A kind of nanoparticle for tumor sonodynamic combined personalized immunotherapy and its preparation method and application

Abstract

The application discloses a kind of nanoparticles for tumor sonodynamic combined individualized immunotherapy and its preparation method and application, belong to the field of biological medicine.The nanoparticles for tumor sonodynamic combined individualized immunotherapy are double-shell core structure nanoparticles formed by inner core and shell, and the inner core is formed by self-assembly of TLR3 agonist Poly (I:C), L-arginine and polylysine, and the shell is hyaluronic acid layer HA-Ce6 conjugated with photosensitizer Ce6.It is found through experiment verification that the nanoparticles can target tumor, generate reactive oxygen species to kill tumor cells under the action of ultrasound, promote M1 macrophage polarization and produce NO release at the same time, play the role of antitumor and immune effect, and realize the combined application of sonodynamic and NO gas therapy.The nanoparticles for tumor sonodynamic combined individualized immunotherapy can trigger systemic immune response, inhibit primary tumor, produce immune memory effect, and prevent tumor metastasis and recurrence.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a nanoparticle for tumor sonodynamic combined with personalized immunotherapy, its preparation method and application. Background Technology

[0002] Colorectal cancer has an extremely high incidence and mortality rate, making it one of the major diseases threatening human life and health. Statistics from the American Cancer Society in 2021 revealed that colorectal cancer ranked third among all cancers in both new cases and mortality, with a 5% increase compared to 2020. The mortality rates for men and women were 9% and 8%, respectively. Surgery, radiation therapy, and chemotherapy are currently the main treatments for colorectal cancer, but the systemic toxicity and drug resistance of chemotherapy drugs remain serious problems. In contrast, immunotherapy, which activates the patient's own immune system to kill tumor cells, can largely solve these problems. Although immunotherapy shows great promise, considering the large differences in patients' immune systems, low immune response rates, and the unfavorable immune microenvironment of tumors, combined immunotherapy strategies, integrating immunotherapy with other therapies, have received widespread attention. Sonodynamic therapy (SDT), as a non-invasive tumor treatment, has significant advantages such as good controllability, high precision, and strong penetration. Under the action of ultrasound, the sonosensitive agent is activated, generating a large amount of reactive oxygen species, promoting tumor cell apoptosis. However, common sound-sensitive agents such as Ce6 have poor water solubility, requiring the design of reasonable delivery strategies.

[0003] NO is a crucial gaseous molecule in the human body, acting as a messenger molecule in the cardiovascular, nervous, and immune systems. In the tumor microenvironment, various cell types are capable of producing NO, including fibroblasts, antigen-presenting cells, macrophages, and NK cells. Among these cells, macrophages are a key source of NO synthesis. The expression of highly inducible nitric oxide synthase (iNOS) in M1 macrophages is a prerequisite for NO production. Endogenous NO produced by iNOS can regulate the immune system in a dose-dependent manner, thereby affecting tumor progression. Studies have found that high concentrations of NO inhibit the development of malignant tumors by inducing apoptosis, delaying angiogenesis, and inhibiting multidrug resistance, while low concentrations of NO can promote T cell infiltration and recruit and activate immune effector cells. L-arginine is a natural NO donor that can release NO in the presence of iNOS or ROS. Since the content of L-arginine in tumor tissue is low, exogenous supplementation of L-arginine can exert an anti-tumor effect by releasing large amounts of NO. In recent years, NO-based anti-tumor therapies have attracted increasing attention. However, there are currently no reports of using sonodynamic therapy combined with NO gas to treat colorectal cancer. Summary of the Invention

[0004] The purpose of this invention is to provide a nanoparticle for combined sonodynamic and personalized immunotherapy of tumors, its preparation method and application, in order to solve the problems existing in the prior art. By constructing a nanoparticle co-loaded with L-arginine and Poly(I:C) coated with HA-Ce6, the combined sonodynamic therapy, NO gas therapy and tumor immunotherapy are realized, which can effectively treat tumors and inhibit tumor metastasis and recurrence.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a nanoparticle for tumor sonodynamic combined with personalized immunotherapy. The nanoparticle is a double-shell core structure nanoparticle consisting of a core and a shell. The core of the nanoparticle includes polylysine, L-arginine and a TLR3 agonist, and the shell of the nanoparticle includes hyaluronic acid and a sonosensitive agent.

[0007] Preferably, the nanoparticle core is self-assembled from polylysine, L-arginine, and a TLR3 agonist, and the nanoparticle shell is constructed by conjugating a sound-sensitizing agent onto hyaluronic acid. More preferably, the TLR3 agonist is Poly(I:C), and the sound-sensitizing agent is Ce6, but the TLR3 agonist and sound-sensitizing agent are not limited to the above compounds.

[0008] The present invention also provides a method for preparing the nanoparticles described above, comprising the following steps:

[0009] Nanoparticle cores were constructed by mixing polylysine, L-arginine and TLR3 agonists and self-assembling them.

[0010] A sound-sensitive agent was conjugated to hyaluronic acid to prepare a nanoparticle shell;

[0011] The nanoparticle core and the nanoparticle shell were dissolved in water, mixed and stirred, centrifuged, washed, resuspended, and then ultrasonically dispersed to obtain multifunctional acoustic-dynamic nanoparticles with a double-shell core-shell structure.

