Self-assembled nano preparation and preparation method thereof

Abstract

The invention relates to a self-assembled nanoparticle, the nanoparticle is formed by self-assembling a hydrophobic antitumor drug and a photosensitizer, the photosensitizer is IR780, and the nanoparticle does not contain a pharmaceutically acceptable auxiliary material carrier. The self-assembled nanoparticles can be administered in an oral way and can be accumulated at a tumor part after being orally taken, the photosensitizer IR780 can be used for in-vivo imaging and photo-thermal treatment, and the'oral administration-diagnosis and treatment integrated 'treatment mode of cooperatively treating tumors with a co-loaded anti-tumor drug is realized.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to a self-assembling nano preparation and a preparation method thereof. Background Art

[0002] Traditional chemotherapy drugs have problems such as poor targeting, low bioavailability and large side effects. They are difficult to effectively concentrate on the tumor site, resulting in unsatisfactory treatment effects and may cause serious adverse reactions. Photothermal therapy (PTT) is a non-invasive treatment method that can effectively destroy tumor tissue by generating local heat under a specific wavelength light source through photosensitizers. However, single photothermal therapy is difficult to completely eliminate tumor cells and can easily lead to tumor recurrence. Therefore, the combination of photothermal therapy and chemotherapy has gradually become a research hotspot.

[0003] Existing photothermal and chemotherapy combined treatment methods usually rely on carrier materials (such as polymers, inorganic nanoparticles, etc.) to carry drugs. However, the safety and metabolic problems of these carrier materials limit their prospects for clinical application. Therefore, the development of a carrier-free drug delivery system that can reduce dependence on carriers and improve therapeutic effects has become a technical problem that needs to be solved in this field.

[0004] As a common method of drug delivery, oral administration has the advantages of convenience, high patient compliance, and no need for professional equipment assistance. It is particularly suitable for long-term treatment and family self-management of cancer patients. Compared with intravenous injection and other methods of administration, oral administration can significantly improve patient compliance, and drugs delivered orally can be absorbed through the gastrointestinal tract, avoiding the risk of infection and tissue damage caused by frequent injections. Therefore, the development of a nanoformulation for combined photothermal and chemotherapy therapy suitable for oral administration is of great significance for improving tumor treatment effects, reducing side effects, and improving patient compliance.

[0005] Currently, there are no reports on carrier-free oral nanoformulations that have synergistic effects of photothermal therapy and chemotherapy. Summary of the invention

[0006] The present invention provides a carrier-free oral nanoformulation and its preparation method and application. The nanoformulation integrates chemotherapy drugs and photosensitizers (such as IR780, etc.) into the same nanostructure through self-assembly technology to form stable self-assembled nanoparticles. The nanoparticles are delivered into the body through oral administration, remain stable in the gastrointestinal tract, can be effectively absorbed into the blood circulation system, and finally target the tumor site, and combine with photothermal therapy to achieve synergistic treatment of tumors.

[0007] To achieve the above object, the present invention includes the following aspects:

[0008] The first aspect of the present invention provides a self-assembled nanoparticle, wherein the nanoparticle is self-assembled from a hydrophobic anti-tumor drug and a photosensitizer, wherein the photosensitizer is IR780, and the nanoparticle does not contain a pharmaceutically acceptable excipient carrier.

[0009] Preferably, the hydrophobic anti-tumor drug is selected from one or more of curcumin, paclitaxel, docetaxel, sirolimus, sorafenib, aclarubicin, idarubicin, triptolide, camptothecin, 10-hydroxycamptothecin, colchicine, vincristine, methotrexate, tamoxifen, teniposide and mitoxantrone. More preferably, the hydrophobic anti-tumor drug is selected from one or more of curcumin, paclitaxel and docetaxel.

[0010] Preferably, the chemical structure of IR780 is The CAS number is 207399-07-3.

[0011] Preferably, the nanoparticles are formed by the hydrophobic anti-tumor drug and the IR780 photosensitizer through π-π stacking, hydrophobic force and electrostatic force.

[0012] Preferably, the molar concentration ratio of IR780 to the anti-tumor drug in the nanoparticles is 1:2-1:30. More preferably, the molar concentration ratio of IR780 to the anti-tumor drug is 1:5-1:25. Further preferably, the molar concentration ratio of IR780 to the anti-tumor drug is 1:8-1:20. Further preferably, the molar concentration ratio of IR780 to the anti-tumor drug is 1:10-1:20.

