure 11 The changes in tumor weight in mice after different drug treatments are shown in Example 15.

[0037] Figure 12 Images showing the tumor size in mice after different drug treatments in Example 15;

[0038] Figure 13A , Figure 13B , Figure 13C and Figure 13D The results of H&E, Cleaved-caspase 3, Ki-67, and GSH staining of excised tumors in mice treated with different drugs in Example 15 are shown below.

[0039] Figure 14Content A shows the changes in body weight of mice after different drug treatments in Example 15, which is used to evaluate the biosafety of different drugs in mice; Figure 14 Contents B to J describe the complete blood count and blood biochemistry tests performed on mice after different drug treatments in Example 15, used to evaluate the biosafety of different drugs in mice. Among them: Figure 14 Content B is the amount of alanine aminotransferase in serum; Figure 14 Content C represents the serum aspartate aminotransferase level; Figure 14 Content D represents the amount of creatine kinase in serum; Figure 14 Content E represents the amount of blood urea nitrogen in the serum; Figure 14 Content F represents the amount of creatinine in serum; Figure 14 Content G represents the amount of red blood cells in the blood; Figure 14 Content H represents the amount of hemoglobin in the blood; Figure 14 Content I describes the amount of platelets in the blood; Figure 14 Content J represents the number of white blood cells in the blood. Detailed Implementation

[0040] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0041] This invention provides an amphiphilic macromolecular compound capable of encapsulating a hydrophobic anthraquinone drug, hydroxyalkyl starch, and a photosensitizer. This macromolecular compound is obtained by chemically coupling hydroxyalkyl starch and a photosensitizer. The photosensitizer is a hydrophobic organic cyanine dye molecule coupled to the hydroxyalkyl starch via a chemical bond; the photosensitizer in the amphiphilic macromolecular compound serves as the hydrophobic end, and the hydroxyalkyl starch as the hydrophilic end; the anthraquinone drug contains anthracene rings and phenolic hydroxyl groups, and the anthraquinone drug is dispersed in an oil phase and then encapsulated by the amphiphilic macromolecular compound in an aqueous phase via emulsification.

[0042] The hydroxyalkyl starch described in this invention includes, but is not limited to, hydroxymethyl starch, hydroxyethyl starch, etc., with hydroxyethyl starch (abbreviated as HES) being a representative component. Hydroxyethyl starch has been used clinically as a plasma substitute for a long time, exhibiting good biocompatibility and safety. This invention, by directly coupling photosensitizer molecules to hydroxyethyl starch, can significantly improve the hydrophilicity of the photosensitizer, optimizing its characteristic absorption peaks while maintaining its stability. Furthermore, the photodynamic properties of the photosensitizer can be utilized for combined therapy with chemotherapy, and the fluorescence imaging properties of the photosensitizer can be used for tumor imaging and real-time monitoring of nanoparticle drug delivery systems.

[0043] In some embodiments, the hydrophobic organic cyanine dye molecule is one or more of IR820, IR783, IR808, IR780, and IR825. As a typical photosensitizer, IR820 has the structure shown in formula (I), exhibiting good near-infrared fluorescence imaging and photothermal properties, but poor biocompatibility and a UV absorption peak at 690 nm, limiting its clinical application in photodynamic therapy. During the experimental process, this invention attempted to encapsulate anthraquinone drug molecules using metal ion coordination to construct a nano-drug delivery system. However, experiments revealed that this method struggled to form stable and uniformly sized nanoparticles, posing a risk of drug leakage. This resulted in significant biotoxicity and poor chemotherapy and photodynamic effects. This invention directly couples photosensitizers, represented by IR820, to hydroxyalkyl starch and simultaneously encapsulates anthraquinone drugs to form a nano-drug delivery system. This significantly improves the stability of the photosensitizer while reducing the toxicity of IR820, and allows for combined chemotherapy and photodynamic therapy to enhance anti-tumor efficacy. Furthermore, cyanide drug molecules can also enable real-time in vivo imaging of the nano-drug delivery system for monitoring.

[0044]

[0045] The anthraquinone drugs described in this invention can be various small molecule drugs with anthraquinone cores, including but not limited to one or more of physcion, rhein, chrysophanol, emodin, and aloe-emodin. This invention utilizes ultrasonic emulsification to encapsulate physcion into a conjugate of a photosensitizer and hydroxyalkyl starch to prepare nanomedicines, thereby improving the drug's half-life in vivo and its accumulation at tumor sites.

[0046] This invention couples a photosensitizer to hydroxyalkyl starch via chemical bonds. The corresponding synthesis steps can be designed according to the molecular structure of the photosensitizer. In some embodiments, the chemical bonds are amide bonds or ester bonds, etc.

[0047] In a preferred embodiment, the photosensitizer is IR820, which is connected to the hydroxyalkyl starch via an amide bond.

[0048] The present invention also provides a method for preparing the aforementioned amphiphilic macromolecular compound, comprising the following steps:

[0049] (1) The hydroxyl groups in hydroxyalkyl starch are replaced by chlorine atoms in a compound containing at least one acyl chloride and a nitro group to obtain hydroxyalkyl starch containing ketone carbonyl and nitro groups;

[0050] (2) The hydroxyalkyl starch containing ketone carbonyl and nitro groups is subjected to a substitution reaction with a primary amine compound containing at least two amino groups, so that the ketone carbonyl group is substituted into an amide bond, thereby obtaining hydroxyalkyl starch containing amino groups.

[0051] (3) The amino-containing hydroxyalkyl starch is subjected to a substitution reaction with the chlorine atom contained in the photosensitizer to generate a hydroxyalkyl starch-photosensitizer conjugate, which is the hydroxyalkyl starch-photosensitizer amphiphilic macromolecular compound of the present invention.

[0052] In some embodiments, step (1) involves a substitution reaction between the hydroxyl groups in the hydroxyalkyl starch and the chlorine atoms in p-nitrophenyl chloroformate to obtain hydroxyalkyl starch containing ketone carbonyl and nitro groups. The hydroxyalkyl starch is hydroxyethyl starch, and step (1) specifically involves: replacing the chlorine atoms in a compound containing at least one acyl chloride and nitro group with the hydroxyl groups in the hydroxyethyl starch to obtain hydroxyethyl starch containing ketone carbonyl and nitro groups. The compound containing at least one acyl chloride and nitro group is p-nitrophenyl chloroformate.

[0053] In a typical embodiment, the hydroxyalkyl starch is hydroxyethyl starch, the photosensitizer is IR820, and step (3) specifically involves: reacting the amino-containing hydroxyethyl starch with the chlorine atom contained in IR820 to generate HES-IR820 conjugate.

[0054] In some embodiments, step (1) specifically involves: weighing HES and placing it in a reaction flask, adding deionized water, sonicating to dissolve, and then placing it in an ice bath. NaOH is quickly added to adjust the pH of the reaction system to alkaline, and then the mixture is vigorously stirred until the hydroxyl groups on the HES are fully activated. Separately, p-nitrophenyl chloroformate (NPC) is weighed and dissolved in dichloromethane. After complete dissolution, it is slowly added dropwise to the reaction flask. After the addition is complete, the system is moved to room temperature and stirred for 2-3 hours. The reaction system is then separated and purified to obtain the target product.

[0055] In some embodiments, the separation and purification in step (1) specifically involves: pouring the reaction solution into isopropanol to obtain a white to yellow flocculent precipitate, centrifuging and washing it multiple times, then washing it once with petroleum ether in the same manner, and drying it under vacuum at low temperature to obtain a white to yellow powder, which is the target product HES-NPC, having the structural formula shown in formula (II).