[0012] Preferably, the preparation method of the nanoparticle core specifically includes the following steps:

[0013] A TLR3 agonist aqueous solution was added dropwise to an L-arginine aqueous solution and a polylysine aqueous solution, and the mixture was shaken at room temperature to perform self-assembly. After centrifugation and washing, the nanoparticle cores were obtained. The volume ratio of the TLR3 agonist aqueous solution, the L-arginine aqueous solution and the polylysine aqueous solution was 60:(80-110):(110-130).

[0014] Preferably, the concentration of the TLR3 agonist aqueous solution is 2 mg / mL, the concentration of the L-arginine aqueous solution is 5 mg / mL, and the concentration of the polylysine aqueous solution is 2 mg / mL.

[0015] The oscillation conditions are: 3000 rpm for 30-60 min; the centrifugation conditions are: 10000 rpm for 10-20 min.

[0016] Preferably, the method for preparing the nanoparticle shell specifically includes the following steps:

[0017] Hyaluronic acid, adipic acid dihydrazide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were mixed and reacted, then dialyzed and lyophilized to obtain intermediate product I.

[0018] N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride were reacted with a sound-sensitive agent, and then reacted with the intermediate product I. The mixture was dialyzed and lyophilized to obtain the nanoparticle shell.

[0019] Preferably, the mass ratio of the hyaluronic acid, the adipic dihydrazide, and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride is 100:(200-240):(20-30);

[0020] The mass ratio of the N-hydroxysuccinimide, the 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, and the sound sensitizer is 30:(35-45):30, and the mass ratio of the sound sensitizer to the intermediate product I is 30:100.

[0021] Preferably, the volume ratio of the nanoparticle core to the nanoparticle shell is 2:3;

[0022] The mixing conditions are: 500 rpm and 10-12 h.

[0023] The ultrasound conditions are as follows: a 3mm probe is used, the power is 20%, the processing time is 1 minute, and ultrasound is performed for 5 seconds every 5 seconds.

[0024] This invention also provides the use of the nanoparticles in any of the following:

[0025] (1) Application in the preparation of products that enhance the body's immune response;

[0026] (2) Application in the preparation of products for treating tumors or inhibiting tumor metastasis and recurrence.

[0027] Furthermore, the aforementioned products include drugs, drug carriers, etc.

[0028] Preferably, the tumor includes colorectal cancer.

[0029] The present invention discloses the following technical effects:

[0030] The nanoparticles prepared in this invention for combined sonodynamic and personalized immunotherapy of tumors have an outer layer of hyaluronic acid (HA-Ce6) conjugated with the sonosensitive agent Ce6, and a core formed by the self-assembly of the TLR3 agonist Poly(I:C), L-arginine, and polylysine. The nanoparticles bind to the CD44 receptor overexpressed by tumor cells through the hyaluronic acid shell to target the tumor. Ce6 generates reactive oxygen species under ultrasound to kill tumor cells and produce specific tumor-associated antigens. Poly(I:C) promotes the polarization of tumor-associated macrophages in the tumor microenvironment to the M1 type. M1 macrophages utilize L-arginine to produce and release NO. NO can inhibit tumor cell growth while further recruiting and activating immune effector cells, thereby achieving combined sonodynamic and NO gas immunotherapy for tumors.

[0031] The nanoparticles prepared in this invention for tumor sonodynamic combined with personalized immunotherapy can induce a systemic immune response, inhibit the primary tumor, generate an immune memory effect, and prevent tumor metastasis and recurrence. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 The particle size distribution (A), zeta potential distribution (B), transmission electron microscope image (C), and ultraviolet scan spectrum (D) of NP, Ce6-NP and free drug in the sonodynamic combined with personalized immunotherapy prepared in Example 1 of the present invention are shown.

[0034] Figure 2 The consumption of DPBF probes caused by different concentrations (A) and different ultrasonic times (B) of Ce6-NP and free Ce6 prepared in Example 1 of the present invention under ultrasonic treatment.

[0035] Figure 3 The cumulative release curves of Ce6(A) and Poly(I:C)(B) prepared by Ce6-NP in Example 1 of the present invention in PBS buffers with and without HAase at pH 7.4 and 6.5 are shown.

[0036] Figure 4 Representative flow cytometry histograms and bar graphs of Ce6 uptake by CT26 cells after co-incubation with Ce6-NP prepared in Example 1 of this invention and free drug for 1, 2 and 6 h.

[0037] Figure 5 The bar chart (A) shows the cell viability of NP, Ce6-NP and free drugs prepared in Example 1 of this invention after co-incubation with CT26 cells at different concentrations with and without ultrasound, the bar chart (B) shows the ATP content in the supernatant, the bar chart (C) shows the HMGB1 expression in CT26 cells, and the representative flow cytometry histogram (D).

[0038] Figure 6 To measure the confocal microscopy results and representative flow cytometry plots of NP, Ce6-NP and free drug prepared in Example 1 of this invention under ultrasound to produce reactive oxygen species (ROS) in CT26 cells, using DCFH-DA fluorescent probes;

[0039] Figure 7 The bar chart and representative flow cytometry histogram of L-Arg uptake by RAW264.7 cells after co-incubation with NP, Ce6-NP prepared in Example 1 of this invention and free drugs for 1, 2 and 6 h.

[0040] Figure 8 Representative flow cytometry plots and corresponding bar charts of NP, Ce6-NP, and BMDMs phenotypes obtained by incubation of free drugs with BMDMs in Example 1 of this invention were obtained by flow cytometry.

[0041] Figure 9 Confocal microscopy results and representative flow cytometry plots were obtained to determine the NP, Ce6-NP, and NO production in RAW264.7 cells induced by free drugs prepared in Example 1 of this invention using DAF-FM DA fluorescent probes.

