Multifunctional carrier-free self-assembled nano-adjuvant, preparation method and application thereof

Abstract

The application discloses a multifunctional carrier-free self-assembled nano adjuvant as well as a preparation method and application thereof. The preparation method comprises the following steps: mixing ferrous chloride tetrahydrate aqueous solution, CpG aqueous solution and deionized water, and then performing reaction, centrifugation and washing to obtain precipitate 1; resuspending the precipitate 1, adding DOTAP aqueous solution, and then performing oscillation reaction, centrifugation and washing to obtain precipitate 2; resuspending the precipitate 2, adding dopamine hydrochloride Tris-HCl solution, and then performing oscillation reaction, centrifugation and washing to collect the precipitate, thereby obtaining the nano adjuvant. The nano adjuvant combined with photothermal therapy can increase the infiltration of dendritic cells, helper T lymphocytes, cytotoxic T lymphocytes and NK cells at a tumor site, reduce the proportion of regulatory T cells and M2 / M1 type tumor-related macrophages, thereby improving an immunosuppressive tumor microenvironment. The nano adjuvant can also induce a systemic immune response, inhibit a primary tumor, produce an immune memory effect and prevent tumor metastasis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a multifunctional carrier-free self-assembled nanoadjuvant, its preparation method, and its application. Background Technology

[0002] Clinically, malignant tumors carry a high risk of lung metastasis and recurrence due to poor prognosis, limited treatment options, and significant treatment resistance. Immunotherapy is a promising approach for treating various malignant tumors; however, the low immunogenicity of tumors, the immunosuppressive tumor microenvironment, and the limited antigen presentation by antigen-presenting cells (APCs) restrict the efficacy of immunotherapy. Combining ablation-based tumor therapy strategies with adjuvants holds promise for enhancing tumor immunogenicity and reversing the immunosuppressive tumor microenvironment. Ablation-based tumor therapy strategies (such as photothermal therapy) can trigger the release of endogenous tumor-associated antigens (TAAs) while eliminating the primary tumor. Utilizing endogenous TAAs to generate vaccines in situ enables personalized immunotherapy. Immunoadjuvants are auxiliary stimulants that significantly enhance antigen immunogenicity by improving the antigen processing and presentation efficiency of APCs. Among various immune adjuvants, phosphate cytosine-guanine oligodeoxynucleotide motifs (CpG) are FDA-approved adjuvants capable of simultaneously inducing humoral and cellular immune responses, demonstrating potent antitumor activity in preclinical and clinical studies. The mechanism of action of CpG involves recognition by Toll-like receptor 9 (TLR9) on the endosomes of adenocarcinoma cells (APCs) in vivo, specifically activating the TLR9-mediated signaling pathway, directly activating APCs, and indirectly activating immune effector cells such as T cells and natural killer cells. Furthermore, studies have shown that CpG can effectively polarize tumor-promoting M2 tumor-associated macrophages (TAMs) into tumor-suppressive M1 TAMs in the tumor microenvironment and reduce the infiltration of regulatory T cells within the tumor, thereby reversing the immunosuppressive tumor microenvironment. However, the clinical application of CpG still faces significant challenges, such as poor stability, suboptimal biodistribution, and low cellular uptake. Nanotechnology can load CpG into nanocarriers, effectively improving these shortcomings.

[0003] Traditional nanomedicine delivery systems have limited their clinical applications due to several drawbacks, such as complex preparation processes, high production costs, low drug loading efficiency, and potential nanomaterial-related toxicity.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] The purpose of this invention is to provide a multifunctional carrier-free self-assembled nanoadjuvant, its preparation method and application. The outermost layer of the nanoadjuvant is a photothermal agent, polydopamine, which can thermally ablate local tumors and generate specific tumor-associated antigens under near-infrared laser irradiation. Then, the exposed nanoadjuvant core forms a vaccine in situ, thereby enabling the sequential application of photothermal-immunotherapy for tumor treatment.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0007] The first aspect of this invention provides a method for preparing a multifunctional carrier-free self-assembled nanoadjuvant, the method comprising the following steps:

[0008] (a) After mixing the aqueous solution of ferrous chloride tetrahydrate, the aqueous solution of CpG and deionized water, the reaction was carried out. After the reaction was completed, the reaction solution was centrifuged and the precipitate was washed to obtain precipitate 1.