[0056]

[0057] In some embodiments, step (2) specifically involves: slowly adding the hydroxyalkyl starch HES-NPC containing ketone carbonyl and nitro groups obtained in step (1) dissolved in an organic solvent to a reaction flask containing a primary amine compound, stirring for 1-2 hours to allow a substitution reaction to occur, whereby the ketone carbonyl group is substituted into an amide bond, thereby obtaining an amino-containing hydroxyalkyl starch. The reaction system is then separated and purified to obtain the target product. The primary amine compound containing at least two amino groups is one or more of 1,4-cyclohexanediamine, 1,6-hexanediamine, 1,2-cyclohexanediamine, and butanediamine.

[0058] In some embodiments, step (2) specifically involves: weighing 1,6-hexanediamine, dissolving it in dimethyl sulfoxide, heating it in a 45°C water bath while maintaining slow stirring for 30 minutes. Then, weighing the hydroxyethyl starch containing ketone carbonyl and nitro groups obtained in step (1), dissolving it in dimethyl sulfoxide, maintaining a 45°C water bath, and sonicating it. The dissolved HES-NPC is slowly added dropwise to a reaction flask containing a primary amine compound, stirred for 1-2 hours, and then the reaction system is separated and purified to obtain the target product. In some embodiments, the separation and purification specifically involves: pouring the reaction solution into a washing solution of isopropanol:petroleum ether = 1:1 to obtain a white to yellow flocculent precipitate, centrifuging and washing it multiple times, then washing it once more with petroleum ether using the same method, and drying it under low temperature and vacuum to obtain a yellow powder, which is the target product HES-NH. 2， It has the structural formula shown in equation (III).

[0059]

[0060] In some embodiments, step (3) includes the following steps: Weigh the amino-containing hydroxyethyl starch obtained in step (2) and dissolve it in N,N-dimethylamide to obtain an HES-NH2 solution; then weigh potassium carbonate and dissolve it in N,N-dimethylamide to obtain a potassium carbonate solution; place the two solutions in a water bath at 50-70°C, then quickly pour the potassium carbonate solution into the HES-NH2 solution, stir and alkalize the reaction solution for 20-40 minutes. Subsequently, cool the reaction solution in an ice bath under light-protected conditions, then weigh IR820 and dissolve it in N,N-dimethylamide, sonicate it in the dark for several minutes, and then quickly add it to the above-mentioned reaction solution that has been cooled in an ice bath. Vacuum is applied, and the reaction solution is placed in a 50-70°C water bath under N2 protection and stirred for 8-15 hours. The reaction system is then separated and purified to obtain the target product. In some embodiments, the separation and purification specifically involves: pouring the reaction solution into a washing solution of isopropanol:petroleum ether = 1:1 to obtain a green precipitate, centrifuging and washing it multiple times, then washing it once more with petroleum ether in the same manner, subsequently redissolving the obtained green precipitate in ultrapure water, dialyzing it with ultrapure water for 2-4 days, and freeze-drying it to obtain a blue-green solid, HES-IR820, which has the structural formula shown in formula (IV); wherein the molecular weight cutoff of the dialysis bag is preferably 3500 Da.

[0061]

[0062] This invention prepares an amphiphilic macromolecular compound of hydroxyalkyl starch-photosensitizer. The initial grafting method involved modifying the hydroxyalkyl starch and photosensitizer separately, followed by a reaction grafting. For example, according to the modification grafting method used in patent document CN114748639A, the chlorine atom in the photosensitizer is first converted to a secondary amino group through a substitution reaction, resulting in a photosensitizer containing a secondary amino group. Then, the photosensitizer containing the amino group undergoes an amide reaction with carboxylated hydroxyalkyl starch to obtain a photosensitizer-carboxylated hydroxyalkyl starch conjugate. However, experiments in this invention have shown that when preparing the photosensitizer IR820 using this method, on the one hand, it is impossible to adjust the position of the ultraviolet absorption peak of the photosensitizer IR820 to perfectly match the near-infrared laser wavelength to achieve its photodynamic therapy properties; on the other hand, the photosensitizer in the resulting conjugate has very poor photostability and has been deactivated during purification and separation, making further grafting with hydroxyalkyl starch impossible. The possible reason is that during the grafting process, only amino groups or their derivatives with strong electron-rejecting ability are used to replace the highly electronegative chlorine atoms. This causes the electron cloud of the photosensitizer to be distributed from the central six-carbon ring to the pyrrole ring, which in turn breaks the stable conjugated electron system of IR820, making it easy to deactivate under light.

[0063] The experiments of this invention have revealed that, following the synthetic approach of hydroxyalkyl starch-photosensitizer proposed in this invention, the hydroxyl groups in hydroxyalkyl starch are first substituted with chlorine atoms in compounds containing at least one acyl chloride and a nitro group to obtain hydroxyalkyl starch containing a ketone carbonyl group and a nitro group. Then, a substitution reaction is carried out with a primary amine compound to obtain hydroxyalkyl starch containing an amino group. Finally, a substitution reaction is carried out with the chlorine atom of the photosensitizer to obtain the hydroxyalkyl starch-photosensitizer conjugate of this invention. Experiments have confirmed that not only is the UV absorption peak position of this photosensitizer adjusted to match the near-infrared laser wavelength of 660 nm, giving it photodynamic therapy properties, but its photosensitizer also exhibits high photosensitizer photostability. Therefore, this invention proposes a modification and grafting method that can maintain the photosensitizer photosensitizer photostability of cyanine dye molecules. This method couples the photosensitizer to hydroxyalkyl starch that has undergone nitro- and primary amino-modification processes. The resulting photosensitizer-hydroxyalkyl starch conjugate not only modulates the UV-Vis absorption peak of the photosensitizer to around 660 nm and solves the problem of poor water solubility of small photosensitizer molecules, but also effectively maintains the photosensitizer's photostability and avoids photosensitizer deactivation.

[0064] In the experimental process of this invention, other synthetic approaches were also explored. For example, a primary amine compound containing at least two amino groups was first substituted with the chlorine atom in the photosensitizer to obtain an amino-modified photosensitizer. However, experiments showed that although this modification grafting method could adjust its UV absorption peak to 660 nm, its photostability was very poor, and it deactivated quickly, failing to achieve a good photodynamic therapy effect and preventing further grafting with hydroxyalkyl starch to prepare a hydroxyalkyl starch-photosensitizer compound. For example, in some embodiments, a primary amine compound containing at least two amino groups was substituted with the chlorine atom in IR820 to generate the IR820-NH2 small molecule product. Specifically, the following steps were included: IR820 was weighed and dissolved in methanol, and sonicated for 5 min in the dark until completely dissolved. Then, 1,6-hexanediamine was weighed and dissolved in methanol, and the reaction solution was cooled in an ice bath in the dark. The IR820 solution was quickly added to the above reaction solution that had been cooled in the ice bath, and the mixture was evacuated and placed in a 50°C water bath under N2 protection and stirred for 3 h. The reaction system was then separated and purified to obtain the target product. In some embodiments, the separation and purification specifically involved: pouring the reaction solution into a washing solution of isopropanol:petroleum ether = 1:1 to obtain a blue precipitate, centrifuging and washing it multiple times, then washing it once more with petroleum ether in the same manner, subsequently redissolving the obtained blue precipitate in ultrapure water, dialyzing it with ultrapure water for 2-4 days, and freeze-drying it to obtain a blue powder IR820-NH2, which has the structural formula shown in formula (V); wherein the molecular weight cutoff of the dialysis bag is preferably 3500 Da.

[0065]

[0066] The photosensitizer is IR820. When it undergoes a substitution reaction with a compound containing two primary amino groups, such as hexamethylenediamine, the substitution of the chloride ion by the primary amino group causes a blue shift in the ultraviolet absorption peak of IR820 to around 660 nm, making it suitable for near-infrared light-induced photodynamic therapy at 660 nm. In some embodiments of the present invention, the photoresponsive properties of the molecule of formula (V) are compared with those of other molecules synthesized in the embodiments.