[0013] Preferably, the molar concentration ratio of IR780 to curcumin in the nanoparticles is 1:10-1:20. More preferably, the molar concentration ratio of IR780 to curcumin in the nanoparticles is 1:15-1:20. Further preferably, the molar concentration ratio of IR780 to curcumin is 1:18-1:20.

[0014] Preferably, the molar concentration ratio of IR780 to paclitaxel in the nanoparticles is 1:1-1:20. More preferably, the molar concentration ratio of IR780 to paclitaxel in the nanoparticles is 1:5-1:10. Further preferably, the molar concentration ratio of IR780 to paclitaxel is 1:6-1:8.

[0015] Preferably, the average particle size of the nanoparticles is 10-500 nm. More preferably, the average particle size of the nanoparticles is 50-300 nm. Further preferably, the average particle size of the nanoparticles is 80-200 nm. Further preferably, the average particle size of the nanoparticles is 100-150 nm.

[0016] The second aspect of the present invention provides a method for preparing a nanoformulation comprising the self-assembled nanoparticles, the preparation method comprising the following steps:

[0017] (1) dissolving IR780 in a benign solvent A to obtain a solution A;

[0018] (2) dissolving the antitumor drug in a benign solvent B to obtain a solution B;

[0019] (3) fully mixing solution A and solution B to obtain a mixed solution C, and adding the mixed solution C dropwise into the poor solvent C under continuous stirring to obtain a mixed solution D;

[0020] (4) removing the organic solvent from the mixed solution D obtained in step (3) to obtain a nanoformulation containing self-assembled nanoparticles.

[0021] Preferably, the concentration of IR780 in step (1) is 0.5 mM-50 mM. More preferably, the concentration of IR780 in step (1) is 5 mM-30 mM. Further preferably, the concentration of IR780 in step (1) is 10 mM-20 mM. Further preferably, the concentration of IR780 in step (1) is 15 mM.

[0022] Preferably, the benign solvent A in step (1) is selected from one or more of dichloromethane, chloroform, methanol, ethanol, ether, ethyl acetate, methyl acetate, acetone, n-propanol, pyridine, N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide. More preferably, the organic solvent A of IR780 is selected from one or more of methanol, ethanol, and n-propanol. Further preferably, the organic solvent A of IR780 is ethanol.

[0023] Preferably, the concentration of the anti-tumor drug in step (2) is 10mM-150mM. More preferably, the concentration of the anti-tumor drug in step (2) is 20mM-100mM. Further preferably, the concentration of the anti-tumor drug in step (2) is 40mM-90mM.

[0024] Preferably, the benign solution B in step (2) is selected from one or more of dichloromethane, chloroform, methanol, ethanol, ether, ethyl acetate, methyl acetate, acetone, n-propanol, pyridine, N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide. More preferably, the benign solution B in step (2) is selected from one or more of dichloromethane, chloroform, ethyl acetate, methyl acetate, and acetone. Further preferably, the benign solution B in step (2) is selected from one or more of dichloromethane and chloroform.

[0025] Preferably, the molar concentration ratio of IR780 to the anti-tumor drug in the mixed solution C of step (3) is 1:2-1:30. More preferably, the molar concentration ratio of IR780 to the anti-tumor drug in the mixed solution C of step (3) is 1:5-1:25. Further preferably, the molar concentration ratio of IR780 to the anti-tumor drug in the mixed solution C of step (3) is 1:8-1:20. Further preferably, the molar concentration ratio of IR780 to curcumin in the mixed solution C of step (3) is 1:15-1:20. Further preferably, the molar concentration ratio of IR780 to curcumin in the mixed solution C of step (3) is 1:18-1:20. Further preferably, the molar concentration ratio of IR780 to paclitaxel in the mixed solution C of step (3) is 1:5-1:10. Further preferably, the molar concentration ratio of IR780 to paclitaxel in the mixed solution C of step (3) is 1:6-1:8.

