Tumor efficient targeting flexible drug-loaded cell microparticle and preparation method thereof

Abstract

The invention belongs to the technical field of anti-tumor drugs, and particularly relates to a flexible drug-loaded cell microparticle capable of efficiently targeting tumors and a preparation method of the flexible drug-loaded cell microparticle. Comprising tumor cell-derived drug-loaded microparticles coated with chemotherapeutic drugs, and a flexible regulator modified on the surfaces of the tumor cell-derived drug-loaded microparticles, the flexible regulator comprises a saponin compound and / or a steroid compound, and can regulate and control the hardness of the tumor cell-derived drug-loaded microparticles. The tumor cell-derived drug-loaded microparticles are modified, so that the enrichment and deep penetration of the drug-loaded microparticles in tumor tissues can be remarkably improved, and the efficient targeted delivery of the tumor tissues is realized. Meanwhile, efficient uptake of tumor stem cells to the medicine is promoted, targeted killing is achieved, the tumor stem cells are effectively removed, and tumor growth is remarkably inhibited. In addition, circulating tumor cells can be efficiently targeted, accurate delivery of tumor metastasis is achieved, and tumor metastasis is remarkably inhibited by inhibiting migration and invasion of tumor cells and reducing formation of lung metastasis.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antitumor drugs, and more particularly relates to a flexible drug-loaded cell microparticle for efficient targeting of tumors and a preparation method thereof. BACKGROUND

[0002] Malignant tumors seriously endanger human life and health. Among them, cancer stem cells (CSCs) play a key role in driving tumor progression, treatment resistance and metastasis and diffusion, seriously affecting the clinical prognosis of patients with malignant tumors. To achieve long-term efficacy of treatment, it is essential to effectively eliminate CSCs. However, CSCs usually exist in a special niche deep in the tumor, protected by a dense stromal barrier and elevated interstitial pressure, making it difficult for therapeutic drugs to reach their location. In addition, the failure to effectively eliminate CSCs leads to the production of circulating tumor cells (CTCs), more than 60% of which are derived from CSCs, making them a key driver of early metastasis. Therefore, the development of an effective drug delivery system that can target and eliminate CSCs and CTCs is crucial for inhibiting tumor recurrence and metastasis.

[0003] Microparticles (MPs) are a class of extracellular vesicles (EVs) with a diameter of 100-1000 nanometers. As anti-tumor drug delivery carriers, they have attracted widespread attention due to their excellent biocompatibility, homologous targeting ability and extensive engineering potential. However, the inherent targeting ability of MPs is not sufficient to overcome the various biophysical barriers present in the CSC microenvironment, so it is urgent to improve it to enhance its delivery efficiency.

[0004] In recent years, research progress in the field of biomechanics and mechanobiology has shown that the mechanical properties of nanocarriers, particularly their stiffness and deformability, play a key role in determining drug delivery efficiency. Modulating these properties can significantly enhance the enrichment of nanocarriers at tumor sites, promote their deep penetration, and improve the uptake efficiency of CSCs. However, current strategies for modulating MP mechanical properties mainly rely on modulating the mechanical response of parent cells, which are often limited to specific types of tumor cells and have poor universality. Therefore, there is an urgent need to develop new strategies to impart good mechanical softness to MPs in order to achieve effective elimination of CSCs and reduce the risk of tumor recurrence and metastasis. SUMMARY

[0005] In view of the defects of the prior art, the present application aims to provide a flexible drug-loaded cell micro-particle for efficient tumor targeting and a preparation method thereof, and aims to solve the problems of the prior art, such as weak enrichment ability of the existing anti-tumor drugs in tumor tissues, poor deep penetration effect, inability to efficiently target and effectively remove tumor stem cells and tumor metastases, and tumor recurrence or metastasis.

[0006] To achieve the above-mentioned purpose, in a first aspect, the present application provides a flexible drug-loaded cell micro-particle for efficient tumor targeting, which comprises tumor cell-derived drug-loaded micro-particles wrapped with a chemotherapeutic drug, and a flexible regulator modified on the surface of the tumor cell-derived drug-loaded micro-particles. The flexible regulator can regulate the hardness of the tumor cell-derived drug-loaded micro-particles, and comprises a saponin compound and / or a steroid compound.

[0007] Preferably, the saponin compound comprises one or more of ginsenoside, dioscin, and digitalis saponin.

[0008] Preferably, the steroid compound comprises one or more of progesterone, cortisone, or a structural derivative thereof.

[0009] Preferably, the loading amount of the flexible regulator in the flexible drug-loaded cell micro-particle is 1% to 20% of the mass of the protein in the tumor cell-derived drug-loaded micro-particle.

[0010] Preferably, the Young's modulus of the flexible drug-loaded cell micro-particle is lower than the Young's modulus of the tumor cell-derived drug-loaded micro-particle.

[0011] Further preferably, the Young's modulus of the flexible drug-loaded cell micro-particle is 1 kPa to 200 kPa.

[0012] Preferably, the tumor cells comprise one or more of hepatoma cells, breast cancer cells, lung cancer cells, colon cancer cells, melanoma cells, and pancreatic cancer cells.

[0013] Preferably, the chemotherapeutic drug comprises one or more of doxorubicin, epirubicin, cisplatin, paclitaxel, oxaliplatin, fluorouracil, methotrexate, and irinotecan.

[0014] In a second aspect, the present application provides a preparation method of the flexible drug-loaded cell micro-particle, which comprises the following steps: S1, after stimulating the tumor cells, incubating the chemotherapeutic drug with the tumor cells, allowing the tumor cells to uptake the chemotherapeutic drug, and collecting the released tumor cell-derived drug-loaded micro-particles; or, after stimulating the tumor cells, collecting the tumor cell-derived micro-particles, then incubating with the chemotherapeutic drug, and collecting the tumor cell-derived drug-loaded micro-particles; S2, incubating the tumor cell-derived drug-loaded microparticles and the flexible regulator to modify the flexible regulator to the surface of the tumor cell-derived drug-loaded microparticles by lipid intercalation, and collecting the flexible drug-loaded cell microparticles.

[0015] Preferably, in step S1, the stimulation includes one or more of physical stimulation, chemical stimulation, biological stimulation, and environmental stimulation.

[0016] Preferably, in step S2, the mass ratio of the protein in the tumor cell-derived drug-loaded microparticles to the flexible regulator is 1:(0.01-0.95).

[0017] Preferably, in step S2, the incubation is performed at 4-50°C for 0.5-12h.

[0018] Preferably, in steps S1 and S2, the collected flexible drug-loaded cell microparticles are collected by centrifugation at a centrifugal force of 500-20000g or separated by tangential flow technology.

[0019] In a third aspect, the present application provides a drug for inhibiting tumor growth and metastasis, which comprises the flexible drug-loaded cell microparticles.