[0067] In some embodiments, the hydroxyalkyl starch is hydroxyethyl starch, with a molecular weight of 25-480 kDa and a degree of substitution of hydroxyethyl of 0.4-0.6. The specifications of the hydroxyethyl starch can be 480 / 0.4, 480 / 0.5, 480 / 0.6, 200 / 0.4, 200 / 0.5, 200 / 0.6, 130 / 0.4, 130 / 0.5, 130 / 0.6, 70 / 0.5, 25 / 0.5, preferably 130 / 0.4, where 130 represents the molecular weight of the hydroxyethyl starch in kDa and 0.4 represents the degree of substitution of hydroxyethyl.

[0068] The present invention also provides a nano-drug delivery system based on the aforementioned amphiphilic macromolecular compound, comprising the aforementioned amphiphilic macromolecular compound and anthraquinone antitumor drugs; the antitumor drugs and the aforementioned amphiphilic macromolecular compounds are assembled into an oil-in-water nanosystem by ultrasonic emulsification to form nanomicelles, which improves the drug's tumor targeting while enabling combined photodynamic and chemotherapy therapy, significantly enhancing antitumor activity.

[0069] In some embodiments, the anthraquinone antitumor drug and the amphiphilic macromolecular compound are constructed into oil-in-water nanoparticles via ultrasonic emulsification, wherein the anthraquinone antitumor drug serves as the oil phase and the amphiphilic macromolecular compound serves as the aqueous phase. Specifically, after separation and purification, the hydroxyalkyl starch-photosensitizer amphiphilic macromolecular compound is dissolved in an aqueous phase and then reacted with the anthraquinone drug dissolved in the organic phase via ultrasonic emulsification to generate oil-in-water nanoparticles. The resulting product is then separated and purified to obtain the hydroxyalkyl starch-photosensitizer nanoparticle drug-loaded system containing the anthraquinone drug.

[0070] The anthraquinone antitumor drugs of the present invention include, but are not limited to, one or more of physcion, rhein, chrysophanol, emodin, and aloe-emodin.

[0071] In some embodiments, the size of the drug-loaded nanoparticles in the nano-drug delivery system provided by the present invention is 180-230 nm, the antitumor drug is emodin methyl ether, and the drug loading of emodin methyl ether is 2.6%-2.8%.

[0072] In some embodiments, the above-mentioned nano-drug delivery system was prepared by the following method: HES-IR820 was weighed and dissolved in ultrapure water, and sonicated until completely dissolved. Then, emodin methyl ether was weighed and dissolved in dichloromethane. A certain amount of HES-IR820 solution was placed in an ultrasonic disruptor, and dichloromethane containing emodin methyl ether was added dropwise while sonicating at a water phase:organic phase volume ratio of 20:1. The ultrasonication was maintained in an ice bath state throughout. After processing in the ultrasonic disruptor at a certain power for several minutes, a dark green emulsion was obtained. The emulsion was then separated and purified to obtain the target product. In some embodiments, the separation and purification were specifically performed as follows: the reaction solution was rotary evaporated at 45°C for 3 minutes, and then the obtained green emulsion was centrifuged at 5000 rpm for 10 minutes. The supernatant was collected and freeze-dried to obtain green powder as Phy@HES-IR820 nanoparticles.

[0073] In some embodiments, the dialysis bags used for dialysis have a molecular weight cutoff of 3500-8000 Da.

[0074] This invention provides an anticancer drug comprising the aforementioned nano-drug delivery system and pharmaceutically acceptable additives. The anticancer drug of this invention is applicable to cancers including, but not limited to, breast cancer, liver cancer, colon cancer, ovarian cancer, or melanoma. The anticancer drug can be in the form of an injection, powder for injection, oral dosage form, spray, capsule, or suppository.

[0075] In a preferred embodiment of the present invention, hydroxyethyl starch is selected as the hydrophilic fragment and IR820 is selected as the hydrophobic fragment, and both are used as drugs for fluorescence imaging and photodynamic therapy. Emodin methyl ether is selected as the tumor therapeutic drug, and drug-loaded nanoparticles with a particle size of approximately 230 nm, uniform distribution, and stable structure are prepared. Compared with the non-nanosystem IR820-NH2 small molecule, the nano-sized particles significantly increase their enrichment at the tumor site, promote the uptake of nanoparticles by tumor cells, and exhibit better anti-tumor effects in the 4T1 breast cancer mouse model. The nano-drug delivery system provided by the present invention can perform real-time imaging of the tumor site through fluorescence. Simultaneously, laser irradiation of the tumor site can rapidly increase the ROS level of the tumor site, realizing the combined treatment of photodynamic strategy and chemotherapy, ultimately enhancing the anti-tumor effect.

[0076] Cyanide-based near-infrared fluorescent dyes possess good biocompatibility and photoresponsive efficacy; however, single photosensitizer molecules have poor solubility, cannot achieve long-term circulation in vivo, and lack tumor targeting. Photodynamic therapy (PDT) is a treatment strategy that uses photosensitizer drugs to transfer energy from laser light to surrounding oxygen molecules, leading to an increase in intracellular reactive oxygen species (ROS) levels, thereby causing tumor cells to die from oxidative damage that is difficult to repair in time. However, ROS accumulation alone is insufficient to achieve broad and effective killing of tumor cells. The pentose phosphate pathway (PPP), as a branch of cellular glycolysis, is responsible for supplying NADPH, maintaining the antioxidant system of tumor cells. Anthraquinone compounds, such as physcion, have been shown in previous studies to induce tumor cell apoptosis, disrupt the cell cycle, and inhibit tumor metastasis, exhibiting strong antitumor activity. However, due to its water solubility, physcion has been difficult to apply clinically in the field of tumor treatment. This invention aims to co-load cyanine-based near-infrared fluorescent dyes and anthraquinone antitumor drugs into a single drug delivery system. This addresses the issues of poor water solubility and low biocompatibility of each drug, while simultaneously adjusting the UV absorption peak position of the photosensitizer to achieve photodynamic therapy effects. This increases the level of reactive oxygen species within tumor cells, leading to tumor cell death from oxidative damage. Furthermore, during the experimental process, it was unexpectedly discovered that anthraquinone compounds are likely specific inhibitors of 6-phosphoglucuronide dehydrogenase (6PGD), potentially disrupting the critical NADPH production node in PPP (phosphoprotein oxidase), making tumor cells more sensitive to oxidative stress, thereby inhibiting the tumor cell's antioxidant system and achieving a synergistic killing effect between the photosensitizer and the anthraquinone drug on tumor cells.

[0077] The following is an example:

[0078] Example 1

[0079] Compounds of formula (IV) were prepared according to the following steps:

[0080] (1) Weigh HES (130kDa, 2g) and place it in a reaction flask. Add 48mL of deionized water, sonicate to dissolve, and then place in an ice bath. Quickly add 2mL of 40mg / mL NaOH solution and stir vigorously for 10min. Separately weigh p-nitrophenyl chloroformate (NPC, 800mg, 4mmol) and dissolve it in 20mL of dichloromethane. Slowly add the solution dropwise to the reaction flask. After the addition is complete, move the system to room temperature and stir for 3h to obtain reaction solution A.

[0081] (2) Pour the reaction solution A described in step (1) into 400 mL of isopropanol and stir slowly to obtain a white to yellow precipitate. Centrifuge to separate the precipitate and wash it twice with 400 mL of petroleum ether.

[0082] (3) Place the white to yellow precipitate obtained in step (2) in a vacuum drying oven and dry it at 50°C for 24 hours. The resulting yellow solid is nitrated hydroxyethyl starch.

[0083] (4) In the reaction flask, weigh 5 mL of 1,6-hexanediamine and dissolve it in 5 mL of DMSO. Heat the solution in a water bath at 45°C for 20 min. Then weigh 200 mg of nitrohydroxyethyl starch (HES-NPC, 130 kDa) from step (3) and dissolve it in 20 mL of DMSO. After sonication, slowly add the solution dropwise to the above reaction flask and stir for 1 h to obtain reaction solution B.

