Drug delivery system of photoactive conjugated molecules as well as preparation method and application of drug delivery system

Abstract

The invention discloses a drug delivery system of photoactive conjugated molecules as well as a preparation method and application of the drug delivery system, and relates to the technical field of optical functional composite materials and biological medicines. The drug delivery system takes red blood cells as a carrier, and photoactive conjugated molecules and anti-tumor drugs are loaded in cells of the red blood cells. The preparation method provided by the invention is simple and easy to implement, effectively integrates the photosensitive performance of conjugated molecules and the oxygen carrier function of red blood cells, and has good biocompatibility and stability. After illumination, the conjugated molecules can effectively generate active oxygen in a tumor hypoxic microenvironment by virtue of the oxygen transport capacity of red blood cells, and meanwhile, the generated active oxygen can destroy the red blood cells, so that on-demand release of a loaded drug is realized, and multifunctional integration of phototherapy effect enhancement, on-demand drug release and imaging is achieved; the synergistic anti-tumor effect of photodynamic therapy and chemotherapy is realized. Therefore, the method has good practical value in the field of biomedicine.

Description

Technical Field

[0001] This invention relates to the fields of photofunctional composite materials and biomedical technology, and in particular to a drug delivery system for photoactive conjugated molecules, its preparation method, and its application. Background Technology

[0002] Photodynamic therapy (PDT) is a promising approach to treating malignant tumors based on photoactivated toxicity. Under external light irradiation, photosensitizers absorb photons and are excited, generating various reactive oxygen species (ROS) through energy or electron transfer. These ROS can kill cancer cells. However, the efficacy of PDT is severely limited by the hypoxic tumor microenvironment. Solid tumors, due to their rapid proliferation and disordered vascular systems, often have oxygen demands far exceeding the blood's oxygen supply, resulting in some tumors being in a hypoxic microenvironment. During PDT, oxygen consumption further exacerbates the tumor's hypoxic state, thereby promoting tumor proliferation, metastasis, and invasion, leading to poor prognosis.

[0003] Currently, various strategies exist to address the problem of hypoxia in the tumor environment. Directly delivering exogenous oxygen to the tumor, while a common method, faces challenges in the effective diffusion and release of oxygen within tumor tissue, and the biocompatibility or stability of the carrier may affect therapeutic efficacy. Strategies for in-situ oxygen generation, including chemical reactions, photocatalytic reactions, or the use of nanozymes, while promising, still face challenges such as demanding reaction conditions, unstable oxygen production efficiency, or adverse effects on normal tissues. Strategies such as inhibiting tumor cell respiration to reduce oxygen consumption and modulating the tumor microenvironment, such as normalizing the tumor vascular system or disrupting the tumor cell extracellular matrix, are theoretically feasible, but in practical applications, precisely controlling the metabolic processes of tumor cells or the microenvironment structure presents significant challenges and may induce adaptive changes in tumor cells, leading to limited therapeutic effects. While oxygen-independent type I PDT is an alternative, it currently faces technical bottlenecks such as quenching caused by aggregation and inefficient ROS production, limiting its widespread clinical application. Therefore, developing an integrated biomimetic material that can simultaneously address these challenges is crucial. Summary of the Invention

[0004] To address the technical problems existing in the prior art, embodiments of the present invention provide a drug delivery system for photoactive conjugated molecules, its preparation method, and its application. The technical solution is as follows:

[0005] A drug delivery system for photoactive conjugated molecules, wherein the drug delivery system uses red blood cells as carriers, and the red blood cells are loaded with photoactive conjugated molecules and antitumor drugs;

[0006] The photoactive conjugated molecule is selected from one or more of the following ①-⑨:

[0007]

[0008]

[0009] .

[0010] Optionally, the drug delivery system is prepared by co-incubating liposomes containing the antitumor drug and the photoactive conjugated molecule with red blood cells. After the liposomes bind to the cell membrane of the red blood cells, the photoactive conjugated molecule and the antitumor drug contained in the liposomes are released into the red blood cells.

[0011] Optionally, the liposomes include phospholipid molecules;

[0012] And / or, the phospholipid molecule is selected from one or more of the following: 1-palmitoyl-sn-glycerol-3-phosphocholine, 1-stearoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine, 1,2-dioleoyl-3-trimethylpropylammonium chloride, 1,2-dioleoyloxy-3-dimethylaminopropane, dodecyl dimethylammonium bromide, and dodecyl trimethylammonium bromide;

[0013] And / or, the molar ratio of the antitumor drug, the photoactive conjugated molecule and the phospholipid molecule is 1:(0-0.4):(1.2-2).

[0014] Optionally, the antitumor drug is selected from one or more of doxorubicin, camptothecin, methylprednisolone, gemcitabine, carmustine, dacarbazine, and etoposide;

[0015] And / or, the red blood cells are selected from one or more of mouse red blood cells, rabbit red blood cells, and sheep red blood cells;

[0016] And / or, the molar ratio of the antitumor drug to the photoactive conjugated molecule is 1:(0-0.4).

[0017] The method for preparing the drug delivery system of the aforementioned photoactive conjugated molecule includes:

[0018] Liposomes containing the antitumor drug and the photoactive conjugated molecule are co-incubated with red blood cells. After the liposomes bind to the cell membrane of the red blood cells, the photoactive conjugated molecule and the antitumor drug contained in the liposomes are released into the red blood cells.

[0019] Optionally, the preparation method includes:

[0020] (1) Dissolve antitumor drugs, photoactive conjugated molecules and phospholipid molecules in an organic solvent to prepare liposome stock solution, wherein the total mass concentration of the antitumor drugs, photoactive conjugated molecules and phospholipids is 5-10 mg / mL;

[0021] (2) The solvent in the liposome mother liquor obtained in step (1) is removed by rotary evaporation to obtain a lipid film;

[0022] (3) Add an aqueous solvent to the lipid film obtained in step (2) to hydrate it and obtain a liposome solution;

[0023] (4) The liposome solution obtained in step (3) is extruded using a liposome extruder and filtered to obtain a liposome dispersion with uniform particle size distribution.

