A ternary carrier-free nanoparticle, a preparation method and application thereof

Abstract

The application discloses a ternary carrier-free nanoparticle and a preparation method and application thereof. The ternary carrier-free nanoparticle comprises a nanoparticle formed by intermolecular interaction force of chlorin e6, lonidamide and RG108. The nanoparticle can improve effective absorption and accumulation of the drug in a tumor, and can also induce tumor-specific pyroptosis under laser irradiation, induce immunogenic effects, promote maturation of antigen-presenting cells and recruitment and activation of cytotoxic T lymphocytes, so as to start adaptive anti-tumor immunity.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a ternary carrier-free nanoparticle, its preparation method, and its application. Background Technology

[0002] Pyroptosis is a unique inflammatory programmed cell death pattern that activates the Caspase-3 / GSDME pathway, forming GSDME-N fragments capable of penetrating the cell membrane and releasing immunostimulatory contents such as damage-associated molecular patterns (DAMPs) and various pro-inflammatory cytokines, leading to immunogenic cell death (ICD) and achieving anti-tumor immunotherapy. Photodynamic therapy (PDT), due to its high spatiotemporal controllability, generates reactive oxygen species (ROS) through laser irradiation, causing oxidative damage to cancer cells. The level of ROS production is closely related to Caspase-3 activation, and PDT has been used to increase the incidence of pyroptosis. However, the tumor's own defense mechanisms limit the application of PDT combined with pyroptosis. In the hypoxic tumor microenvironment, tumor cells rely more on glycolysis to maintain sufficient energy supply, further limiting ROS production and thus hindering PDT activation. Simultaneously, this abnormal metabolic process creates an immunosuppressive tumor microenvironment (ITM), severely impairing the tumor's immune response. GSDME expression is reduced in tumors due to promoter methylation. Therefore, it is necessary to develop a method that can enhance photodynamic-induced pyroptosis while improving the immunosuppressive microenvironment to improve the efficacy of antitumor immunotherapy.

[0003] The high glycolysis rate within tumors converts pyruvate to lactate (LA), which is one reason why immunosuppression limits pyroptosis immunotherapy. The acidic tumor microenvironment often causes tumor-associated macrophages (TAMs) to transform into the tumor-promoting M2 type. Regulatory T cells (Tregs) possess metabolic flexibility and can use "alternative" metabolites of LA in the tumor microenvironment to maintain their inhibitory properties against effector T cells. The resulting ITM (intracytoplasmic renal migration) poses a significant challenge to the efficacy of immunotherapy. Hexokinase II (HKII) plays a crucial role in the regulation of tumor glycolysis. Therefore, HKII inhibitors show great potential in reducing lactate production in tumor cells and regulating ITM caused by tumor metabolism.

[0004] Nanomaterials, with their unique physicochemical properties, have shown remarkable application prospects in the field of drug delivery. Using nanomaterials as carriers can increase the cumulative concentration of drugs at tumor sites and optimize drug bioavailability. Most existing nanomedicines employ methods such as encapsulating hydrophobic drugs with responsive polymer carriers or adsorbing drugs with mesoporous materials, which present problems such as uncontrollable drug release and potential biotoxicity of the carrier materials. Summary of the Invention

[0005] The present invention aims to at least solve one of the aforementioned technical problems existing in the prior art. Therefore, the object of the present invention is to provide a ternary carrier-free nanoparticle, its preparation method, and its application.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] In one aspect, the present invention provides a ternary carrier-free nanoparticle comprising dihydroporphyrin e6, chlordamine and RG108 formed by intermolecular interaction forces.

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

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

[0010] In some embodiments of the present invention, the mass ratio of dihydroporphyrin E6, chlordamine, and RG108 is (1–15):(3–10):(3–15), such as (3–15):(3–8):(3–12). In this invention, limiting the mass ratio of the three components can effectively improve the stability of the nanoparticles.