[0020] Overall, compared with the prior art, the above technical solutions conceived by the present application mainly have the following technical advantages: (1) The flexible drug-loaded cell microparticles provided by the present application modify the flexible regulator on the surface of the tumor cell-derived drug-loaded microparticles wrapped with chemotherapeutic drugs, wherein the flexible regulator includes saponins and / or steroid compounds, which can regulate the hardness of the tumor cell-derived drug-loaded microparticles. Compared with existing anti-tumor drugs, the flexible drug-loaded cell microparticles provided by the present application can significantly improve the enrichment ability and deep penetration ability of the drug-loaded cell microparticles in tumor tissues, achieving efficient targeted delivery of tumor tissues. At the same time, it can promote the efficient uptake of tumor stem cells to drugs, target and kill and effectively eliminate tumor stem cells, significantly inhibit the growth of tumors. In addition, the flexible drug-loaded cell microparticles can also efficiently target circulating tumor cells, achieve precise delivery of tumor metastases, inhibit the migration and invasion of tumor cells and reduce the formation of lung metastases, thereby significantly inhibiting tumor metastasis.

[0021] (2) Compared with single tail vein injection of tumor cell-derived microparticles (MP), Rh2-modified tumor cell-derived microparticles (RM), chemotherapy drugs (free DOX), free DOX combined with Rh2 (DOX / Rh2), and tumor cell-derived microparticles loaded with chemotherapy drugs (D@M), the flexible drug-loaded cell microparticles (D@RM) provided in this application show a synergistic effect in anti-tumor therapy. Its anti-tumor effect is significantly better than other treatment groups, and even better than the simple sum of the anti-tumor effects of RM, DOX, DOX / Rh2, and D@M.

[0022] (3) This application modifies the drug-loaded microparticles derived from tumor cells and modifies their surface with a flexible modifier, which can effectively optimize the mechanical properties and mechanical characteristics of the microparticles and construct a universal microparticle mechanical property regulation strategy. Attached Figure Description

[0023] Figure 1 Characterization of flexible tumor cell-derived drug-loaded cell microparticles (D@RM) engineered with ginsenoside Rh2; where content a represents particle size distribution and content b represents zeta potential; Figure 2 The mechanical properties of flexible tumor cell-derived drug-loaded cell microparticles engineered with ginsenoside Rh2 are shown; content a and content b are representative force-moment curves of D@M and D@RM, respectively, and content c and content d are Young's modulus and anisotropy of D@RM, respectively. Figure 3 Young's modulus of flexible tumor cell-derived drug-loaded cell microparticles engineered with digitalisin / diosgenin; Figure 4 Young's modulus of flexible tumor cell-derived drug-loaded cell microparticles engineered with cortisone / hydrocortisone / prednisone / dexamethasone; Figure 5 Young's modulus of flexible tumor cell-derived drug-loaded cell microparticles engineered with progesterone / medroxyprogesterone acetate / dydrogesterone; Figure 6 The accumulation of ginsenoside Rh2-engineered flexible tumor cell-derived drug-loaded cell microparticles in tumor tissue; Figure 7 This study investigates the penetration of ginsenoside Rh2-engineered flexible tumor cell-derived drug-loaded cell microparticles into 3D tumor spheres and tumor tissues. Content a shows the fluorescence distribution of DOX in 3D tumor spheres detected by confocal microscopy; content b shows the fluorescence intensity of DOX in 3D tumor spheres analyzed by ImageJ fluorescence quantitative analysis; content c shows the fluorescence distribution of DOX in tumor tissues detected by confocal microscopy; and content d shows the fluorescence intensity of DOX in tumor tissues analyzed by ImageJ fluorescence quantitative analysis. Figure 8 This study describes the uptake and killing of tumor cells by ginsenoside Rh2-engineered flexible tumor cell-derived drug-loaded cell microparticles by tumor cells; content a shows the average fluorescence intensity of relative DOX after treating H22 cells with different DOX concentrations of D@RM, and content b shows the cell viability of H22 cells after treating with different DOX concentrations of D@RM as detected by CCK-8 assay. Figure 9 This study describes the uptake and killing of tumor stem cells by flexible tumor cell-derived drug-loaded cell microparticles engineered with ginsenoside Rh2 by tumor stem cells; content a represents the average relative DOX fluorescence intensity in tumor stem cells treated with D@RM, and content b, c, and d represent the morphology, relative number, and size of 3D tumor spheres after D@RM treatment, respectively. Figure 10 The antitumor effect of ginsenoside Rh2 engineered flexible tumor cell-derived drug-loaded cell microparticles; content a is the tumor volume curve during tail vein administration in H22 subcutaneous hepatocellular carcinoma-bearing mice, and content b is the tumor weight after treatment; Figure 11 The ability of ginsenoside Rh2-engineered flexible tumor cell-derived drug-loaded cell microparticles to target circulating tumor cells; wherein content a is the relative average fluorescence intensity of DiD in circulating tumor cells and leukocytes after co-culturing circulating tumor cells with DiD-labeled RM in a simulated circulating tumor cell blood environment; content b is the relative average fluorescence intensity of DiD in circulating tumor cells and leukocytes after tail vein injection of circulating tumor cells and DiD-labeled RM in mice. Figure 12 The study aimed to assess the ability of ginsenoside Rh2-engineered flexible tumor cell-derived drug-loaded cell microparticles to target lung metastases. Content a represents the quantitative analysis of in vitro bioluminescence imaging signal intensity in the major organs of tumor-bearing mice after tail vein injection of 4T1-Luc cells and IR870-labeled RM; content b represents the quantitative analysis of in vitro IR780 fluorescence intensity in the major organs of tumor-bearing mice after tail vein injection of 4T1-Luc cells and IR870-labeled RM; and content c represents the quantitative analysis of in vitro IR780 fluorescence intensity in the major organs of tumor-bearing mice after tail vein injection of IR870-labeled RM only. Figure 13 The study investigated the antitumor metastasis effect of ginsenoside Rh2-engineered flexible tumor cell-derived drug-loaded cell microparticles; content a shows the tumor lung metastasis progression curve during intravenous administration in 4T1 metastatic breast cancer mice; content b shows the in vitro bioluminescence signal intensity of mouse lung tissue after treatment; and content c shows the quantitative analysis of the number of lung metastases in mice after treatment. Figure 14This study investigates the uptake and killing of paclitaxel-loaded flexible breast cancer cell-derived drug-loaded cell microparticles (P@RM) engineered with ginsenoside Rh2 by tumor cells. Content a shows the average fluorescence intensity relative to DiD after treatment of 4T1 cells with DiD-labeled P@RM, and content b shows the cell viability of 4T1 cells after P@RM treatment as detected by CCK-8 assay. Figure 15 This study investigates the uptake and killing of tumor cells by ginsenoside Rh2-engineered irinotecan-loaded flexible colon cancer cell microparticles (I@RM). Content a shows the average fluorescence intensity relative to DiD after treatment of CT26 cells with DiD-labeled I@RM, and content b shows the cell viability of CT26 cells after treatment with I@RM as detected by CCK-8 assay. Figure 16 This study investigates the uptake and killing of tumor cells by ginsenoside Rh2-engineered oxaliplatin-loaded flexible colon cancer cell microparticles (O@RM); content a shows the average fluorescence intensity relative to DiD after treatment of CT26 cells with DiD-labeled O@RM, and content b shows the cell viability of CT26 cells after O@RM treatment as detected by CCK-8 assay. In all the figures, the same figure labels are used to indicate the same level of significance, where: * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, and ns represents no statistical difference. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0025] In the description of this application, it should be understood that the term "and / or" describes a relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. The symbol " / " in this document indicates that the related objects are in an "or" relationship; for example, A / B means A or B.