[0084] (5) Pour the reaction solution B obtained in step (4) into a 400 mL mixture of isopropanol and petroleum ether in a volume ratio of 1:1, stir slowly to obtain a white to yellow flocculent precipitate, centrifuge and wash twice, and then wash once with petroleum ether in the same way.

[0085] (6) Place the white to yellow precipitate obtained in step (5) in a vacuum drying oven and dry it at 50°C for 24 hours. The resulting yellow solid is the aminated hydroxyethyl starch.

[0086] (7) Weigh 400 mg of the aminated hydroxyethyl starch HES-NH2 obtained in step (6), and weigh 50 mg of potassium carbonate. Dissolve the mixture in 20 mL of DMF and alkalize the reaction solution by stirring slowly in a 60 °C water bath for 1 h. Then, cool the reaction solution in an ice bath under light-protected conditions, and then quickly add 45 mg of IR820 (5 mL of DMF). Vacuum the solution and transfer it to a 60 °C water bath under N2 protection. Stir the solution for 12 h under light-protected conditions to obtain reaction solution C.

[0087] (8) Pour the reaction solution C into a washing solution with a volume ratio of isopropanol:petroleum ether of 1:1 and stir slowly to obtain a green precipitate. Centrifuge and wash twice, then wash once with petroleum ether in the same way and collect the precipitate.

[0088] (9) The green precipitate obtained in step (8) is redissolved in ultrapure water, dialyzed with ultrapure water for 3 days, and freeze-dried to obtain a blue-green solid as HES-IR820, wherein the molecular weight cutoff of the dialysis bag is preferably 3500 Da.

[0089] Compound (V) was prepared according to the following steps:

[0090] (10) Weigh 40 mg of IR820 and dissolve it in methanol, then sonicate it in the dark for 5 min until completely dissolved. Weigh 5 mL of 1,6-hexanediamine and dissolve it in methanol, then cool the reaction solution in an ice bath in the dark. Quickly add the IR820 solution to the above reaction solution that has been cooled in an ice bath, evacuate the vacuum, and place the reaction solution in a 50°C water bath under N2 protection and stir for 3 h to obtain reaction solution D.

[0091] (11) Pour the reaction solution D into a washing solution with a volume ratio of isopropanol:petroleum ether of 1:1 and stir slowly to obtain a green precipitate. Centrifuge and wash twice, then wash once with petroleum ether in the same way and collect the precipitate.

[0092] (12) The green precipitate obtained in step (11) is redissolved in ultrapure water, dialyzed with ultrapure water for 3 days, and then freeze-dried to obtain a blue solid IR820-NH2, wherein the molecular weight cutoff of the dialysis bag is preferably 3500 Da.

[0093] See the schematic diagram of the above preparation process. Figure 2A The 1H NMR (600MHz) data of the target compound are as follows: Figure 2B , Figure 2C As shown. Figure 1 The UV-Vis absorption spectra of free IR820, the amino-substituted IR820 prepared in this embodiment (IR820-NH2 as shown in formula (V)), and the coupling compound of IR820 and hydroxyethyl starch (HES-IR820) are shown. It can be seen that the UV-Vis absorption peak of free IR820 is located at 690 nm, while the UV-Vis absorption peaks of the amino-substituted IR820 (IR820-NH2 as shown in formula (V)) and the coupling compound of IR820 and hydroxyethyl starch (HES-IR820) prepared in this embodiment both show a significant blue shift compared to free IR820, especially the HES-IR820, whose UV-Vis absorption peak is close to 660 nm.

[0094] Compared to HES, the 1H NMR spectrum of hydroxyethyl starch co-loaded with IR820 (using D2O as solvent) showed new peaks in the chemical shift range of 1.2–2.8 ppm, which are attributed to the methylene hydrogen, secondary amine bond hydrogen, and methylene hydrogen of hexamethylenediamine used to replace the chlorine atom of IR820 in the IR820 spectrum; the peaks in the NMR spectrum at 7.2–8.6 ppm correspond to the aromatic hydrogens in IR820; proving that IR820 was successfully grafted onto HES.

[0095] The 1H NMR spectrum of IR820 with only amino groups attached (using deuterated DMSO as solvent) shows peaks at 7.2–8.6 ppm corresponding to aromatic hydrogens in IR820, and peaks at chemical shifts between 1.2 and 2.8 ppm corresponding to methylene hydrogens and amino hydrogens in IR820, proving that the chlorine atom in IR820 has been successfully replaced by an amino group.

[0096] Fourier transform infrared spectrum as Figure 2D The results showed that hydroxyethyl starch grafted with IR820 was at 1698 cm⁻¹. -1 A small peak appears, attributed to the deformation vibrations of the NH and CN bonds in the amide bond. The small peak at 1594 cm⁻¹ is attributed to the deformation vibrations of the NH bond in the secondary amine; the peaks at 1539 and 1516 cm⁻¹ are also attributed to these vibrations. -1 The mid-peak is caused by the vibration of the aromatic ring skeleton of IR820 itself, and the peaks are at 1293 and 1234 cm⁻¹. -1 The mid-peak is attributed to the stretching vibration of the sulfonate S=O bond, 679 cm⁻¹ -1 The sulfonate SO fingerprint peak of Xiaofeng indicates the successful grafting of IR820 onto HES.

[0097] Determination of IR820 grafting rate: Dissolve 1 mg of IR820-NH2 in 1 mL of ultrapure water to prepare a 1 mg / mL standard stock solution. Dilute the stock solution with ultrapure water to a series of concentrations: 100, 50, 25, 10, and 5 μg / mL. Analyze using a UV-Vis spectrophotometer, recording the visible spectrum in the 300-800 nm range for each concentration, and recording the absorbance (Abs) at 655 nm. Figure 2E As shown. Plotting the concentration of the IR820 standard on the x-axis and the absorbance at 655 nm on the y-axis, the standard curve Y = 0.03440X + 0.06332(R) is obtained. 2 =0.9988). Dissolve 2 mg of HES-IR820 in ultrapure water and bring the volume to 2 mL. Measure the absorbance (Abs) at 655 nm using a UV-Vis spectrophotometer. Substitute the absorbance into the standard curve to obtain the IR820 content in HES. Calculate the mass fraction of IR820 in the total HES-IR820, and the grafting rate of IR820 is found to be 2.88%.

[0098] Example 2

[0099] Preparation and characterization of Phy@HES-IR820:

[0100] Phy@HES-IR820 nanoparticles were prepared using an ultrasonic emulsification method, the specific method of which is as follows:

[0101] (1) Weigh 10 mg of HES-IR820 and dissolve it in 4 mL of ultrapure water. Sonicate the solution in the dark until completely dissolved. Weigh 2.84 mg of emodin methyl ether and dissolve it in 1 mL of dichloromethane. Place the HES-IR820 solution in an ice bath and sonicate it using a 220 W ultrasonic probe at a frequency of 2 seconds of operation followed by 2 seconds of rest. At the 10th second of treatment, rapidly add 200 μL of emodin methyl ether solution. After sonicating for 3 minutes, collect the solution to obtain a green emulsion containing Phy@HES-IR820 nanoparticles.

[0102] (2) The green emulsion obtained in step (1) was rotary evaporated at 45°C for 3 min. After the dichloromethane was removed, the supernatant was collected by centrifugation at 5000 rpm for 5 min. The supernatant was then filtered through a 450 nm filter membrane to obtain purified nanoparticles, which were a clear green solution. After freeze-drying, green powder Phy@HES-IR820 nanoparticles were obtained.

[0103] The particle size distribution and surface zeta potential of Phy@HES-IR820 nanoparticles were detected using a dynamic light scattering particle size analyzer (DLS), and the results are as follows: Figure 3 Contents A and B are shown. 20 μL of diluted Phy@HES-IR820 nanoparticles was dropped onto a copper grid, stained with 0.1% phosphotungstic acid, and allowed to air dry at room temperature overnight. The morphology was observed using a transmission electron microscope (TEM), and the results are as follows. Figure 3 As shown in content C. The hydrated particle size of Phy@HES-IR820 nanoparticles was measured daily for one week using a dynamic light scattering particle size analyzer. The results are as follows. Figure 3 As shown in content D.

