Ternary carrier-free nanoparticle as well as preparation method and application thereof

Abstract

The invention discloses a ternary carrier-free nanoparticle as well as a preparation method and application thereof. The ternary carrier-free nanoparticles comprise nanoparticles which are formed by the chlorine e6, the lonidamine and the RG108 through an intermolecular interaction force. The nanoparticles can improve effective absorption and accumulation of drugs in tumors, can induce tumor specific pyroptosis and immunogenic effect under laser irradiation, and can promote maturation of antigen presenting cells and recruitment and activation of cytotoxic T lymphocytes, thereby starting adaptive anti-tumor immunity.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a ternary carrier-free nanoparticle and a preparation method and application thereof. Background Art

[0002] Pyroptosis is a unique inflammatory programmed cell death mode. It activates the Caspase-3 / GSDME pathway to form GSDME-N fragments that can penetrate the cell membrane, release immunostimulatory contents such as damage-associated molecular patterns (DAMPs) and various proinflammatory cytokines, and thus lead to immunogenic cell death (ICD) and achieve anti-tumor immunotherapy. Photodynamic therapy (PDT) is used to increase the incidence of pyroptosis because of its high spatiotemporal controllability. It produces reactive oxygen species (ROS) by laser irradiation to cause oxidative damage to cancer cells. At the same time, the level of ROS production is closely related to the activation of Caspase-3. However, the tumor's own defense mechanism limits the application of single photodynamic therapy combined with pyroptosis. In the hypoxic microenvironment of the tumor, tumor cells rely more on the glycolysis process to maintain sufficient energy supply, which further limits the conditions for the production of ROS, thereby hindering the activation of PDT. At the same time, this abnormal metabolic process will form an immunosuppressive tumor microenvironment (ITM), which seriously hinders the immune response of the tumor. Due to promoter methylation, the expression of GSDME in tumors is reduced. Therefore, it is necessary to develop a method that can enhance photodynamic-induced pyroptosis and improve the immunosuppressive microenvironment to enhance the effect of anti-tumor immunotherapy.

[0003] High glycolysis rates within tumors convert pyruvate into lactate (LA), which is one of the reasons why immunosuppression limits pyroptotic immunotherapy. The acidic tumor microenvironment often makes tumor-associated macrophages (TAMs) more inclined to transform into the pro-tumor M2 type. Regulatory T cells (Tregs) have metabolic flexibility and can use the "alternative" metabolite LA in the tumor microenvironment to maintain their inhibitory properties on effector T cells. The resulting ITM poses a huge challenge to the efficacy of immunotherapy. Hexokinase II (HKII) plays a key role in the regulation of glycolysis in tumors. Therefore, HKII inhibitors have great potential in reducing lactate production in tumor cells and regulating ITM caused by tumor metabolism.

[0004] Nanomaterials have shown extraordinary application prospects in the field of drug delivery due to their unique physical and chemical properties. Using nanomaterials as carriers can increase the cumulative concentration of drugs in tumor sites and optimize the bioavailability of drugs. Most existing nanomedicines use responsive polymer carriers to encapsulate hydrophobic drugs, mesoporous materials to adsorb drugs, etc., which have problems such as the inability to control the release of drugs and the potential biological toxicity of carrier materials. Summary of the invention

[0005] The present invention aims to solve at least one of the above technical problems existing in the prior art. To this end, the present invention aims to provide a ternary carrier-free nanoparticle and a preparation method and application thereof.

[0006] In order to achieve the above object, the technical solution adopted by the present invention is:

[0007] One aspect of the present invention provides a ternary carrier-free nanoparticle, comprising nanoparticles formed by intermolecular interaction forces of dihydrochlorin e6, lonidamine and RG108.

[0008] In the present invention, the photosensitizer Ce6, the glycolysis inhibitor LND and the DNA methyltransferase inhibitor RG108 can self-organize to form carrier-free ternary nanoparticles through hydrophobic interaction, π-π stacking and hydrogen bonding interaction, and the nanoparticles have uniform particle size, good stability and high drug loading rate.

[0009] In some embodiments of the present invention, the intermolecular interaction force includes at least one of a hydrophobic force, a π-π stacking force and a hydrogen bonding force.

[0010] In some embodiments of the present invention, the mass ratio of chlorin e6, lonidamine and RG108 is (1-15):(3-10):(3-15), such as (3-15):(3-8):(3-12). In the present invention, limiting the mass ratio of the three can effectively improve the stability of the nanoparticles.