[0026] In the description of the embodiments in this application, the words "exemplary" or "for example" are used to indicate that they are examples, illustrations, or descriptions. Any embodiment or design that is described as "exemplary" or "for example" in the embodiments of this application should not be construed as being more preferred or advantageous than other embodiments or design options. Specifically, the use of the words "exemplary" or "for example" is intended to present the relevant concepts in a specific manner.

[0027] The term "effective dose" refers to the amount that, when administered to a subject, demonstrates an effect of improving, treating, detecting and diagnosing cancer, or inhibiting or slowing the progression of cancer. The term "subject" can refer to an animal, preferably a mammal, particularly an animal including humans, or to cells, tissues, and organs derived from an animal. A subject can be a patient who requires these effects.

[0028] This application provides a flexible drug-loaded cell microparticle for highly efficient tumor targeting, comprising tumor cell-derived drug-loaded microparticles encapsulating chemotherapeutic drugs, and a flexible modifier modified on the surface of the tumor cell-derived drug-loaded microparticles. Among them, the aforementioned flexibility modulators can regulate the stiffness of drug-loaded microparticles derived from tumor cells, and include saponin compounds and / or steroid compounds.

[0029] In some embodiments, the above-mentioned saponin compounds include one or more of ginsenosides, diosgenin, and digitalis saponins, wherein ginsenosides include protopanaxadiol (PPD) saponins, such as ginsenoside Rh2, ginsenoside Rg3, ginsenoside Rd, etc.

[0030] In some embodiments, the aforementioned steroidal compounds include one or more of progesterone, cortisone, or their structural derivatives, with a core structure consisting of a fused tetracyclic compound composed of three hexacarbon cyclohexanes (A, B, C) and a five-carbon ring (D). The progesterone structural derivatives include those with modifications to the A ring, C17 position, etc., exemplified by medroxyprogesterone acetate and dydrogesterone. The cortisone structural derivatives include hydrocortisone, prednisone, and dexamethasone. Those skilled in the art are unaware whether the aforementioned saponin compounds and / or steroidal compounds can be modified onto the surface of microparticles to regulate microparticle stiffness, and whether they can enhance the targeting of drug-loaded cell microparticles to tumor stem cells and their ability to kill tumor stem cells. Therefore, the synergistic effect of combining tumor cell-derived microparticles encapsulated with chemotherapeutic drugs with a flexible modulator modified on the surface of the cell microparticles is a technical feature of this application.

[0031] In some embodiments, the loading of the flexible modulator in the above-mentioned flexible drug-loaded cell microparticles is 1% to 20% of the protein mass in the above-mentioned tumor cell-derived drug-loaded microparticles.

[0032] In some embodiments, the Young's modulus of the aforementioned flexible drug-loaded cell microparticles is lower than that of the aforementioned tumor cell-derived drug-loaded microparticles. Preferably, the Young's modulus of the aforementioned flexible drug-loaded cell microparticles is 1 kPa to 200 kPa. The flexible drug-loaded cell microparticles provided in this application have excellent mechanical properties. Compared with existing drugs, the flexible drug-loaded cell microparticles provided in this application can significantly enhance the accumulation ability of drug-loaded cell microparticles at tumor sites, the deep penetration effect, the targeted killing and effective elimination of tumor stem cells and circulating tumor cells, and effectively inhibit tumor growth and metastasis.

[0033] It should be understood that this application does not impose any special limitation on the amount of chemotherapy drugs loaded in the flexible microparticles. In practical applications, those skilled in the art can determine the amount of chemotherapy drugs loaded in the flexible drug-loaded cell microparticles based on the subject's age, weight, gender, disease severity, diet and excretion, route of administration and number of treatments, while ensuring that the Young's modulus of the drug-loaded cell microparticles is controlled within the above-mentioned range.

[0034] This application does not limit the types of tumor cells mentioned above, and they can be various types of cancer cells. Considering that microparticles prepared from parental tumor cells have the best uptake and killing ability for parental tumor cells, those skilled in the art can select any parental tumor cells according to actual application, all of which are within the scope of protection of this application. The aforementioned tumor cells include, but are not limited to, liver cancer cells, breast cancer cells, lung cancer cells, colon cancer cells, melanoma cells, pancreatic cancer cells, etc.

[0035] It is understood that this application does not specifically limit the aforementioned chemotherapeutic drugs. Any chemotherapeutic drug reported in the prior art can be encapsulated within tumor cell-derived microparticles to form drug-loaded microparticles. Modifying the surface of the drug-loaded microparticles with the aforementioned flexible modifier enables the drug-loaded microparticles to accumulate in tumor tissue, deeply penetrate the tumor, and be efficiently taken up by tumor cells, tumor stem cells, and circulating tumor cells. This allows the chemotherapeutic drugs encapsulated in the drug-loaded microparticles to exert their effects more effectively without affecting the anti-tumor efficacy of the chemotherapeutic drugs themselves. The aforementioned chemotherapeutic drugs include, but are not limited to, doxorubicin, epirubicin, cisplatin, paclitaxel, oxaliplatin, fluorouracil, methotrexate, irinotecan, etc.

[0036] On the other hand, this application also provides a method for preparing the above-mentioned flexible drug-loaded cell microparticles, comprising the following steps: S1. After stimulating the tumor cells, the chemotherapy drug is co-incubated with the tumor cells to allow the chemotherapy drug to be taken up by the tumor cells, and the released drug-loaded microparticles from the tumor cells are collected. Alternatively, tumor cell-derived microparticles can be collected after stimulating tumor cells, and then co-incubated with chemotherapy drugs to collect drug-loaded microparticles derived from tumor cells. S2. The above-mentioned tumor cell-derived drug-loaded microparticles and flexible modifiers are incubated, and the flexible modifiers are modified onto the surface of the tumor cell-derived drug-loaded microparticles through lipid intercalation, and the flexible drug-loaded cell microparticles are collected.

[0037] In some embodiments, in step S1, the stimulation includes one or more of physical, chemical, biological, and environmental stimuli, with the aim of inducing tumor cells to release microparticles. The physical stimuli include, but are not limited to, shear force, temperature stimulation, and radiation. For example, shear force, thermal stimulation, cold stimulation, or ionizing radiation (e.g., microwave radiation, ultraviolet radiation, X-ray radiation, etc.) can be applied to tumor cells. The chemical stimuli include, but are not limited to, chemotherapeutic drugs, oxidants, calcium ion carriers, and low-pH solution treatment. For example, chemotherapeutic drugs (e.g., cisplatin, paclitaxel, doxorubicin, etc.), oxidants (H2O2), or lactate-induced acidic environments can be used to treat tumor cells. The biological stimuli include, but are not limited to, cytokines, co-culture of immune cells, and pathogen stimulation. For example, cytokines such as TNF-α, IL-6, TGF-β, and IFN-γ can be used to treat tumor cells, or tumor cells can be co-cultured with T cells, NK cells, and macrophages. The environmental stimuli include, but are not limited to, hypoxia, nutrient restriction, and acidic microenvironments. For example, tumor cells can be cultured under hypoxic, low-glucose, or low-pH conditions.

[0038] In some embodiments, in step S2, the mass ratio of the protein in the tumor cell-derived drug-loaded microparticles to the flexible modulator is 1:(0.01~0.95).

[0039] In some embodiments, in step S2, the incubation conditions are: 4℃~50℃ for 0.5h~12h.