[0104] Experimental results show that the Phy@HES-IR820 nanoparticles have a particle size of about 230 nm and a surface potential of about +5-10 mV. The nanoparticles remain basically stable within one week, with no obvious fluctuations in particle size and PDI value.

[0105] Example 3

[0106] The UV-Vis absorption spectrum and Fourier transform infrared spectrum of the Phy@HES-IR820 nanoparticles prepared in Example 2 are as follows:

[0107] Phy@HES-IR820 nanoparticles were diluted with ultrapure water to an IR820 concentration of 20 μg / mL. The IR820, HES-IR820, and IR820-NH2 groups were all diluted with ultrapure water to an IR820 concentration of 20 μg / mL. Phy was diluted with NaOH solution. Ultrapure water was used as a reference. The absorption spectra of the five samples were measured using a UV-Vis spectrophotometer, with a scanning wavelength range of 300-800 nm and a scanning step size of 0.5 nm. The results are as follows: Figure 3 Content E is shown.

[0108] UV-Vis spectroscopy results showed that free IR820, after dissolving in water, exhibited maximum absorption at 690 nm, while free Phy showed maximum absorption at 505 nm. HES-IR820 and IR820-NH2 showed maximum absorption between 640 and 660 nm. This indicates that the chlorine atoms in IR820 have been substituted, causing a shift in its absorption peak position to accommodate the photodynamic effect of laser excitation at 660 nm. Simultaneously, Phy@HES-IR820 also exhibited its strongest absorption peak near 660 nm, perfectly consistent with HES-IR820, and an absorption peak for Phy was observed at 500 nm, indicating that HES-IR820 effectively encapsulated Phy, forming nanoparticles. The slight redshift and blueshift of the maximum absorption wavelength may be related to the interaction between Phy, IR820, and the carbohydrate macromolecule hydroxyethyl starch.

[0109] Fourier transform infrared spectra of each sample are as follows Figure 3 As shown in content F, hydroxyethyl starch grafted with IR820 was at 1698 cm⁻¹. -1 A small peak appears, attributed to the deformation vibrations of the NH and CN bonds in the amide bond. 1594 cm⁻¹ -1 The small peaks at 1539 and 1516 cm⁻¹ belong to the deformation vibrations of the NH bond in secondary amines; -1 The mid-peak is caused by the vibration of the aromatic ring skeleton of IR820 itself, and the mid-peaks at 1293 and 1234 cm⁻¹ are attributed to the stretching vibration of the sulfonate S=O bond, while the mid-peak at 679 cm⁻¹ is attributed to the stretching vibration of the sulfonate group S=O bond. -1 The presence of a small peak in the sulfonate SO fingerprint indicates successful grafting of IR820 onto HES. (3300-3500 cm⁻¹) -1 The nitrogen-hydrogen stretching vibration produces a distinct peak in the amino group, indicating successful amino attachment to IR820-NH2. (1626 cm⁻¹) -1 The peak at 1676 cm⁻¹ represents the carbonyl group associated with one of the phenolic hydroxyl groups. -1 The presence of another peak without associated carbonyl groups indicates the presence of Phy in the nanoparticles. The characteristic peaks of the remaining phenolic hydroxyl groups and anthraquinones are masked by the original peaks of HES and IR820, respectively, making them difficult to identify.

[0110] Example 4

[0111] Fluorescence emission spectrum of Phy@HES-IR820 nanoparticles prepared in Example 2:

[0112] Dimethyl sulfoxide (DMSO) solutions of IR820, HES-IR820, IR820-NH2, and Phy@HES-IR820 were prepared. The fluorescence spectra of the four samples in DMSO were measured using fluorescence spectroscopy, with excitation wavelengths of 405 nm and 488 nm, respectively; the emission spectral scan ranges were 420-780 nm and 500-780 nm, respectively. The results are as follows: Figure 3 Content G and Figure 3 Content H is shown.

[0113] From the fluorescence spectra, in dimethyl sulfoxide (DMSO), the IR780 fluorescence was not quenched because the Phy@HES-IR820 nanoparticles were all in a completely dissolved state. The characteristic fluorescence spectrum of IR820 was observed in all four samples, indicating that IR820 maintained its properties during preparation and was successfully loaded onto HES and thus present within the nanoparticles.

[0114] Example 5

[0115] Drug release behavior of the Phy@HES-IR820 nanoparticles prepared in Example 2 under different conditions:

[0116] (1) Preparation of release media: Prepare 300 mL of PBS buffer containing 0.5% Tween-80 (pH 7.4); 300 mL of PBS buffer containing 0.5% Tween-80 (pH 6.8); and 300 mL of acetate buffer containing 0.5% Tween-80 (pH 5.0);

[0117] (2) Prepare 12 mL Phy@HES-IR820 nanoparticle solution (Phy: 20 μg / mL), take 1 mL Phy@HES-IR82 nanoparticle solution and put it into a dialysis bag with a molecular weight cutoff of 3500 Da. Set up 3 parallels for each group. Then immerse the dialysis bag in a 50 mL centrifuge tube containing 30 mL of release solution and shake it in a shaker at a temperature of 37 °C and a rotation speed of 180 rpm.

[0118] (3) At predetermined time points (0, 1, 2, 4, 6, 8, 12, 24, and 48 hours), 1 mL of the release solution was collected, and 1 mL of blank release solution was added. The collected release solution was alkalized with NaOH solution and then the Phy content was determined using a multifunctional microplate reader. The excitation wavelength was 485 nm, and the detection emission wavelength was 492 nm. The results are as follows: Figure 3As shown in Content I, the release rates of Phy@HES-IR820 nanoparticles at pH 5.0, pH 6.8, and pH 7.4 were 73.7%, 62.1%, and 51.3%, respectively. The high release rate under acidic conditions is attributed to the presence of two weakly acidic phenolic hydroxyl groups in Phy. In a typical weakly alkaline environment, the phenolic hydroxyl groups dissociate, giving Phy good water solubility. However, with increasing proton levels in the environment, the phenolic hydroxyl groups gradually protonate and lose their water solubility, precipitating as a yellow, flocculent, insoluble substance. This water solubility can be restored by alkalizing the environment. Given the acidic environment of tumor sites, this drug release behavior of the nanoparticles helps ensure the relative stability of the drug during storage and blood circulation, while its rapid release after entering the lysosomes of tumor cells achieves a good therapeutic effect.

[0119] Example 6

[0120] In vitro photostability characterization of IR820 components at different concentrations and in different groups:

[0121] Lyophilized HES-IR820 powder, IR820-NH2 powder, and Phy@HES-IR820 nanoparticles prepared in Example 2 were dissolved in ultrapure water and diluted to 20 μg / mL according to the concentration of IR820. 4 mL of the solution was placed in a 10 mL EP tube and heated with a 660 nm laser at 0.2 W / cm². 2 Irradiation was performed, and the absorbance at 660 nm was recorded at specific time points, with continuous recording of absorbance changes for 180 seconds. The results are as follows: Figure 4 As shown in content A.

[0122] The results showed that when the IR820 concentration was 20 μg / mL and the laser power was 0.2 W / cm², the effect was optimal. 2 Both Phy@HES-IR820 nanoparticle solution and HES-IR820 exhibited good photostability, with absorbance at 660 nm decreasing by 19% and 16%, respectively, after 3 minutes of illumination. However, the small molecule IR820-NH2 showed poor photostability, with absorbance at 660 nm decreasing by 87% after 3 minutes of illumination.