[0042] Figure 10 Representative flow cytometry plots and corresponding bar charts are shown for the NP, Ce6-NP prepared in Example 1 of this invention, and the flow cytometry analysis of the expression levels of CD40, CD80, CD86 and MHC I molecules in BMDCs by incubation of free drugs with BMDCs.

[0043] Figure 11 To observe the Ce6-NP prepared in Example 1 after tail vein injection and the in vivo imaging results after free drug, as well as the in vitro fluorescence signals of each tissue 24 h after drug administration (A), and the fluorescence intensity of Ce6 in mouse tumor tissue at different time points after drug administration (B), a small animal in vivo imaging instrument was used.

[0044] Figure 12 The tumor growth curves (A), survival curves (B), and body weight curves (C) of NP, Ce6-NP, and free drugs prepared in Example 1 after treatment with and without ultrasound in mice bearing unilateral tumors.

[0045] Figure 13 The growth curves, survival curves (C) and body weight curves (D) of the proximal tumor (A) and distal tumor (B) of NP, Ce6-NP and free drugs prepared in Example 1 after treatment with and without ultrasound in mice bearing bilateral tumors.

[0046] Figure 14 H&E staining images of major organs in mice after treatment;

[0047] Figure 15 The effector memory CD4 in spleen cells of tumor-bearing mice after 85 days of ultrasound treatment with Ce6-NP prepared in Example 1. + TEM(A) and CD8 + TEM(B) proportional representativeness flow cytometry and bar chart;

[0048] Figure 16 Photographs (A), bar charts (B), and H&E staining results (C) of lung tumor nodules 14 days after Ce6-NP-treated CT26 tumor-bearing mice were injected via the tail vein. Detailed Implementation

[0049] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0051] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0052] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0053] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0054] All raw materials used in the following examples can be purchased through commercial channels.

[0055] Unless otherwise specified, "room temperature" refers to 20-30℃.

[0056] This invention provides a method for preparing and applying nanoparticles for combined sonodynamic and personalized immunotherapy of tumors. The nanoparticles have an outer layer of hyaluronic acid conjugated with the sonosensitive agent Ce6, and a core formed by the self-assembly of the TLR3 agonist Poly(I:C), L-arginine (L-Arg), and polylysine (PLL). Ce6 conjugation to the hyaluronic acid molecule enhances tumor targeting ability. Under ultrasound, Ce6 can generate reactive oxygen species to kill tumors and produce specific tumor-associated antigens. Poly(I:C) can promote the transformation of tumor-associated macrophages from the immunosuppressive M2 type to the anti-tumor M1 type. M1 macrophages overexpress iNOS and can utilize L-arginine to produce the anti-tumor small molecule NO, thereby achieving combined sonodynamic and NO gas-based tumor immunotherapy. Specific embodiments further illustrate the above technical solution below.

[0057] Example 1: A method for preparing nanoparticles for sonodynamic combined personalized immunotherapy

[0058] (1) Prepare polylysine (MW30000-70000) aqueous solution (2 mg / mL), L-arginine aqueous solution (5 mg / mL), and Poly(I:C) aqueous solution (2 mg / mL). Take 100 μL of L-arginine aqueous solution and mix it with 120 μL of polylysine aqueous solution. Then add 60 μL of Poly(I:C) aqueous solution. Shake at 3000 rpm for 45 min at room temperature. Centrifuge the solution (10000 rpm, 10 min) and wash it with deionized water. The resulting precipitate is nanoparticle core (NP).

[0059] (2) Dissolve 100 mg of hyaluronic acid (10 kDa) and 220 mg of adipic acid dihydrazide (ADH) in 10 mL of ultrapure water and stir for 30 min. Then add 25 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl). Maintain the pH at 4.8 with HCl during the reaction. After 24 h of reaction, adjust the pH to 7.0 by adding NaOH to stop the reaction. Purify HA-ADH by dialyzing in ultrapure water for 24 h using a dialysis bag (MW = 3500D) and freeze-dry.

[0060] 30 mg of N-hydroxysuccinimide (NHS) and 40 mg of EDC·HCl were added to a Ce6 dimethylformamide (DMF) solution (10 mL, 3 mg / mL), and the mixture was stirred at room temperature for 4 h to obtain a Ce6-NHS solution. Then, 10 mL of HA-ADH aqueous solution (10 mg / mL) was added to the Ce6-NHS solution, and the mixture was stirred at room temperature for another 24 h. Finally, the entire solution was dialyzed sequentially in DMF and ultrapure water for 24 h each, and then lyophilized to obtain HA-Ce6.

[0061] (3) Mix 300 μL of HA-Ce6 aqueous solution (containing 0.5 mg / mL of Ce6) with 200 μL of NP aqueous solution (containing 1 mg / mL of Poly(I:C)), stir at room temperature for 10-12 h at 500 rpm, centrifuge (10000 rpm, 10 min) and wash with deionized water, resuspend in ultrapure water, and sonicate for 1 min (using a 3 mm probe, power of 20%, and sonicate for 5 s every 5 s) to completely disperse it in the solution to obtain Ce6-NP.

[0062] Experimental Example 1

[0063] 1. The particle size, potential, and morphology of the Ce6-NP nanoparticles prepared in Example 1 were measured.