[0024] (5) The liposome dispersion obtained in step (4) is co-incubated with erythrocytes to obtain a drug delivery system for erythrocytes modified with photoactive conjugated molecules.

[0025] Optionally, in step (1), the antitumor drug is selected from one or more of doxorubicin, camptothecin, methylprednisolone, gemcitabine, carmustine, dacarbazine, and etoposide;

[0026] And / or, the phospholipid molecule is selected from one or more of the following: 1-palmitoyl-sn-glycerol-3-phosphocholine, 1-stearoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine, 1,2-dioleoyl-3-trimethylpropylammonium chloride, 1,2-dioleoyloxy-3-dimethylaminopropane, dodecyl dimethylammonium bromide, and dodecyl trimethylammonium bromide;

[0027] And / or, the molar ratio of the antitumor drug, the photoactive conjugated molecule, and the phospholipid molecule is 1:(0-0.4):(1.2-2);

[0028] And / or, the organic phase solvent is selected from one or more of chloroform, dichloromethane, and methanol;

[0029] And / or, in step (2), the rotary evaporation is carried out at a temperature of 35-45°C;

[0030] And / or, in step (2), after the rotary evaporation, the film surface is purged with nitrogen or left to stand overnight;

[0031] And / or, in step (3), the aqueous solvent is selected from ultrapure water, phosphate buffer, and physiological saline;

[0032] And / or, in step (3), the hydration is carried out under a vortex at 30-50°C;

[0033] And / or, in step (3), after hydration, sonicate for 5-15 minutes.

[0034] Optionally, in step (4), the filtration is a gradient through a polycarbonate membrane;

[0035] And / or, in step (4), the pore size of the polycarbonate membrane is selected from one or more of 1000 nm, 800 nm, 400 nm, 200 nm and 100 nm;

[0036] And / or, in step (4), each aperture is extruded 11-21 times, and the number of times is odd.

[0037] And / or, in step (5), the red blood cells are selected from one or more of mouse red blood cells, rabbit red blood cells, and sheep red blood cells;

[0038] And / or, in step (5), the volume concentration of the red blood cells is 2%-4%;

[0039] And / or, in step (5), the volume ratio of the liposome dispersion to the suspension of red blood cells is 1:5-1:12;

[0040] And / or, in step (5), the co-incubation time is 1-6 hours.

[0041] The application of the aforementioned drug delivery system of photoactive conjugated molecules in the preparation of drugs for treating tumors.

[0042] A pharmaceutical composition for treating tumors, the pharmaceutical composition comprising: a drug delivery system for the photoactive conjugated molecule and pharmaceutically acceptable excipients.

[0043] The beneficial effects of the technical solutions provided in the embodiments of the present invention include at least the following:

[0044] This invention discloses a drug-loaded system for erythrocytes modified with photoactive conjugated molecules, its preparation method, and its applications. The preparation method involves combining hydrophobic photosensitizing conjugated molecules with phospholipids to prepare liposomes loaded with hydrophobic drugs. The hydrophobic photosensitizing conjugated molecules and the hydrophobic drug aggregate within the liposomes, and then assemble with erythrocytes through a membrane fusion mechanism to prepare a photofunctional drug-loaded system. The preparation method provided by this invention is simple and easy to implement, effectively integrating the photosensitizing properties of the conjugated molecules with the oxygen-carrying function of erythrocytes, exhibiting good biocompatibility and stability. After light irradiation, utilizing the oxygen transport capacity of erythrocytes, the conjugated molecules can effectively generate reactive oxygen species (ROS) in the hypoxic microenvironment of tumors. Simultaneously, the generated ROS can destroy erythrocytes, achieving on-demand release of the loaded drug, thus achieving a multifunctional integration of enhanced phototherapy efficacy, on-demand drug release, and imaging, realizing a synergistic anti-tumor effect of photodynamic therapy and chemotherapy. Therefore, it has significant practical value in the biomedical field.

[0045] Compared with existing technologies, the advantages and beneficial effects of this invention are as follows: Traditional photodynamic therapy (PDT) faces the problem of limited ROS generation efficiency due to tumor microenvironment hypoxia. Red blood cells, as natural oxygen carriers, possess excellent self-oxygenation capacity, long circulation characteristics, and good biocompatibility. This invention utilizes red blood cells to construct a biomimetic drug delivery system. Its lipid bilayer membrane structure can be efficiently assembled with artificial liposomes through membrane fusion strategies, enabling convenient loading of hydrophobic drugs or functional molecules. It simultaneously encapsulates photoactive conjugated molecules and antitumor drugs at the single-cell scale, constructing a multifunctional synergistic system. The ROS generated by photoactivation can both kill tumor cells and trigger a ROS-responsive drug release mechanism, overcoming the tumor hypoxia barrier. Simultaneously, the spatiotemporal coupling of photodynamic therapy and chemotherapy can significantly enhance the antitumor effect. Attached Figure Description

[0046] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0047] Figure 1 These are cryo-transmission electron microscope (Cryo-TEM) images of liposomes provided in Example 1 of this invention;

[0048] Figure 2 This is a liposome hydration particle size distribution diagram provided in Example 1 of the present invention;

[0049] Figure 3 This is a graph of liposome ROS generation capacity quantified by the change trend of DCF fluorescence intensity over time, provided in Example 1 of the present invention.