[0123] Example 7

[0124] In vitro photodynamic performance characterization of samples with different concentrations and groups containing IR820:

[0125] HES-IR820 powder, IR820-NH2 powder, and Phy@HES-IR820 nanoparticles prepared in Example 2 were dissolved in ultrapure water and diluted to 10 μg / mL according to the concentration of IR820. First, 1 mg of the singlet oxygen probe DPBF (1,3-diphenylisobenzofuran) was dissolved in 1 mL of ethanol to obtain a DPBF stock solution. Then, 80 μL of the DPBF solution was added to the IR820-NH2 and Phy@HES-IR820 nanoparticle solutions (3920 μL, both calculated as 10 μg / mL of IR820). Then, a 660 nm laser at 0.2 W / cm² was used. 2 The mixed solution was irradiated for 0, 10, 20, 30, 60, 90, 120, and 180 seconds. The absorbance of DPBF at 405 nm was recorded over time using a UV-Vis spectrophotometer, representing the ROS generation capacity. Results are as follows... Figure 4 As shown in content B.

[0126] The radical scavenger TEMP (100 mM) was then pre-added to the EP tube, followed by the addition of IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles (each 20 μg / mL, 1 mL, calculated as IR820). The tube was then treated with a 660 nm laser at 0.2 W / cm². 2 Irradiation was performed for 2 minutes, then the mixture was transferred to a quartz capillary tube, and the ESR signal was detected at room temperature. Results are as follows: Figure 4 As shown in content C.

[0127] The results showed that HES-IR820 and Phy@HES-IR820 nanoparticles had better ability to generate singlet oxygen in vitro by photodynamic therapy, while IR820-NH2 had poorer photodynamic ability due to the lack of stability.

[0128] Example 8

[0129] Study of nanoparticle uptake by 4T1 tumor cells using confocal microscopy:

[0130] First, 4T1 cells were placed at a density of 2 × 10⁶ cells per well. 5 Cells were seeded at a density of 1000 mcg / mL in confocal culture dishes and incubated overnight. Then, 4T1 cells were co-incubated for 12 h with IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles (16 μg / mL, 1.5 mL, based on IR820). The culture medium was then removed, and the cells were washed three times with PBS. Subsequently, the cells were fixed with paraformaldehyde for 15 min and stained with DAPI for 20 min. After washing three times with PBS, the cells were observed using CLSM. The excitation wavelengths for DAPI and IR820 were 405 nm and 561 nm, respectively. Results are as follows: Figure 5As shown in content A.

[0131] The results showed that IR820-NH2, HES-IR820 and Phy@HES-IR820 nanoparticles were all distributed in the cytoplasm of cells. The uptake of Phy@HES-IR820 nanoparticles was the highest, followed by the small molecule IR820-NH2, and the lowest was HES-IR820.

[0132] Example 9

[0133] Flow cytometry was used to study the uptake of nanoparticles by 4T1 tumor cells.

[0134] 4T1 cells were fed at a rate of 2 × 10 5 Cells were seeded at a density of 100 cells per well in 6-well plates and incubated overnight. Cells were then co-incubated with Phy@HES-IR820 nanoparticles (16 μg / mL, 2 mL) for 0, 0.5, 1, 2, 4, 8, and 12 hours, respectively. Another set of 6-well plates was prepared and co-incubated with IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles (16 μg / mL, 2 mL) for 12 hours. Cells were then digested with EDTA-free trypsin and collected by centrifugation at 300g for 5 minutes. Cells were then washed with PBS and centrifuged twice. Finally, 300 μl of PBS buffer was added to the solution, and flow cytometry was used for analysis. Results are shown below. Figure 5 Content B and Figure 5 As shown in content C.

[0135] The results showed that the cellular uptake of Phy@HES-IR820 nanoparticles gradually increased over time, reaching a plateau at 8-12 hours. The uptake was highest for Phy@HES-IR820 nanoparticles, followed by small molecule IR820-NH2, and lowest for HES-IR820, consistent with the CLSM results.

[0136] Example 10

[0137] The killing effect of Phy, IR820-NH2, HES-IR820 and Phy@HES-IR820 nanoparticles on 4T1 tumor cells was detected using the CCK-8 assay:

[0138] 4T1 cells were seeded at a density of 5000 cells per well in 96-well plates and incubated overnight. Then, the 4T1 cells were co-incubated for 12 hours with Phy, IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles (0, 1, 2, 4, 8, 16, 32 μg / mL of IR820, with Phy concentration at 95% of IR820). The drug-containing medium was then replaced with fresh medium. After 24 and 48 hours, 100 μL of 10% CCK-8 assay solution was added to each well and the cells were incubated at 37°C for 1–2 hours in the dark. Cell viability was calculated by measuring absorbance at 450 nm using a microplate reader. Results are shown below. Figure 6 Content A and Content B are shown.

[0139] The results showed that IR820 concentrations above 16 μg / mL produced significant cytotoxicity, but overall cell viability remained above 80% below 16 μg / mL, indicating that the modified IR820 drug has good biosafety.

[0140] Example 11

[0141] The killing effect of Phy, IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles on 4T1 tumor cells under laser irradiation was detected using the CCK-8 assay:

[0142] 4T1 cells were seeded at a density of 5000 cells per well in 96-well plates and incubated overnight. Then, the 4T1 cells were co-incubated for 12 hours with Phy, IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles (0, 1, 2, 4, 8, 16, 32 μg / mL of IR820, and Phy concentration of 95% of IR820). The drug-containing medium was then replaced with fresh medium. Each well was then treated with a 660 nm laser at 0.2 W / cm². 2 Irradiate for 2 min. After 24 and 48 hours, add 100 μL of 10% CCK-8 assay solution to each well and incubate at 37°C for 1–2 h in the dark. Cell viability is calculated by measuring absorbance at 450 nm using a microplate reader. Results are as follows. Figure 6 Contents C and D are shown.

[0143] The results showed that HES-IR820 and Phy@HES-IR820 nanoparticles, under laser irradiation, significantly enhanced their ability to kill tumor cells through the generation of reactive oxygen species via photodynamic effects. However, IR820-NH2, due to its poor photostability, struggled to achieve prolonged ROS generation, with a 24-hour IC50 value. 50The concentration decreased only from 28.71 μg / mL to 26.81 μg / mL. The IC50 concentration of HES-IR820 over 24 hours decreased. 50 The concentration decreased from 22.61 μg / mL to 12.86 μg / mL. The IC50 concentration of Phy@HES-IR820 nanoparticles after 24 hours was [not specified]. 50 The concentration decreased from 21.33 μg / mL to 8.25 μg / mL. Furthermore, with increasing drug concentration, the photodynamic ability to generate ROS became stronger, and the drug's toxicity improved.

[0144] Example 12

[0145] Apoptosis and intracellular ROS production in 4T1 tumor cells after different sample treatments were investigated using flow cytometry and confocal microscopy.

[0146] CLSM assay first involves loading 4T1 cells at a density of 2 × 10⁶ cells per well. 5 4T1 cells were seeded at a density of 10 μg / mL in confocal culture dishes and incubated overnight. Then, the cells were co-incubated for 12 hours with IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles (16 μg / mL, 1.5 mL, based on IR820). The culture medium was then removed, and the cells were washed three times with PBS. Afterward, the cells were co-incubated for 30 min with 1.5 mL (10 μg / mL) of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Then, the cells were incubated with 0.2 W / cm² solution. 2 Cells were irradiated with a 660nm laser for 2 minutes. After washing three times with PBS, the cells were fixed with paraformaldehyde and then observed using CLSM. The excitation wavelength of DCF was set to 488nm. The fluorescence intensity of DCF represents the intracellular ROS level. Cells were treated and drugged in the same manner, and after irradiation, the culture medium was removed, and the cells were washed three times with PBS. Cells were then treated with an apoptosis kit, with 500μL of 1% (v / v) pyridine iodide and annexin-V staining solution added to each dish. After staining for 10 minutes, cell apoptosis was observed using CLSM. The results are as follows: Figure 7 Content A and Content B are shown.