[0064] The particle size and potential were analyzed using a particle size analyzer; the morphology of the nanoparticles was characterized using a transmission electron microscope; and the successful encapsulation of Poly(I:C) and Ce6 was verified by scanning NP and HA-Ce6 aqueous solutions in the wavelength range of 200-800 nm using a UV-Vis spectrophotometer.

[0065] like Figure 1 As shown in Figure A, the average particle size of NP is 161.50 ± 3.12 nm, and the average particle size of Ce6-NP is 187.90 ± 4.38 nm. The particle size exhibits a normal distribution. The morphology of the nanoparticles was observed using transmission electron microscopy (see Figure A). Figure 1 The results (C) were consistent with those measured by the particle size analyzer, indicating that Ce6-NPs are uniformly spherical, appropriately sized, and possess a clear core-shell structure. Further particle size analysis revealed that the zeta potential of the NPs was 27.67 ± 1.22 mV. After being coated with a negatively charged HA-Ce6 shell, the zeta potential of Ce6-NPs changed to -2.802 ± 0.58 mV (see [reference needed]). Figure 1 (B)

[0066] 2. Determination of encapsulation efficiency and drug loading of Ce6, Poly(I:C), and L-Arg in the Ce6-NP nanoparticles prepared in Example 1.

[0067] The mass of Ce6-NP was obtained by freeze-drying, and the absorbance of the supernatant obtained by centrifuging Ce6-NP was measured at the characteristic peaks of Ce6 and Poly(I:C) using a UV-Vis spectrophotometer (see [reference]). Figure 1 (D).

[0068] The L-Arg content was determined by the α-naphthol method: 1 mL each of NaOH solution (40 g / L), naphthol-n-propanol solution (80 g / L), and diacetyl-n-propanol solution (0.5 mg / L) were added sequentially to a test tube, followed by 1 μL of the sample. After color development in a 30°C water bath for 5 min, the absorbance was measured at 545 nm using a UV-Vis spectrophotometer (see [link to sample]). Figure 1 (D).

[0069] The formula for calculating the encapsulation ratio is:

[0070] Drug encapsulation efficiency (%) = (Drug dosage - Mass of drug in supernatant) / Drug dosage × 100%

[0071] Drug loading (%) = (Drug dosage - Mass of drug in supernatant) / Total mass of nanoparticles × 100%

[0072] The results showed that the encapsulation efficiency of Ce6 in Ce6-NP was 49.0±3.96%, and the drug loading was 3.3%±0.44%; the encapsulation efficiency of Poly(I:C) was 56.0±0.82%, and the drug loading was 7.5%±0.53%; and the encapsulation efficiency of L-Arg was 51.7±2.71%, and the drug loading was 28.7%±1.5%. These results indicate that the nanoparticles possess high drug loading capacity.

[0073] Experimental Example 2

[0074] The in vitro sonodynamic effects of the Ce6-NP nanoparticles prepared in Example 1 of this invention were investigated: the generation of reactive oxygen species (ROS) in free Ce6 and Ce6-NP under ultrasonic and non-ultrasonic conditions was analyzed using a DPBF probe. Different equivalent concentrations of free drug and Ce6-NP were prepared, and after adding a DPBF probe (50 μM), the mixture was sonicated in the dark (1.0 MHz, 1.0 W / cm²). -2 The sample was treated for 5 minutes at a 50% duty cycle. Finally, the absorbance intensity of each group at 398 nm was measured using a UV-Vis spectrophotometer. DPBF consumption was calculated using the following formula: DPBF consumption (%) = (Absorbance before DPBF irradiation - Absorbance after DPBF irradiation) / Absorbance before DPBF irradiation × 100%

[0075] Results under different Ce6 concentrations and different sonication times are as follows Figure 2 As shown, Ce6-NP exhibits better reactive oxygen species (ROS) generation capacity compared to free Ce6, resulting in better sonodynamic performance. Because Ce6 is a hydrophobic molecule, it is unevenly dispersed in solution. However, after being coupled with HA and coated onto the surface of nanoparticles, it can be more uniformly dispersed in solution and more stably subjected to ultrasound. With increasing ultrasound time and Ce6 concentration, more ROS are generated, leading to greater DPBF consumption. At a Ce6 concentration of 8 μg / mL and an ultrasound time of 5 minutes, the DPBF consumption rate in the Ce6-NP group reached over 35%, significantly higher than that in the free Ce6 group. Experimental investigation revealed that further increasing the Ce6 concentration to 10 μg / mL or extending the ultrasound time to 10 minutes did not significantly increase DPBF consumption. Therefore, this invention determined the subsequent Ce6 dosage to be 8 μg / mL and the ultrasound treatment time to be 5 minutes.

[0076] Experimental Example 3

[0077] The drug release from Ce6-NP nanoparticles was determined using the dialysis bag method. Specifically, 200 μL of Ce6-NP solution was placed in a dialysis bag (MW = 3500D) and sealed. The bag was then placed in 10 mL of PBS buffer solution (pH = 6.5 or pH = 7.4) with or without hyaluronidase (HAase), and the solution was shaken at 37°C and 150 rpm. At regular intervals, all the released solution was collected and the same volume of fresh buffer solution was added. The absorbance values ​​of Ce6 and Poly(I:C) in the test solution were measured using a UV-Vis spectrophotometer, and the drug release amount was calculated using a standard curve.

