Antigen-free biodegradable nano preparation and application thereof

Abstract

The invention relates to the field of tumor treatment preparations, in particular to an antigen-free biodegradable nano preparation and application. Near-infrared light response molecule indocyanine green which is clinically used in batches is used as a phototherapy active component, and a polymer PLGA which is approved by FDA and is widely used in the field of medicine is used as a delivery auxiliary material to design an antigen-free nano system. PLGA is composed of copolymers of lactic acid and glycollic acid synthesized in different proportions, and has the application advantages of biocompatibility, high biodegradability, low toxicity and the like compared with an inorganic nano system. On the other hand, in consideration of the fact that ICG is mainly used for clinical diagnosis through imaging molecules and the phototherapy response is relatively limited, natural polysaccharide pachymaran with an immunostimulatory effect is used as an adjuvant component to enhance the overall immunostimulatory effect, and finally the phototherapy anti-tumor effectiveness is improved; and a good treatment effect on large tumors and recurrent metastatic tumors is expected to be achieved.

Description

Technical Field

[0001] The present invention relates to the field of tumor treatment preparations, and in particular to a biodegradable nano preparation that does not require an antigen, and its anti-cancer application in the combined effect of phototherapy / immunotherapy. Background Art

[0002] Tumors are major diseases that seriously endanger human health. Their morbidity and mortality rates are increasing year by year, and there is currently a lack of effective treatment methods. Tumor phototherapy is a minimally invasive treatment method that uses light activation to controllably induce apoptosis of tumor cells, and it is relatively safe. Phototherapy mainly includes photodynamic therapy (PDT) and photothermal therapy (PTT). At present, many anti-tumor phototherapy studies use the near-infrared light-responsive molecule indocyanine green (ICG) as the active ingredient to implement phototherapy. In fact, it is difficult to effectively control the occurrence and development of tumors through phototherapy alone, because the singlet oxygen in PDT therapy has a short lifetime (μs level) and insufficient diffusion distance (<200nm), which makes it difficult to play a therapeutic role on larger tumors and recurrent metastatic tumors. For photothermal anti-tumor treatment, excessively high temperatures during phototherapy will affect normal tissue function and even cause immune tolerance.

[0003] As research progresses, researchers have discovered that phototherapy also has a certain anti-tumor potential as an immunotherapy, and will have a good therapeutic effect on large tumors and recurrent metastatic tumors. Currently, common tumor immunotherapy nanoformulations need to encapsulate tumor-associated antigens or tumor-specific antigens to activate the immune system's recognition of tumor tissue. Its anti-tumor effect is closely related to the antigen type, dose, and antigen stability in the nanoformulation. Therefore, new nanoformulations that do not require antigen encapsulation have good innovation for universal anti-cancer and simplified nanoformulation preparation.

[0004] Activating tumor immunogenic death (ICD) is an effective strategy for implementing anti-tumor immunotherapy without antigen packaging. After ICD is induced in tumor tissue, tumor-associated antigens can be released, thereby completing the recruitment and activation of antigen-presenting cells (APCs), and exerting a therapeutic effect against tumor metastasis and recurrence. Dying tumor cells and cell fragments that have undergone phototherapy will play the role of tumor-associated antigens, stimulate the maturation of dendritic cells (DCs), migrate to lymph nodes and differentiate into antigen-presenting cells, and induce the occurrence of anti-tumor adaptive immune responses; on the other hand, photodynamic therapy (PDT) in phototherapy can also induce oxidative stress by producing reactive oxygen species (ROS), thereby inducing the tumor immunogenic death (ICD) process and activating the anti-tumor effect of the immune system; in addition, photothermal therapy (PTT) molecules can convert light into heat under light activation, stimulate endoplasmic reticulum stress, and trigger the ICD effect. In summary, the implementation of phototherapy is expected to enhance the ICD process in a multi-pronged manner and stimulate anti-tumor immunotherapy. Summary of the invention

[0005] Aiming at the main application goal of anti-tumor immunotherapy, based on the current biosafety and limited application potential of phototherapy delivery systems, this technology uses the clinically approved near-infrared light-responsive molecule indocyanine green (ICG) as the phototherapy active component, and uses the FDA-approved polymer PLGA (polylactic-co-glycolic acid) widely used in the medical field as a delivery excipient to design an antigen-free nanosystem. PLGA is composed of copolymers of lactic acid and glycolic acid synthesized in different proportions, and has application advantages such as biocompatibility, strong biodegradability, and low toxicity compared to inorganic nanosystems. On the other hand, considering that ICG is mainly used as an imaging molecule for clinical diagnosis, its phototherapy response is relatively limited. Therefore, the natural polysaccharide Poria cocos polysaccharide (PPS) with immunostimulatory effect is used as an adjuvant component to enhance the ICD process in a multi-pronged manner to enhance the overall immunostimulatory effect, and ultimately improve the effectiveness of phototherapy against tumors, which is expected to have a good therapeutic effect on large tumors and recurrent metastatic tumors.