[0147] Flow cytometry first involves loading 4T1 cells at a concentration of 2 × 10⁻⁶. 5 4T1 cells were seeded at a density of 10 μg / well in 6-well plates and incubated overnight. The cells were then co-incubated for 12 hours with IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticles (16 μg / mL, 1.5 mL, based on IR820). The culture medium was then removed, and the cells were washed three times with PBS. Afterward, the cells were co-incubated for 30 min with 1.5 mL (10 μg / mL) of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Then, the cells were incubated with 0.2 W / cm² solution. 2Cells were irradiated with a 660nm laser for 2 minutes. After washing three times with PBS, the cells were digested with EDTA-free trypsin and collected by centrifugation at 300g for 5 minutes. The cells were then washed with PBS and centrifuged twice. Finally, 300μl of PBS buffer was added to the solution, and flow cytometry was used for analysis. Cells were treated in the same manner and administered the drug. After irradiation, the culture medium was removed, and the cells were washed three times with PBS. Cells were then treated with an apoptosis kit. After collecting cells from each dish, 1mL of 1% pyridine iodide and annexin-V staining solution was added. After staining for 10 minutes, apoptosis was analyzed by flow cytometry. The results are as follows: Figure 7 Contents C and D are shown.

[0148] The results showed that ROS levels in the PBS and Phy groups did not significantly increase after light exposure, nor did the corresponding apoptosis levels increase substantially, indicating that single chemotherapy was not highly cytotoxic. The IR820-NH2 group, however, produced a certain amount of ROS, which improved the drug's efficacy. The HES-IR820 group showed further improvement, and the Phy@HES-IR820 group exhibited the most significant ROS production and apoptosis-inducing effect due to the synergistic effect of Phy's chemotherapy and IR820's photodynamic effect.

[0149] Example 13

[0150] In vitro study of the effects of emodin methyl ether on the pentose phosphate pathway in 4T1 tumor cells:

[0151] 4T1 cells were fed at a rate of 2 × 10 5 Cells were seeded at a density of 100 cells per well in 6-well plates and incubated overnight. Then, the cells were co-incubated for 12 hours with different concentrations of Phy and Phy@HES-IR820 nanoparticles (maximum concentration: 15 μg / mL for Phy, 16 μg / mL for IR820, 2 mL each). The culture medium was then removed. After washing three times with PBS, the cells were digested with EDTA-free trypsin and collected by centrifugation at 300g for 5 minutes. Measurements were performed using G6PD, 6PGD, NADP(H), and GSH / GSSG assay kits, respectively. The experimental procedures were performed according to the instructions of each kit. Results are shown below. Figure 8 Contents A, B, C, and D are shown.

[0152] The results showed that Phy had no significant effect on G6PD in the pentose phosphate pathway, but exhibited high inhibitory activity against its downstream target 6PGD, which was Phy's specific target, and this inhibitory activity increased with increasing drug concentration. Further downstream, NADPH and GSH levels also decreased with the inhibitory effect of Phy, indicating that emodin methyl ether has a good ability to weaken the intratumoral antioxidant system. However, the effect of Phy in Phy@HES-IR820 nanoparticles was slightly less than that of the free group due to the difficulty in complete release of Phy.

[0153] Example 14

[0154] In vivo fluorescence imaging and drug distribution in tissues of Phy@HES-IR820 nanoparticles:

[0155] Establishing a 4T1 subcutaneous tumor model: Purchase six-week-old BALB / c female mice weighing 18-21g. ​​After one week of acclimatization in the laboratory animal facility, shave the fur around the right hind limb of the mice. Culture 4T1 cells, and when the cell number is sufficient and in the logarithmic growth phase, digest and centrifuge to collect the cells. Wash once with PBS and resuspend in PBS to prepare 2x10⁻¹⁰ cells / mL. 7 Prepare a 50 μL / mL cell suspension and store it in an ice box until needed. Inject 50 μL of the cell suspension subcutaneously into the area above the right hind limb of each mouse using a syringe. Continue feeding the mice after injection.

[0156] The formula for calculating tumor volume is: V = (L × W) 2 ) / 2, where V represents the tumor volume, L represents the long axis of the tumor, and W represents the short axis. When the tumor volume reaches 200 mm... 3 Subsequently, tumor-bearing mice were randomly divided into three groups of five each. Free IR820-NH2, HES-IR820, and Phy@HES-IR820 nanoparticle solutions were administered via tail vein, respectively, at a dose of 4 mg / kg based on IR820. Mice were anesthetized before administration and at 1, 2, 4, 8, 12, and 24 hours post-administration, and fluorescence imaging was performed using a small animal in vivo imaging system with the Cy5.5 channel selected. To further investigate the distribution behavior of the nanomedicine in vivo, mice were sacrificed 24 hours post-administration, and the heart, liver, spleen, lung, kidney, and tumor were removed for fluorescence imaging using a small animal in vivo imaging system. The results are as follows: Figure 9 Contents A, B, and C are shown.

[0157] The results showed that IR820 endowed the nanoparticles with in vivo imaging capabilities, enabling real-time tumor imaging. The fluorescence intensity at the tumor sites in all three mouse groups increased with increasing administration time, while the HES-IR820 and Phy@HES-IR820 nanoparticle groups showed no significant change after 8 hours. Therefore, the irradiation time selected for the photodynamic experiment was 8 hours after administration. At various time points when acquiring fluorescence images, the fluorescence of the free IR820-NH2 group was relatively weak. This is likely because free IR820-NH2 is a small molecule, easily cleared by the liver or kidneys after direct injection, with a very short blood circulation half-life, thus only a small amount of IR820-NH2 reaches the tumor site to produce fluorescence. In contrast, the Phy@HES-IR820 group showed the highest enrichment at the tumor site. This is likely because the hydrophilic hydroxyethyl starch macromolecules on the surface of Phy@HES-IR820 increase the blood circulation time of the nanoparticles, and the EPR effect enhances the enrichment of nanoparticles at the tumor site.

[0158] Figure 9 Contents B and C are fluorescence photographs of various organs from mice in each group, taken 24 hours after drug administration, and semi-quantitative results of fluorescence in each organ. The results show that, compared to free IR820-NH2, the Phy@HES-IR820 nanoparticles exhibit higher enrichment at tumor sites and should therefore possess better imaging and therapeutic effects.

[0159] Example 15

[0160] In vivo pharmacodynamic studies of Phy@HES-IR820 nanoparticles:

[0161] Establishing a 4T1 subcutaneous tumor model: Purchase six-week-old BALB / c female mice weighing 18-21g. ​​After one week of acclimatization in the laboratory animal facility, shave the fur around the right hind limb of the mice. Culture 4T1 cells, and when the cell number is sufficient and in the logarithmic growth phase, digest and centrifuge to collect the cells. Wash once with PBS and resuspend in PBS to prepare 2x10⁻¹⁰ cells / mL. 7 Prepare a 50 μL / mL cell suspension and store it in an ice box until needed. Inject 50 μL of the cell suspension subcutaneously into the area above the right hind limb of each mouse using a syringe. Continue feeding the mice after injection.