[0040] In some embodiments, in steps S1 and S2, the above collection method is: collection with centrifugal force of 500g to 20000g or separation collection using tangential flow technology.

[0041] This application utilizes saponin and / or steroid compounds as flexibility modifiers on the surface of drug-loaded microparticles derived from tumor cells to effectively optimize the mechanical properties / mechanical characteristics of drug-loaded microparticles, reduce their hardness, and construct a universally applicable strategy for regulating the mechanical properties of microparticles.

[0042] This application also provides a drug for inhibiting tumor growth and metastasis, comprising the aforementioned flexible drug-loaded cell microparticles.

[0043] It is understood that the aforementioned drugs for inhibiting tumor growth and metastasis may include flexible drug-loaded cell microparticles formulated in a suitable form with pharmaceutically acceptable carriers, excipients and / or diluents.

[0044] The drug for inhibiting tumor growth and metastasis provided in this application can be administered to mammals, including humans, by any method, exemplarily including but not limited to oral or parenteral administration. The total effective dose of the drug can be administered to the patient in a single dose, or, according to a fractionated treatment regimen, in multiple doses over a long period. Those skilled in the art can determine the appropriate effective dose of the drug based on the patient's age, weight, sex, disease severity, diet, route of administration, and number of treatments, as long as the effects of this application are demonstrated; therefore, the total dose of the drug is not limited.

[0045] In some embodiments, the tumor may be liver cancer, breast cancer, lung cancer, colon cancer, melanoma, pancreatic cancer, etc., but is not limited to these.

[0046] It should be understood that materials of the same or similar type, model, quality, properties, or function as the reagents and instruments used in the following embodiments can be used to implement this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0047] The following is an example: The following examples use various cells, reagents, and experimental animals: H22 cells, 4T1 cells, and CT26 cells were all purchased from the China Center for Type Culture Collection; doxorubicin, paclitaxel, irinotecan, oxaliplatin, ginsenoside Rh2, progesterone, medroxyprogesterone acetate, dydrogesterone, hydrocortisone, prednisone, and dexamethasone were all purchased from Meilun Biotechnology Co., Ltd.; digitalis saponins, diosgenin, and cortisone were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; BALB / c mice were purchased from Liaoning Changsheng Animal Technology Co., Ltd.

[0048] Example 1: Flexible drug-loaded cell microparticles engineered with ginsenoside Rh2 (1) Preparation of flexible drug-loaded cell microparticles H22 cells were resuspended in serum-free medium and then exposed to ultraviolet light (UVB, 300 J / m²). 2Cells were irradiated for 1 h, then co-incubated with 200 μg / mL doxorubicin (DOX) for 12 h. The cell suspension was collected and centrifuged at 600g for 10 min to remove cells and cell debris. The supernatant was collected again and centrifuged at 18,000g for 60 min to collect the precipitate. The precipitate was washed twice with PBS to obtain DOX-loaded drug-loaded microparticles derived from liver cancer cells (D@M). Blank microparticles (MP) were prepared using the same steps, but without the addition of DOX. Ginsenoside Rh2 (final concentration 15 μg / mL) was incubated with D@M and MP (final protein concentration 75 μg / mL) at 37°C for 3 h to modify Rh2. Unbound Rh2 was then removed by centrifugation at 18,000g for 60 min, and the cells were washed twice with PBS to obtain ginsenoside Rh2-engineered flexible drug-loaded cell microparticles (D@RM) and ginsenoside Rh2-engineered cell microparticles (RM), respectively. D@RM was resuspended in PBS and stored at -80°C for later use. The particle size and zeta potential of D@RM were determined using dynamic light scattering (DLS).

[0049] The results are as follows Figure 1 As shown in content a, the hydrated particle size of D@RM is approximately 200-600 nm, enabling it to passively target tumor regions through enhanced permeability and retention effects (EPR effect). Figure 1 As shown in section b, the zeta potential of D@RM is approximately -10.5 mV, which remains stable under physiological conditions. In summary, this indicates that drug loading and modification with ginsenoside Rh2 on tumor cell-derived microparticles have no significant effect on their particle size and zeta potential.

[0050] (2) Measure Young's modulus The Young's modulus of D@M and D@RM was measured under liquid phase conditions using atomic force microscopy (AFM). Specific parameters were as follows: ScanAsyst mode; MLCT (Bruker) probe type; spring constant of 0.01 N / m; scan frequency of 0.977 Hz; NanoScope Analysis software; and Hertzian (Spherical) model used for calculation. For each microparticle analysis, 10–15 force curves were recorded and averaged to ensure data accuracy.

[0051] The results are as follows Figure 2 Content a. Figure 2 Content b and Figure 2 As shown in content c, compared with D@M, D@RM has a significantly lower Young's modulus, approximately 30~40 kPa, and D@RM is more flexible.

[0052] (3) Measuring anisotropy The anisotropy of D@M and D@RM was determined using the Laurdan fluorescence method. Specifically, D@M and D@RM were incubated with Laurdan dye (1 μM) at 37°C for 1 h, and then the fluorescence anisotropy was measured using a polarized fluorescence spectrometer (FP-6500) at excitation / emission wavelengths of 360 nm and 440 nm, respectively. The fluorescence anisotropy was calculated using the formula: r = (I VV -GI VH ) / (I VV +2GI VH ), where G=I HV / I HH Used to correct differences in transmittance in optical systems.

[0053] The results are as follows Figure 2 As shown in section d, the anisotropy of D@RM also shows a decreasing trend, and the membrane fluidity of D@RM is enhanced. In summary, modification of drug-loaded microparticles with the saponin compound ginsenoside Rh2 can improve the softness and fluidity of the microparticles.

[0054] Example 2: Flexible drug-loaded cell microparticles engineered with digitalis saponins / diosgenin (1) Digitin (DIG, final concentration 5 μM) and D@M (final protein concentration 500 μg / mL) prepared in Example 1 were incubated at 37°C for 1 h to modify the digitin. Then, the unbound DIG was removed by centrifugation at 18,000g for 60 min. After washing twice with PBS, flexible drug-loaded cell microparticles (D@GM) engineered with digitin were obtained.

[0055] (2) Dioscin (DIO, final concentration 10 μM) and D@M (final protein concentration 500 μg / mL) prepared in Example 1 were incubated at 37°C for 1 h to modify dioscin. Then, the unbound DIO was removed by centrifugation at 18,000g for 60 min. After washing twice with PBS, flexible drug-loaded cell microparticles (D@OM) engineered with dioscin were obtained.

[0056] The Young's modulus of D@M, D@GM, and D@OM was measured using the method provided in step (2) of Example 1. The results are as follows: Figure 3 As shown, compared with D@M, D@GM and D@OM both have significantly lower Young's modulus, indicating that modification of drug-loaded microparticles with saponin compounds such as digitoxin and diosgenin can also affect their mechanical properties, resulting in soft drug-loaded cell microparticles.

[0057] Example 3: Flexible drug-loaded cell microparticles engineered with cortisone / hydrocortisone / prednisone / dexamethasone (1) Cortisone (COR, final concentration 15 μg / mL) and D@M (final protein concentration 75 μg / mL) prepared in Example 1 were incubated at 37°C for 3 h to modify cortisone. Then, the unbound COR was removed by centrifugation at 18,000g for 60 min. After washing twice with PBS, cortisone-engineered flexible drug-loaded cell microparticles (D@CM) were obtained.