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

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

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

[0014] The ternary carrier-free nanoparticles were prepared by ultrasonication of an organic solution of dihydroporphyrin E6, chlordamine, and RG108.

[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 ratio of dihydroporphyrin E6, chlordamine 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 dihydroporphyrin E6 is 5 to 15 mg / mL.

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

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

[0020] In some embodiments of the present invention, the frequency of the ultrasound is 20 to 60 kHz; the duration 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 includes adding an organic solution of dihydroporphyrin E6, chlordamine and RG108 dropwise into water while simultaneously sonicating, and continuing sonication 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, sterile water, and distilled water.

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

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

[0025] In another aspect, 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 includes pharmaceutically acceptable excipients.

[0027] In some embodiments of the present invention, the drug can be prepared into dosage forms such as powder, suppository, injection, emulsion, patch, spray, and aerosol, and a suitable drug carrier in the art can be selected for different dosage forms.

[0028] In some embodiments of the invention, the drug carrier used may be solid, liquid, or gaseous. Examples of solid carriers include lactose, kaolin, sucrose, talc, 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 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, coloring agents, etc., can be used to form oral liquid dosage forms, such as suspensions, solutions, and precipitates; while carriers, such as starch, sugars, microcrystalline cellulose, diluents, granulators, emulsifiers, lubricants, binders, and disintegrants, can be used to form oral solid dosage forms, such as powders, capsules, and tablets. Tablets and capsules are preferred oral dosage units using solid drug carriers due to their ease of administration. Standard aqueous or non-aqueous coating techniques can be used to coat the tablets.

[0030] In some embodiments of the invention, tablets containing the nanoparticles of the invention can be prepared by compression or molding, optionally using one or more excipients or adjuvants. The active ingredient can be compressed in a free-flowing form (e.g., powder or granules) in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, surfactant, or dispersant. Molded tablets can 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 a suitable and convenient carrier material, which may comprise about 5% to 95% of the total composition. Unit dosage forms typically contain approximately 1 mg to approximately 2 g of nanoparticles, usually in doses of 25 mg, 50 mg, 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 800 mg, or 1000 mg.

[0031] The present invention can be prepared as an aqueous solution or suspension comprising nanoparticles for parenteral administration. Suitable surfactants, such as hydroxypropyl cellulose, may be included. Dispersions can also be prepared in a mixture of glycerol, liquid polyethylene glycol, and its oil. Furthermore, preservatives may be added to prevent harmful microbial growth.

[0032] The pharmaceutical products suitable for injection in this invention include sterile aqueous solutions or dispersions. Furthermore, the pharmaceutical product can be in the form of a sterile powder for the ad hoc preparation of such sterile injection solutions or dispersions. In all cases, the final injectable form must be sterile and must be an effective liquid so that the injectable pharmaceutical ingredient must remain stable under the production and storage conditions; therefore, it is best to preserve it to prevent contamination by microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, such as containing water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), vegetable oils, and suitable mixtures thereof.

[0033] The medicaments of the present invention can be in forms suitable for topical use, such as aerosols, creams, ointments, lotions, powders, or the like. Furthermore, nanoparticles can be suitable for use in transdermal drug delivery devices. These formulations can be prepared using the nanoparticles of the present invention via conventional processing methods. For example, a cream or ointment with a desired consistency can be prepared by mixing a hydrophilic material and water, and about 5 wt% to about 10 wt% of nanoparticles.

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

[0035] In addition to the carrier components described above, the pharmaceutical formulations may include (if applicable) one or more additional carrier components, such as diluents, buffers, flavoring agents, binders, surfactants, thickeners, lubricants, preservatives (including antioxidants), etc. Furthermore, other excipients may be added, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., colorants, and flavoring agents, etc., to make the formulation isotonic with the blood of the intended receptor. Components containing the nanoparticles of this invention can also be prepared in powder or concentrated form.

[0036] In another aspect, 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, colorectal cancer, lung cancer, and pancreatic cancer.