[0026] Preferably, the concentration of the antitumor drug in the mixed solution C of step (3) is 10mM-100mM. More preferably, the concentration of the antitumor drug in the mixed solution C of step (3) is 20mM-80mM. Further preferably, the concentration of the antitumor drug in the mixed solution C of step (3) is 30mM-70mM.

[0027] Preferably, the poor solvent C in step (3) is selected from one or more of water, phosphate buffer, physiological saline, and glucose solution. More preferably, the poor solvent C in step (3) is selected from one or more of water and phosphate buffer. Further preferably, the poor solvent C in step (3) is phosphate buffer.

[0028] Preferably, the volume ratio of the mixed solution C to the poor solvent C in the step (3) is 1:5-1:100. More preferably, the volume ratio of the mixed solution C to the poor solvent C in the step (3) is 1:10-1:50. Further preferably, the volume ratio of the mixed solution C to the poor solvent C in the step (3) is 1:20-1:30. Further preferably, the volume ratio of the mixed solution C to the poor solvent C in the step (3) is 1:25.

[0029] Preferably, the concentration range of IR780 in the mixed solution D in step (3) is 0.05mM-5mM. More preferably, the concentration range of IR780 in the mixed solution D in step (3) is 0.1mM-1mM. Further preferably, the concentration range of IR780 in the mixed solution D in step (3) is 0.14mM-0.5mM. Further preferably, the concentration range of IR780 in the mixed solution D in step (3) is 0.14mM.

[0030] Preferably, the concentration of IR780 in the nanoformulation of step (4) is greater than or equal to 0.1 μg / mL, or greater than or equal to 0.5 μg / mL, or greater than or equal to 1 μg / mL, or greater than or equal to 2 μg / mL, or greater than or equal to 5 μg / mL; the concentration of curcumin is greater than or equal to 1 μg / mL, or greater than or equal to 5 μg / mL, or greater than or equal to 10 μg / mL, or greater than or equal to 20 μg / mL, or greater than or equal to 50 μg / mL. More preferably, the concentration of IR780 in the nanoformulation of step (4) is 0.1-10 μg / mL, and the concentration of curcumin is 1-100 μg / mL. Further preferably, the concentration of IR780 in the nanoformulation of step (4) is 1-10 μg / mL, and the concentration of curcumin is 10-100 μg / mL. Further preferably, the concentration of IR780 in the nanoformulation of step (4) is 2-10 μg / mL, and the concentration of curcumin is 20-100 μg / mL. Further preferably, the concentration of IR780 in the nanoformulation of step (4) is 5-10 μg / mL, and the concentration of curcumin is 50-100 μg / mL.

[0031] Preferably, the concentration of IR780 in the nanoformulation of step (4) is greater than or equal to 2ng / mL, or greater than or equal to 10ng / mL, or greater than or equal to 20ng / mL, or greater than or equal to 40ng / mL, or greater than or equal to 100ng / mL; the concentration of paclitaxel is greater than or equal to 20ng / mL, or greater than or equal to 100ng / mL, or greater than or equal to 200ng / mL, or greater than or equal to 400ng / mL, or greater than or equal to 1000ng / mL. More preferably, the concentration of IR780 in the nanoformulation of step (4) is 2-200ng / mL, and the concentration of paclitaxel is 20-2000ng / mL. Further preferably, the concentration of IR780 in the nanoformulation of step (4) is 20-200ng / mL, and the concentration of paclitaxel is 200-2000ng / mL. More preferably, the concentration of IR780 in the nanoformulation of step (4) is 40-200 ng / mL, and the concentration of paclitaxel is 400-2000 ng / mL. More preferably, the concentration of IR780 in the nanoformulation of step (4) is 100-200 ng / mL, and the concentration of paclitaxel is 1000-2000 ng / mL.

[0032] Preferably, the method for removing the organic solvent in step (4) is dialysis or volatilization.

[0033] Preferably, the step (4) further comprises a concentration step after removing the organic solvent.

[0034] The third aspect of the present invention provides a nanoformulation, which comprises the above-mentioned nanoparticles and a pharmaceutically acceptable excipient.

[0035] Preferably, the pharmaceutically acceptable excipient is selected from one or more of water, phosphate buffer, physiological saline, and glucose solution.

[0036] Preferably, the nano preparation is an oral preparation. Further preferably, the nano preparation is an oral liquid.