[0058] (2) Hydrocortisone (HC, final concentration 15 μg / mL) and D@M (final protein concentration 75 μg / mL) prepared in Example 1 were incubated at 37°C for 1 h to modify hydrocortisone. Then, the unbound HC was removed by centrifugation at 18,000g for 60 min. After washing twice with PBS, hydrocortisone-engineered flexible drug-loaded cell microparticles (D@HM) were obtained.

[0059] (3) Prednisolone (PDN, final concentration of 15 μg / mL) and D@M (final protein concentration of 75 μg / mL) prepared in Example 1 were incubated at 37°C for 1 h to modify prednisolone. Then, the unbound PDN was removed by centrifugation at 18,000g for 60 min. After washing twice with PBS, prednisolone-engineered flexible drug-loaded cell microparticles (D@NM) were obtained.

[0060] (4) Dexamethasone (DXMS, final concentration 15 μg / mL) and D@M (final protein concentration 75 μg / mL) prepared in Example 1 were incubated at 37°C for 1 h to modify dexamethasone. Then, the unbound DXMS was removed by centrifugation at 18,000g for 60 min. After washing twice with PBS, dexamethasone-engineered flexible drug-loaded cell microparticles (D@DM) were obtained.

[0061] The Young's modulus of D@M, D@CM, D@HM, D@NM, and D@DM was measured using the method provided in step (2) of Example 1. The results are as follows: Figure 4 As shown, compared with D@M, D@CM, D@HM, D@NM, and D@DM all have significantly lower Young's modulus, indicating that modification of cell microparticles with steroid compounds such as cortisone, hydrocortisone, prednisone, and dexamethasone can also affect their mechanical properties, resulting in soft drug-loaded cell microparticles.

[0062] Example 4: Flexible drug-loaded cell microparticles engineered with progesterone / medroxyprogesterone acetate / dydrogesterone (1) Progesterone (P4, final concentration 15 μg / mL) was incubated with D@M (final protein concentration 75 μg / mL) prepared in Example 1 at 37°C for 3 h to modify progesterone. Then, the unbound P4 was removed by centrifugation at 18,000g for 60 min. After washing twice with PBS, progesterone-engineered flexible drug-loaded cell microparticles (D@PM) were obtained.

[0063] (2) Medroxyprogesterone acetate (MPA, final concentration 15 μg / mL) was incubated with D@M (final protein concentration 75 μg / mL) prepared in Example 1 at 37°C for 3 h to modify with medroxyprogesterone acetate. Then, the unbound MPA was removed by centrifugation at 18,000g for 60 min. After washing twice with PBS, flexible drug-loaded cell microparticles modified with medroxyprogesterone acetate (D@MM) were obtained.

[0064] (3) Dydrogesterone (DYD, final concentration 15 μg / mL) and D@M (final protein concentration 75 μg / mL) prepared in Example 1 were incubated at 37°C for 1 h to modify dydrogesterone. Then, the unbound DYD was removed by centrifugation at 18,000g for 60 min. After washing twice with PBS, flexible drug-loaded cell microparticles (D@YM) engineered with dydrogesterone were obtained.

[0065] The Young's modulus of D@M, D@PM, D@MM, and D@YM was measured using the method provided in step (2) of Example 1. The results are as follows: Figure 5 As shown, compared with D@M, D@PM, D@MM, and D@YM all have significantly lower Young's modulus, indicating that modification of cell microparticles with steroid compounds such as progesterone, medroxyprogesterone acetate, and dydrogesterone can also affect their mechanical properties, resulting in soft drug-loaded cell microparticles.

[0066] Example 5: Accumulation of flexible drug-loaded cell microparticles engineered with ginsenoside Rh2 in tumor tissue (1) Constructing a subcutaneous tumor model of hepatocellular carcinoma H22 2×10 6 H22 cells were inoculated into the lower right back near the right hind limb of male BALB / c mice to construct a subcutaneous hepatocellular carcinoma model of H22 cells.

[0067] (2) Wait until the subcutaneous tumor grows to approximately 250 mm in size. 3In this study, tumor-bearing mice were randomly divided into three groups of three. Equal volumes of free DOX, D@M, and D@RM were injected into the tumor-bearing mice via the tail vein at a dose of 0.75 mg / kg (based on DOX equivalent). After 24 hours, the mice were sacrificed, and the tumor tissue was collected, homogenized, and DOX was extracted by adding methanol. The concentration of DOX in the tumor tissue of each group was measured using a FlexStation3 microplate reader under conditions of excitation wavelength 480 nm and emission wavelength 585 nm.

[0068] The results are as follows Figure 6 As shown, the concentration of DOX in tumor tissue treated with D@RM was significantly higher than that in the free DOX treatment group and the D@M treatment group, indicating that D@RM has better tumor targeting and can accumulate in tumor tissue.

[0069] Example 6: Deep penetration of flexible drug-loaded cell microparticles engineered with ginsenoside Rh2 into 3D tumor spheres and tumor tissues. (1) Deep penetration in 3D tumor spheres H22 cells were diluted to 1×10⁻⁶ using RPMI 1640 medium. 4 H22 cell suspension was prepared by diluting 3D soft fibrin gel to 2 mg / mL using T7 buffer, and then mixing it with the H22 cell suspension at a volume ratio of 1:1. In a pre-chilled 96-well plate, 1 μL of thrombin (0.1 U / μL) was added, followed by 50 μL of the above mixture, and the mixture was incubated at 37°C for 30 min. Then, 200 μL of RPMI 1640 complete medium was added to each well, and the plate was cultured for another 6 days to obtain tumor stem cell clones (3D tumor spheres). Equal volumes of free DOX, D@M, and D@RM (DOX concentration 1 μg / mL) were added and incubated for 4 h. After washing three times with PBS, the cells were fixed with 4% paraformaldehyde for 30 min, and then observed using a laser confocal microscope along the Z-axis. The DOX was observed through a filter with Ex=488 nm and Em=560 nm.

[0070] The results are as follows Figure 7 Content a and Figure 7 As shown in section b, compared with DOX and D@M, D@RM significantly increased the fluorescence intensity of DOX at different levels of 3D tumor spheres, indicating that D@RM has a better penetration effect in 3D tumor spheres.

[0071] (2) Construct a subcutaneous tumor model of liver cancer H22 according to step (1) of Example 4, and wait for the subcutaneous tumor volume to grow to about 250 mm. 3Tumor-bearing mice were randomly divided into three groups of three. Equal volumes of free DOX, D@M, and D@RM were injected intravenously into the mice via the tail vein at a dose of 0.75 mg / kg (based on DOX equivalent). Mice were sacrificed 24 hours later, and tumor tissues from each group were collected, fixed in 4% paraformaldehyde, and subjected to paraffin sectioning and immunofluorescence staining. First, the tumor was incubated with anti-CD31 primary antibody, followed by incubation with Alexa Fluor® 488-labeled goat anti-rabbit IgG secondary antibody for vascular labeling, and DAPI was used to label cell nuclei. Subsequently, DOX autofluorescence (red), Alexa Fluor® 488 fluorescence (green), and DAPI (blue) were observed using confocal microscopy. Tumor penetration efficiency was quantitatively analyzed by the ratio of DOX fluorescence intensity in the parenchymal region to the signal intensity within blood vessels.