[0011] In some embodiments of the present invention, the average particle size of the nanoparticles is 100 nm to 180 nm, such as 110 nm to 155 nm.

[0012] In some embodiments of the present invention, the surface of the nanoparticle is negatively charged.

[0013] Another aspect of the present invention provides a method for preparing the ternary carrier-free nanoparticles, comprising the following steps:

[0014] The organic solution of dihydrochlorin e6, lonidamine and RG108 is ultrasonically treated to prepare the ternary carrier-free nanoparticles.

[0015] In some embodiments of the present invention, the organic solvent of the organic solution includes at least one of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and tetrahydrofuran (THF).

[0016] In some embodiments of the present invention, the mass concentration feed ratio of the dihydrochlorin e6, lonidamine and RG108 is 1:0.5-5:0.5-5.

[0017] In some embodiments of the present invention, the concentration of the organic solution of chlorin e6 is 5 to 15 mg / mL.

[0018] In some embodiments of the present invention, the concentration of the organic solution of lonidamine is 5 to 15 mg / mL.

[0019] In some embodiments of the present invention, the concentration of the organic solution of RG108 is 10 to 30 mg / mL.

[0020] In some embodiments of the present invention, the frequency of the ultrasound is 20 to 60 kHz; the time of the ultrasound is 1 to 10 min.

[0021] In some embodiments of the present invention, the method for preparing the ternary carrier-free nanoparticles comprises dropwise adding an organic solution of dihydrochlorin e6, lonidamine and RG108 into water while ultrasonicating, and continuing ultrasonicating to obtain the ternary carrier-free nanoparticles.

[0022] In some embodiments of the present invention, the water includes at least one of distilled water, deionized water, ultrapure water, purified water, purified water, and sterile water.

[0023] In some embodiments of the present invention, the method for preparing the ternary carrier-free nanoparticles further comprises dialyzing the ultrasonic product after ultrasonication to remove the organic solvent to obtain the ternary carrier-free nanoparticles.

[0024] In some embodiments of the present invention, the dialysis uses a dialysis bag of 800-1200 Da; the dialysis time is 3-6 hours.

[0025] Another aspect of the present invention provides a pharmaceutical composition comprising the ternary carrier-free nanoparticles.

[0026] In some embodiments of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

[0027] In some embodiments of the present invention, the drug can be prepared into dosage forms such as powders, suppositories, injections, emulsions, patches, sprays, aerosols, etc., and suitable drug carriers in the art can be selected for different dosage forms.

[0028] In some embodiments of the present invention, the pharmaceutical carrier used can be solid, liquid or gas. Examples of solid carriers include lactose, kaolin, sucrose, talcum powder, gelatin, agar, pectin, gum arabic, magnesium stearate and stearic acid. Examples of liquid carriers include syrup, peanut oil, olive oil and water. Examples of gaseous carriers include carbon dioxide and nitrogen.

[0029] In some embodiments of the present invention, any convenient pharmaceutical medium can be used when preparing oral dosage forms of the drug. For example, water, ethanol, oil, alcohol, flavoring agents, preservatives, colorants, etc. can be used to form oral liquid preparations, such as suspensions, elixirs and solutions; and carriers, such as starch, sugars, microcrystalline cellulose, diluents, granulators, emulsifiers, lubricants, binders, disintegrants can be used to form oral solid preparations, such as powders, capsules and tablets. Tablets and capsules are preferred oral dosage units using solid pharmaceutical carriers due to their ease of administration. Tablets can be coated using standard aqueous or non-aqueous techniques.

[0030] In some embodiments of the invention, tablets containing nanoparticles of the invention may be prepared by tableting or molding, optionally with the use of one or more auxiliary ingredients or adjuvants. Compressed tablets may be prepared by tableting the active ingredient in a free-flowing form (such as a powder or granules) in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, surfactant or dispersant. Molded tablets may be molded in a suitable machine, i.e., powdered nanoparticles moistened with an inert liquid diluent. Each tablet preferably contains about 0.05 mg to about 5 g of nanoparticles, and each sachet or capsule preferably contains about 0.05 mg to about 5 g of nanoparticles. For example, a formulation intended for oral administration to humans may contain about 0.5 mg to about 5 g of nanoparticles, mixed with an appropriate and convenient carrier material, which may account for about 5% to 95% of the total composition. Unit dosage forms typically contain from about 1 mg to about 2 g of nanoparticles, typically 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg or 1000 mg.

[0031] The invention can be prepared as an aqueous solution or suspension comprising nanoparticles for parenteral administration. Suitable surfactants such as hydroxypropylcellulose can be included. Dispersions can also be prepared in glycerol, liquid polyethylene glycol and oil mixtures thereof. In addition, preservatives can be added to prevent harmful growth of microorganisms.