[0037] Preferably, the concentration of IR780 in the nanoformulation is greater than or equal to 0.1 μg / mL, or greater than or equal to 0.5 μg / mL, or greater than or equal to 1 μg / mL, or greater than or equal to 2 μg / mL, or greater than or equal to 5 μg / mL; the concentration of curcumin is greater than or equal to 1 μg / mL, or greater than or equal to 5 μg / mL, or greater than or equal to 10 μg / mL, or greater than or equal to 20 μg / mL, or greater than or equal to 50 μg / mL. More preferably, the concentration of IR780 in the nanoformulation is 0.1-10 μg / mL, and the concentration of curcumin is 1-100 μg / mL. Further preferably, the concentration of IR780 in the nanoformulation is 1-10 μg / mL, and the concentration of curcumin is 10-100 μg / mL. Further preferably, the concentration of IR780 in the nanoformulation is 2-10 μg / mL, and the concentration of curcumin is 20-100 μg / mL. Further preferably, the concentration of IR780 in the nanoformulation is 5-10 μg / mL, and the concentration of curcumin is 50-100 μg / mL.

[0038] Preferably, the concentration of IR780 in the nanoformulation is greater than or equal to 2ng / mL, or greater than or equal to 10ng / mL, or greater than or equal to 20ng / mL, or greater than or equal to 40ng / mL, or greater than or equal to 100ng / mL; the concentration of paclitaxel is greater than or equal to 20ng / mL, or greater than or equal to 100ng / mL, or greater than or equal to 200ng / mL, or greater than or equal to 400ng / mL, or greater than or equal to 1000ng / mL. More preferably, the concentration of IR780 in the nanoformulation is 2-200ng / mL, and the concentration of paclitaxel is 20-2000ng / mL. Further preferably, the concentration of IR780 in the nanoformulation is 20-200ng / mL, and the concentration of paclitaxel is 200-2000ng / mL. Further preferably, the concentration of IR780 in the nanoformulation is 40-200ng / mL, and the concentration of paclitaxel is 400-2000ng / mL. More preferably, the concentration of IR780 in the nanoformulation is 100-200 ng / mL, and the concentration of paclitaxel is 1000-2000 ng / mL.

[0039] The fourth aspect of the present invention provides the use of the self-assembled nanoparticles in the preparation of anti-tumor drugs.

[0040] Beneficial effects of the present invention:

[0041] 1. The present invention utilizes the interaction between IR780 and hydrophobic anti-tumor drugs in a water-soluble system, and aggregates to form nano-sized particles through π-π stacking, hydrophobic forces and electrostatic forces, effectively solving the water solubility and bioavailability problems of IR780 and hydrophobic drugs. In addition, the self-assembled nanoparticles of the present invention do not contain any carrier excipients, overcome the complex metabolism of traditional nano drug delivery carriers in the body, and avoid the clinical safety risks brought by artificial synthetic carriers.

[0042] 2. The self-assembled nanoparticles of the present invention can be accumulated in the tumor site after oral administration, wherein the photosensitizer IR780 can perform in vivo imaging and photothermal therapy, and realize an "oral-integrated diagnosis and treatment" treatment mode of synergistically treating tumors with co-loaded anti-tumor drugs.

[0043] 3. The self-assembled nanoparticles of the present invention can be administered orally, which greatly improves the patient's compliance and effectively reduces medical costs.

[0044] 4. The self-assembled nanoparticles of the present invention have a simple preparation process and convenient operation, and are suitable for industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is the particle size distribution diagram of IR780-CCM nanoparticles in Example 1;

[0046] Figure 2 is a transmission electron microscopy image of IR780-CCM nanoparticles in Example 1;

[0047] Figure 3 is the particle size distribution diagram of IR780-PTX nanoparticles in Example 2;

[0048] Figure 4 The photothermal-chemotherapy combined treatment effect of IR780-CCM nanoparticles in CT26 cells in Experimental Example 1;

[0049] Figure 5 This is the tumor targeting test effect of IR780-CCM nanoparticles after oral administration in Test Example 2;

[0050] Figure 6 This is the combined photothermal and chemotherapeutic effect of IR780-PTX nanoparticles in CT26 cells in Experimental Example 3. DETAILED DESCRIPTION

[0051] The technical scheme of the present invention is further described in detail below through specific embodiments. It should be understood that the examples listed are only for the purpose of illustrating the present invention, not for limiting the present invention. Without departing from the spirit and technical principles of the present invention, any modification made thereto, and any equivalent substitution or improvement made based on the conventional knowledge and common means of those skilled in the art, shall be regarded as part of the protection scope of the present invention.