[0078] like Figure 3 As shown, at 12 h, the cumulative release rates of Ce6 and Poly(I:C) in PBS buffer solution containing HAase at pH 6.5 reached 20% and 40%, respectively, which were significantly higher than the drug release in PBS buffer solution at pH 7.4, demonstrating a clear response to the tumor microenvironment (acidic and containing HAase). The release of Ce6 was relatively low, which may be because Ce6 is chemically linked to high molecular weight hyaluronic acid. Even after being degraded by HAase, some HA-Ce6 fragments still cannot pass through the dialysis bag, while Poly(I:C) is a small molecule and can pass through the dialysis bag more easily.

[0079] Test Example 4

[0080] Assay of tumor cell uptake of Ce6-NP nanoparticles prepared in Example 1:

[0081] CT26 mouse colon cancer cells were digested, centrifuged, and then subjected to 5×10⁻⁶ mol / L. 5 Cells were seeded at a density of 1 / mL in 12-well plates and cultured overnight. The culture medium was then discarded, and a new medium containing Free (a mixture of free Ce6, Poly(I:C), and L-Arg) and Ce6-NP (equivalent Ce6 concentration of 8 μg / mL) was added. The cells were co-incubated with the formulation for 1, 2, and 6 hours. The culture medium was then discarded, and the cells were washed twice with PBS. The cells were then collected, sieved, and flow cytometry was used to detect the drug uptake efficiency of CT26 cells.

[0082] Representative flow cytometry histograms of Ce6 uptake by CT26 cells at different incubation times are shown below. Figure 4 As shown, the results indicate that, compared to the Free group, the Ce6-NP group resulted in a higher uptake rate of Ce6 by CT26 cells, reaching 87.7% within 6 hours after administration, demonstrating the tumor targeting effect of the HA shell.

[0083] Experimental Example 5

[0084] The effects of Ce6-NP nanoparticles prepared in Example 1 on tumor cell killing and induction of immunogenic cell death (ICD) under ultrasound were investigated.

[0085] Free (a mixture of free Ce6, Poly(I:C), and L-Arg), NP, and Ce6-NP were co-incubated with CT26 cells at different Ce6 equivalent concentrations for 24 hours. The US group received 5 minutes of sonication (1.0 MHz, 1.0 W cm⁻¹) after 6 hours of co-incubation. -2 (50% duty cycle). The effects of various treatments on the viability of CT26 cells were analyzed using the CCK8 assay. Simultaneously, cells were stained with HMGB1-PE antibody, and the expression of HMGB1 in cells was analyzed by flow cytometry. The level of ATP secreted by cells was measured using an ATP kit to investigate the immunogenic cell death (ICD) induced by nanoparticles under ultrasound.

[0086] like Figure 5 As shown in Figure A, without ultrasound treatment, the survival rate of CT26 cells remained high with increasing drug concentration in each group, indicating that the drug was non-toxic to cells without ultrasound. The cell survival rate in the NP group was slightly lower than that in the Free group, possibly due to the slight toxicity caused by the strong positive charge of the nanoparticles. Ce6-NP also showed slight toxicity with increasing Ce6 dosage. This is partly due to the dose-dependent cytotoxicity of Ce6 itself, and partly because the targeting function of hyaluronic acid led to cells taking up more Ce6 than in the Free group. Additionally, HAase in the tumor microenvironment degrades the HA shell, exposing the strongly positively charged NP core, which explains why the cytotoxic trend of Ce6-NP is consistent with that of NP overall. After ultrasound treatment, the survival rate of CT26 cells in the Ce6-NP group dropped below 40% at a Ce6 concentration of 8 μg / mL, indicating that Ce6-NP-based sonodynamic therapy can significantly kill tumor cells. Extracellular ATP, as a recognized "find me" signal for dendritic cells (DCs) and macrophages, can recruit immune cells to tumor sites, thereby regulating tumor antigen cross-presentation and macrophage polarization. HMGB1 can bind to pattern recognition receptors expressed by various immune cells, including TLR4, and is an important component of ICD-induced immunogenicity. Representative flow cytometry and quantitative analysis results are as follows: Figure 5 As shown in the figure, under ultrasound, the positive rate of HMGB1 in the Ce6-NP group can reach 55.8%, and the ATP secretion level can reach 26.7 nM, which is significantly higher than that in the Free group. This is consistent with the cytotoxic results of CCK8, proving that the sonodynamic killing effect of Ce6-NP can induce ICD in tumor cells, thereby further inducing the anti-tumor effect of immune cells.

[0087] Experimental Example 6

[0088] Measurement of reactive oxygen species (ROS) generated in tumor cells by Ce6-NP nanoparticles prepared in Example 1 under ultrasound:

[0089] DCFH-DA was used as a ROS fluorescent probe. CT26 cells were cultured at 5 × 10⁶ cells per well. 5 CT26 cells were seeded at a density of [number] cells per well in 12-well plates. After 24 h, CT26 cells were co-incubated with PBS, Free (a mixture of free Ce6, Poly(I:C), and L-Arg), NP, and Ce6-NP. After 6 h of incubation, the medium was replaced with 1 mM DCFH-DA and incubated for 30 min. The cells were washed three times with PBS and then sonicated for 5 min (1.0 MHz, 1.0 W cm⁻¹). -2 The intracellular ROS fluorescence intensity was detected using flow cytometry and laser confocal microscopy (50% duty cycle).

[0090] Representative flow cytometry plots and microscope images for each group are as follows: Figure 6 As shown, Ce6-NP can promote the generation of more reactive oxygen species in tumor cells under ultrasound conditions compared to the Free group, resulting in better sonodynamic effects. At the same time, the fluorescence intensity decreased after the addition of the antioxidant vitamin C.