[0032] The drugs suitable for injection use in the present invention include sterile aqueous solutions or dispersions. In addition, the drug can be in the form of sterile powders for the extemporaneous preparation of such sterile injections or dispersions. In all cases, the final injection form must be sterile and must be an effective liquid so that the injectable drug component must remain stable under production and storage conditions; therefore, it is best to preserve it to prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium, for example, containing water, ethanol, polyols (such as glycerol, propylene glycol and liquid polyethylene glycol), vegetable oils and suitable mixtures thereof.

[0033] Medicine of the present invention can be in a form suitable for topical use, such as aerosol, cream, ointment, lotion, powder or the like. In addition, nanoparticles can be in a suitable form for transdermal administration devices. Nanoparticles of the present invention can be used to prepare these prescriptions by conventional processing methods. For example, by mixing hydrophilic materials and water, and nanoparticles of about 5wt% to about 10wt%, creams or ointments with desired consistency are prepared.

[0034] The medicine of the present invention can be in a form suitable for rectal administration, wherein the carrier is a solid. Preferably, the mixture is made into a unit dose suppository. Suitable carriers include cocoa butter and other materials commonly used in the art. Suppositories can be prepared by first forming a composition containing a softened or melted carrier, followed by cooling and shaping in a mold.

[0035] In addition to the above-mentioned carrier components, the above-mentioned pharmaceutical preparations may include (if applicable) one or more additional carrier components, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. In addition, other excipients, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., colorants and flavoring agents, etc., may be added. The preparation is made isotonic with the blood of the intended recipient. The components containing the nanoparticles of the present invention can also be prepared in the form of powder or concentrate.

[0036] Another aspect of the present invention provides the use of the ternary carrier-free nanoparticles and / or the pharmaceutical composition in the preparation of tumor therapeutic drugs.

[0037] In some embodiments of the present invention, the tumor treatment includes at least one of photodynamic therapy, immunotherapy, pyroptosis therapy, and metabolic regulation therapy.

[0038] In some embodiments of the present invention, the tumor includes at least one of breast cancer, prostate cancer, ovarian cancer, intestinal cancer, lung cancer, and pancreatic cancer.

[0039] In some embodiments of the present invention, the photodynamic therapy uses a laser of 500 to 750 nm (such as 600 to 700 nm) at 0.1 to 1.0 W / cm 2 The light intensity is irradiated for 1 to 20 minutes; such as 1 to 15 minutes, 1 to 10 minutes, etc.

[0040] The beneficial effects of the present invention are:

[0041] Photosensitizer Ce6, glycolysis inhibitor LND and DNA methyltransferase inhibitor RG108 self-assembled into CLRN nanoparticles with high drug loading and stability through hydrophobic interactions, π-π stacking and hydrogen bonding interactions, realizing carrier-free self-assembly of more than two drugs. CLRN can improve the effective absorption and accumulation of drugs in tumors, and can also induce tumor-specific pyroptosis under laser irradiation, induce immunogenic effects, promote the maturation of antigen-presenting cells and the recruitment and activation of cytotoxic T lymphocytes, thereby initiating adaptive anti-tumor immunity. In addition, CLRN can inhibit glycolysis and relieve tumor hypoxia, thereby amplifying the effect of photodynamic activation of pyroptosis in a synergistic manner. At the same time, CLRN can reduce lactate production by inhibiting glycolysis, promote M1 macrophage polarization, reprogram the acidic immunosuppressive microenvironment caused by metabolic abnormalities, and synergistically amplify the anti-tumor immune effect, ultimately leading to the regression of primary and distant tumors. In summary, combining photodynamic therapy to achieve controllable activation of pyroptosis is a new strategy for the treatment of triple-negative breast cancer. This approach has strong immunological efficacy and has the potential to alleviate immunosuppression, amplifying the immunotherapeutic effect in a synergistic manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The particle size and polydispersity index of the nanoparticles in Example 1.

[0043] Figure 2 The particle size and polydispersity index of the nanoparticles in Example 2.

[0044] Figure 3 The particle size and polydispersity index of the nanoparticles in Example 3.

[0045] Figure 4 2 is the potential diagram of the nanoparticles of Examples 1 to 3.

[0046] Figure 5 The stability test results of the nanoparticles of Example 1 in water and PBS.

[0047] Figure 6 These are laser confocal microscopy images of 4T1 cells taking up CLRN for 6 h and 8 h in Example 4.