[0009] (b) Resuspend precipitate 1, add DOTAP aqueous solution and shake to react. After the reaction is complete, centrifuge and wash the reaction solution to obtain precipitate 2.

[0010] (c) Resuspend precipitate 2, add dopamine hydrochloride Tris-HCl solution and shake to react. After the reaction is complete, centrifuge the reaction solution, wash and collect the precipitate to obtain the multifunctional carrier-free self-assembled nanoadjuvant.

[0011] Preferably, in step (a), the concentration of the ferrous chloride tetrahydrate aqueous solution is 3-5 mg / mL; the concentration of the CpG aqueous solution is 8-12 μL / OD.

[0012] The volume ratio of ferrous chloride tetrahydrate aqueous solution, CpG aqueous solution and deionized water is 3:(6-10):(45-55).

[0013] Preferably, in step (a), the reaction temperature is 90–100°C and the reaction time is 2.5–4 h.

[0014] Preferably, in step (b), the volume ratio of the solvent used for resuspension to the deionized water used in step (a) is 1:(0.8-1.2), and the solvent is deionized water.

[0015] Preferably, in step (b), the volume ratio of the DOTAP aqueous solution to the deionized water in step (a) is 1:(0.8-1.2); and the concentration of the DOTAP aqueous solution is 3-5 mg / mL.

[0016] Preferably, in step (b), the shaking reaction time is 1.5 to 2.5 hours, and the reaction temperature is room temperature.

[0017] Preferably, in step (c), the volume ratio of the solvent used for resuspension to the deionized water in step (a) is 1:(0.8-1.2), and the solvent is a 10mM Tris-HCl buffer solution with pH 8.0-8.5.

[0018] Preferably, in step (c), the volume ratio of dopamine hydrochloride Tris-HCl solution to deionized water in step (a) is 1:(0.8-1.2); the concentration of dopamine hydrochloride Tris-HCl solution is 3-5 mg / mL.

[0019] The shaking reaction time is 6-8 hours; the reaction temperature is room temperature.

[0020] A second aspect of the present invention provides a multifunctional carrier-free self-assembled nanoadjuvant prepared by the above-described preparation method.

[0021] A third aspect of this invention provides the application of the multifunctional carrier-free self-assembled nanoadjuvant prepared by the above-described method in the preparation of tumor treatment products.

[0022] The fourth aspect of this invention provides the application of the multifunctional carrier-free self-assembled nanoadjuvant prepared by the above-mentioned preparation method in the preparation of products that activate DCs and T cells to generate systemic immune responses, improve the immunosuppressive tumor microenvironment, or inhibit tumor metastasis.

[0023] Compared with the prior art, the beneficial effects of the present invention include at least the following:

[0024] The preparation method of this invention forms a multifunctional carrier-free nanoadjuvant through self-assembly. It can first kill the in situ tumor with PTT and then further inhibit the tumor metastasis and recurrence through immunotherapy, thus realizing the sequential nature of photothermal combined immunotherapy.

[0025] The self-assembled nanoadjuvant of this invention can capture TAAs generated by photothermal therapy, enhance the uptake of antigens and adjuvants by DCs, promote the activation and maturation of BMDCs, achieve cross-presentation of antigens, and induce the activation and proliferation of T cells.

[0026] The self-assembled nanoadjuvant of this invention can increase the infiltration of helper T lymphocytes, cytotoxic T lymphocytes, and NK cells at the tumor site with a single treatment, while reducing the proportion of regulatory T cells and M2 / M1 tumor-associated macrophages, thereby improving the immunosuppressive tumor microenvironment. In addition, the self-assembled nanoadjuvant prepared by this invention can induce a systemic immune response, inhibit the primary tumor, generate an immune memory effect, and prevent tumor metastasis. Attached Figure Description

[0027] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0028] Figure 1 The particle size distribution, transmission electron microscopy image, zeta potential diagram, ultraviolet spectrum, and temperature rise curve after laser irradiation of the self-assembled nano-adjuvant FCDP-NPs prepared in Example 1 of this invention are shown.