[0050] Figure 4 These are confocal fluorescence microscopy images of the drug delivery system for erythrocytes modified with photoactive conjugated molecules provided in Example 1 of this invention;

[0051] Figure 5 This is a bar chart showing the survival rate of HeLa cells treated with a drug delivery system for erythrocytes modified with photoactive conjugated molecules, as provided in Example 1 of this invention.

[0052] Figure 6 This is a liposome hydration particle size distribution diagram provided in Example 2 of the present invention;

[0053] Figure 7 This is a graph of liposome ROS generation capacity quantified by the change trend of DCF fluorescence intensity over time, provided in Example 2 of the present invention.

[0054] Figure 8 These are confocal fluorescence microscopy images of the drug delivery system for erythrocytes modified with photoactive conjugated molecules provided in Example 2 of this invention;

[0055] Figure 9 This is a liposome hydration particle size distribution diagram provided in Example 3 of the present invention;

[0056] Figure 10 This is a graph of liposome ROS generation capacity quantified by the trend of DCF fluorescence intensity changing over time, provided in Example 3 of the present invention.

[0057] Figure 11 These are confocal fluorescence microscopy images of the drug delivery system for erythrocytes modified with photoactive conjugated molecules provided in Example 3 of this invention;

[0058] Figure 12 This is a liposome hydration particle size distribution diagram provided in Example 4 of the present invention;

[0059] Figure 13 This is a graph of liposome ROS generation capacity quantified by the trend of DCF fluorescence intensity changing over time, provided in Example 4 of the present invention.

[0060] Figure 14 These are confocal fluorescence microscopy images of the drug delivery system for erythrocytes modified with photoactive conjugated molecules provided in Example 4 of this invention;

[0061] Figure 15 This is a liposome hydration particle size distribution diagram provided in Example 5 of the present invention;

[0062] Figure 16 This is a graph of liposome ROS generation capacity quantified by the trend of DCF fluorescence intensity changing over time, provided in Example 5 of the present invention.

[0063] Figure 17These are confocal fluorescence microscopy images of the drug delivery system for erythrocytes modified with photoactive conjugated molecules provided in Example 5 of this invention;

[0064] Figure 18 This is a bar chart showing the survival rate of HeLa cells treated with a single antitumor drug, provided in Comparative Example 1 of the present invention.

[0065] Figure 19 This is a bar chart showing the survival rate of HeLa cells treated with a single photoactive conjugated molecule provided in Comparative Example 2 of the present invention. Detailed Implementation

[0066] The technical solution of the present invention will now be described with reference to the accompanying drawings.

[0067] In embodiments of the present invention, words such as "exemplarily," "for example," etc., are used to indicate that something is an example, illustration, or description. Any embodiment or design described as "exemplary" in the present invention should not be construed as being more preferred or advantageous than other embodiments or designs. Specifically, the use of the word "exemplary" is intended to present the concept in a concrete manner. Furthermore, in embodiments of the present invention, the meaning expressed by "and / or" can be both, or either one.

[0068] In this invention, liposomes, also known as lipid microspheres or liquid crystal microcapsules, are a novel dosage form similar to microcapsules. They are microvesicles formed by encapsulating drugs within a lipid bilayer. Because their structure resembles a biological membrane, they are also called artificial biological membranes.

[0069] The inventors have discovered that self-assembly technology is a powerful method for constructing multifunctional biomimetic materials. Red blood cells are naturally biocompatible oxygen carriers. Through a fusion mechanism, liposomes assemble with red blood cells, loading photosensitizers or drugs onto the cells. Furthermore, chemotherapeutic drugs can be stably assembled with liposomes, ultimately allowing a single red blood cell to encapsulate multiple therapeutic agents. Therefore, self-assembly technology can be used to prepare multifunctional biomimetic materials that enhance phototherapy efficacy, provide on-demand drug release, and enable imaging, which has practical value in tumor treatment.

[0070] The purpose of this invention is to overcome the shortcomings of photodynamic therapy, such as tumor microenvironment hypoxia, photosensitizer quenching, and uncontrollable treatment process, and to provide a drug delivery system for red blood cells modified with photoactive conjugated molecules and its preparation method, so as to realize multiple functions of photodynamic therapy, on-demand drug release, and fluorescence imaging.

[0071] The technical solution of this invention involves preparing photoactive drug-loaded liposomes by combining antitumor drugs, photoactive conjugated molecules, and phospholipid molecules. These liposomes are then assembled with red blood cells to create a biomimetic material with self-oxygenating capabilities. Under light irradiation, the liposomes can release drugs on demand, synergistically enhancing the therapeutic efficacy of chemotherapy and photodynamic therapy. Furthermore, by adjusting the types and ratios of antitumor drugs, photoactive conjugated molecules, and phospholipid molecules, this invention can prepare liposome solutions that simultaneously encapsulate hydrophobic antitumor drug molecules and photoactive conjugated molecules. After co-incubation with red blood cells, a drug-loaded system modified with photoactive conjugated molecules on red blood cells is obtained. Under light irradiation, the photoactive conjugated molecules within this red blood cell drug-loaded system sensitize the loaded oxygen, efficiently generating reactive oxygen species (ROS), which disrupt the structure of the red blood cells, thereby achieving the on-demand release of the antitumor drug.

[0072] For example, in one specific embodiment of the present invention, the specific steps are as follows:

[0073] (1) Mix antitumor drugs, photoactive conjugated molecules and phospholipid molecules in a certain proportion and dissolve them in an organic solvent to prepare a liposome stock solution with a concentration of 5-10 mg / mL;

[0074] (2) Add the liposome mother liquor obtained in step (1) into a pear-shaped flask, remove the solvent by rotary evaporation at 35-45℃, purge the membrane surface with nitrogen or let it stand overnight to obtain a dry liposome membrane.