[0048] Figure 7 Schematic diagram of the reactive oxygen production capacity of each group after treatment under normoxic conditions.

[0049] Figure 8 Schematic diagram of the reactive oxygen production capacity of each group after treatment under hypoxic conditions.

[0050] Fig. 9 Analysis of lactate content in cells and culture supernatant.

[0051] Fig.10Cytotoxicity analysis of 4T1 cells under normoxic conditions.

[0052] Fig.11 This is a cytotoxicity analysis of 4T1 cells under hypoxic conditions.

[0053] Fig.12 This is a 14-day tumor growth curve of mice with unilateral triple-negative breast cancer model.

[0054] Fig.13 This is the 14-day tumor growth curve of the proximal and distal tumors in mice with bilateral triple-negative breast cancer model.

[0055] Fig.14 Determination of mature antigen-presenting cells in the lymph nodes of triple-negative breast cancer model mice.

[0056] Fig.15 CD8 proximal and distal tumors in triple-negative breast cancer model mice + Cytotoxic T lymphocyte cell assay.

[0057] Fig.16 Analysis of the ratio of M1 macrophages to M2 macrophages in triple-negative breast cancer model mice.

[0058] Fig.17 To determine the suppressive T lymphocytes (Tregs) in triple-negative breast cancer model mice. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0060] Example 1

[0061] This example prepares a CLRN nanoparticle, and the specific process is as follows:

[0062] Accurately weigh 10 mg of chlorin e6 (Ce6) and lonidamine (LND) and dissolve them in 1 mL of dimethyl sulfoxide to prepare a 10 mg / mL mother solution. Accurately weigh 10 mg of RG108 and dissolve it in 500 μL of dimethyl sulfoxide to prepare a 20 mg / mL mother solution. Take 11.7 μL of chlorin e6 mother solution and lonidamine mother solution, take 5.8 μL of RG108 mother solution, add 70.8 μL of dimethyl sulfoxide, mix well, add dropwise to 1 mL of ultrapure water under 40kHz ultrasound, shake slowly until all drops, and continue ultrasound for 3 minutes. The resulting solution is transferred to a 1000Da dialysis bag, placed in 1L ultrapure water for 4 hours, and collected after removing the organic solvent to obtain a CLRN nanoparticle solution with a feed ratio of Ce6, LND, and RG108 of 1:1:1.

[0063] 200 μL of the nanoparticle solution was taken into a particle size dish and its particle size was measured using a Malvern particle size analyzer; the results are as follows: Figure 1 The obtained CLRN nanoparticles have uniform particle size, with an average particle size of 149.2±3.4nm; the mass ratio of Ce6, LND, and RG108 in the prepared CLRN nanoparticles is 11:5:4.

[0064] Example 2

[0065] This example prepares a CLRN nanoparticle, and the specific process is as follows:

[0066] 7 μL of chlorin e6 mother solution, 14 μL of lonidamine mother solution, 7 μL of RG108 mother solution, 72 μL of dimethyl sulfoxide were added, mixed evenly, and then added dropwise to 1 mL of ultrapure water under 40 kHz ultrasound, and slowly shaken until all the solution was dropped, and the ultrasound was continued for 3 min. The resulting solution was transferred to a 1000 Da dialysis bag, dialyzed in 1 L of ultrapure water for 4 h, and collected after removing the organic solvent to obtain a CLRN nanoparticle solution with a feed ratio of Ce6, LND, and RG108 of 1:2:2.

[0067] 200 μL of the nanoparticle solution was taken into a particle size dish and the particle size was measured using a Malvern particle size analyzer. The results are as follows: Figure 2 The average particle size of the obtained CLRN nanoparticles is 139.8±2.0nm. The mass ratio of Ce6, LND and RG108 in the prepared CLRN nanoparticles is 7:7:6.

[0068] Example 3

[0069] This example prepares a CLRN nanoparticle, and the specific process is as follows:

[0070] 5 μL of chlorin e6 mother liquor, 15 μL of lonidamine mother liquor, 7.5 μL of RG108 mother liquor were taken, 72.5 μL of dimethyl sulfoxide was added, mixed evenly, and then added dropwise to 1 mL of ultrapure water under 40kHz ultrasound, and slowly shaken until all the drops were added, and the ultrasound was continued for 3 minutes. The resulting solution was transferred to a 1000Da dialysis bag, dialyzed in 1L of ultrapure water for 4 hours, and collected after removing the organic solvent to obtain a CLRN nanoparticle solution with a feed ratio of Ce6, LND, and RG108 of 1:3:3.