[0039] In some embodiments of the present invention, the photodynamic therapy uses a 500–750 nm (e.g., 600–700 nm) laser at a frequency of 0.1–1.0 W / cm². 2 Irradiate with light intensity for 1–20 minutes; such as 1–15 minutes, 1–10 minutes, etc.

[0040] The beneficial effects of this invention are:

[0041] Photosensitizer Ce6, glycolysis inhibitor LND, and DNA methyltransferase inhibitor RG108 self-assembled into stable CLRN nanoparticles with high drug loading capacity through hydrophobic interactions, π-π stacking, and hydrogen bonding, achieving carrier-free self-assembly of two or more drugs. CLRN can enhance drug absorption and accumulation in tumors, and simultaneously induce tumor-specific pyroptosis under laser irradiation, inducing immunogenic effects, promoting the maturation of antigen-presenting cells, and recruiting and activating cytotoxic T lymphocytes, thereby initiating adaptive anti-tumor immunity. Furthermore, CLRN can inhibit glycolysis, alleviating tumor hypoxia, thus synergistically amplifying the photodynamic activation of pyroptosis. Simultaneously, 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 regression of primary and distant tumors. In conclusion, combining photodynamic therapy to achieve controllable activation of pyroptosis is a novel strategy for treating triple-negative breast cancer. This method has strong immunomodulatory effects and may alleviate immunosuppression, thus synergistically amplifying the effects of immunotherapy. Attached Figure Description

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

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

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

[0045] Figure 4 The potential diagrams are for nanoparticles in Examples 1-3.

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

[0047] Figure 6 The images are laser confocal microscope images of CLRN uptake by 4T1 cells at 6h and 8h in Example 4.

[0048] Figure 7 This is a schematic diagram showing the reactive oxygen species production capacity of each group after treatment under normal oxygen conditions.

[0049] Figure 8 This is a schematic diagram showing the reactive oxygen species production capacity of each group after treatment under hypoxic conditions.

[0050] Figure 9 To analyze the lactate content in cells and culture supernatant.

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

[0052] Figure 11 Cytotoxicity analysis of 4T1 cells under hypoxic conditions.

[0053] Figure 12 This is a 14-day tumor growth curve in a mouse model of unilateral triple-negative breast cancer.

[0054] Figure 13 Tumor growth curves of proximal and distal tumors in a mouse model of bilateral triple-negative breast cancer at 14 days.

[0055] Figure 14 This study aimed to determine the presence of mature antigen-presenting cells in the lymph nodes of triple-negative breast cancer model mice.

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

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

[0058] Figure 17 This study measured the suppressor T lymphocytes (Tregs) in a triple-negative breast cancer model mouse. Detailed Implementation

[0059] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments and comparative examples are all available from conventional commercial sources or can be obtained by existing technical methods. Unless otherwise specified, the test or experimental methods are conventional methods in the art.

[0060] Example 1

[0061] This embodiment prepares CLRN nanoparticles, and the specific process is as follows:

[0062] Accurately weigh 10 mg of chlorin E6 (Ce6) and chlordamine (LND) and dissolve them separately in 1 mL of dimethyl sulfoxide to prepare a stock solution of 10 mg / mL. Accurately weigh 10 mg of RG108 and dissolve it in 500 μL of dimethyl sulfoxide to prepare a stock solution of 20 mg / mL. Take 11.7 μL of the chlorin E6 stock solution and 11.7 μL of the chlordamine stock solution, and 5.8 μL of the RG108 stock solution, add 70.8 μL of dimethyl sulfoxide, mix well, and then add dropwise to 1 mL of ultrapure water while sonicating at 40 kHz. Gently shake until all the solution is added, and continue sonicating for 3 min. Transfer the resulting solution to a 1000 Da dialysis bag and dialyze it in 1 L of ultrapure water for 4 h. After removing the organic solvent, collect the solution to obtain a CLRN nanoparticle solution with a Ce6, LND, and RG108 feed ratio of 1:1:1.