[0029] Figure 2 The diagram shows a flow cytometry scatter plot of tumor cell apoptosis of the self-assembled nanoadjuvant FCDP-NPs prepared in Example 1 of this invention, a schematic diagram of antigen capture by FCD-NPs prepared in Example 1, a zeta potential diagram before and after antigen capture, and a bar chart of DC cytotoxicity and antigen capture amount of FCD-NPs.

[0030] Figure 3 The graphs show the flow cytotoxicity of BMDCs to the self-assembled nanoadjuvant FCDP-NPs prepared in Example 1 of this invention, the flow cytometry histograms of DC uptake and ROS generation of FCD-NPs prepared in Example 1, the laser confocal microscopy (CLSM) graph of DC uptake of FCD-NPs@TAAs obtained after antigen capture, and the bar graphs of expression levels of co-stimulatory molecules CD40, CD80, and CD86 on the surface of BMDCs.

[0031] Figure 4 The following is a description of the treatment regimen for tumor-bearing mice with the self-assembled nanoadjuvant FCDP-NPs prepared in Example 1 of this invention: infrared thermographic images of the highest temperature reached after intratumoral injection and local laser irradiation of the tumor; tumor growth curve, survival curve, and body weight change curve of the mice during the observation period; and tumor cell apoptosis on the first day after treatment.

[0032] Figure 5 The expression of co-stimulatory molecules CD40, CD80, CD86 and major histocompatibility complex MHC I on the surface of DCs in the tumor drainage lymph nodes of tumor-bearing mice on day 7 after treatment with the self-assembled nanoadjuvant FCDP-NPs prepared in Example 1 of this invention.

[0033] Figure 6 The self-assembled nano-adjuvant FCDP-NPs prepared in Example 1 of this invention infiltrated the tumors of tumor-bearing mice on day 7 after treatment. + T cells, CD4 +Bar charts of T cells, DCs, M1 / M2 macrophage ratio, and regulatory T cells, as well as CLSM charts of tumor-infiltrating NK cells;

[0034] Figure 7 The self-assembled nano-adjuvant FCDP-NPs prepared in Example 1 of this invention were used to treat tumor-bearing mice. On day 7 after treatment, CD8 levels in the spleen of these mice were... + T cells, CD4 + Flow cytometry diagram of T cells and macrophages;

[0035] Figure 8 Immune memory analysis and anti-tumor lung metastasis regimen of the self-assembled nano-adjuvant FCDP-NPs prepared in Example 1 of this invention were performed on tumor-bearing mice after treatment. CD8+ in the spleen was also analyzed. + T cells and CD4 + Flow cytometry scatter plots and bar charts of effector memory T cells in T cells, as well as photographs of lung tissue collected in anti-tumor lung metastasis experiments. Detailed Implementation

[0036] The embodiments of the technical solution of the present invention will be described in detail below with reference to the examples. The following embodiments are only used to illustrate the technical solution of the present invention more clearly, and are therefore only examples, and should not be used to limit the scope of protection of the present invention.

[0037] It should be noted that, unless otherwise stated, the technical or scientific terms used in this application should have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0038] Example 1

[0039] This embodiment describes a method for preparing a multifunctional carrier-free self-assembled nanoadjuvant, which includes the following steps:

[0040] (a) Take 30 μL of FeCl2·4H2O aqueous solution with a concentration of 4 mg / mL and 80 μL of CpG aqueous solution with a concentration of 10 μL / OD and add them to a 1.5 mL centrifuge tube containing 490 μL of deionized water. Vortex for 10 s and then place the centrifuge tube in a 95 °C oven for 3 h. Centrifuge the reaction solution at 10000 rpm for 10 min and wash it with deionized water. The precipitate obtained is denoted as FC-NPs.

[0041] (b) Resuspend FC-NPs in 0.5 mL of deionized water, then add 0.5 mL of DOTAP aqueous solution with a concentration of 4 mg / mL, shake and react for 2 h, centrifuge the reaction solution at 10000 rpm for 5 min and wash with deionized water to obtain the precipitate, which is denoted as FCD-NPs.