[0091] Experimental Example 7

[0092] Example 1: Study on the promotion of macrophage uptake by Ce6-NP nanoparticles prepared in Example 1:

[0093] RAW264.7 macrophages were digested, centrifuged, and then subjected to a 5×10⁻⁶ mol / L hydrochloride solution. 5 Cells were seeded at a density of 12-well plates. After overnight culture, the culture medium was discarded, and new medium containing Free (a mixture of free Ce6, Poly(I:C), and L-Arg), NP, and Ce6-NP was added (L-Arg was FITC-labeled, with an equivalent L-Arg concentration of 70 μg / mL). Cells were co-incubated for 1, 2, and 6 hours, respectively. The medium was then discarded, and the cells were washed twice with PBS. Cells were then collected, sieved, and flow cytometry was used to detect the drug uptake efficiency of RAW264.7 cells. Representative flow cytometry histograms and bar graphs are shown below. Figure 7 As shown, compared to the free drug group, RAW264.7 cells took up more NP and Ce6-NP. The Ce6-NP group had a slightly lower uptake ratio than the NP group, which may be because the protective shell of HA-Ce6 shielded the positive charge on the surface of the nanoparticles.

[0094] Experimental Example 8

[0095] Example 1: Study on the promotion of macrophage M1 polarization by Ce6-NP nanoparticles prepared in Example 1.

[0096] BMDMs (5×10⁶) that were rearranged and adhered to the walls on day 6 of culture. 5 Add BMDMs-specific medium containing PBS, Free (a mixture of free Ce6, Poly(I:C), and L-Arg), NP, and Ce6-NP to macrophages (cells / mL), and culture for 24 h. Collect cells and wash once with PBS. Incubate with CD11b-PE and F4 / 80-eFlour450 antibodies at 4°C for 30 min, wash once with PBS, add 0.1 mL of IC50 fixation buffer, incubate at room temperature in the dark for 60 min, wash once with 1 mL of freshly prepared 1× permeabilization buffer, centrifuge and discard the supernatant, add CD206-APC antibody diluted with 1× permeabilization buffer, incubate at room temperature in the dark for 60 min, wash once with 1× permeabilization buffer, centrifuge and discard the supernatant, resuspend in 0.3 mL of PBS, and detect macrophages (CD11b) using flow cytometry. + The expression of surface markers F4 / 80 and CD206 in the sample, with F4 / 80 as the primary indicator. + CD206 + M2 macrophage population, F4 / 80 + CD206- represents the M1 macrophage population. (Example) Figure 8 As shown, both NP and Ce6-NP can induce an increase in the proportion of M1 macrophages and a decrease in the proportion of M2 macrophages, thereby inducing the ability of macrophages to kill tumor cells by reversing the macrophage phenotype.

[0097] Experimental Example 9

[0098] Assay 1: Determination of the effect of Ce6-NP nanoparticles prepared in promoting NO production in macrophages:

[0099] DAF-FM DA was used as the NO fluorescent probe. RAW264.7 cells were cultured at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 12-well plates. After 24 hours, PBS, a mixture of free Ce6, Poly(I:C), and L-Arg, NP, and Ce6-NP were added and co-incubated with the cells. After 6 hours of incubation, the medium was replaced with 20 μM containing DAF-FM DA and incubated for 30 minutes. After washing the cells three times with PBS, the intracellular NO fluorescence intensity was detected using flow cytometry and laser confocal microscopy. Microscopic images and representative flow cytometry plots for each group are shown below. Figure 9 As shown, compared with the Free group, the NP containing L-Arg and the Ce6-NP group can promote macrophages to produce more NO and exert anti-tumor effects.

[0100] Experimental Example 10

[0101] Assay 1: Determination of the ability of Ce6-NP nanoparticles prepared to promote the maturation and activation of bone marrow-derived dendritic cells (BMDCs):

[0102] Collect BMDCs cultured to day 6 and plate them in 12-well plates (5 × 10⁻⁶). 5 Cells were cultured at 12 h (cells / mL). After 12 h, BMDCs-specific culture medium containing PBS, Free (a mixture of free Ce6, Poly(I:C), and L-Arg), NP, and Ce6-NP was added, and the cells were cultured for another 24 h. Cells were then collected and washed once with PBS. Staining with CD40, CD80, CD86, and MHC I antibodies was performed at 4 °C for 30 min. After centrifugation and washing, the cells were resuspended in 300 μL of 4% paraformaldehyde, sieved, and the expression levels of CD40, CD80, CD86, and MHC I molecules on the surface of BMDCs were determined by flow cytometry.

[0103] Representative flow cytometry plots and corresponding bar charts are as follows: Figure 10 As shown, compared with the PBS group and the Free group, NP and Ce6-NP significantly enhanced the expression of CD40 and CD80 molecules, with the proportions of double-positive cells reaching 72.4% and 78.9%, respectively, and the proportions of CD86-positive cells reaching 52.7% and 55.2%, respectively. This indicates that NP and Ce6-NP loaded with Poly(I:C) and L-Arg have a good ability to induce the activation and maturation of BMDCs. In addition, the expression level of MHC I molecules was also significantly increased under the induction of NP and Ce6-NP, from 3.84% in the PBS group to about 20%, reflecting the role of NP and Ce6-NP in promoting antigen presentation of BMDCs.