[0052] Example 1. Preparation of IR780-Curcumin Self-Assembled Nanoparticles (IR780-CCM NPs)

[0053] Weigh 10 mg IR780 and dissolve it in 1 mL ethanol to a concentration of 15 mM. At the same time, weigh 100 mg curcumin (CCM) and dissolve it in 3 mL chloroform to a concentration of 90 mM. Mix the two solutions thoroughly. Under continuous stirring, slowly drop the mixed solution into a beaker containing 100 mL PBS solution. The concentration of IR780 in the mixed solution is 0.14 mM, and the concentration of curcumin is 2.6 mM. Use dialysis to remove free drugs and organic solvents to obtain carrier-free self-assembled nanoparticles IR780-CCMNPs, which are concentrated to obtain.

[0054] The average particle size of the carrier-free self-assembled nanoparticles IR780-CCMNPs prepared in Example 1 is 150 nm, and the particle size distribution is as follows: Figure 1 The morphology of the IR780-CCM NPs prepared in this embodiment was observed using a transmission electron microscope (TEM). Figure 2 shown.

[0055] Since IR780 and curcumin have low solubility in aqueous solution, precipitation occurs after standing, and stable nanoparticles cannot be formed alone. The above experimental results show that through the preparation process of the present invention, IR780 and curcumin form stable self-assembled nanoparticles by π-π stacking, hydrophobic force and electrostatic force.

[0056] Example 2. Preparation of IR780-paclitaxel self-assembled nanoparticles (IR780-PTX NPs)

[0057] Weigh 10 mg of IR780 and dissolve it in 1 mL of ethanol to a concentration of 15 mM. At the same time, weigh 100 mg of PTX and dissolve it in 3 mL of dichloromethane to a concentration of 39 mM. Mix the two solutions thoroughly. Under continuous stirring, slowly drop the mixed solution into a beaker containing 100 mL of PBS solution. The concentration of IR780 in the mixed solution is 0.14 mM, and the concentration of paclitaxel is 1.1 mM. Use dialysis to remove free drugs and organic solvents to obtain carrier-free self-assembled nanoparticles IR780-PTX NPs, which are concentrated to obtain.

[0058] The average particle size of the IR780-PTX NPs prepared in Example 2 is 135 nm, and the particle size distribution is as follows: Figure 3 The above experimental results show that, through the preparation process of the present invention, IR780 and paclitaxel form stable self-assembled nanoparticles by π-π stacking, hydrophobic force and electrostatic force.

[0059] Experimental Example 1: Anti-tumor effect test of photothermal combined chemotherapy of self-assembled nanoparticles IR780-CCM NPs

[0060] 1. Test materials

[0061] The commonly used MTT method in the laboratory was used to determine the proliferation inhibitory activity of free IR780 (free IR780 group), IR780-curcumin nanoparticles (IR780-CCM NPs group), curcumin nanoparticles (CCM NPs group), and free IR780 and IR780-curcumin nanoparticles under laser irradiation ("free IR780+Laser group" and "IR780-CCM NPs+Laser group", respectively) on CT-26 cells.

[0062] The preparation method of IR780-CCM NPs is the same as that of Example 1 of the present invention. The preparation method of CCM NPs is as follows: referring to the preparation method of IR780-curcumin nanoparticles in Example 1, 100 mg of curcumin is dissolved in 3 mL of chloroform and 1 mL of ethanol, and the remaining steps are the same as those of Example 1. The preparation method of free IR780 is as follows: 50 mg of IR780 is dissolved in 1 mL of DMSO for later use, and diluted with PBS to the desired concentration.