[0042] (c) Resuspend FCD-NPs in 0.5 mL of 10 mM Tris-HCl buffer (pH 8.0–8.5), then add 0.5 mL of 4 mg / mL dopamine hydrochloride Tris-HCl solution, and shake at room temperature for 6 h. Centrifuge the reaction solution at 10,000 rpm for 10 min and wash with deionized water to obtain the precipitate, which is the multifunctional carrier-free self-assembled nanoadjuvant, denoted as FCDP-NPs.

[0043] Comparative Example 1

[0044] This comparative example demonstrates the preparation of FCP-NPs:

[0045] (a) Take 30 μL of FeCl2·4H2O aqueous solution with a concentration of 4 mg / mL and 80 μL of CpG aqueous solution with a concentration of 10 μL / OD and add them to a 1.5 mL centrifuge tube containing 490 μL of deionized water. Vortex for 10 s and then place the centrifuge tube in a 95 °C oven for 3 h. Centrifuge the reaction solution at 10000 rpm for 10 min and wash it with deionized water. The precipitate obtained is denoted as FC-NPs.

[0046] (b) Resuspend FC-NPs in 0.5 mL of 10 mM Tris-HCl buffer (pH 8.0–8.5), then add 0.5 mL of 4 mg / mL dopamine hydrochloride Tris-HCl solution, and shake at room temperature for 6 h. Centrifuge the reaction solution at 10,000 rpm for 10 min and wash with deionized water to obtain the precipitate, which is FCP-NPs.

[0047] Experimental Example 1

[0048] The self-assembled nano-adjuvants prepared in Example 1 were subjected to particle size and distribution, and zeta potential determined using a particle size potentiometer; for the FCDP-NPs+NIR group, the FCDP-NPs solution was subjected to a 1.5 W / cm² test. 2 Irradiation with 808nm laser for 5 min followed by centrifugation and resuspending for zeta potential measurement; for FCDP-NPs+H + / NIR group, FCDP-NPs were resuspended in PBS at pH 6.5 and subjected to 1.5W / cm 2 After irradiation with an 808 nm laser for 5 min, the mixture was left to stand for 24 h, centrifuged, and resuspended for zeta potential determination. The morphology of the self-assembled nanoadjuvants was observed using transmission electron microscopy. The absorption spectra of each drug and nanoparticle aqueous solution in the wavelength range of 220–400 nm were scanned using a UV-Vis spectrophotometer to verify the encapsulation of CpG in the nanoadjuvants. The encapsulation efficiency of CpG was determined by measuring the absorbance of the supernatant obtained from centrifugation in step S1 at a wavelength of 262 nm. Infrared thermal imaging was used to monitor the absorption of each sample at 1.5 W / cm². 2Temperature change within 5 minutes of irradiation with an 808nm laser.

[0049] Figure 1 In Figure A, the particle size and distribution of FCDP-NPs prepared in Example 1 of this invention are shown, with a particle size of 215.4 nm and uniform distribution. The attached transmission electron microscope image shows that the nano-adjuvant is a regular sphere with a distinct core-shell structure. Figure 1 In Figure B, the zeta potentials of the various formulations prepared in Example 1 of this invention are shown. The zeta potentials of FC-NPs, FCD-NPs, and FCDP-NPs are -34.1 mV, +21.5 mV, and -31.0 mV, respectively. The adsorption of DOTAP makes the potential positive, and the coating of the PDA film makes the potential of the nanoparticles negative and gives them strong electrostatic stability. The zeta potential of FCDP-NPs after laser irradiation is +14.4 mV, and after further incubation under acidic conditions, the zeta potential is +18.6 mV, indicating that the PDA film is basically destroyed, exposing the positively charged FCD-NPs. Figure 1 C represents the UV spectra of each formulation. Free CpG has a characteristic absorption peak in the range of 230–300 nm and reaches its maximum absorbance at 262 nm. FC-NPs and FCD-NPs have consistent characteristic absorption peaks, and FCDP-NPs also have relatively obvious characteristic absorption peaks, but all of them show a significant red shift compared to free CpG. Free FeCl2·4H2O has no characteristic absorption peak in this range, proving the successful loading of CpG. The measured CpG encapsulation efficiency is 61.5 ± 7.9%. Figure 1 In the figure, D represents the formulations prepared in Example 1 and Comparative Example 1 of this invention under laser irradiation (808 nm, 1.5 W / cm²). 2 The recorded temperature-time curves show that PDA is an excellent photothermal agent. Its successful loading enabled FCP-NPs and FCDP-NPs to rise to 47.0 and 50.3 °C respectively within 5 minutes, while the temperatures of the other groups remained basically unchanged.