[0072] The results are as follows Figure 7 Content c and Figure 7 As shown in section d, the DOX in the free DOX group was mainly retained in the blood vessels of the tumor tissue, while the DOX in D@RM was most widely distributed in the tumor tissue. In summary, this indicates that D@RM can not only effectively accumulate at the tumor site but also penetrate deep into the tumor parenchyma, reaching the tumor stem cell-rich microenvironment that is difficult for conventional anticancer drugs to access.

[0073] Example 7: Uptake and killing of tumor cells by flexible drug-loaded cell microparticles engineered with ginsenoside Rh2. (1) H22 cells were fed at a concentration of 2.0 × 10⁻⁶ 4 The cells were seeded at a density of cells / well in 12-well plates and incubated at 37°C for 24 h. Then, equal volumes of free DOX, D@M, and D@RM (DOX concentration 0.25 μg / mL) were added, and incubation continued at 37°C for another 4 h. Tumor cells were collected, and the drug uptake levels in the cells were analyzed by flow cytometry. This procedure was repeated twice, with the DOX concentration in each treatment group increased to 0.5 μg / mL and 1 μg / mL, respectively, to assess the uptake of D@RM by H22 tumor cells.

[0074] The results are as follows Figure 8 As shown in content a, the fluorescence intensity of DOX in tumor cells was significantly higher than that of free DOX and D@M, and the fluorescence intensity of DOX showed a dose-dependent increase, indicating that there is a synergistic effect between the components, which can effectively promote the uptake of D@RM by tumor cells.

[0075] (2) H22 cells were fed at 8×10 3Cells were seeded at a density of [number] cells / well in 96-well plates and incubated at 37°C for 24 h. Then, equal volumes of free DOX, D@M, and D@RM (DOX concentration 0.25 μg / mL) were added to each well, and incubation continued at 37°C for another 24 h. Finally, 10 μL of LCK-8 solution was added to each well and incubated for 2 h. The absorbance of each well at 450 nm was measured using a microplate reader. This process was repeated twice, with the DOX concentration in each treatment group increased to 0.5 μg / mL and 1 μg / mL, respectively, to assess the killing effect of D@RM on H22 tumor cells.

[0076] The results are as follows Figure 8 As shown in section b, compared with the DOX and D@M groups, D@RM showed the strongest killing effect on H22 tumor cells, further confirming the significant improvement of D@RM in drug delivery efficiency and therapeutic effect.

[0077] Example 8: Uptake and killing of tumor stem cells by flexible drug-loaded cell microparticles engineered with ginsenoside Rh2. (1) Prepare H22 tumor stem cells according to step (1) of Example 5, then add equal volumes of free DOX, D@M, and D@RM (DOX concentration is 1 μg / mL), incubate at 37°C and 5% CO2 for 4 h, collect cells and wash them 3 times with PBS, resuspend the cells in 500 μL of pre-cooled PBS, pass them through a 200-mesh sieve, and use flow cytometry to detect DOX fluorescence in tumor stem cells to assess the uptake of D@RM by H22 tumor stem cells.

[0078] The results are as follows Figure 9 As shown in content a, D@RM exhibited the strongest intracellular DOX fluorescence signal in H22 tumor stem cells, which was 29.6 times and 1.5 times higher than that of free DOX and D@M, respectively, indicating that there is a synergistic effect between the components, which can effectively promote the uptake of D@RM by tumor stem cells.

[0079] (2) H22 cells were seeded at a density of 500 cells / well in 96-well plates coated with 1 mg / mL 3D soft fiber protein glue and cultured at 37°C and 5% CO2 for 5 days. The culture medium was discarded and then fresh culture medium containing equal volumes of free DOX, D@M and D@RM (DOX concentration of 0.5 μg / mL) was added. The cells were incubated at 37°C for 48 h. The tumor spheroids were photographed using an optical microscope and the number and size of live tumor cell spheroids were counted to evaluate the killing effect of D@RM on H22 tumor stem cells.

[0080] The results are as follows Figure 9 Content b Figure 9 Content c and Figure 9As shown in d, compared with other groups, D@RM significantly reduced the size, number and volume of tumor spheroids formed by H22 tumor stem cells, and had a more significant tumor stem cell killing effect.

[0081] Example 9: Antitumor effect of drug-loaded cell microparticles engineered with ginsenoside Rh2 A subcutaneous hepatocellular carcinoma (H22) model was constructed according to step (1) of Example 4. The subcutaneous tumor was allowed to grow to approximately 100 mm. 3 At that time, tumor-bearing mice were randomly divided into 7 groups, with 6 mice in each group. On days 0, 2, 4, 6, 8, and 10, mice were injected via tail vein with PBS, MP, RM, free DOX, DOX / Rh2 (free DOX and Rh2 combined treatment), D@M, and D@RM, respectively. The protein concentration in the treatment groups was 25 mg / kg, and the Rh2 concentration was 2.25 mg / kg. During the treatment period, the longest (a) and widest (b) points of the tumor were measured daily using calipers, and the tumor volume V = a × b was calculated. 2 / 2. On day 11, the mice were sacrificed and the tumor tissue was weighed.

[0082] The results are as follows Figure 10 Content a and Figure 10 As shown in section b, in the PBS, MP, RM, and DOX / Rh2 treatment groups, tumors grew rapidly in mice, and no significant tumor inhibition was observed. The anti-tumor effect of D@RM was significantly better than that of the other treatment groups, and it could effectively inhibit tumor growth, confirming the synergistic effect among the three components of D@RM.

[0083] Example 10: Ginsenoside Rh2 engineered drug-loaded cell microparticles can target CTCs In addition to effectively targeting and eliminating the primary tumor, the ability of drugs to target circulating tumor cells (CTCs) is crucial for improving cancer treatment outcomes, especially for inhibiting tumor metastasis.

[0084] (1) H22 tumor stem cells were prepared according to step (1) of Example 5 and cultured in RPMI 1640 complete medium. CFSE-labeled H22 CTCs (2.5 × 10⁻⁶) were then cultured. 4 H22 CTCs were added to 100 μL of fresh blood and incubated with DiD-labeled MP or RM (protein concentration of 10 μg / mL) at 37°C and 120 rpm for 4 h with shaking. Cells were then collected, erythrocytes were lysed, and stained with PE-labeled anti-CD45 antibody. Finally, flow cytometry was used to analyze the DiD fluorescence intensity in H22 CTCs and white blood cells (WBCs) to assess the targeting ability of RM to CTCs.

[0085] The results are as follows Figure 11As shown in section a, compared with MP, RM exhibited higher DiD fluorescence intensity in H22 CTCs, indicating that RM has a stronger CTC capture ability. Furthermore, there was no significant difference in the uptake levels of MP and RM in leukocytes, suggesting that the enhanced targeting effect of RM on CTCs is specific.

[0086] (2) 1×10 via tail vein injection 7 CFSE-labeled H22 CSCs were injected into healthy BALB / c mice, followed immediately by a tail vein injection of DiD-labeled MP or RM derived from H22 cells (protein dose of 12.5 mg / kg). Four hours after injection, blood was collected from the orbital sinus, erythrocytes were lysed, and stained with PE-labeled anti-CD45 antibody. The DiD fluorescence intensity in H22 CTCs and WBCs was detected by flow cytometry to assess the in vivo targeting ability of RM to CTCs.