[0063] 2. Test methods

[0064] CT-26 cells were plated at 1×10 3 Cells were seeded in 96-well plates at a density of 100 cells and cultured in RPMI 1640 medium containing 10% fetal bovine serum. Fresh RPMI1640 medium containing different concentrations of IR780-CCM NPs and control groups was prepared. The IR780 concentrations of IR780-CCM NPs and their control groups were set to 0, 0.1, 0.5, 1.0, 2.0, 5.0 μg / mL, and the CCM concentrations were set to 0, 1.0, 5.0, 10.0, 20.0, 50.0 μg / mL. These culture media were replaced with cells that had been cultured overnight in the plate, and the cells were incubated in these new culture media for 2 hours. After the incubation period, the culture medium was removed and the cells were washed with PBS to remove the unabsorbed nanoparticles and residual culture medium. The cells in the experimental group that needed laser irradiation were then exposed to 808 nm and a power of 1 W / cm2 The laser was irradiated for 3 minutes to stimulate the photothermal effect of the nanoparticles and evaluate their photothermal therapeutic effect.

[0065] Add 20 μL of MTT solution to each well and continue incubation for 4 hours. After 4 hours, remove the solution, add 150 μL of dimethyl sulfoxide (DMSO) to each well to dissolve the methylamine blue crystals, measure the absorbance of each well at a wavelength of 570 nm using an ELISA reader, and calculate the cell survival rate using the following formula: Cell survival rate = (absorbance of the treatment group / absorbance of the control group) × 100%.

[0066] 3. Test results

[0067] The coefficient of drug interaction (CDI) was used to evaluate the nature of the drug-drug interaction, which was calculated according to the following formula: CDI = AB / (A×B). In this test example, the survival rate of tumor cells in different test groups was used to calculate CDI, where AB was the survival rate of tumor cells in the combination group of the two drugs; A or B was the survival rate of tumor cells in the group where each drug was used alone. When CDI < 1, it means that the interaction between A and B is synergistic; when CDI < 0.7, it means that the synergistic effect between A and B is significant; when CDI = 1, it means that the interaction between A and B is additive; when CDI > 1, it means that the interaction between A and B is antagonistic.

[0068] The experimental results of the combined use of drugs plus laser irradiation (IR780-CCM NPs+Laser group) and the control group (free IR780+Laser group and CCM NPs group) are as follows Figure 4 As shown in Table 1 below, the cell viability was calculated as the average value at each dose. When IR780 was 5 μg / mL and CCM was 50 μg / mL, the CDI value of the combination of the two drugs was 0.4, indicating that the combination of IR780 and curcumin had a significant synergistic effect.

[0069] Table 1 CDI values ​​of different concentration ratios of IR780 and CCM combinations

[0070] IR780 concentration (μg / mL) 0.1 0.5 1 2 5 CCM concentration (μg / mL) 1 5 10 20 50 CDI 0.98 0.99 0.98 0.48 0.4

[0071] In addition, from Figure 4 The test results show that the cytotoxicity of the IR780-CCM NPs group is significantly improved, and the cytotoxicity of the IR780-CCM NPs+Laser group after laser irradiation is more significant, which proves the effect of photothermal therapy. Among them, the IR780-CCM NPs+Laser group has the best anti-tumor effect. Combined with the CDI value calculated above, it shows that the photothermal therapy of the nanoparticles of the present invention has a synergistic effect with chemotherapy.

[0072] Experimental Example 2: In vivo targeting test after oral administration of IR780-CCM NPs

[0073] 1. Test methods

[0074] To verify the in vivo tumor targeting of IR780-CCM NPs after oral administration, tumor-bearing mice were given the drug by oral gavage, and the distribution of IR780 in the body at different time points after administration was photographed using a small animal near-infrared imaging system.

[0075] Among them, the preparation method of IR780-CCM NPs is the same as that of Example 1 of the present invention. The preparation method of free IR780 is the same as that of Experimental Example 1. The preparation method of lipid nanocarrier loaded IR780 (IR780@NLCs) is as follows: soybean lecithin (260 mg), triglyceride (20 mg), Labrafac CC (70 mg) and IR780 (8 mg) are dissolved in 40 mL of ethanol / dichloromethane mixed solvent (volume ratio 4: 1). The solvent is evaporated under vacuum conditions at 37 ° C by a rotary evaporator to form a thin film on the surface of a round-bottom flask. The dried film is then hydrated at 40 ° C, treated with 100 W ultrasound, and extruded through a 0.22 μm filter membrane to remove free IR780, thereby obtaining IR780@NLCs.