[0071] 200 μL of the nanoparticle solution was taken into a particle size dish and the particle size was measured using a Malvern particle size analyzer. The results are as follows: Figure 3 The average particle size of the obtained CLRN nanoparticles is 121.6±12.3nm. The mass ratio of Ce6, LND and RG108 in the prepared CLRN nanoparticles is 4:3:12.

[0072] Test Example 1

[0073] In this test example, 200 μL of the nanoparticle solution obtained in the example was taken into a particle size dish, and the potential was measured using a Malvern particle size analyzer. The results are as follows: Figure 4 The nanoparticles obtained in different proportions are all negatively charged.

[0074] Test Example 2

[0075] In this test example, 200 μL of the nanoparticles obtained in Example 1 were taken in a particle size dish at 1, 3, 5, and 7 days respectively, and the particle sizes in water and PBS solution were measured using a Malvern particle size analyzer. The results are as follows: Figure 5 ,It can be seen that the CLRN nanoparticles with a feed ratio of 1:1:1 had good stability for seven days and did not change significantly.

[0076] In the following examples, the CLRN nanoparticles prepared in Example 1 were tested.

[0077] The CLR is formed by mixing Ce6, LND and RG108 in a free state at a feed ratio of 1:1:1.

[0078] The concentrations of CLR and CLRN are represented by the concentration of Ce6.

[0079] Example 4

[0080] This example tests the uptake capacity of CLRN nanoparticles, and the specific process is as follows:

[0081] 4T1 cells were cultured at 10 6 / well were inoculated in a 6-well plate and placed in a 5% CO2, 37°C incubator for 24 hours. The old culture medium was removed and the following drug-containing culture medium was added: Ce6 and CLRN, where the concentration of Ce6 was 3 μg / mL. The culture was continued for 6 hours, the drug-containing culture medium was removed, and the cells were observed and photographed under a laser confocal microscope.

[0082] The results are as follows Figure 6 As shown, the results show that Ce6 has spontaneous fluorescence (red), which can be used to detect uptake. It can be seen from the figure that compared with free Ce6, CLRN nanoparticles have a stronger ability to be taken up by cells, and the spontaneous red fluorescence intensity is higher under the same incubation time, providing a basis for exerting stronger anti-tumor properties.

[0083] Example 5

[0084] This example tests the ability of CLRN nanoparticles to produce reactive oxygen species (ROS), and the specific process is as follows:

[0085] 4T1 cells were cultured at 10 6 / well were inoculated in a 6-well plate and placed in a 5% CO2, 37°C incubator for further culturing for 24 hours. The old culture medium was removed and the following drug-containing culture media were added: PBS, LND, RG108, Ce6+L, CLR+L, CLRN, CLRN+L, where the concentration of Ce6 was 0.25μg / mL, the concentration of LND was 0.19μg / mL, and the concentration of RG108 was 0.17μg / mL. The cells were further cultured in a 5% CO2, 37°C incubator or hypoxic chamber for 8 hours. The light-added group was then irradiated with a 660nm laser for 1 minute, the drug-containing culture medium was removed, and the basal culture medium containing DCFH-DA dye was added. The cells were further incubated in a 37°C incubator for 1 hour and observed and photographed under an inverted fluorescence microscope.

[0086] The LND group was free LND in PBS buffer; the RG108 group was free RG108 in PBS buffer; the Ce6+L group was free Ce6 in PBS buffer, and was cultured in a 5% CO2, 37°C incubator or hypoxic chamber for 8 hours and irradiated with a 660nm laser for 1 minute; the CLR+L group was free Ce6, free LND and free RG108 mixed in PBS buffer in the proportion of the content in the nanoparticles described in Example 1, and was cultured in a 5% CO2, 37°C incubator or hypoxic chamber for 8 hours and irradiated with a 660nm laser for 1 minute; the CLRN group was the nanoparticles described in Example 1 mixed in PBS buffer; the CLRN+L group was the nanoparticles described in Example 1 mixed in PBS buffer, and was cultured in a 5% CO2, 37°C incubator or hypoxic chamber for 8 hours and irradiated with a 660nm laser for 1 minute.