[0075] (3) Add an aqueous solvent to the lipid membrane obtained in step (2), hydrate it by vortexing at 40°C in a water bath, and sonicate for 5-15 minutes to obtain a liposome solution;

[0076] (4) The liposome solution obtained in step (3) is extruded using a liposome extruder and passed through a polycarbonate membrane with a certain pore size in a gradient manner to obtain a liposome dispersion with uniform particle size distribution.

[0077] (5) The liposome dispersion obtained in step (4) is co-incubated with red blood cells in a certain proportion to obtain a drug delivery system for red blood cells modified with photoactive conjugated molecules.

[0078] Further, the antitumor drug mentioned in step (1) is one or more of doxorubicin, camptothecin, methylprednisolone, gemcitabine, carmustine, dacarbazine, and etoposide.

[0079] Furthermore, the photoactive conjugated molecule mentioned in step (1) is one or more of the ①-⑨ type molecules.

[0080] Further, the phospholipid molecule mentioned in step (1) is one or more of the following: 1-palmitoyl-sn-glycerol-3-phosphocholine, 1-stearoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine, 1,2-dioleoyl-3-trimethylpropylammonium chloride, 1,2-dioleoyloxy-3-dimethylaminopropane, dodecyl dimethylammonium bromide, and dodecyl trimethylammonium bromide.

[0081] Further, the molar ratio of the antitumor drug, photoactive conjugated molecule, and phospholipid molecule in step (1) is 1:(0-0.4):(1.2-2).

[0082] Further, the organic solvent mentioned in step (1) is one or more of chloroform, dichloromethane, and methanol.

[0083] Furthermore, the aqueous solvent mentioned in step (3) is one of ultrapure water, phosphate buffer, or physiological saline.

[0084] Further, the polycarbonate membrane in step (4) has one or more of the following pore sizes: 1000 nm, 800 nm, 400 nm, 200 nm, and 100 nm, and each pore size is extruded 11-21 times, with an odd number of times.

[0085] Furthermore, the red blood cells mentioned in step (5) are either rabbit red blood cells or mouse red blood cells, with a cell volume concentration of 2%-4%, and the volume ratio of liposome dispersion to red blood cells is 1:5-1:12.

[0086] Furthermore, the co-incubation time in step (5) is 1-6 hours.

[0087] The drug delivery system for photoactive conjugated molecules modified into erythrocytes obtained by the method of this invention has the ability to supply its own oxygen and can generate reactive oxygen species under light. It triggers the release of drugs in response to reactive oxygen species during photodynamic therapy, and enhances the efficacy of phototherapy in conjunction with chemotherapy.

[0088] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0089] Unless otherwise specified, the experimental methods described in the following embodiments are conventional experimental methods well known to those skilled in the art, and are performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Where specific conditions are not specified in the experimental methods, they are generally operated according to conventional conditions.

[0090] Unless otherwise specified, all materials and reagents described in the following examples are commercially available.

[0091] Example 1

[0092] (1) Mix 23.16 mg doxorubicin (available from Shanghai Mairui Biochemical Technology Co., Ltd.), 33.23 mg photoactive conjugated molecule ①, 31.70 mg 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.), and 11.91 mg 1,2-dioleoyl-3-trimethylpropylammonium chloride (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.) (the molar ratio of antitumor drug, photoactive conjugated molecule, and phospholipid molecule is 1:0.4:1.4), dissolve in 10 mL of chloroform / methanol solvent with a volume ratio of 9:1, and prepare a liposome stock solution with a concentration of 10 mg / mL; the chemical formula of photoactive conjugated molecule ① is as follows:

[0093] ;

[0094] (2) Add the liposome mother liquor obtained in step (1) into a pear-shaped flask, remove the solvent by rotary evaporation at 40°C, and purge the surface of the film with nitrogen to obtain a dry liposome film.

[0095] (3) Add ultrapure water to the lipid membrane obtained in step (2), vortex in a water bath at 40°C for 30 minutes, and sonicate for 15 minutes to obtain a liposome solution;

[0096] (4) The liposome solution obtained in step (3) is extruded through a liposome extruder and passed through polycarbonate membranes of 800, 400 and 200 nm in sequence, with each pore size being extruded 21 times to obtain a liposome dispersion with uniform particle size distribution.

[0097] (5) The liposome dispersion obtained in step (4) is incubated with 4% mouse red blood cells (available from Shanghai Yuanye Biotechnology Co., Ltd.) at a volume ratio of 1:10 for 2 hours to obtain a drug delivery system for red blood cells modified with photoactive conjugated molecules.

[0098] Performance testing:

[0099] The ROS generation capacity of the above photoactive drug-loaded liposomes (liposomes prepared in step (4)) was tested using the following method: DCFH was used as the reactive oxygen species fluorescent probe. 10 μL of an aqueous solution of liposomes containing 480 μg / mL of photoactive conjugated molecules was added to 1 mL of probe solution (0.04 mM). A light intensity of 10.5 mW / cm² was used. 2The solution was irradiated with a white light source, and the fluorescence spectrum under 488 nm excitation was recorded every minute during the irradiation process to monitor the change in fluorescence intensity at 522 nm.

[0100] The antitumor drug release capacity of the drug delivery system modified with the above-mentioned photoactive conjugated molecules on erythrocytes was tested. The specific method is as follows: using a confocal microscope, fluorescence images were recorded for specific channels of the photoactive conjugated molecules (λex=405 nm, λem=460-520 nm).