[0006] To achieve the above object, the present invention is implemented through the following scheme:

[0007] In one aspect, a method for preparing a biodegradable nanoformulation without the need for an antigen is provided, comprising the following steps:

[0008] S1. PLGA is dissolved in dichloromethane to prepare an oil phase; Poria cocos polysaccharide and indocyanine green are dissolved in water to obtain an inner water phase; the inner water phase is added to the oil phase and ultrasonically vibrated for 1-3 minutes to form a stable W / O type colostrum;

[0009] S2. Adding W / O colostrum to a 0.1-1% F68 aqueous solution, ultrasonically vibrating for 4-6 minutes, to form a W / O / W emulsion;

[0010] S3. Stir the W / O / W type emulsion for 4-7 hours, centrifuge and collect the supernatant to obtain.

[0011] Furthermore, in the oil phase of S1, the concentration of PLGA is 10-50 mg / ml; or 10-40 mg / ml; or 10-30 mg / ml; or 20-30 mg / ml.

[0012] Furthermore, in the inner aqueous phase of S1, the concentration of indocyanine green is 0.1-5 mg / ml; or 0.1-3 mg / ml; or 0.1-2 mg / ml; or 0.1-1 mg / ml; or 0.3-1 mg / ml; or 0.5-0.8 mg / ml.

[0013] Furthermore, in the inner aqueous phase of S1, the concentration of Poria cocos polysaccharide is 1-20 mg / ml; or 1-15 mg / ml; or 5-15 mg / ml; or 8-15 mg / ml; or 8-12 mg / ml.

[0014] Furthermore, the volume ratio of the oil phase to the inner water phase is (2-10):1; or (2-8):1; or (2-6):1; or (3-5):1.

[0015] Furthermore, in S2, the mass fraction of the F68 aqueous solution is 0.1-0.8%; or 0.1-0.6%; or 0.2-0.6%; or 0.3-0.5%.

[0016] In the second aspect, a biodegradable nanoformulation that does not require an antigen is provided. The nanoformulation is a core-shell structure, the core of the core-shell structure is a mixed aqueous solution of Poria cocos polysaccharide and indocyanine green, and the outer shell is a PLGA membrane; the loading amount of Poria cocos polysaccharide is 31±5%, and the loading amount of indocyanine green is 24±5%.

[0017] Furthermore, the particle size of the nano preparation is 80-150nm.

[0018] In a third aspect, a nanoformulation prepared based on the above method and the use of the above nanoformulation in the preparation of tumor drugs are provided.

[0019] In a fourth aspect, a pharmaceutical composition is provided, comprising an active ingredient and a pharmaceutically acceptable excipient; the active ingredient is the above-mentioned nanoformulation or the nanoformulation prepared by the above-mentioned method.

[0020] Furthermore, the auxiliary materials include one or more of solvents, disintegrants, flavoring agents, preservatives, colorants, adhesives, lubricants, diluents and drug carriers.

[0021] Furthermore, the drug uses the nano preparation as an active ingredient and is prepared into a pharmaceutically acceptable dosage form using pharmaceutically acceptable excipients.

[0022] It is well understood by those skilled in the art that excipients (e.g., pharmaceutical excipients) can be selected from one or more of the following: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesive agents, integrities, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, and release retardants.

[0023] Furthermore, the dosage form includes any one of tablets, capsules, pills, powders, ointments, pills, suspensions, powders, injections, sustained-release preparations, controlled-release preparations or targeted preparations.