[0063] Pipette 200 μL of the nanoparticle solution into a particle size analyzer and measure its particle size 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±3.4 nm; the mass ratio of Ce6, LND and RG108 in the prepared CLRN nanoparticles is 11:5:4.

[0064] Example 2

[0065] This embodiment prepares CLRN nanoparticles, and the specific process is as follows:

[0066] Take 7 μL of dihydroporphyrin (e6) stock solution, 14 μL of chlordamine stock solution, and 7 μL of RG108 stock solution, add 72 μL of dimethyl sulfoxide, mix thoroughly, and then add dropwise to 1 mL of ultrapure water while sonicating at 40 kHz, gently shaking until all is added, and continue sonicating for 3 min. Transfer the resulting solution to a 1000 Da dialysis bag, place it in 1 L of ultrapure water, and dialyze for 4 h. After removing the organic solvent, collect the solution to obtain a CLRN nanoparticle solution with a Ce6, LND, and RG108 feed ratio of 1:2:2.

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

[0068] Example 3

[0069] This embodiment prepares CLRN nanoparticles, and the specific process is as follows:

[0070] Take 5 μL of dihydroporphyrin (e6) stock solution, 15 μL of chlordamine stock solution, and 7.5 μL of RG108 stock solution. Add 72.5 μL of dimethyl sulfoxide and mix thoroughly. Then, add the mixture dropwise to 1 mL of ultrapure water while sonicating at 40 kHz, gently shaking until all the solution is added. Continue sonicating for 3 min. Transfer the resulting solution to a 1000 Da dialysis bag and dialyze it in 1 L of ultrapure water for 4 h. After removing the organic solvent, collect the solution to obtain a CLRN nanoparticle solution with a Ce6, LND, and RG108 feed ratio of 1:3:3.

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

[0072] Experimental Example 1

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

[0074] Experimental Example 2

[0075] In this experiment, 200 μL of the nanoparticles obtained in Example 1 were placed in particle size dishes on days 1, 3, 5, and 7, and the particle size in water and PBS solution was 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 have good stability after seven days and there is no significant change.

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

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

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

[0079] Example 4

[0080] This embodiment tests the uptake capacity of CLRN nanoparticles. The specific process is as follows:

[0081] 4T1 cells were used at 10 6The culture medium was seeded at a density of / wells in 6-well plates and incubated for 24 hours in a 5% CO2, 37°C incubator. The old culture medium was removed, and the following drug-containing media were added respectively: Ce6 and CLRN, with Ce6 concentration of 3 μg / mL in both media. The plates were incubated for another 6 hours, the drug-containing media were removed, and the plates were observed and photographed using a laser confocal microscope.

[0082] The results are as follows Figure 6 As shown in the figure, the results indicate that Ce6 exhibits autofluorescence (red), which can be used to detect uptake. The figure shows that compared to free Ce6, CLRN nanoparticles have a stronger ability to be taken up by cells, and the intensity of autofluorescence is higher at the same incubation time, providing a basis for exerting stronger anti-tumor properties.

[0083] Example 5

[0084] This embodiment tests the reactive oxygen species (ROS) generation capacity of CLRN nanoparticles. The specific process is as follows:

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

[0086] The LND group consisted of free LND in PBS buffer; the RG108 group consisted of free RG108 in PBS buffer; the Ce6+L group consisted of free Ce6 in PBS buffer, and cultured for 8 hours in an incubator or hypoxic chamber at 37°C with 5% CO2, followed by 1 minute of 660nm laser irradiation; the CLR+L group consisted of free Ce6, free LND, and free RG108 mixed in PBS buffer at the proportions described in the nanoparticles of Example 1, and cultured for 8 hours in an incubator or hypoxic chamber at 37°C with 5% CO2, followed by 1 minute of 660nm laser irradiation; the CLRN group consisted of nanoparticles as described in Example 1 mixed in PBS buffer; the CLRN+L group consisted of nanoparticles as described in Example 1 mixed in PBS buffer, and cultured for 8 hours in an incubator or hypoxic chamber at 37°C with 5% CO2, followed by 1 minute of 660nm laser irradiation.