[0101] The tumor cell killing ability of the above-mentioned photoactive conjugated molecule modified erythrocyte drug delivery system was tested. The specific method is as follows: 100 μL of HeLa cells (5 × 10⁶ cells / year) were loaded onto the erythrocytes. 4 HeLa cells (available from the Cell Bank of the Chinese Academy of Sciences) were seeded in 96-well plates. Subsequently, the DMEM medium containing 10% fetal bovine serum was replaced with a drug-loaded system of 100 μL of photoactive conjugated erythrocytes. One group of HeLa cells was cultured at 0.1 W / cm². 2 One group of cells was irradiated at a specific intensity for 5 min, followed by incubation for 24 hours; another group of cells was incubated for 24 hours without irradiation. After incubation, HeLa cells were washed at least twice with PBS, then the culture medium was replaced with 0.5 mg / mL MTT solution, and incubated for 4 hours. After removing the MTT solution, 100 μL of DMSO was added, and absorbance was measured at 550 nm to assess cell viability.

[0102] Experimental results:

[0103] See the experimental results. Figures 1 to 5 .

[0104] Figure 1 This is a cryo-transmission electron microscope (Cryo-TEM) image of the liposomes prepared in step (4) of Example 1. From Figure 1 As can be seen, the liposomes have a uniform particle size of about 150 nm and are well dispersed.

[0105] Figure 2 This is a hydrated particle size distribution diagram of the liposomes prepared in step (4) of Example 1. From Figure 2 As can be seen, the hydrated particle size of the liposomes is 150.0 nm and the polydispersity index is 0.166, indicating that the liposomes have a narrow particle size distribution and good uniformity.

[0106] Figure 3 This is the emission intensity curve of DCF in the photoactive drug-loaded liposomes containing the DCFH reactive oxygen species fluorescent probe prepared in Example 1 under light irradiation. Figure 3As can be seen, the fluorescence intensity of the photoactive drug-loaded liposomes increased significantly compared to the control group, confirming that the drug-loaded liposomes can effectively generate reactive oxygen species under photoexcitation.

[0107] Figure 4 This is a confocal fluorescence microscopy image of the drug delivery system for erythrocytes modified with photoactive conjugated molecules prepared in Example 1. Figure 4 As can be seen, the photoactive conjugated molecule ① has entered the interior of the erythrocyte and exhibits a random, dotted distribution. This result demonstrates that the drug-loaded liposomes have successfully fused with the erythrocyte membrane and released their contents into the cytoplasm.

[0108] Figure 5 This is a bar chart showing the survival rate of HeLa cells treated with the drug delivery system for photoactive conjugated molecules modified into erythrocytes prepared in Example 1. From... Figure 5 As can be seen, under no-light conditions, over 90% of HeLa cells remained viable after co-culturing with the drug-loaded system, confirming the system's excellent biocompatibility. With light exposure, the drug-loaded system significantly reduced the cell-killing effect on HeLa cells, decreasing cell viability to 8.4%. These results demonstrate that the drug-loaded system modified with photoactive conjugated molecules on erythrocytes can combine photodynamic therapy and chemotherapy to achieve a good anti-tumor effect.

[0109] Example 2

[0110] (1) Mix 19.80 mg camptothecin (available from Shanghai Mairui Biochemical Technology Co., Ltd.), 20.95 mg photoactive conjugated molecule ②, 40.82 mg 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.), and 18.42 mg 1,2-dioleoyloxy-3-dimethylaminopropane (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.) (the molar ratio of antitumor drug, photoactive conjugated molecule, and phospholipid molecule is 1:0.3:1.5), dissolve in 10 mL of chloroform / methanol solvent with a volume ratio of 9:1, and prepare a liposome stock solution with a concentration of 10 mg / mL; the chemical formula of photoactive conjugated molecule ② is as follows:

[0111] ;

[0112] (2) Add the liposome mother liquor obtained in step (1) into a pear-shaped flask, remove the solvent by rotary evaporation at 40°C, and purge the surface of the film with nitrogen to obtain a dry liposome film.

[0113] (3) Add ultrapure water to the lipid membrane obtained in step (2), vortex in a water bath at 40°C for 30 minutes, and sonicate for 15 minutes to obtain a liposome solution;

[0114] (4) The liposome solution obtained in step (3) is extruded through a liposome extruder and passed through polycarbonate membranes of 800, 400 and 200 nm in sequence, with each pore size being extruded 21 times to obtain a liposome dispersion with uniform particle size distribution.

[0115] (5) The liposome dispersion obtained in step (4) was incubated with 4% mouse red blood cells at a volume ratio of 1:10 for 2 hours to obtain a drug delivery system for red blood cells modified with photoactive conjugated molecules.

[0116] The corresponding performance tests were performed according to the method in Example 1.

[0117] Experimental results:

[0118] See the experimental results. Figures 6 to 8 .

[0119] The liposomes have a particle size of approximately 180 nm. The survival rate of HeLa cells treated with a drug carrier modified with photoactive conjugated molecules on red blood cells after light exposure was 9.6%.

[0120] Figure 6 This is a hydration particle size distribution diagram of the liposomes prepared in Example 2. From... Figure 6 As can be seen, the hydrated particle size of the liposomes is 199.9 nm and the polydispersity index is 0.154, indicating that the liposomes have a narrow particle size distribution and good uniformity.

[0121] Figure 7 This is the emission intensity curve of DCF in the photoactive drug-loaded liposomes containing the DCFH reactive oxygen species fluorescent probe prepared in Example 2 under light irradiation. Figure 7 As can be seen, the fluorescence intensity of the photoactive drug-loaded liposomes increased significantly compared to the control group, confirming that the drug-loaded liposomes can effectively generate reactive oxygen species under photoexcitation.

[0122] Figure 8 This is a confocal fluorescence microscopy image of the drug delivery system for erythrocytes modified with photoactive conjugated molecules prepared in Example 2. Figure 8 As can be seen, photoactive conjugated molecule ② has entered the interior of erythrocytes and exhibits a random, dotted distribution. This result demonstrates that the drug-loaded liposomes have successfully fused with the erythrocyte membrane and released their contents into the cytoplasm.