[0024] Compared with the prior art, the antigen-free biodegradable nanoformulation of the present invention uses molecules with PTT / PDT phototherapy effects and polysaccharides as adjuvants to prepare a nano delivery system. Through light stimulation, tumor apoptosis and necrosis are induced, tumor antigens are produced and anti-tumor immune effects are activated, lymphocyte proliferation and cytokine secretion are promoted, the tumor infiltration ratio of CD4+T lymphocytes and CD8+T lymphocytes is increased, the ratio of Treg cells in the tumor site is reduced, and tumor-associated macrophages are promoted to secrete cytokines (IL-6, TNF-α), effectively inducing PTT / PDT dual-mode phototherapy, promoting dendritic cell maturation, reshaping the tumor immunosuppressive microenvironment, and enhancing the responsiveness of tumor cells to phototherapy and immunotherapy, which is expected to improve the clinical treatment effect of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Characterization diagram of PPS-ICG@PLGA NPs;

[0026] Among them, 1a is the transmission electron microscope image of the nanoformulation; 1b is the dynamic light scattering detection image of the nanoformulation;

[0027] Figure 2 To detect the heat generation ability of PPS-ICG@PLGA NPs under laser irradiation;

[0028] Figure 3 The ICD response induced by incubation of 4T1 mouse breast cancer cells with different nanoparticles;

[0029] Figure 4 The production of cytokines in RAW 264.7 cells after being treated in different ways;

[0030] Figure 5 Tumor enrichment fluorescence imaging and biodistribution after intravenous injection of ICG solution and PPS-ICG@PLGA NPs in mice;

[0031] Figure 6 The regulation of the tumor microenvironment after intravenous injection of different nanoparticles into mice;

[0032] Figure 7 To investigate the therapeutic effects of different nanoparticles on tumor model mice in vivo;

[0033] Figure 8 The preparation process of nanoparticles and the in vivo treatment model diagram for tumor model mice;

[0034] 8a is a flow chart of the preparation method of nanoparticles; 8b is a schematic diagram of the experimental process of the nanoparticles of the present invention on tumor treatment animal models. DETAILED DESCRIPTION

[0035] In order to make those skilled in the art better understand the technical scheme of the present invention, the present invention is described in detail below in conjunction with specific embodiments. The experimental methods for which specific conditions are not indicated in the following examples are usually carried out under normal conditions or according to the conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meaning as those familiar to those skilled in the art. In addition, any method and material similar to or equal to the recorded content can all be applied to the present invention. The preferred implementation methods and materials described in the text are only for demonstration purposes.

[0036] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0037] The present invention is based on the screening of the immunopotentiating effect of polysaccharide natural products, which are used as adjuvant components, and the near-infrared light-responsive molecule indocyanine green (ICG) is used as the phototherapy active component to construct a nano-preparation for light-activated anti-cancer immunotherapy without antigens, providing a new candidate system for nano-delivery for the clinical application of anti-cancer immunotherapy, and also proposing a practical way and strategy for improving the effect of combined tumor immunotherapy, which specifically includes the following steps:

[0038] S1. PLGA is dissolved in dichloromethane to prepare an oil phase; Poria cocos polysaccharide and indocyanine green are dissolved in water to obtain an inner water phase; the inner water phase is added to the oil phase and ultrasonically vibrated for 1-3 minutes to form a stable W / O type colostrum;

[0039] S2. Adding W / O colostrum to a 0.1-1% F68 aqueous solution, ultrasonically vibrating for 4-6 minutes, to form a W / O / W emulsion;

[0040] S3. Stir the W / O / W type emulsion for 4-7 hours, centrifuge and collect the supernatant to obtain.

[0041] In some specific embodiments, the PLGA concentration in the oil phase of S1 is 10-50 mg / ml, or 10-40 mg / ml, or 10-30 mg / ml, or 20-30 mg / ml. In specific experiments, reasonable selection can be made according to the situation, and will not be repeated here.

[0042] In some specific embodiments, the concentration of indocyanine green in the inner aqueous phase of S1 is 0.1-5 mg / ml; or 0.1-3 mg / ml; or 0.1-2 mg / ml; or 0.1-1 mg / ml; or 0.3-1 mg / ml; or 0.5-0.8 mg / ml. In specific experiments, reasonable selection can be made according to the situation, and will not be repeated here.

[0043] In some specific embodiments, the concentration of pachymaran in the inner aqueous phase of S1 is 1-20 mg / ml, or 1-15 mg / ml, or 5-15 mg / ml, or 8-15 mg / ml, or 8-12 mg / ml. In specific experiments, reasonable selection can be made according to the situation, and will not be repeated here.