[0087] Ce6, as a highly efficient photosensitizer, 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 serves as an indicator of cellular ROS generation. Clearly, 4T1 cells treated with Ce6, CLR, and CLRN exhibited significant green fluorescence after laser irradiation, confirming the increased intracellular ROS levels during phototherapy treatment (PDT). Figure 7 ).like Figure 8 As shown, in a hypoxic environment, CLR+L and CLRN+L also exhibited better ROS generation capacity 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 embodiment tests the intracellular and extracellular lactate levels after the action of CLRN nanoparticles. The specific process is as follows:

[0090] 4T1 cells were used at 10 6 Cells were seeded at a density of / wells in 6-well plates and incubated for 24 hours in a 5% CO2, 37°C incubator. The old culture medium was removed, and the following drug-containing media were added: PBS, LND, CLR, and CLRN, with LND concentration at 10 μg / mL. Cells were incubated for another 12 hours, and the cells and drug-containing media were collected. Lactate levels were measured using a lactate assay kit.

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

[0092] Lactate content in 4T1 cells and culture supernatant after different treatments was collected and analyzed. Figure 9 As shown, compared with PBS-treated cells, CLRN-treated cells exhibited a 43% reduction in intracellular lactate levels. Extracellular lactate levels remained lower with CLRN than with PBS. These results indicate that CLRN plays a role in reducing lactate production.

[0093] Example 7

[0094] The cytotoxicity of CLRN nanoparticles after treatment in this embodiment is specifically as follows:

[0095] 4T1 cells were seeded at a density of 8000 / well in 96-well plates and cultured for 24 hours in an incubator with 5% CO2 at 37°C. Remove the old culture medium and add the following drug-containing media: PBS, LND, RG108, Ce6+L, CLR+L, CLRN, and CLRN+L. The Ce6 concentration gradient is 0.03 μg / mL, 0.06 μg / mL, 0.13 μg / mL, 0.25 μg / mL, 0.5 μg / mL, and 1.0 μg / mL. The corresponding LND concentration gradients are 0.02 μg / mL, 0.05 μg / mL, 0.10 μg / mL, 0.19 μg / mL, 0.38 μg / mL, and 0.76 μg / mL. The corresponding RG108 concentration gradients are 0.02 μg / mL, 0.04 μg / mL, 0.09 μg / mL, 0.17 μg / mL, 0.35 μg / mL, and 0.69 μg / mL. Each concentration is used in 5 replicates. 5% of the media is returned to the culture medium. Cells were cultured for 12 hours in a CO2 incubator at 37°C or a hypoxic chamber. Then, the cells were irradiated with a 660nm laser for 90 seconds and cultured for another 12 hours in a 5% CO2 incubator at 37°C or a hypoxic chamber. The drug-containing medium was removed, and 100μL of basal medium containing CCK8 was added to each well. The cells were then incubated again in a 5% CO2 incubator at 37°C for 1 hour. Cell viability was then measured at 450nm using a microplate reader.

[0096] The LND group (II) consisted of free LND in PBS buffer; the RG108 group (III) consisted of free RG108 in PBS buffer; the Ce6+L group (IV) consisted of free Ce6 in PBS buffer, and irradiated with a 660nm laser for 90 seconds 12 hours after drug administration; the CLR+L group (V) consisted of free Ce6, free LND, and free RG108 mixed in PBS buffer at the proportions described in the nanoparticles of Example 1, and irradiated with a 660nm laser for 90 seconds 12 hours after drug administration; the CLRN group (VI) consisted of the nanoparticles described in Example 1 mixed in PBS buffer; and the CLRN+L group (VII) consisted of the nanoparticles described in Example 1 mixed in PBS buffer, and irradiated with a 660nm laser for 90 seconds 12 hours after drug administration.