[0076] The tumor volume was about 800 mm 3 CT-26 tumor-bearing mice were used as an experimental model to evaluate the effect of drug distribution in tumors. IR780-CCMNPs, lipid nanocarrier-loaded IR780, and free IR780 were administered to mice by oral gavage, and the dosage of IR780 in the three test samples was 1.0 mg / kg. Near-infrared fluorescence imaging of mice was performed at multiple time points from 1 hour after oral administration to 72 hours to cover the entire process of drug absorption, distribution, and metabolism. Images were taken using a small animal in vivo imager with an excitation wavelength set to 745 nm and an emission wavelength of 780 nm, which is designed to maximize the detection of IR780 fluorescence signals.

[0077] 2. Test results

[0078] Figure 5The near-infrared fluorescence images at different time points after oral administration of IR780-CCMNPs, lipid nanocarriers loaded with IR780 (NLCs group) and free IR780 to tumor-bearing mice. From 1 hour to 72 hours of monitoring, it can be seen that the IR780 fluorescence is almost invisible at the tumor site in the mice in the oral free IR780 group, while the IR780-CCM NPs group shows strong tumor accumulation. IR780 fluorescence can be seen after 3 hours, and the fluorescence reaches the strongest at 24 hours. Although the NLCs group can also accumulate in the tumor site for 24 hours, the fluorescence is weaker than that of the IR780-CCM NPs group. The above experimental results show that IR780-CCM NPs have excellent tumor targeting after oral administration and have good oral imaging effects.

[0079] Experimental Example 3: Anti-tumor effect test of photothermal combined chemotherapy of self-assembled nanoparticles IR780-PTX NPs

[0080] 1. Test materials

[0081] The commonly used MTT method in the laboratory was used to determine the proliferation inhibitory activity of PBS solution control (PBS group), free IR780 (free IR780 group), IR780-paclitaxel nanoparticles (IR780-CCM NPs group), free paclitaxel (free PTX), and PBS solution, free IR780 and IR780-paclitaxel nanoparticles under laser irradiation ("PBS Laser group", "free IR780+Laser group" and "IR780-PTXNPs+Laser group", respectively) on CT-26 cells.

[0082] The preparation method of IR780-paclitaxel nanoparticles is the same as that of Example 2 of the present invention. The preparation method of free IR780 is the same as that of Experimental Example 1. The preparation method of free paclitaxel is as follows: 20 mg of paclitaxel is dissolved in 1 mL of a mixed solvent of polyoxyethylene castor oil and ethanol (volume ratio 1:1), and diluted with PBS to a desired concentration as needed.

[0083] 2. Test methods

[0084] CT-26 cells were plated at 1×10 3Cells were seeded in 96-well plates at a density of 10 cells and cultured in RPMI 1640 medium containing 10% fetal bovine serum. Fresh RPMI1640 medium containing different concentrations of IR780-PTX NPs and control groups was prepared. The IR780 concentrations of the nanoparticles and their control groups were set to 0, 2.0, 10.0, 20.0, 40.0, 100.0 ng / mL, and the PTX concentrations were set to 0, 20.0, 100.0, 200.0, 400.0, 1000.0 ng / mL. These culture media were replaced with cells that had been cultured overnight in the plate, and the cells were incubated in these new culture media for 2 hours. After the incubation, the culture medium was removed and the cells were washed with PBS to remove the unabsorbed nanoparticles and residual culture medium. The cells in each experimental group that required laser irradiation were then exposed to 808nm and a power of 1W / cm 2 The laser was irradiated for 3 minutes to stimulate the photothermal effect of the nanoparticles and evaluate their photothermal therapeutic effect.

[0085] Add 20 μL MTT solution to each well and continue incubation for 4 hours. After 4 hours, remove the solution and add 150 μL dimethyl sulfoxide (DMSO) to each well to dissolve the methylamine blue crystals. Use an ELISA reader to measure the absorbance value of each well at a wavelength of 570 nm, and calculate the cell survival rate using the following formula: Cell survival rate = (absorbance of the treatment group / absorbance of the control group) × 100%.