[0087] The results are as follows Figure 11 As shown in section b, the DiD fluorescence intensity of CSCs in the RM group was significantly higher than that of MP, while the uptake levels of MP and RM in leukocytes remained comparable, further confirming that RM has excellent CTC targeting ability.

[0088] Example 11: Ginsenoside Rh2 engineered drug-loaded cell microparticles enhance lung metastasis targeting ability by targeting CTCs. 1×10 7 After injecting 4T1 cells overexpressing luciferase (4T1-Luc cells), mice were injected with IR780-labeled 4T1 cell-derived MP and IR780-labeled 4T1 cell-derived RM (protein dose 12.5 mg / kg). Four hours after injection, mice were sacrificed, and tissues from various organs (heart, liver, spleen, lung, and kidney) were collected. Bioluminescence imaging (BLI) and IR780 fluorescence imaging were performed on the isolated mouse tissues using an in vivo imaging system to assess the impact of RM's targeting ability for CTCs on its enrichment in metastatic lesions. Furthermore, in mice not injected with 4T1-Luc cells, another in vivo targeting experiment was conducted under the same conditions to eliminate the influence of the physicochemical properties of RM itself on enrichment.

[0089] Bioluminescence imaging results showed that ( Figure 12 Content a), after injection of 4T1-Luc cells followed by administration of MP or RM, the 4T1-Luc cells primarily metastasized to the lungs. IR780 fluorescence imaging showed ( Figure 12 Content b), RM accumulation in the lungs was significantly higher than MP, while in mice not injected with 4T1-Luc cells, there was no significant difference in the distribution of MP and RM in the lungs. Figure 12Content c). In summary, the more efficient enrichment of RM in metastatic lesions may stem from its specific targeting ability for CTCs.

[0090] Example 12: Antitumor metastasis effect of drug-loaded cell microparticles engineered with ginsenoside Rh2 (1) Construct 4T1 cell-derived D@RM and 4T1 cell-derived D@M according to step (1) of Example 1.

[0091] (2) 4×10 5 A mouse model of metastatic breast cancer was established by intravenous injection of 4T1-Luc cells (4T1 cells overexpressing luciferase) into BALB / c mice via tail vein. The mice were then randomly divided into three groups of five mice each. On day 0, equal volumes of PBS, 4T1 cell-derived D@M, and 4T1 cell-derived D@RM were administered intravenously to the tumor-bearing mice at a dose of 0.5 mg / kg (DOX equivalent), once every two days for a total of seven administrations. Bioluminescence imaging (BLI) was performed every four days during treatment to monitor lung metastasis. On day 16, the mice were sacrificed, and lung tissue was harvested and imaged using the in vivo imaging system. The ex vivo lung tissue was fixed in Bouin's solution, and metastatic lung tumor nodules were counted.

[0092] The results are as follows Figure 13 Content a: Bioluminescence imaging results showed that as the treatment time progressed, the bioluminescence signal of lung metastases in each treatment group gradually increased, while the bioluminescence signal of the D@RM treatment group was significantly weaker than that of other treatment groups. Figure 13 Content b: Ex vivo lung tissue imaging further confirmed that the D@RM treatment group had the lowest bioluminescent signal in the lungs. In summary, this indicates that, compared to PBS and D@M, D@RM, through synergistic effects between its components, can effectively inhibit the metastasis of 4T1-Luc cells injected via tail vein to the lungs. Furthermore, compared to other treatment groups, the D@RM treatment group showed a significant reduction in the number of metastatic lung nodules (…). Figure 13 Content c) indicates that D@RM has better anti-tumor metastasis ability.

[0093] Example 13: Uptake and killing of tumor cells by flexible drug-loaded (paclitaxel) cell microparticles engineered with ginsenoside Rh2. 4T1 cells were resuspended in serum-free medium and then exposed to ultraviolet light (UVB, 300 J / m²). 2The cells were irradiated for 1 h, then co-incubated with 400 μg / mL paclitaxel (PTX) for 12 h. The cell suspension was collected and centrifuged at 600g for 10 min to remove cells and cell debris. The supernatant was collected and centrifuged at 18,000g for 60 min to collect the precipitate. The precipitate was washed twice with PBS to obtain PTX-loaded breast cancer cell-derived drug-loaded microparticles (P@M). Ginsenoside Rh2 (final concentration 15 μg / mL) was incubated with P@M (final protein concentration 75 μg / mL) at 37°C for 3 h, then centrifuged at 18,000g for 60 min to remove unbound Rh2. The precipitate was washed twice with PBS to obtain Rh2-engineered flexible drug-loaded cell microparticles (P@RM). The P@RM was resuspended in PBS and stored at -80°C for later use.

[0094] (1) 4T1 cells were subjected to a concentration of 2.0 × 10⁻⁶ cells. 4 The cells were seeded at a density of cells / well in 12-well plates and incubated at 37°C for 24 hours. Then, equal volumes of DiD-labeled P@M and P@RM (protein concentration 10 μg / mL) were added, and the cells were incubated at 37°C for another 4 hours. Tumor cells were collected and the DiD fluorescence intensity in the cells was detected by flow cytometry to assess the uptake of P@RM by 4T1 tumor cells.

[0095] The results are as follows Figure 14 As shown in section a, compared with P@M, P@RM treatment showed a stronger DiD fluorescence signal in 4T1 cells, indicating that P@RM can be effectively taken up by tumor cells.

[0096] (2) 4T1 cells were fed at 8×10 3 The cells were seeded at a density of 1 cell / well in a 96-well plate and incubated at 37°C for 24 h. Then, equal volumes of free PTX, P@M, and P@RM (PTX concentration of 2 μg / mL) were added to each well. After incubation at 37°C for another 24 h, 10 μL of CCK-8 solution was added to each well and incubated for 2 h. The absorbance of each well at 450 nm was measured using a microplate reader to assess the killing effect of P@RM on 4T1 tumor cells.

[0097] The results are as follows Figure 14 As shown in section b, compared with free PTX and P@M, P@RM showed the strongest killing effect on 4T1 cells, indicating that Rh2 modification of tumor cell-derived drug-loaded microparticles loaded with the chemotherapeutic drug paclitaxel can also significantly improve the drug delivery efficiency and therapeutic effect of drug-loaded cell microparticles.

[0098] Example 14: Uptake and killing of tumor cells by ginsenoside Rh2-engineered flexible drug-loaded (irinotecan) cell microparticles. CT26 cells were resuspended in serum-free medium and then exposed to ultraviolet light (UVB, 300 J / m²). 2Cells were irradiated for 1 h, then co-incubated with 500 μg / mL irinotecan (IRT) for 12 h. The cell suspension was collected and centrifuged at 600g for 10 min to remove cells and cell debris. The supernatant was collected and centrifuged at 18,000g for 60 min to collect the precipitate. The precipitate was washed twice with PBS to obtain IRT-loaded colon cancer cell-derived drug-loaded microparticles (I@M). Ginsenoside Rh2 (final concentration 15 μg / mL) was incubated with I@M (final protein concentration 75 μg / mL) at 37°C for 3 h, followed by centrifugation at 18,000g for 60 min to remove unbound Rh2. The precipitate was washed twice with PBS to obtain Rh2-engineered flexible drug-loaded cell microparticles (P@RM). I@RM was resuspended in PBS and stored at -80°C for later use.