[0044] In some specific embodiments, Poria cocos polysaccharide is crudely prepared by water extraction and alcohol precipitation, specifically: 1 kg of Poria cocos diced is refluxed in a water bath at 80°C for 3 times with water as the extraction solvent, 10 times the amount of water is refluxed for 2h for the first extraction, 8 times the amount of water is refluxed for 1h for the second extraction, and 6 times the amount of water is refluxed for 1h for the third extraction, and the filtrates of the 3 times are combined and concentrated to about 10L at 50°C under reduced pressure, and the obtained filtrate is subjected to subsequent alcohol precipitation. Ethanol is added under stirring to make the alcohol content reach 80%, and the mixture is allowed to stand for 12h, centrifuged, and the precipitate is collected, 60ml of distilled water is added to dissolve and boil, and the insoluble matter is filtered out while hot, and ethanol is added to the filtrate under stirring to make the alcohol content reach 80%, and the mixture is allowed to stand, and after the brown precipitate is precipitated, it is dried at low temperature to obtain the crude Poria cocos polysaccharide.

[0045] In some specific embodiments, the crude product of Poria cocos polysaccharide is refined by the following method: the crude product is dissolved in 150ml of distilled water, boiled, 1% tannic acid solution is added under stirring, boiled, centrifuged, and the supernatant is added to the tannic acid solution until it is not turbid. Add 2% activated carbon, stir for 10min, filter with a Buchner funnel while hot, cool the filtrate, add ethanol to make the alcohol content reach 70%, stand for 24h, filter the precipitate, use 70% ethanol 100ml, repeatedly wash the precipitate, and check that it does not contain tannic acid. Dissolve the wet product in 583ml of 20% hot ethanol, place it in a Buchner funnel with 50g neutral alumina, and after decompression, add 60℃ hot distilled water for continuous elution, and the effluent is concentrated under reduced pressure to 62.5ml, add ethanol to make the alcohol content reach 70%, stand, filter the precipitate and dry it at low temperature (or freeze), and the pure product of Poria cocos polysaccharide is obtained.

[0046] In some specific embodiments, the volume ratio of the oil phase to the internal water phase is (2-10): 1; or (2-8): 1; or (2-6): 1; or (3-5): 1. In specific experiments, reasonable selection can be made according to the situation, and will not be repeated here.

[0047] In some specific embodiments, the mass fraction of the F68 aqueous solution in S2 is 0.1-0.8%; or 0.1-0.6%; or 0.2-0.6%; or 0.3-0.5%. In specific experiments, reasonable selection can be made according to the situation, and will not be repeated here.

[0048] Based on the present invention, the core-shell structured biodegradable nanoformulation without antigen has a mixed aqueous solution of tuckahoe polysaccharide and indocyanine green as the core and a PLGA membrane as the shell; the loading amount of tuckahoe polysaccharide is 31±5%, the loading amount of indocyanine green is 24±5%, and the particle size is 80-150nm. Nanoparticles within this particle size range have high penetration in the tumor site and high passive targeting, and the phototherapy active component indocyanine green (ICG) and the adjuvant component tuckahoe polysaccharide (PPS) have a high loading amount, thereby further increasing CD4 + T lymphocytes and CD8 + It reduces the tumor infiltration rate of T lymphocytes, reduces the proportion of Treg cells in the tumor site, promotes the secretion of cytokines (IL-6, TNF-α) by tumor-associated macrophages, promotes the maturation of dendritic cells, reshapes the tumor immunosuppressive microenvironment, and enhances the responsiveness of tumor cells to phototherapy and immunotherapy.

[0049] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0050] Example 1 Preparation of Nanoformulation PPS-ICG@PLGA NPs

[0051] 25 mg PLGA was dissolved in 1 mL of dichloromethane to prepare the oil phase, 40 μL of 4 mg / mL indocyanine green (ICG) and 200 μL of 12.5 mg / mL Poria cocos polysaccharide (PPS) were used to prepare the inner aqueous phase. Using probe ultrasound, an oil-in-water emulsion (w / o) was formed by ultrasound at an amplitude of 20% for 1 min in an ice bath. Subsequently, the primary emulsion was added to 4 mL of the outer aqueous phase containing 0.4% F68, and ultrasound was performed at an amplitude of 20% for 5 min in an ice bath to obtain a stable w / o / w emulsion. The final emulsion was magnetically stirred at 37 ° C for 5 hours, and finally, centrifuged at 5510 rpm for 10 min, and the supernatant was taken to obtain PPS-ICG@PLGA NPs.