[0097] like Figure 10 As shown, even under laser irradiation, free Ce6 did not significantly inhibit the proliferation of 4T1 cells, which may be related to insufficient internalization. Compared with CLR+L, CLRN+L exhibited extremely strong antitumor activity at low concentrations, due to increased stability after self-assembly and increased accumulation via endocytosis. Simultaneously, thanks to the alleviating effect of LND on the tumor hypoxic microenvironment, CLRN+L maintained remarkable cytotoxicity under hypoxic conditions, which will be beneficial for exerting a synergistic antitumor effect in hypoxic breast cancer. Figure 11 ).

[0098] Example 8

[0099] This embodiment tests the antitumor effect of CLRN nanoparticles. The specific process is as follows:

[0100] 4T1 tumor-bearing mice were constructed by subcutaneously injecting 4T1 cells into 4-5 week old female BALB / c mice. The tumor volume (V = 1 / 2 × L × W × W (L: tumor length, W: tumor width)) reached 100 mm. 3 BALB / c mice were randomly divided into 7 groups (n=5 per group). During a 14-day observation period, PBS, LND, RG108, Ce6+L, CLR+L, CLRN, and CLRN+L were administered via tail vein injection three times on days 0, 3, and 6 (Ce6 1 mg / kg, LND 0.8 mg / kg, and RG108 0.4 mg / kg, respectively). Eight hours after injection, the Ce6+L, CLR+L, and CLRN+L groups underwent laser irradiation of the tumor site (660 nm, 0.5 W / cm²). 2 (5 min). Starting from day 0, the tumor volume of mice was recorded every 2 days to obtain the tumor growth curve.

[0101] A unilateral tumor was constructed by subcutaneously injecting 4T1 cells into the right side of 4-5 week old female BALB / c mice. Three days later, a tumor was established by subcutaneously injecting 4T1 cells into the left side of the mice to obtain a bilateral 4T1-mediated breast cancer model. The tumor on the right side reached approximately 100 mm in size. 3 BALB / c mice were divided into 7 groups (n=5 per group). During a 14-day observation period, the right-side tumors of mice in the PBS, LND, RG108, Ce6+L, CLR+L, CLRN, and CLRN+L groups were directly injected three times on days 0, 3, and 6 (Ce6 1 mg / kg, LND 0.8 mg / kg, and RG108 0.4 mg / kg). Eight hours after injection, the right-side tumors of the Ce6+L, CLR+L, and CLRN+L groups were irradiated with laser (660 nm, 0.5 W / cm²). 2 (5 min). Starting from day 0, the tumor volume of mice was recorded every 2 days to obtain the tumor growth curve.

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

[0103] Tumor growth curves showed that the average primary tumor volume in mice in the PBS group (I) reached 1200 mm² after 14 days. 3 In the CLRN+L group mice, the average tumor volume of primary and distal tumors was controlled at 100 mm. 3 Within this range. Meanwhile, the CLR group showed better tumor growth inhibition than Ce6 under laser irradiation, which may be related to LND's ability to reverse ITM. These results all indicate that CLRN+L has an objective ability to inhibit tumor growth. Figure 12 , Figure 13 ).

[0104] Example 9

[0105] This embodiment tests the ability of CLRN nanoparticles to induce the maturation of antigen-presenting cells (DCs). The specific process is as follows:

[0106] On day 14, unilateral tumor model mice were sacrificed, and tumor-draining lymph nodes were collected. The tumor-draining lymph nodes were ground, dissolved, and filtered through a 70 μm cell filter to obtain a single-cell suspension for determining dendritic cell maturation. In short, single-cell suspensions from different mice were stained with anti-CD11c-BV450, anti-CD80-FITC, and anti-CD86-APC antibodies, respectively, incubated at 4°C in the dark for 1 hour, fixed with PBS:4% paraformaldehyde 1:1 solution, washed with PBS and resuspended before testing, and finally analyzed by flow cytometry. The results were statistically analyzed using one-way ANOVA.