[0104] Experimental Example 11

[0105] Study on the intracellular distribution and tumor targeting ability of Ce6-NP nanoparticles prepared in Example 1:

[0106] Female BALB / c mice aged 6-8 weeks were selected, and after hair removal treatment, CT26 cells (1×10⁻⁶) were inoculated into the right back near the hind limb. 6 A tumor-bearing mouse model was constructed (number of mice per mouse); when the tumor volume reached 100 mm², the model was established. 3 Mice were randomly divided into FreeCe6 group and Ce6-NP group, with 3 mice in each group. At 6, 12, 24, 48, 72 and 96 h after tail vein injection, the in vivo fluorescence distribution of Ce6 was observed using a small animal in vivo imaging system. At the same time, 24 h after administration, the mice were dissected to observe the fluorescence intensity of Ce6 in various organs and tumor tissues of the mice.

[0107] In vivo imaging results and in vitro fluorescence signals of various tissues, such as Figure 11 As shown, Ce6-NP showed significant accumulation at the tumor site 12 hours after tail vein administration. Quantitative analysis indicated that the drug accumulation reached its peak 24 hours after administration, possibly because the HA coating enabled the nanoparticles to bind to the CD44 receptor overexpressed by tumor cells, thereby effectively targeting the tumor. The drug was not completely metabolized until 96 hours after administration; therefore, this invention determined that the drug should be administered every 4 days, with ultrasound treatment performed at 12 and 24 hours after administration.

[0108] Experimental Example 12

[0109] The Ce6-NP nanoparticles obtained in Example 1 of this invention were used to test their in vivo antitumor effects. Specifically, 6-8 week old female BALB / C mice were selected for the efficacy experiment. After hair removal, CT26 cells (1×10⁻⁶) were inoculated into the right back near the hind limb. 6 A tumor-bearing mouse model was constructed (number of mice per mouse), with the right-sided tumor volume being 50 mm². 3 Mice were randomly divided into 7 groups after administration of the drug: PBS, Free (a mixture of free Ce6, Poly(I:C), and L-Arg), NP, Ce6-NP, Free+US, NP+US, and Ce6-NP+US groups, with 9 mice in each group. The equivalent Ce6 dose was 2.5 mg / kg, and the equivalent Poly(I:C) dose was 5.7 mg / kg. The ultrasound conditions for the ultrasound group were 1.0 MHz and 1.0 W cm⁻¹. -2 50% duty cycle, continuous ultrasound of the tumor site for 5 minutes. The day of administration is recorded as day 0. The formulations for each group are injected via tail vein on days 0, 4, 8, and 12. Ultrasound therapy is administered 12 hours and 24 hours after each injection. Mice are observed every two days, and the long and short diameters of the tumor are measured using calipers. When the tumor volume exceeds 2000 mm², the tumor is considered a tumor. 3 The formula for calculating tumor volume when mice are euthanized is as follows:

[0110]

[0111] The results are as follows Figure 12 As shown, from Figure 12 As shown in Figure A, neither the no-ultrasound treatment group nor the NP+US group could inhibit tumor growth; however, the tumor volume in the Free group mice increased rapidly even under ultrasound treatment, because the free drug could not target the tumor site and was rapidly metabolized in the mice. The Ce6-NP+US group showed a significant inhibitory effect on tumor growth. Figure 12 As can be seen from Figure B, all mice in the other groups died within 38 days, while all nine mice in the Ce6-NP+US group survived until day 48. Figure 12 As shown in Figure C, the mouse weight curves indicate that there were no significant changes in weight among the various formulation groups, suggesting that each formulation has good biocompatibility.

[0112] Experimental Example 13

[0113] The inhibitory effect of Ce6-NP nanoparticles obtained in Example 1 on distal tumors in mice was further investigated. A mouse model bearing bilateral tumors was constructed in the same manner as in Example 9: CT26 cells (1.5 × 10⁻⁶) were seeded on the right side of the back near the hind limb. 6 (each tumor), when the right-side tumor volume is 80mm 3 CT26 cells (1.5 × 10⁻⁶) were inoculated into the left back near the hind limb one day prior to administration. 6 (6 mice per group), and the right-sided tumors of mice in each group were treated with the same method as in Experiment 9. When the tumor volume was greater than 2000 mm², 3 Mice were sacrificed, and the growth of proximal and distal tumors was observed every two days.

[0114] The results are as follows Figure 13 As shown, treatment with Ce6-NP+US significantly inhibited both proximal and distal tumors in tumor-bearing mice, with the distal tumor showing almost no growth, reflecting that the combination of nanoparticles and ultrasound can significantly suppress tumor recurrence. Even when the tumor volume was large, four mice in the Ce6-NP+US group were completely cured, and the remaining two survived until day 32 without significant weight change, further demonstrating that the nanoparticles have good biocompatibility and can prolong the survival of tumor-bearing mice.

[0115] Test Example 14

[0116] Safety evaluation of Ce6-NP nanoparticles obtained in Example 1:

[0117] To further evaluate the safety of the nanoparticles to tissues and organs, mice in each group were dissected on day 14 after administration in Experiment 9, and their hearts, livers, spleens, lungs, and kidneys were collected for H&E staining.

[0118] like Figure 14 As shown, no lesions were observed in the major organs of mice in each group, indicating that the nanoparticles used for tumor sonodynamic combined with personalized immunotherapy have good biocompatibility and no toxic side effects on normal tissues and organs.