[0052] After measurement, the loading amount of tuckahoe polysaccharide in the nanoformulation prepared in this embodiment is 31±5%, and the loading amount of indocyanine green is 24±5%. The performance of the nanoformulation prepared in this embodiment is characterized, and the transmission electron microscopy test results are as follows: Figure 1As shown in a, the results show that PPS-ICG@PLGA NPs are distributed in a monodisperse state in water, indicating that the particle size of the PPS-ICG@PLGA NPs prepared by the present invention is uniform and well dispersed in water; the results of dynamic light scattering detection are shown in Figure 1 As shown in b, the results showed that PPS-ICG@PLGA NPs 2 O, PBS, cell culture medium, etc.), the particle size is mainly concentrated around 100nm, and experiments at the living body level can be carried out.

[0053] Example 2 Nanoformulation PPS-ICG@PLGA NPs

[0054] 20 mg PLGA was dissolved in 1 mL of dichloromethane to prepare the oil phase, 50 μL of 3 mg / mL indocyanine green (ICG) and 200 μL of 15 mg / mL Poria cocos polysaccharide (PPS) were used to prepare the inner aqueous phase. Using probe ultrasound, an oil-in-water emulsion (w / o) was formed by ultrasound at an amplitude of 20% for 1 min in an ice bath. Subsequently, the primary emulsion was added to 4 mL of the outer aqueous phase containing 0.2% F68, and ultrasound was performed at an amplitude of 20% for 5 min in an ice bath to obtain a stable w / o / w emulsion. The final emulsion was magnetically stirred at 37 ° C for 5 hours, and finally, centrifuged at 5510 rpm for 10 min, and the supernatant was taken to obtain PPS-ICG@PLGA NPs.

[0055] Example 3 Nanoformulation PPS-ICG@PLGA NPs

[0056] 30 mg PLGA was dissolved in 1 mL of dichloromethane to prepare the oil phase, 40 μL of 12 mg / mL indocyanine green (ICG) and 200 μL of 18 mg / mL Poria cocos polysaccharide (PPS) were used to prepare the inner aqueous phase. Using probe ultrasound, an oil-in-water emulsion (w / o) was formed by ultrasound at an amplitude of 20% for 2 min in an ice bath. Subsequently, the primary emulsion was added to 4 mL of the outer aqueous phase containing 0.3% F68, and ultrasound was performed at an amplitude of 20% for 5 min in an ice bath to obtain a stable w / o / w emulsion. The final emulsion was magnetically stirred at 37 ° C for 5 hours, and finally, centrifuged at 5510 rpm for 10 min, and the supernatant was taken to obtain PPS-ICG@PLGA NPs.

[0057] Example 4 Nanoformulation PPS-ICG@PLGA NPs

[0058] 10 mg PLGA was dissolved in 1 mL of dichloromethane to prepare the oil phase, 50 μL of 4 mg / mL indocyanine green (ICG) and 250 μL of 9.6 mg / mL Poria cocos polysaccharide (PPS) were prepared as the inner aqueous phase. Using probe ultrasound, an oil-in-water emulsion (w / o) was formed by ultrasound at an amplitude of 20% for 1 min in an ice bath. Subsequently, the primary emulsion was added to 4 mL of the outer aqueous phase containing 0.3% F68, and ultrasound was performed at an amplitude of 20% for 5 min in an ice bath to obtain a stable w / o / w emulsion. The final emulsion was magnetically stirred at 37 ° C for 5 hours, and finally, centrifuged at 5510 rpm for 10 min, and the supernatant was taken to obtain PPS-ICG@PLGA NPs.

[0059] Example 5 Nanoformulation PPS-ICG@PLGA NPs

[0060] 50 mg PLGA was dissolved in 1 mL of dichloromethane to prepare the oil phase, 50 μL of 15 mg / mL indocyanine green (ICG) and 250 μL of 24 mg / mL Poria cocos polysaccharide (PPS) were prepared as the inner aqueous phase. Using probe ultrasound, an oil-in-water emulsion (w / o) was formed by ultrasound at an amplitude of 20% for 3 min in an ice bath. Subsequently, the primary emulsion was added to 4 mL of the outer aqueous phase containing 0.8% F68, and ultrasound was performed at an amplitude of 20% for 5 min in an ice bath to obtain a stable w / o / w emulsion. The final emulsion was magnetically stirred at 37 ° C for 5 hours, and finally, centrifuged at 5510 rpm for 10 min, and the supernatant was taken to obtain PPS-ICG@PLGA NPs.