[0107] Under laser irradiation, CLRN+L treatment group, CD80 + CD86 +Mature dendritic cells (DCs) accounted for 29.6% of the total DCs in the lymph nodes, more than twice the number observed after Ce6+L treatment alone. These results indicate that CLRN+L-mediated PDT increases oxidative stress, triggers pyroptosis leading to significant cell death and enhanced ICD effects, ultimately increasing tumor immunogenicity. Figure 14 ).

[0108] Example 10

[0109] This embodiment tests the immune-promoting effect of CLRN nanoparticles. The specific process is as follows:

[0110] Mice were sacrificed on day 14, and tumors were collected. The tumors were ground and filtered through a 70μm cell filter to obtain a single-cell suspension. The single-cell suspension from each mouse was divided into different fractions to determine 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, and anti-Foxp3-PE are used to detect regulatory T lymphocytes (CD45-FITC, anti-CD3-PerCP / Cy5.5, anti-CD4-PE / Cy7, anti-CD25-APC, and anti-Foxp3-PE.) + 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. Cells were incubated at 4°C in the dark for 1 hour, then fixed with a 1:1 solution of PBS:4% paraformaldehyde. Cells were washed with PBS and resuspended before testing. Flow cytometry analysis was performed, and the results were statistically analyzed using one-way ANOVA.

[0111] Depend on Figure 15 It can be seen that the CD8+ of the primary tumor treated with CLRN+L... + The proportion of T cells (31.9±5.77%) was 9 times that of PBS (3.44±1.18%), while the CD8+ of distant tumors... + The T-cell infiltration rate reached 30.6±4.86%, significantly higher than that of other control groups. After CLRN+L treatment, CD4+ in primary and distant tumors... +T cells also increased. Under light and dark conditions, the M1 / M2 ratio of CLRN was 6-fold and 4-fold higher than 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. Figure 16 The infiltration rate of Tregs in CLRN+L (7.07±0.63%) was significantly lower than that in PBS (22.57±2.15%). Figure 17 CLRN enhanced the pyroptosis-induced ICD effect under laser irradiation, effectively promoted the maturation of DCs, recruited cytotoxic T lymphocytes (CTLs), and ultimately induced a strong anti-tumor immune response.

[0112] The above results indicate 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 subjected to laser irradiation, showed a stronger anti-tumor effect. This is because the nanoparticles can be more effectively taken up into tumor cells, relieving hypoxia and suppressing the tumor microenvironment, and synergistically activating the immune response caused by photoinduced pyroptosis, thereby amplifying and enhancing the anti-tumor immune effect.

[0113] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of ternary carrier-free nanoparticles in the preparation of tumor therapeutic drugs, characterized in that: The ternary carrier-free nanoparticles are nanoparticles formed by intermolecular interactions of dihydroporphyrin E6, chlordamine, and RG108; the mass ratio of dihydroporphyrin E6, chlordamine, and RG108 is (1~15):(3~10):(3~15); the tumor is breast cancer.

2. The application of the ternary carrier-free nanoparticles according to claim 1 in the preparation of tumor therapeutic drugs, characterized in that: The average particle size of the nanoparticles is 100 nm to 180 nm.

3. The application of the ternary carrier-free nanoparticles according to claim 1 in the preparation of tumor therapeutic drugs, characterized in that: The intermolecular interaction forces include at least one of hydrophobic interaction forces, π-π stacking forces, and hydrogen bonding forces.

4. The application of the ternary carrier-free nanoparticles according to claim 1 in the preparation of tumor therapeutic drugs, characterized in that: The ternary carrier-free nanoparticles are prepared by a method comprising the following steps: ultrasonicating an organic solution of dihydroporphyrin E6, chlordamine and RG108 to obtain the ternary carrier-free nanoparticles.

5. The application of the ternary carrier-free nanoparticles according to claim 4 in the preparation of tumor therapeutic drugs, characterized in that: The frequency of the ultrasound is 20~60kHz; the duration of the ultrasound is 1~10min.

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