[0099] (1) CT26 cells were subjected to a concentration of 2.0 × 10⁻⁶. 4 The cells were seeded at a density of cells / well in 12-well plates and incubated at 37°C for 24 hours. Then, equal volumes of DiD-labeled I@M and I@RM (protein concentration 10 μg / mL) were added, and the cells were incubated at 37°C for another 4 hours. Tumor cells were collected and the DiD fluorescence intensity in the cells was detected by flow cytometry to assess the uptake of P@RM by CT26 tumor cells.

[0100] The results are as follows Figure 15 As shown in section a, compared with I@M, CT26 cells treated with I@RM showed a stronger DiD fluorescence signal, indicating that I@RM can be effectively taken up by tumor cells.

[0101] (2) CT26 cells were prepared at 8×10 3 The cells were seeded at a density of 1 cell / well in a 96-well plate and incubated at 37°C for 24 h. Then, equal volumes of free IRT, I@M, and I@RM (IRT concentration of 2 μg / mL) were added to each well. After incubation at 37°C for another 24 h, 10 μL of CCK-8 solution was added to each well and incubated for 2 h. The absorbance of each well at 450 nm was measured using a microplate reader to assess the killing effect of P@RM on CT26 tumor cells.

[0102] The results are as follows Figure 15 As shown in section b, compared with free IRT and I@M, I@RM showed the strongest killing effect on CT26 cells, indicating that Rh2 modification of the drug-loaded microparticles derived from colon cancer cells loaded with the chemotherapy drug irinotecan can also significantly improve the drug delivery efficiency and therapeutic effect of the drug-loaded cell microparticles.

[0103] Example 15: Uptake and killing of tumor cells by ginsenoside Rh2-engineered flexible drug-loaded (oxaliplatin) cell microparticles. CT26 cells were resuspended in serum-free medium and then exposed to ultraviolet light (UVB, 300 J / m²).2 After irradiation for 1 h, the cell suspension was collected and centrifuged at 600g for 10 min to remove cells and cell debris. The supernatant was collected and centrifuged at 18,000g for 60 min to collect the precipitate. The precipitate was washed twice with PBS to obtain blank microparticles (MP) derived from colon cancer cells. MP was then incubated with free oxaliplatin (OXA) at a mass ratio of 2:1 at 37°C for 3 h. After centrifugation at 18,000g for 60 min to remove free OXA, the precipitate was collected and washed twice with PBS to obtain OXA-loaded drug-loaded microparticles (O@M) derived from colon cancer cells. Ginsenoside Rh2 (final concentration 15 μg / mL) was incubated with O@M (final protein concentration 75 μg / mL) at 37°C for 3 h. After centrifugation at 18,000g for 60 min to remove unbound Rh2, the precipitate was washed twice with PBS to obtain Rh2-engineered flexible drug-loaded cell microparticles (O@RM). Resuspend O@RM in PBS and store at -80°C for later use.

[0104] (1) The uptake of O@M and P@RM by CT26 tumor cells was evaluated according to the method provided in step (1) of Example 14, wherein equal volumes of DiD-labeled O@M and O@RM (protein concentration 10 μg / mL) were added to the treatment group respectively.

[0105] The results are as follows Figure 16 As shown in content a, compared with O@M, CT26 cells treated with O@RM showed a stronger DiD fluorescence signal, indicating that O@RM can be effectively taken up by tumor cells.

[0106] (2) The killing effect of free OXA, O@M, and O@RM on CT26 tumor cells was evaluated according to the method provided in step (2) of Example 14, wherein equal volumes of free OXA, O@M, and O@RM (OXA concentration of 10 μM) were added to the treatment group respectively.

[0107] The results are as follows Figure 16 As shown in section b, compared with free OXA and O@M, O@RM exhibited the strongest killing effect on CT26 cells, indicating that Rh2 modification of drug-loaded microparticles derived from colon cancer cells loaded with the chemotherapeutic drug oxaliplatin can also significantly improve the drug delivery efficiency and therapeutic effect of drug-loaded cell microparticles.

[0108] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A flexible drug-loaded cell microparticle for highly efficient tumor targeting, characterized in that, It includes tumor cell-derived drug-loaded microparticles encapsulating chemotherapy drugs, and flexible modifiers modified on the surface of the tumor cell-derived drug-loaded microparticles; The flexibility modulator can regulate the stiffness of the drug-loaded microparticles derived from tumor cells, including saponin compounds and / or steroid compounds.

2. The flexible drug-loaded cell microparticles according to claim 1, characterized in that, The saponin compounds include one or more of ginsenosides, diosgenin, and digitalis saponins; and / or, The steroid compounds include one or more of progesterone, cortisone, or their structural derivatives.

3. The flexible drug-loaded cell microparticles according to claim 1 or 2, characterized in that, In the flexible drug-loaded cell microparticles, the loading amount of the flexible regulator is 1% to 20% of the protein mass in the tumor cell-derived drug-loaded microparticles.

4. The flexible drug-loaded cell microparticles according to claim 3, characterized in that, The Young's modulus of the flexible drug-loaded cell microparticles is lower than that of the tumor cell-derived drug-loaded microparticles. Preferably, the Young's modulus of the flexible drug-loaded cell microparticles is 1 kPa to 200 kPa.

5. The flexible drug-loaded cell microparticles according to claim 1, characterized in that, The tumor cells include one or more of the following: liver cancer cells, breast cancer cells, lung cancer cells, colon cancer cells, melanoma cells, and pancreatic cancer cells; and / or, The chemotherapy drugs include one or more of doxorubicin, epirubicin, cisplatin, paclitaxel, oxaliplatin, fluorouracil, methotrexate, and irinotecan.

6. A method for preparing flexible drug-loaded cell microparticles as described in any one of claims 1 to 5, characterized in that, Includes the following steps: S1. After stimulating the tumor cells, the chemotherapy drug is co-incubated with the tumor cells to allow the chemotherapy drug to be taken up by the tumor cells, and the released drug-loaded microparticles from the tumor cells are collected. Alternatively, tumor cell-derived microparticles can be collected after stimulating tumor cells, and then co-incubated with chemotherapy drugs to collect drug-loaded microparticles derived from tumor cells. S2. The tumor cell-derived drug-loaded microparticles and the flexible modifier are incubated. The flexible modifier is modified onto the surface of the tumor cell-derived drug-loaded microparticles by lipid intercalation, and the flexible drug-loaded microparticles are collected.

7. The preparation method according to claim 6, characterized in that, In step S1, the stimulus includes one or more of physical stimuli, chemical stimuli, biological stimuli, and environmental stimuli.

8. The preparation method according to claim 6, characterized in that, In step S2, the mass ratio of protein in the tumor cell-derived drug-loaded microparticles to the flexible regulator is 1:(0.01~0.95); The incubation conditions are: 4℃~50℃ for 0.5h~12h.

9. The preparation method according to claim 6, characterized in that, In steps S1 and S2, the collection method is as follows: collection with centrifugal force of 500g to 20000g or separation and collection using tangential flow technology.

10. A drug for inhibiting tumor growth and metastasis, characterized in that, Includes the flexible drug-loaded cell microparticles as described in any one of claims 1 to 5.

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