[0119] Experimental Example 15

[0120] Assay of antitumor immune memory of Ce6-NP nanoparticles obtained in Example 1:

[0121] In the observation experiment, mice cured in the Ce6-NP+US group were treated. On day 85 after drug administration, spleens were collected, and single-cell suspensions were obtained by grinding erythroblasts. Then, CD4-FITC, CD8a-PE, CD62L-PerCP-Cy5.5, CD44-APC, and CD3e-eflour450 antibodies were added and incubated at 4°C for 30 min. The cells were washed once with PBS and finally resuspended in 4% paraformaldehyde. Flow cytometry was used to detect effector memory T cells (CD44...). + CD62L - )Proportion.

[0122] The results are as follows Figure 15 As shown in Figures A and B, in the control group, CD4 + TEM and CD8 + The TEM rates were 42.6% and 38.4%, respectively, while the treatment group had CD4... + TEM and CD8 + The proportion of TEM was significantly higher than that of the control group, at 56.5% and 52.9%, respectively, indicating that the nanoparticles of Example 1 can induce an effective immune memory effect under ultrasound.

[0123] Experimental Example 16

[0124] Study on the anti-tumor lung metastasis of Ce6-NP nanoparticles obtained in Example 1:

[0125] Hair was removed from the skin near the right hind limb on the back of 6-8 week old female BALB / c mice, and CT26 cells (1×10⁻⁶) were subcutaneously injected into this area. 6 (cells / each), when the tumor volume grows to 50mm 3 Administration was performed, with the day of administration designated as day 0. Ce6-NP was injected via tail vein on days 0, 4, 8, and 12, followed by ultrasound therapy 12 and 24 hours after each injection. On day 30 of treatment, three fully cured mice and the same-week-old female BALB / c mice were used as a control group, receiving a tail vein injection of 3 × 10⁻⁶ NP. 5 CT26 cells were collected, and lung tissue from mice was collected 14 days later. Lung nodules were observed, photographed, and H&E staining was performed.

[0126] H&E results are as follows Figure 16 As shown, the control group exhibited numerous obvious tumor metastases, while the cured mice in the Ce6-NP+US group showed almost no visible tumor metastases. This demonstrates that Ce6-NP+US treatment in mice induces an immune memory effect, inhibiting tumor metastasis.

[0127] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A nanoparticle for tumor sonodynamic combined personalized immunotherapy, characterized in that, The nanoparticle is a double-shell core structure nanoparticle composed of an inner core and an outer shell, the nanoparticle inner core comprises polylysine, L-arginine and a TLR3 agonist, and the nanoparticle outer shell comprises hyaluronic acid and a sonosensitizer. The TLR3 agonist is Ploy(I:C), and the sonosensitizer is Ce6.

2. The nanoparticle of claim 1, wherein, The nanoparticle inner core is self-assembled by polylysine, L-arginine and a TLR3 agonist, and the nanoparticle outer shell is constructed by conjugating a sonosensitizer to hyaluronic acid.

3. A method of producing nanoparticles as claimed in claim 1 or 2, characterized in that, The method comprises the following steps: Self-assembling polylysine, L-arginine and a TLR3 agonist to construct a nanoparticle inner core; Conjugating a sonosensitizer to hyaluronic acid to prepare a nanoparticle outer shell; After the nanoparticle inner core and the nanoparticle outer shell are dissolved in water respectively, mixing and stirring, centrifugation, washing, resuspension, and then ultrasonic dispersion, a multifunctional sonodynamic nanoparticle with a double-shell core structure composed of an inner core and an outer shell is prepared.

4. The production method according to claim 3, wherein The preparation method of the nanoparticle inner core comprises the following steps: The TLR3 agonist aqueous solution is added dropwise into the L-arginine aqueous solution and the polylysine aqueous solution, and self-assembled at room temperature with shaking, centrifugation and washing to obtain the nanoparticle inner core; wherein the volume ratio of the TLR3 agonist aqueous solution, the L-arginine aqueous solution and the polylysine aqueous solution is 60:(80-110):(110-130).

5. The production method according to claim 4, wherein The concentration of the TLR3 agonist aqueous solution is 2mg / mL, the concentration of the L-arginine aqueous solution is 5mg / mL, and the concentration of the polylysine aqueous solution is 2mg / mL. The shaking condition is: rotation speed 3000rpm, time 30-60min; the centrifugation condition is: rotation speed 10000rpm, time 10-20min.

6. The production method according to claim 3, wherein The preparation method of the nanoparticle outer shell comprises the following steps: Mixing hyaluronic acid, adipic acid dihydrazide and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride for reaction, dialysis and freeze-drying to obtain an intermediate product I; Reacting N-hydroxysuccinimide, 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and a sonosensitizer, and then reacting with the intermediate product I, dialysis and freeze-drying to obtain the nanoparticle outer shell.

7. The production method according to claim 6, wherein The mass ratio of the hyaluronic acid, the adipic acid dihydrazide and the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is 100:(200-240):(20-30). The mass ratio of the N-hydroxysuccinimide, the 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride and the sonosensitizer is 30:(35-45):30, and the mass ratio of the sonosensitizer and the intermediate product I is 30:

100.

8. The production method according to claim 3, wherein The volume ratio of the nanoparticle inner core and the nanoparticle outer shell is 2:

3. The mixing and stirring condition is: rotation speed 500rpm, stirring time 10-12h; The ultrasonic condition is: using a 3mm probe, power 20%, processing time 1min, and ultrasonic treatment for 5s every 5s.

9. The nanoparticle of claim 1 or 2 is used in any one of the following: (1) use in the preparation of a product for enhancing the immune response of the body; (2) use in the preparation of a product for treating or inhibiting metastasis and recurrence of a tumor.

10. Use according to claim 9, wherein The tumor includes colorectal cancer.

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