[0162] The formula for calculating tumor volume is: V = (L × W) 2 ) / 2, where V represents the tumor volume, L represents the long axis of the tumor, and W represents the short axis. When the tumor grows to 60mm... 3Mice were randomly divided into 8 groups of 10 mice each. The drug administration groups were: (G1) PBS, (G2) Phy, (G3) IR820-NH2, (G4) HES-IR820, (G5) Phy@HES-IR820 nanoparticles, (G6) IR820-NH2+NIR, (G7) HES-IR820+NIR, and (G8) Phy@HES-IR820 nanoparticles+NIR. NIR represents near-infrared laser. Each group received intravenous injection of the drug (4 mg / kg of IR820, Phy concentration of 95% of IR820) on days 1, 4, 7, and 10. Mice in groups 6, 7, and 8 received PDT treatment 8 hours after intravenous injection. A 660 nm near-infrared laser was used at a concentration of 0.6 W / cm². 2 The mice were irradiated with a specific power for 10 minutes. Over 16 days, the length and width of the tumors were recorded every two days using a smart caliper, and the mice were weighed and recorded every two days using an electronic scale. At the end of treatment, all mice were euthanized, and the tumors were collected for weighing and photographing. Results are as follows... Figure 10 As shown. Mice were sacrificed on day 16 after drug administration. Two whole blood samples were collected from each mouse: one for routine blood tests (anticoagulation), and the other for blood biochemistry tests (non-anticoagulation). The results of weighing and photographing the ex vivo tumor are shown below. Figure 11 and Figure 12 As shown.

[0163] from Figure 6 The results of experiments C and D show that Phy alone does not possess effective killing ability at the experimental drug concentration and is not suitable for photodynamic therapy under near-infrared light irradiation. However, the presence of Phy enhances the killing ability of nanoparticles against tumor cells after light irradiation. Figure 7 The results of Content A and Content C indicate that Phy can further increase the level of reactive oxygen species in the tumor area, thus having better efficacy. Therefore, it is speculated that it may have the effect of weakening the antioxidant capacity of tumors. Figure 8 Content D investigated the level of GSH, an antioxidant that directly scavenges reactive oxygen species within tumors. It was found that Phy has the ability to inhibit GSH production and exhibits a concentration-dependent effect. Therefore, it is further speculated that Phy is an upstream of GSH, and the level of its precursor NADPH is inhibited. Figure 8 Content C confirms this conjecture. We further speculate that in the pentose phosphate pathway, at least one of the enzymes responsible for the first and third steps of NADPH production, especially the enzymes catalyzing these two steps, is a target of Phy inhibition. Figure 8Contents A and B investigated the effects of Phy on the activities of two enzymes (6PGD and G6PD). They found that it specifically inhibited 6PGD, but had no significant effect on G6PD. Therefore, it is inferred that Phy targets 6PGD, the third step in the PPP process, thus inhibiting NADPH and GSH production and ultimately weakening tumor antioxidant capacity. This may corroborate the literature report that "emodin methyl ether is a specific inhibitor of 6-phosphoglucuronide dehydrogenase (6PGD), which can interrupt the key NADPH production node in PPP, making tumor cells more sensitive to oxidative stress." Therefore, from... Figure 10 , Figure 11 and Figure 12 It can be seen that the G8 group achieved the best anti-tumor effect, and there were significant differences compared with the control group, especially the G7 group, which fully demonstrates the synergistic killing effect of photosensitizer IR820 and Phy.

[0164] The tumor was fixed with 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E, Cleaved-caspase 3, Ki-67, and GSH. The results are as follows: Figure 13A , Figure 13B , Figure 13C and Figure 13D As shown. Figure 13A The H&E results showed that the Phy@HES-IR820 nanoparticles + NIR group exhibited the largest tumor necrosis area. Simultaneously, the Phy@HES-IR820 nanoparticles + NIR group showed the lowest Ki67 fluorescence, indicating the weakest intratumoral cell proliferation. Furthermore, the Phy@HES-IR820 nanoparticles + NIR group showed the highest Cleaved-caspase3 fluorescence, indicating the strongest intratumoral cell apoptosis. These results consistently demonstrate that the Phy@HES-IR820 nanoparticles + NIR group possesses the best antitumor effect.

[0165] A hematology analyzer can directly detect routine blood parameters in anticoagulated blood. Whole blood used for measuring blood biochemical parameters should be incubated overnight at 4°C, centrifuged at 3000 rpm for 5 minutes, and the serum collected before testing. Results are as follows: Figure 14 As shown. Figure 14 Content A shows the changes in body weight of mice after different drug treatments in Example 15, which is used to evaluate the biosafety of different drugs in mice; Figure 14 Content B to Figure 14 Content J describes the complete blood count and blood biochemistry tests performed on mice after different drug treatments, as described in Example 15, to evaluate the biosafety of different drugs in mice. Among them: Figure 14 Content B is the amount of alanine aminotransferase in serum; Figure 14 Content C represents the serum aspartate aminotransferase level; Figure 14 Content D represents the amount of creatine kinase in serum; Figure 14 Content E represents the amount of blood urea nitrogen in the serum; Figure 14 Content F represents the amount of creatinine in serum; Figure 14 Content G represents the amount of red blood cells in the blood; Figure 14 Content H represents the amount of hemoglobin in the blood; Figure 14 Content I describes the amount of platelets in the blood; Figure 14 Content J represents the number of white blood cells in the blood.

[0166] The results showed that both the HES-IR820 group and the Phy@HES-IR820 nanoparticle group had a good inhibitory effect on tumor growth, which may be attributed to the cytotoxic effect of IR820 accumulation. The Phy@HES-IR820 group had a better tumor inhibition effect than the HES-IR820 group, which may be due to the nanosystem of Phy@HES-IR820 and the interference of Phy on the PPP pathway. Compared with the chemotherapy alone, both the HES-IR820+NIR group and the Phy@HES-IR820 nanoparticle+NIR group showed better tumor inhibition effects due to the synergistic effect of chemotherapy and photodynamic therapy. Under laser irradiation, the rapidly increasing reactive oxygen species level at the tumor site can not only directly kill tumor cells, but also synergistically enhance the effect of chemotherapy drugs and increase the cytotoxicity of chemotherapy drugs.

[0167] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a hydroxyalkyl starch-photosensitizer coupled amphiphilic macromolecular compound, characterized in that, This macromolecular compound is obtained by chemically coupling hydroxyalkyl starch and a photosensitizer; in this amphiphilic macromolecular compound, the hydroxyalkyl starch serves as the hydrophilic end, and the photosensitizer serves as the hydrophobic end; the photosensitizer is a hydrophobic organic cyanine dye molecule; the hydrophobic organic cyanine dye molecule is one or more of IR820, IR780, IR808, IR783, and IR825; the chemical bond is an amide bond or an ester bond; the preparation method includes the following steps: (1) The hydroxyl group in hydroxyalkyl starch is replaced by a chlorine atom in a compound containing at least one acyl chloride and a nitro group to obtain hydroxyalkyl starch containing a ketone carbonyl group and a nitro group; the compound containing at least one acyl chloride and a nitro group is p-nitrophenyl chloroformate; (2) The hydroxyalkyl starch containing ketone carbonyl and nitro groups is subjected to a substitution reaction with a primary amine compound containing at least two amino groups, so that the ketone carbonyl group is replaced with an amide bond, thereby obtaining hydroxyalkyl starch containing amino groups; the primary amine compound containing at least two amino groups is 1,6-hexanediamine. (3) The amino-containing hydroxyalkyl starch is subjected to a substitution reaction with the chlorine atom contained in the photosensitizer to obtain the amphiphilic macromolecular compound coupled with the hydroxyalkyl starch-photosensitizer.

2. The preparation method according to claim 1, characterized in that, The photosensitizer is IR820, and IR820 is coupled to the hydroxyalkyl starch via an amide bond.

3. A nano-drug delivery system based on amphiphilic macromolecular compounds, characterized in that, The compound comprises an amphiphilic macromolecular compound prepared by the preparation method described in claim 1 or 2, and further comprises an anthraquinone antitumor drug, wherein the anthraquinone antitumor drug is emodin methyl ether.

4. The nano-drug delivery system as described in claim 3, characterized in that, The anthraquinone antitumor drug and the amphiphilic macromolecular compound are used to construct an oil-in-water nanoparticle system through ultrasonic emulsification, wherein the anthraquinone antitumor drug is the oil phase and the amphiphilic macromolecular compound is the aqueous phase.

5. An antitumor drug, characterized in that, It includes the nano-drug delivery system as described in claim 3 or 4 and pharmaceutically acceptable additives.

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