[0061] Experimental Example 1 Investigating the heat generation ability of PPS-ICG@PLGA NPs under laser irradiation

[0062] 808nm, 1W / cm 2 , 10min irradiation of PPS-ICG@PLGA NPs, it can be seen that with the extension of laser irradiation time, the temperature of PPS-ICG@PLGA NPs gradually increased ( Figure 2 ).

[0063] Experimental Example 2 Investigating the ICD response induced by 4T1 cells after incubation with different nanoparticles

[0064] Detection of CRT exposure on the surface of 4T1 cells, HMGB1 outflow from the nucleus, and ATP secretion induced by different treatments under laser irradiation ( Figure 3 ae). We found that laser-irradiated PPS-ICG@PLGA NPs significantly promoted CRT exposure on the surface of 4T1 cells and HMGB1 efflux from the nucleus. In addition, the ATP secretion of 4T1 cells treated with irradiated PPS-ICG@PLGA NPs was significantly higher than that of other groups.

[0065] Experimental Example 3: Investigating the production of cytokines in RAW 264.7 cells after treatment in different ways

[0066] 4T1 cells were seeded in the upper chamber and cultured overnight. RAW 264.7 cells were cultured in the lower chamber for 24 h. Then, 4T1 cells and RAW264.7 cells were incubated with PPS-ICG@PLGA NPs (final ICG concentration was 10 μg / mL). After incubation for 5 h, 808 nm laser (1 W / cm 2 , 5min) irradiated 4T1 cells. Then, RAW 264.7 cells were co-cultured with 4T1 cells for 12h. Finally, the supernatant of the culture medium was collected and quantitatively analyzed using an ELISA kit to measure cytokines (TNF-α and IL-6). Under laser irradiation, PPS-ICG@PLGA NPs accelerated the secretion of IL-6 and TNF-α ( Figure 4 ). These results indicate that laser-irradiated PPS-ICG@PLGA NPs promoted the secretion of cytokines (TNF-α and IL-6) by tumor-associated macrophages, i.e., possessed immunomodulatory potential on mouse macrophages.

[0067] Experimental Example 4: Behavioral detection of PPS-ICG@PLGA NPs in mice

[0068] After intravenous injection of PPS-ICG@PLGA NPs prepared in Example 1, the results of tumor enrichment are as follows: Figure 5 As shown in a. The results show that after intravenous injection of the PPS-ICG@PLGA NPs, they will gradually accumulate in the tumor site over time. The biodistribution test results are shown in Figure 5 As shown in b, from left to right are heart, liver, spleen, lung, kidney, and tumor. The results show that the PPS-ICG@PLGA NPs prepared in Example 1 are highly enriched in the tumor site 24 hours after intravenous injection.

[0069] Experimental Example 5: Investigating the regulation of tumor microenvironment after intravenous injection of different nanoparticles into mice

[0070] 4T1 cells were inoculated subcutaneously on the right flank of 5-week-old BALB / C female mice. 3 The mice were divided into 6 groups (n = 3) and injected intravenously with 100 μL of PBS, ICG solution (150 μg / mL), 200 μg / mL PPS solution or PPS-ICG@PLGA NPs (150 μg / mL) on days 7 and 9. 6 h after injection, the mice in the laser group were exposed to laser (808 nm, 1 W / cm 2) for 5 min. On the 11th day, the spleen and tumor were separated and processed into single cell suspensions, and the content of CD3﹢CD4﹢T cells, CD3﹢CD8﹢T cells, CD80﹢CD86﹢ dendritic cells and Treg cells was detected by flow cytometry. Figure 6 As shown, PPS-ICG@PLGA NPs (+) induced more CD4+CD8+T cells than other treatments. Compared with the ICG (+) group, the proportions of CD3+CD4+T cells, CD3+CD8+T cells, and CD80+CD86+ dendritic cells in mice in the PPS-ICG@PLGA NPs (+) group increased significantly, while the percentage of Tregs decreased significantly. This result shows that PPS-ICG@PLGA NPs combined with PTT and PDT has the advantage of improving immune response, promoting the maturation of dendritic cells, and significantly reducing tumor-related immunosuppression.