[0087] As a highly efficient photosensitizer, Ce6 generates reactive oxygen species (ROS) under laser irradiation, further enhancing intracellular oxidative stress and exerting cytotoxic effects, and has been widely used in medical research. DCFH-DA can be oxidized to DCF by ROS, and green fluorescence is used as an indicator of cellular ROS generation. Obviously, 4T1 cells treated with Ce6, CLR, and CLRN showed obvious green fluorescence after laser irradiation, confirming that intracellular ROS levels increased during PDT ( Figure 7 ).like Figure 8 As shown in the figure, in a hypoxic environment, CLR+L and CLRN+L also showed better ROS production ability than Ce6+L, indicating that LND alleviated the hypoxic tumor microenvironment and was more conducive to the effect of photodynamic therapy.

[0088] Example 6

[0089] This example tests the intracellular and extracellular lactic acid content after the action of CLRN nanoparticles, and the specific process is as follows:

[0090] 4T1 cells were cultured at 10 6 / well were inoculated in a 6-well plate and placed in a 5% CO2, 37°C incubator for 24 hours. The old culture medium was removed and the following drug-containing culture media PBS, LND, CLR, and CLRN were added respectively, where the concentration of LND was 10 μg / mL. The cells and drug-containing culture media were collected and the lactic acid content was determined using a lactic acid detection kit.

[0091] The LND group was free LND in PBS buffer; the CLR group was free Ce6, free LND and free RG108 mixed in PBS buffer at the content ratio of the nanoparticles described in Example 1; and the CLRN group was the nanoparticles described in Example 1 mixed in PBS buffer.

[0092] The lactate content of 4T1 cells and culture supernatant after different treatments was collected and analyzed. Fig. 9 As shown, the intracellular lactate content of CLRN-treated cells was reduced by 43% compared with that of PBS-treated cells. In terms of extracellular lactate content, CLRN was still lower than PBS. These results suggest that CLRN plays a role in reducing lactate production.

[0093] Example 7

[0094] The cytotoxicity after the CLRN nanoparticles in this example is as follows:

[0095] 4T1 cells were seeded in a 96-well plate at a density of 8000 cells / well and cultured in a 5% CO2, 37°C incubator for 24 hours. The old culture medium was removed, and the following drug-containing culture media were added respectively: PBS, LND, RG108, Ce6+L, CLR+L, CLRN, CLRN+L, among which the concentration gradient of Ce6 was 0.03μg / mL, 0.06μg / mL, 0.13μg / mL, 0.25μg / mL, 0.5μg / mL, and 1.0μg / mL, and the corresponding concentration gradient of LND was 0.02μg / mL, 0.05μg / mL, 0.10μg / mL, 0.19μg / mL, 0.38μg / mL, and 0.76μg / mL, and the corresponding concentration gradient of RG108 was 0.02μg / mL, 0.04μg / mL, 0.09μg / mL, 0.17μg / mL, 0.35μg / mL, and 0.69μg / mL. Five replicate wells were set for each concentration, and 5% was put back in the wells. CO2, 37℃ incubator or hypoxic chamber for 12h, then irradiate the light-added group with 660nm laser for 90s, and continue to culture in 5% CO2, 37℃ incubator or hypoxic chamber for 12h. Remove the drug-containing culture medium, add 100μL of basal culture medium containing CCK8 to each well, return to 5% CO2, 37℃ incubator for 1h, and then select 450nm wavelength on the microplate reader to measure cell viability.

[0096] Among them, the LND group (II) is free LND in PBS buffer; the RG108 group (III) is free RG108 in PBS buffer; the Ce6+L group (IV) is free Ce6 in PBS buffer, and irradiated with 660nm laser for 90 seconds 12 hours after drug addition; the CLR+L group (V) is free Ce6, free LND and free RG108 mixed in PBS buffer in the content ratio of the nanoparticles described in Example 1, and irradiated with 660nm laser for 90 seconds 12 hours after drug addition; the CLRN group (VI) is the nanoparticles described in Example 1 mixed in PBS buffer; the CLRN+L group (VII) is the nanoparticles described in Example 1 mixed in PBS buffer, and irradiated with 660nm laser for 90 seconds 12 hours after drug addition.

[0097] like Fig.10 As shown in the figure, even under laser irradiation, free Ce6 cannot significantly inhibit the proliferation of 4T1 cells, which may be related to insufficient cell internalization. Compared with CLR+L, CLRN+L showed a strong anti-tumor effect at low concentrations, which was due to the improved stability after self-assembly and increased cellular endocytosis accumulation. At the same time, thanks to the relief of the tumor hypoxic microenvironment by the action of LND, CLRN+L can still maintain an amazing cytotoxic effect under hypoxic conditions, which will be beneficial to play a synergistic anti-tumor effect in hypoxic breast cancer ( Fig.11 ).