[0123] Example 3

[0124] (1) Mix 32.42 mg doxorubicin (available from Shanghai Mairui Biochemical Technology Co., Ltd.), 13.08 mg photoactive conjugated molecule ③, 45.33 mg 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.), and 9.17 mg dodecyl dimethyl ammonium bromide (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.) (the molar ratio of antitumor drug, photoactive conjugated molecule, and phospholipid molecule is 1:0.4:1.4), dissolve in 10 mL of chloroform / methanol solvent with a volume ratio of 9:1, and prepare a liposome stock solution with a concentration of 10 mg / mL; the chemical formula of photoactive conjugated molecule ③ is as follows:

[0125] ;

[0126] (2) Add the liposome mother liquor obtained in step (1) into a pear-shaped flask, remove the solvent by rotary evaporation at 40°C, and purge the surface of the film with nitrogen to obtain a dry liposome film.

[0127] (3) Add ultrapure water to the lipid membrane obtained in step (2), vortex in a water bath at 40°C for 30 minutes, and sonicate for 15 minutes to obtain a liposome solution;

[0128] (4) The liposome solution obtained in step (3) is extruded through a liposome extruder and passed through polycarbonate membranes of 800, 400 and 200 nm in sequence, with each pore size being extruded 21 times to obtain a liposome dispersion with uniform particle size distribution.

[0129] (5) The liposome dispersion obtained in step (4) was incubated with 4% mouse red blood cells at a volume ratio of 1:10 for 2 hours to obtain a drug delivery system for red blood cells modified with photoactive conjugated molecules.

[0130] The corresponding performance tests were performed according to the method in Example 1.

[0131] Experimental results:

[0132] The liposomes have a particle size of approximately 150 nm. The survival rate of HeLa cells treated with a drug delivery system that modifies red blood cells with photoactive conjugated molecules after light exposure was 7.4%.

[0133] See the experimental results. Figures 9 to 11 .

[0134] Figure 9 This is a hydration particle size distribution diagram of the liposomes prepared in Example 3. From... Figure 9 As can be seen, the hydrated particle size of the liposomes is 169.3 nm and the polydispersity index is 0.220, indicating that the liposomes have a narrow particle size distribution and good uniformity.

[0135] Figure 10 This is the emission intensity curve of DCF in the photoactive drug-loaded liposomes containing the DCFH reactive oxygen species fluorescent probe prepared in Example 3 under light irradiation. Figure 10 As can be seen, the fluorescence intensity of the photoactive drug-loaded liposomes increased significantly compared to the control group, confirming that the drug-loaded liposomes can effectively generate reactive oxygen species under photoexcitation.

[0136] Figure 11 This is a confocal fluorescence microscopy image of the drug delivery system for erythrocytes modified with photoactive conjugated molecules prepared in Example 3. Figure 11 As can be seen, photoactive conjugated molecule ③ has entered the interior of erythrocytes and exhibits a random, dotted distribution. This result demonstrates that the drug-loaded liposomes have successfully fused with the erythrocyte membrane and released their contents into the cytoplasm.

[0137] Example 4

[0138] (1) Mix 24.81 mg doxorubicin (available from Shanghai Mairui Biochemical Technology Co., Ltd.), 9.36 mg photoactive conjugated molecule ④, 33.96 mg 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.), and 31.87 mg 1,2-dioleoyl-3-trimethylpropylammonium chloride (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.) (the molar ratio of antitumor drug, photoactive conjugated molecule, and phospholipid molecule is 1:0.4:2), and dissolve in 10 mL of chloroform / methanol solvent with a volume ratio of 9:1 to prepare a liposome stock solution with a concentration of 10 mg / mL; the chemical formula of photoactive conjugated molecule ④ is as follows:

[0139] ;

[0140] (2) Add the liposome mother liquor obtained in step (1) into a pear-shaped flask, remove the solvent by rotary evaporation at 40°C, and purge the surface of the film with nitrogen to obtain a dry liposome film.

[0141] (3) Add ultrapure water to the lipid membrane obtained in step (2), vortex in a water bath at 40°C for 30 minutes, and sonicate for 15 minutes to obtain a liposome solution;

[0142] (4) The liposome solution obtained in step (3) is extruded through a liposome extruder and passed through polycarbonate membranes of 800, 400 and 200 nm in sequence, with each pore size being extruded 21 times to obtain a liposome dispersion with uniform particle size distribution.

[0143] (5) The liposome dispersion obtained in step (4) was incubated with 4% mouse red blood cells at a volume ratio of 1:10 for 2 hours to obtain a drug delivery system for red blood cells modified with photoactive conjugated molecules.

[0144] The corresponding performance tests were performed according to the method in Example 1.

[0145] Experimental results:

[0146] The liposomes have a particle size of approximately 170 nm. The survival rate of HeLa cells treated with a drug delivery system that modifies red blood cells with photoactive conjugated molecules after light exposure was 5.6%.

[0147] See the experimental results. Figures 12 to 14 .

[0148] Figure 12 This is a hydration particle size distribution diagram of the liposomes prepared in Example 4. From... Figure 12 As can be seen, the hydrated particle size of the liposomes is 181.4 nm and the polydispersity index is 0.173, indicating that the liposomes have a narrow particle size distribution and good uniformity.

[0149] Figure 13 This is the emission intensity curve of DCF under illumination for the photoactive drug-loaded liposomes containing the DCFH reactive oxygen species fluorescent probe prepared in Example 4. Figure 13 As can be seen, the fluorescence intensity of the photoactive drug-loaded liposomes increased significantly compared to the control group, confirming that the drug-loaded liposomes can effectively generate reactive oxygen species under photoexcitation.