[0050] Experimental Example 2

[0051] Each formulation was co-incubated with 4T1 mouse breast cancer cells and then irradiated with laser (808 nm, 1.5 W / cm²). 2 After culturing for 5 minutes, cells were collected, stained with Annexin V-FITC / PI apoptosis detection kit, and the apoptosis rate of cells after different treatments was determined by flow cytometry. Figure 2 A in the middle is a flow cytometry scatter plot, showing the apoptosis rates (Annexin V-FITC) in the FCP-NPs+NIR and FCDP-NPs+NIR groups. + PI - and Annexin V-FITC+ PI + The percentages in the quadrants were 20.17% and 36.6%, respectively. This is because the load of the PDA effectively increased the temperature after laser irradiation, killing tumor cells.

[0052] 4T1 breast cancer cells were co-incubated with PDA nanoparticles and irradiated with an 808 nm laser to induce tumor cell apoptosis. After 24 hours, tumor-associated antigens (TAAs) were collected by centrifugation and co-incubated with FCD-NPs prepared in step S2 of Example 1 of this invention. Unadsorbed TAAs were removed by ultrafiltration to obtain FCD-NPs@TAAs. Figure 2 (B) The potential changes of FCD-NPs prepared with DOTAP (0.5 mg and 2.0 mg respectively) before and after co-incubation with TAAs were measured. Figure 2 As shown in Figure C, both carried a positive charge before incubation, with the latter having a larger value. However, after co-incubation with TAAs, both became negatively charged, with the latter exhibiting a higher degree of negative charge. This is because the amount of TAAs adsorbed by the latter (266.04±11.26 μg) was significantly higher than that of the former (149.32±26.54 μg). Figure 2 (D). Furthermore, the toxicity of both to dendritic cells (DC2.4) was also determined; both showed excellent biocompatibility and no significant toxic effects on DC2.4 cells. Figure 2 (D).

[0053] Experimental Example 3

[0054] The preparations obtained in Example 1, Comparative Example 1, and Experimental Example 2 of this invention were co-incubated with immature mouse bone marrow-derived dendritic cells (BMDCs) for 24 hours. Cells were then collected, stained with an Annexin V-APC apoptosis detection kit containing 7-AAD, and flow cytometry was used to detect whether each preparation affected the activity of BMDCs. Figure 3 As shown in Figure A, the activity of BMDCs in each group exceeded 95%, indicating that each formulation has good biocompatibility.

[0055] Next, FC-NPs and FCD-NPs were prepared using CpG labeled with Alexa Fluor 488. After co-incubation with DC2.4 cells for 12 hours, cells were collected and the proportion of cells taking up CpG-Alexa Fluor 488 in each group was detected by flow cytometry. Figure 3As shown in Figure B, the proportions of positive cells in the free CpG, FC-NPs, and FCD-NPs groups were 80.8%, 38.1%, and 69.7%, respectively. The positive rate of the FCD-NPs group was significantly higher than that of the FC-NPs group. This is because the positively charged DOTAPs interact with the cell membrane, enhancing the cell's ability to take up nanoparticles. Additionally, the production of intracellular ROS in DC2.4 cells after co-incubation with each formulation was detected using Carboxy-H2DCFDA fluorescent dye. Figure 3 As shown in Figure C, the proportions of ROS production in the free CpG, FC-NPs, and FCD-NPs groups were 3.5%, 11.0%, and 19.0%, respectively. The free CpG group produced virtually no ROS, while the other two groups showed significantly increased ROS production rates due to the presence of metallic iron, particularly the FCD-NPs group, which is attributed to the higher nanoparticle uptake in this group. Furthermore, TAAs were labeled with the fluorescent dye FITC, and antigen-adsorbed nanoparticles FCD-NPs@TAAs were prepared. Free TAAs and FCD-NPs@TAAs were co-incubated with DC2.4 cells for 24 h, and then stained with lysosomal dye Lyso-Tracker Red and nuclear dye Hoechst. The co-localization level of TAAs with lysosomes was observed using CLSM. Figure 3 As shown in Figure D, the green fluorescence of most TAAs in the free TAAs group overlaps with the red fluorescence of lysosomes, while the overlap between green and red fluorescence in the FCD-NPs@TAAs group is very low. This indicates that most TAAs in this group have achieved lysosomal escape, which is caused by ROS-induced lysosomal rupture and the "proton sponge effect" caused by DOTAP.