[0098] Example 8

[0099] This example tests the anti-tumor effect of CLRN nanoparticles, and the specific process is as follows:

[0100] 4T1 cells were injected subcutaneously into 4- to 5-week-old BALB / C female mice to construct 4T1 tumor-bearing mice. When the tumor volume (V = 1 / 2 × L × W × W (L: tumor length, W: tumor width)) of the mice reached 100 mm 3 BALB / C mice were randomly divided into 7 groups (5 mice in each group). During the 14-day observation period, PBS, LND, RG108, Ce6+L, CLR+L, CLRN, and CLRN+L were injected with drugs (Ce6 1 mg / kg, LND 0.8 mg / kg, and RG108 0.4 mg / kg) three times through the tail vein on days 0, 3, and 6, respectively. Then, laser irradiation (660 nm, 0.5 W / cm 2 , 5min). Starting from day 0, the tumor volume of mice was recorded every 2 days to obtain the tumor growth curve.

[0101] 4T1 cells were injected subcutaneously on the right side of 4-5 week old BALB / C female mice to establish a unilateral tumor. Three days later, 4T1 cells were injected subcutaneously on the left side of the mice to establish a tumor to obtain a 4T1 bilateral tumor breast cancer model. When the right tumor size reached about 100 mm 3 BALB / C mice were divided into 7 groups (5 mice in each group). During the 14-day observation period, drugs (Ce6 1 mg / kg, LND 0.8 mg / kg, and RG108 0.4 mg / kg) were directly injected into the right tumor of PBS, LND, RG108, Ce6+L, CLR+L, CLRN, and CLRN+L three times on days 0, 3, and 6, respectively. Then, laser irradiation (660 nm, 0.5 W / cm 2 , 5min). Starting from day 0, the tumor volume of mice was recorded every 2 days to obtain the tumor growth curve.

[0102] Group I is the PBS group; LND group (II) is free LND in PBS buffer; RG108 group (III) is free RG108 in PBS buffer; Ce6+L group (IV) is free Ce6 in PBS buffer, and 8 hours after injection, the right tumor site was irradiated with a 660nm laser for 5 minutes; CLR+L group (V) is free Ce6, free LND and free RG108 mixed in PBS buffer in the proportion of the content in the nanoparticles described in Example 1, and 8 hours after injection, the right tumor site was irradiated with a 660nm laser for 5 minutes; CLRN group (VI) is the nanoparticles described in Example 1 mixed in PBS buffer; CLRN+L group (VII) is the nanoparticles described in Example 1 mixed in PBS buffer, and 8 hours after injection, the right tumor site was irradiated with a 660nm laser for 5 minutes.

[0103] The tumor growth curve showed that the average tumor volume of the primary tumor in mice in the PBS group (I) reached 1200mm after 14 days. 3 The average tumor volume of primary tumors and distal tumors in mice in the CLRN+L group was controlled at 100 mm 3 At the same time, the CLR group showed better tumor growth inhibition than Ce6 under laser irradiation, which may be related to the ability of LND to reverse ITM. These results all indicate that CLRN+L has an objective ability to inhibit tumor growth ( Fig.12 , Fig.13 ).

[0104] Example 9

[0105] This example tests the ability of CLRN nanoparticles to mature antigen presenting cells (DCs), and the specific process is as follows:

[0106] On the 14th day, the unilateral tumor model mice were killed and the tumor-draining lymph nodes were collected. The tumor-draining lymph nodes were ground, digested, and filtered with a 70μm cell strainer to obtain a single cell suspension for the determination of dendritic cell maturation. Briefly, the single cell suspensions of different mice were stained with anti-CD11c-BV450, anti-CD80-FITC, and anti-CD86-APC antibodies, incubated at 4°C in the dark for 1h, and fixed with PBS: 4% paraformaldehyde 1:1 solution. When tested, they were washed with PBS and resuspended, and finally analyzed by flow cytometry. The results were statistically analyzed using ONE-WAY ANOVA.

[0107] Under laser irradiation, CD80 + CD86 +Mature DCs accounted for 29.6% of the total DCs in the lymph nodes, more than two times higher than that observed after Ce6+L treatment alone. These results suggest that CLRN+L-mediated PDT increases oxidative stress, triggers pyroptosis, leads to significant cell death and enhanced ICD, ultimately increasing tumor immunogenicity ( Fig.14 ).