[0071] Experimental Example 6: Investigating the therapeutic effects of different nanoparticles on tumor model mice

[0072] The 4T1 breast cancer tumor-bearing mice were divided into six groups (n﹦5), including: Group 1, control group (only injected with normal saline); Group 2, group injected with Poria polysaccharide solution; Group 3, group injected with ICG solution; Group 4, group injected with ICG solution and combined with laser treatment; Group 5, group injected with PPS-ICG@PLGA NPs; Group 6, group injected with PPS-ICG@PLGA NPs and combined with laser treatment. After the mice were treated accordingly, their tumor growth was measured. On the 16th day, the weight of the tumor in each group of mice was measured. Figure 7 It can be seen that the tumor size of the other groups grew to ≈1100 mm on day 16. 3 However, the tumor size in the PPS-ICG@PLGA NPs (+) group gradually shrank and almost completely disappeared. The tumor volume and weight were also the smallest compared with the other treatment groups, further indicating that PPS-ICG@PLGA NPs combined with laser irradiation can enhance the anti-tumor therapeutic effect.

[0073] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the protection scope of the present invention.

Claims

1. A method for preparing a biodegradable nanoformulation without the need for an antigen, characterized in that: The following steps are involved: S1. PLGA is dissolved in dichloromethane to prepare an oil phase; Poria cocos polysaccharide and indocyanine green are dissolved in water to obtain an inner water phase; the inner water phase is added to the oil phase and ultrasonically vibrated for 1-3 minutes to form a stable W / O type colostrum; S2. Adding W / O colostrum to a 0.1-1% F68 aqueous solution, ultrasonically vibrating for 4-6 minutes, to form a W / O / W emulsion; S3. Stir the W / O / W type emulsion for 4-7 hours, centrifuge and collect the supernatant to obtain.

2. The method for preparing a biodegradable nanoformulation without the need for an antigen according to claim 1, characterized in that: In the oil phase of S1, the concentration of PLGA is 10-50 mg / ml; or 10-40 mg / ml; or 10-30 mg / ml; or 20-30 mg / ml.

3. The method for preparing a biodegradable nanoformulation without the need for an antigen according to claim 1, characterized in that: In the inner aqueous phase of S1, the concentration of indocyanine green is 0.1-5 mg / ml; or 0.1-3 mg / ml; or 0.1-2 mg / ml; or 0.1-1 mg / ml; or 0.3-1 mg / ml; or 0.5-0.8 mg / ml.

4. The method for preparing a biodegradable nanoformulation without the need for an antigen according to claim 1, characterized in that: In the inner aqueous phase of S1, the concentration of Poria cocos polysaccharide is 1-20 mg / ml; or 1-15 mg / ml; or 5-15 mg / ml; or 8-15 mg / ml; or 8-12 mg / ml.

5. The method for preparing a biodegradable nanoformulation without the need for an antigen according to claim 1, characterized in that: The volume ratio of the oil phase to the internal water phase is (2-10):1; or (2-8):1; or (2-6):1; or (3-5):

1.

6. The method for preparing a biodegradable nanoformulation without the need for an antigen according to claim 1, characterized in that: In the S2, the mass fraction of the F68 aqueous solution is 0.1-0.8%; or 0.1-0.6%; or 0.2-0.6%; or 0.3-0.5%.

7. A biodegradable nanoformulation without the need for an antigen, characterized in that: The nano preparation is a core-shell structure, the core of the core-shell structure is a mixed aqueous solution of tuckahoe polysaccharide and indocyanine green, and the shell is a PLGA membrane; the loading amount of tuckahoe polysaccharide is 31±5%, and the loading amount of indocyanine green is 24±5%.

8. The nanoformulation prepared according to claim 7, characterized in that The particle size of the nano preparation is 80-150nm.

9. Use of the nanoformulation obtained by the method according to any one of claims 1 to 6, or the nanoformulation according to claim 7 or 8 in the preparation of tumor drugs.

10. A pharmaceutical composition, characterized in that The invention comprises an active component and a pharmaceutically acceptable excipient; the active component is the nano preparation according to claim 7 or 8, or the nano preparation prepared by the method according to any one of claims 1 to 6.

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