[0056] After co-incubating each formulation with BMDCs for 24 hours, cells were collected and stained with fluorescently labeled CD11c, CD40, CD80, and CD86 antibodies. The expression levels of various co-stimulatory molecules on the surface of BMDCs were detected by flow cytometry. Figure 3 As shown in the EG, the expression levels of CD40, CD80, and CD86 on the surface of BMDCs co-incubated with FCD-NPs@TAAs were significantly higher than those in other groups, indicating that a greater degree of BMDC maturation and activation was induced.

[0057] Experiment Example 4

[0058] The formulations prepared in Example 1 and Comparative Example 1 of this invention were subjected to in vivo antitumor efficacy tests, and the treatment regimens were as follows: Figure 4 As shown in Figure A.

[0059] Female Balb / c mice aged 6-8 weeks were selected for the efficacy experiment. After hair removal treatment, 4T1 cells (1×10⁻⁶) were inoculated into the right back near the hind limb. 6A tumor-bearing mouse model was constructed (number of mice per mouse), with a tumor volume of ~50 mm². 3 Day 0 was designated as the starting point. Mice were randomly divided into 7 groups: PBS+NIR, free CpG+NIR, FC-NPs+NIR, FCD-NPs+NIR, FCP-NPs+NIR, FCDP-NPs, and FCDP-NPs+NIR, with 9 mice in each group. The dosage was 20 μg CpG per mouse. On day 0, tumor-bearing mice in each group underwent intratumoral administration. The laser irradiation group received 0.5 W / cm² of CpG after administration. 2 Mice were irradiated with an 808nm laser for 5 minutes, and images of the highest temperature were recorded using an infrared thermal imager. Starting from day 0, tumor volume and body weight were recorded every two days. When the tumor volume exceeded 1000 mm², the tumor was considered full. 3 Recorded as death.

[0060] like Figure 4 As shown in Figure B, the highest temperatures reached after laser irradiation in the PBS+NIR, free CpG+NIR, FC-NPs+NIR, FCD-NPs+NIR, FCP-NPs+NIR, and FCDP-NPs+NIR groups were 35.4℃, 36.2℃, 35.7℃, 37.0℃, 49.0℃, and 53.5℃, respectively. The latter two groups, due to the loading of the photothermal agent PDA, exhibited excellent photothermal effects, sufficient to kill tumor cells, while the temperatures of the other groups remained basically unchanged, consistent with body temperature.

[0061] Figure 4 C and Figure 4 The results showed that both FCP-NPs+NIR and FCDP-NPs+NIR groups had good tumor treatment effects and effectively inhibited tumor growth. The latter had a better anti-tumor growth effect, with a survival rate of 66.67%. Six out of nine tumor-bearing mice achieved complete tumor elimination. Figure 4 The results showed that the mice did not experience significant weight loss during the observation period. To examine the cytotoxic effect of photothermal therapy on tumor cells, tumor tissue was collected on day 1, and apoptosis was detected using a terminal deoxyribonucleotide transferase-mediated nick-end labeling (TUNEL) apoptosis assay kit. Figure 4 The results showed that, compared to the other groups, the FCP-NPs+NIR and FCDP-NPs+NIR groups induced more tumor cell apoptosis, which was due to the effective increase in temperature.

[0062] Experimental Example 5

[0063] The formulations prepared in Example 1 and Comparative Example 1 of this invention were subjected to in vivo experiments to promote DC maturation.