[0150] Figure 14 This is a confocal fluorescence microscopy image of the drug delivery system for erythrocytes modified with photoactive conjugated molecules prepared in Example 4. Figure 14 As can be seen, photoactive conjugated molecule ④ has entered the interior of the erythrocyte and exhibits a random, dotted distribution. This result demonstrates that the drug-loaded liposomes have successfully fused with the erythrocyte membrane and released their contents into the cytoplasm.

[0151] Example 5

[0152] (1) Mix 31.06 mg doxorubicin (available from Shanghai Mairui Biochemical Technology Co., Ltd.), 10.46 mg photoactive conjugated molecule ⑤, 42.52 mg 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.), and 15.97 mg 1,2-dioleoyl-3-trimethylpropylammonium chloride (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.) (the molar ratio of antitumor drug, photoactive conjugated molecule, and phospholipid molecule is 1:0.4:1.4), dissolve in 10 mL of chloroform / methanol solvent with a volume ratio of 9:1, and prepare a liposome stock solution with a concentration of 10 mg / mL; the chemical formula of photoactive conjugated molecule ⑤ is as follows:

[0153] ;

[0154] (2) Add the liposome mother liquor obtained in step (1) into a pear-shaped flask, remove the solvent by rotary evaporation at 40°C, and purge the surface of the film with nitrogen to obtain a dry liposome film.

[0155] (3) Add ultrapure water to the lipid membrane obtained in step (2), vortex in a water bath at 40°C for 30 minutes, and sonicate for 15 minutes to obtain a liposome solution;

[0156] (4) The liposome solution obtained in step (3) is extruded through a liposome extruder and passed through polycarbonate membranes of 800, 400, 200 and 100 nm in sequence, extruding 17 times for each pore size to obtain a liposome dispersion with uniform particle size distribution.

[0157] (5) The liposome dispersion obtained in step (4) was incubated with 4% mouse red blood cells at a volume ratio of 1:10 for 2 hours to obtain a drug delivery system for red blood cells modified with photoactive conjugated molecules.

[0158] The corresponding performance tests were performed according to the method in Example 1.

[0159] Experimental results:

[0160] The liposomes have a particle size of approximately 150 nm. The survival rate of HeLa cells treated with a drug delivery system that modifies red blood cells with photoactive conjugated molecules after light exposure was 9.8%.

[0161] See the experimental results. Figures 15 to 17 .

[0162] Figure 15 This is a hydrated particle size distribution diagram of the liposomes prepared in Example 5. From... Figure 15 As can be seen, the hydrated particle size of the liposomes is 156.5 nm and the polydispersity index is 0.131, indicating that the liposomes have a narrow particle size distribution and good uniformity.

[0163] Figure 16 This is the emission intensity curve of DCF in the photoactive drug-loaded liposomes containing the DCFH reactive oxygen species fluorescent probe prepared in Example 5 under light irradiation. Figure 16 As can be seen, the fluorescence intensity of the photoactive drug-loaded liposomes increased significantly compared to the control group, confirming that the drug-loaded liposomes can effectively generate reactive oxygen species under photoexcitation.

[0164] Figure 17 This is a confocal fluorescence microscopy image of the drug delivery system for erythrocytes modified with photoactive conjugated molecules prepared in Example 5. Figure 17 As can be seen, photoactive conjugated molecule ⑤ has entered the interior of erythrocytes and exhibits a random, dotted distribution. This result demonstrates that the drug-loaded liposomes have successfully fused with the erythrocyte membrane and released their contents into the cytoplasm.

[0165] The inventors also conducted corresponding tests on other types of antitumor drugs, photoactive conjugated molecules, and phospholipid molecules mentioned in this specification to obtain photoactive conjugated molecule-modified red blood cell drug delivery systems. The experimental results were similar to those in Examples 1 to 5, and will not be repeated here.

[0166] Comparative Example 1

[0167] Weigh 23.16 mg of doxorubicin (available from Shanghai Mairui Biochemical Technology Co., Ltd.), 31.70 mg of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.), and 11.91 mg of 1,2-dioleoyl-3-trimethylpropylammonium chloride (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.) and mix them (the molar ratio of antitumor drug to phospholipid molecule is 1:1.4). Dissolve them in 10 mL of chloroform / methanol (9:1) and prepare a red blood cell drug loading system loaded only with the antitumor drug doxorubicin according to steps (2)-(5) of Example 1.

[0168] The corresponding performance tests were performed according to the method in Example 1.

[0169] Experimental results:

[0170] See the experimental results. Figure 18 .

[0171] Figure 18 This is a bar chart showing the survival rate of HeLa cells treated with the antitumor drug alone, as provided in Comparative Example 1. From... Figure 18 As can be seen, the HeLa cells co-cultured with the anti-tumor drug maintained a cell viability of approximately 80% regardless of whether they were exposed to light. The results indicate that, compared to the anti-tumor drug alone, the drug-loaded system constructed in this invention has a significantly enhanced killing effect on tumor cells.

[0172] Comparative Example 2

[0173] Weigh 33.23 mg of photoactive conjugated molecule ①, 31.70 mg of 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.), and 11.91 mg of 1,2-dioleoyl-3-trimethylpropylammonium chloride (available from Beijing Huawi Ruike Chemical Technology Co., Ltd.) and mix them (the molar ratio of photoactive conjugated molecule to phospholipid molecule is 0.4:1.4). Dissolve them in 10 mL of chloroform / methanol (9:1) and prepare a red blood cell system loaded only with photoactive conjugated molecule according to steps (2)-(5) of Example 1.

[0174] The corresponding performance tests were performed according to the method in Example 1.

[0175] Experimental results:

[0176] See the experimental results. Figure 19 .

[0177] Figure 19 This is a bar chart showing the survival rate of HeLa cells treated with the red blood cell system loaded only with photoactive conjugated molecules provided in Comparative Example 2. Figure 19 As can be seen, under no-light conditions, over 90% of HeLa cells survived. With light exposure, the cell survival rate decreased to 40.5%, significantly higher than the cell survival rate after treatment with the drug-loaded system in Example 1. The results show that, compared to the erythrocyte system loaded only with photoactive conjugated molecules, the drug-loaded system constructed in this invention has a significantly enhanced killing effect on tumor cells.