[0108] Example 10

[0109] This example tests the immune-promoting effect of CLRN nanoparticles, and the specific process is as follows:

[0110] On the 14th day, the mice were killed and the tumors were collected. The tumors were ground and filtered through a 70 μm cell strainer to obtain a single cell suspension, and the single cell suspension of each mouse was divided into different portions for determining the proportion of different immune cells in the tumor tissue. Anti-CD45-FITC, anti-CD3-PerCP / Cy5.5, anti-CD4-PE / Cy7, and anti-CD8a-Pacific blue were used to detect cytotoxic T lymphocytes CD8 (CD3 + CD8 + ). Anti-CD45-FITC, anti-CD3-PerCP / Cy5.5, anti-CD4-PE / Cy7, anti-CD25-APC, anti-Foxp3-PE were used to detect regulatory T lymphocytes (CD4 + Foxp3 + ). Anti-F4180-BV450, anti-CD11b-ER780, anti-CD86-PerCP / Cy5.5, and anti-CD206-PE were used to detect M1 and M2 tumor-associated macrophages. The cells were incubated at 4°C in the dark for 1 h, fixed with PBS:4% paraformaldehyde 1:1 solution, washed with PBS and resuspended for testing, and finally analyzed by flow cytometry. The results were statistically analyzed using ONE-WAY ANOVA.

[0111] Depend on Fig.15 It can be seen that the primary tumor CD8 + The proportion of T cells (31.9±5.77%) was 9 times that of PBS (3.44±1.18%), and the CD8 + The infiltration rate of T cells reached 30.6±4.86%, which was significantly higher than that of other control groups. +T cells also increased. Under light or no light conditions, the M1 / M2 ratio of CLRN was 6 times and 4 times that of PBS, respectively. The M1 / M2 ratio of CLR+L (1.31±0.17%) was also higher than that of Ce6+L (0.76±0.16%), indicating that LND plays a role in macrophage polarization regulation through lactate regulation, promoting the polarization of M2 tumor-associated macrophages to M1 tumor-associated macrophages ( Fig.16 The infiltration rate of Tregs in CLRN+L (7.07±0.63%) was significantly lower than that in PBS (22.57±2.15%) ( Fig.17 CLRN enhanced the ICD effect induced by laser irradiation and effectively promoted the maturation of DCs, recruited cytotoxic T lymphocytes (CTLs), and ultimately induced a strong anti-tumor immune response.

[0112] The above results show that compared with the three free drugs plus light group CLR+L, the CLRN+L group, in which the three drugs were prepared into carrier-free nanoparticles and then irradiated with laser, showed a stronger anti-tumor effect. This is because the nanoparticles can be more effectively taken up into tumor cells, alleviate the effects of hypoxia and immunosuppression in the tumor microenvironment, and synergize with the immune activation caused by light-induced pyroptosis to amplify and enhance the anti-tumor immune effect.

[0113] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A ternary carrier-free nanoparticle, characterized in that: The nanoparticles include dihydrochlorin e6, lonidamine and RG108 formed by intermolecular interaction forces.

2. The ternary carrier-free nanoparticles according to claim 1, characterized in that: The mass ratio of the dihydrochlorin e6, lonidamine and RG108 is (1-15):(3-10):(3-15).

3. The ternary carrier-free nanoparticles according to claim 1, characterized in that: The average particle size of the nanoparticles is 100nm to 180nm.

4. The ternary carrier-free nanoparticles according to claim 1, characterized in that: The intermolecular interaction force includes at least one of a hydrophobic force, a π-π stacking force and a hydrogen bonding force.

5. A method for preparing the ternary carrier-free nanoparticles according to any one of claims 1 to 4, characterized in that: The following steps are involved: The organic solution of dihydrochlorin e6, lonidamine and RG108 is ultrasonically treated to prepare the ternary carrier-free nanoparticles.

6. The method for preparing ternary carrier-free nanoparticles according to claim 5, characterized in that: The mass concentration feed ratio of the dihydrochlorin e6, lonidamine and RG108 is 1:0.5-5:0.5-5.

7. The method for preparing ternary carrier-free nanoparticles according to claim 5, characterized in that: The frequency of the ultrasound is 20 to 60 kHz; the time of the ultrasound is 1 to 10 minutes.

8. A pharmaceutical composition, characterized in that: It comprises the ternary carrier-free nanoparticles as described in any one of claims 1 to 4.

9. Use of the ternary carrier-free nanoparticles according to any one of claims 1 to 4 and / or the pharmaceutical composition according to claim 8 in the preparation of tumor therapeutic drugs.

10. The use according to claim 9, characterized in that: The tumor includes at least one of breast cancer, prostate cancer, ovarian cancer, intestinal cancer, lung cancer, and pancreatic cancer.

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