[0086] 3. Test results

[0087] The drug interaction index (CDI) was calculated by referring to the method in Experimental Example 1. The experimental results of the combined use of drugs plus laser irradiation (IR780-PTXNPs+Laser group) and the control group (free IR780+Laser group and free PTX group) are as follows Figure 6 As shown in Table 2 below, when IR780 was 100 ng / mL and PTX was 1000 ng / mL, the CDI value of the combination of the two drugs was 0.57, indicating that the combination of IR780 and paclitaxel has a significant synergistic effect.

[0088] Table 2 CDI values ​​of different concentration ratios of IR780 and PTX combinations

[0089] IR780 concentration (ng / mL) 2 10 20 40 100 PTX concentration (ng / mL) 20 100 200 400 1000 CDI 0.92 0.90 0.91 0.69 0.57

[0090] In addition, from Figure 6The data show that the cytotoxicity of the IR780-PTX NPs group is significantly improved, and the cytotoxicity of the IR780-PTX NPs+Laser group irradiated by laser is more significant, proving the effect of photothermal therapy. Among them, the IR780-PTX NPs+Laser group has the best anti-tumor effect. Combined with the CDI value calculated above, it also shows that the photothermal therapy of the nanoparticles of the present invention has a synergistic effect with chemotherapy.

[0091] Although specific embodiments of the present invention have been described, it will be appreciated by those skilled in the art that various changes and modifications may be made to the present invention without departing from the scope or spirit of the present invention. Therefore, the present invention is intended to cover all such changes and modifications that fall within the scope of the appended claims and their equivalents.

Claims

1. A self-assembled nanoparticle, characterized in that: The nanoparticles are self-assembled from a hydrophobic anti-tumor drug and a photosensitizer, the photosensitizer is IR780, and the nanoparticles do not contain pharmaceutically acceptable auxiliary carriers.

2. The self-assembled nanoparticles according to claim 1, characterized in that The hydrophobic anti-tumor drug is selected from one or more of curcumin, paclitaxel, docetaxel, sirolimus, sorafenib, aclarubicin, idarubicin, triptolide, camptothecin, 10-hydroxycamptothecin, colchicine, vincristine, methotrexate, tamoxifen, teniposide and mitoxantrone.

3. The self-assembled nanoparticles according to claim 2, characterized in that: The hydrophobic anti-tumor drug is selected from one or more of curcumin, paclitaxel and docetaxel.

4. The self-assembled nanoparticles according to claim 1, characterized in that The nanoparticles are formed by hydrophobic anti-tumor drugs and IR780 photosensitizers through π-π stacking, hydrophobic forces and electrostatic forces.

5. The self-assembled nanoparticles according to claim 1, characterized in that: The molar ratio of the hydrophobic anti-tumor drug to the photosensitizer in the nanoparticles is.

6. A method for preparing a nanoformulation comprising the self-assembled nanoparticles according to any one of claims 1 to 5, characterized in that: The preparation method comprises the following steps: (1) dissolving IR780 in a benign solvent A to obtain a solution A; (2) dissolving the antitumor drug in a benign solvent B to obtain a solution B; (3) fully mixing solution A and solution B to obtain a mixed solution C, and adding the mixed solution C dropwise into the poor solvent C under continuous stirring to obtain a mixed solution D; (4) removing the organic solvent from the mixed solution D obtained in step (3) to obtain a nanoformulation containing self-assembled nanoparticles.

7. The method for preparing the self-assembled nanoparticles according to claim 6, characterized in that: The benign solvent A in step (1) is selected from one or more of dichloromethane, chloroform, methanol, ethanol, ether, ethyl acetate, methyl acetate, acetone, n-propanol, pyridine, N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide.

8. The method for preparing the self-assembled nanoparticles according to claim 6, characterized in that: The benign solution B in step (2) is selected from one or more of dichloromethane, chloroform, methanol, ethanol, ether, ethyl acetate, methyl acetate, acetone, n-propanol, pyridine, N,N-dimethylformamide, tetrahydrofuran, and dimethyl sulfoxide.

9. The method for preparing the self-assembled nanoparticles according to claim 6, characterized in that: The poor solvent C in step (3) is selected from one or more of water, phosphate buffer, physiological saline, and glucose solution.

10. Use of the self-assembled nanoparticles according to any one of claims 1 to 5 in the preparation of anti-tumor drugs.

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