[0178] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A photoactive conjugated molecule drug delivery system, characterized in that, The drug delivery system uses red blood cells as carriers, and the red blood cells are loaded with photoactive conjugated molecules and antitumor drugs. The photoactive conjugated molecule is selected from one or more of the following ①-⑨: ； ； 。 2. The photoactive conjugated molecule drug delivery system of claim 1, wherein, The drug delivery system is prepared by co-incubating liposomes containing the antitumor drug and the photoactive conjugated molecule with red blood cells. After the liposomes bind to the cell membrane of the red blood cells, the photoactive conjugated molecule and the antitumor drug contained in the liposomes are released into the red blood cells.

3. The photoactive conjugated molecule drug delivery system of claim 2, wherein, The liposomes include phospholipid molecules; And / or, the phospholipid molecule is selected from one or more of the following: 1-palmitoyl-sn-glycerol-3-phosphocholine, 1-stearoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine, 1,2-dioleoyl-3-trimethylpropylammonium chloride, 1,2-dioleoyloxy-3-dimethylaminopropane, dodecyl dimethylammonium bromide, and dodecyl trimethylammonium bromide; And / or, the molar ratio of the antitumor drug, the photoactive conjugated molecule and the phospholipid molecule is 1:(0-0.4):(1.2-2).

4. The photoactive conjugated molecule drug delivery system of claim 1, wherein, The antitumor drug is selected from one or more of doxorubicin, camptothecin, methylprednisolone, gemcitabine, carmustine, dacarbazine, and etoposide. And / or, the red blood cells are selected from one or more of mouse red blood cells, rabbit red blood cells, and sheep red blood cells; And / or, the molar ratio of the antitumor drug to the photoactive conjugated molecule is 1:(0-0.4).

5. The method of claim 1-4, wherein the method is for the preparation of a drug delivery system of a photoactive conjugated molecule, characterized in that, The preparation method includes: Liposomes containing the antitumor drug and the photoactive conjugated molecule are co-incubated with red blood cells. After the liposomes bind to the cell membrane of the red blood cells, the photoactive conjugated molecule and the antitumor drug contained in the liposomes are released into the red blood cells.

6. The production method according to claim 5, wherein The preparation method includes: (1) Dissolve antitumor drugs, photoactive conjugated molecules and phospholipid molecules in an organic solvent to prepare liposome stock solution, wherein the total mass concentration of the antitumor drugs, photoactive conjugated molecules and phospholipids is 5-10 mg / mL; (2) The solvent in the liposome mother liquor obtained in step (1) is removed by rotary evaporation to obtain a lipid film; (3) Add an aqueous solvent to the lipid film obtained in step (2) to hydrate it and obtain a liposome solution; (4) The liposome solution obtained in step (3) is extruded using a liposome extruder and filtered to obtain a liposome dispersion with uniform particle size distribution. (5) The liposome dispersion obtained in step (4) is co-incubated with erythrocytes to obtain a drug delivery system for erythrocytes modified with photoactive conjugated molecules.

7. The production method according to claim 6, characterized by, In step (1), the antitumor drug is selected from one or more of doxorubicin, camptothecin, methylprednisolone, gemcitabine, carmustine, dacarbazine, and etoposide; And / or, the phospholipid molecule is selected from one or more of the following: 1-palmitoyl-sn-glycerol-3-phosphocholine, 1-stearoyl-sn-glycerol-3-phosphocholine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylethanolamine, 1,2-dioleoyl-sn-glycerol-3-phosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphatidylcholine, 1,2-dioleoyl-3-trimethylpropylammonium chloride, 1,2-dioleoyloxy-3-dimethylaminopropane, dodecyl dimethylammonium bromide, and dodecyl trimethylammonium bromide; And / or, the molar ratio of the antitumor drug, the photoactive conjugated molecule, and the phospholipid molecule is 1:(0-0.4):(1.2-2); And / or, the organic phase solvent is selected from one or more of chloroform, dichloromethane, and methanol; And / or, in step (2), the rotary evaporation is carried out at a temperature of 35-45°C; And / or, in step (2), after the rotary evaporation, the film surface is purged with nitrogen or left to stand overnight; And / or, in step (3), the aqueous solvent is selected from ultrapure water, phosphate buffer, and physiological saline; And / or, in step (3), the hydration is carried out under a vortex at 30-50°C; And / or, in step (3), after hydration, sonicate for 5-15 minutes.

8. The preparation method according to claim 6, characterized in that, In step (4), the filtration is performed by gradient passing through a polycarbonate membrane; And / or, in step (4), the pore size of the polycarbonate membrane is selected from one or more of 1000 nm, 800 nm, 400 nm, 200 nm and 100 nm; And / or, in step (4), the number of extrusions per aperture is 11-21 times and is an odd number; And / or, in step (5), the red blood cells are selected from one or more of mouse red blood cells, rabbit red blood cells, and sheep red blood cells; And / or, in step (5), the volume concentration of the red blood cells is 2%-4%; And / or, in step (5), the volume ratio of the liposome dispersion to the suspension of red blood cells is 1:5-1:12; And / or, in step (5), the co-incubation time is 1-6 hours.

9. The use of the drug delivery system of the photoactive conjugated molecule according to any one of claims 1-4 in the preparation of a drug for treating tumors.

10. A pharmaceutical composition for treating tumors, characterized in that, The pharmaceutical composition comprises: a drug delivery system for a photoactive conjugated molecule according to any one of claims 1-4 and a pharmaceutically acceptable excipient.

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