[0064] On the 7th day after treatment, inguinal lymph nodes were collected and ground into single-cell suspensions. After centrifugation at 450g for 5 min, the suspensions were incubated at 4℃ for 30 min with antibodies against CD86, CD40, CD11c, CD80, and MHC I (H-2kb). The suspensions were washed once with PBS, resuspended in 0.3 mL of 4% tissue cell fixative, and the expression levels of CD40, CD80, CD86, and MHC I were measured by flow cytometry.

[0065] Figure 5 The results from the study showed that, compared to other groups, the DCs in the tumor-draining lymph nodes of mice in the FCP-NPs+NIR group highly expressed CD40, CD80, CD86, and MHC I molecules, demonstrating an advantage in inducing DC maturation and antigen cross-presentation. The FCDP-NPs+NIR group had a higher proportion of activated DCs, indicating that the formation of in-situ personalized vaccines after antigen capture also plays a role in promoting DC maturation and antigen presentation.

[0066] Experimental Example 6

[0067] Experiments were conducted to improve the tumor immunosuppressive microenvironment using the preparations obtained in Example 1 and Comparative Example 1 of this invention.

[0068] On day 7 post-treatment, tumor cells were harvested and prepared into single-cell suspensions. After centrifugation at 450g for 5 min, the suspensions were incubated at 4℃ for 30 min with CD3e, CD4, CD8a, and CD11c antibodies. Flow cytometry was used to determine the infiltration levels of antigen-specific T cells and dendritic cells within the tumor. After incubation at 4℃ for 30 min with CD11b and F4 / 80 antibodies, fixation and membrane perforation were followed by incubation at room temperature in the dark with CD206 antibody for 60 min. Flow cytometry was used to determine the M1 / M2 TAM ratio. After incubation at 4℃ for 30 min with CD3e, CD8a, and CD4 antibodies, fixation and membrane perforation were followed by incubation at room temperature in the dark with Foxp3 antibody for 30 min. Flow cytometry was used to determine the level of regulatory T cells in the tumor environment. Additionally, tumor tissue was collected, fixed, dehydrated, frozen sectioned, stained with fluorescently labeled NK1.1 antibody, and mounted with a DAPI-containing anti-fluorescence quenching mounting medium. Intratumoral NK cell infiltration was then observed under a laser confocal microscope.

[0069] Figure 6 AC is CD3 in mouse tumors after treatment with different formulations. + CD4 + T cells (Ths), CD3 + CD8 + A bar chart showing the ratio of T cells (CTLs) to dendritic cells (DCs) indicates that photothermal therapy can effectively promote the production of CD4+ in tumors. + CD8 +The infiltration of T cells and dendritic cells (DCs), especially FCDP-NPs+NIR, can maximally activate antigen-specific T cells at the tumor site. Furthermore, the FCP-NPs+NIR and FCDP-NPs+NIR groups also have the ability to increase the M1 / M2 TAMs ratio and reduce tumor-infiltrating regulatory T cells (Tregs), improving the immunosuppressive microenvironment at the tumor site and enhancing tumor immunogenicity. Figure 6 (Dual DF). CLSM diagram ( Figure 6 The results showed that the FCDP-NPs+NIR group had a large area of ​​NK cell (red) infiltration, while the FCP-NPs+NIR group only had scattered red spots.

[0070] Experimental Example 7

[0071] The formulations prepared in Example 1 and Comparative Example 1 of this invention were subjected to experiments on their systemic immune effects in vivo.

[0072] On the 7th day after treatment, spleen was harvested and prepared into a single-cell suspension. After centrifugation at 450g for 5 min, the suspension was incubated at 4℃ for 30 min with CD3e, CD4, and CD8a antibodies. The level of tumor antigen-specific T cells was measured by flow cytometry. After incubation at 4℃ for 30 min with CD11b and F4 / 80 antibodies, the suspension was fixed and ruptured. Then, the suspension was incubated at room temperature in the dark with CD206 antibody for 60 min. The ratio of M1 / M2 TAMs was measured by flow cytometry.

[0073] Figure 7 A and B represent the levels of CD3 in the spleen of mice after treatment with different formulations. + CD4 + T cells (Ths), CD3 + CD8 + Representative flow cytometry plots of T cells (CTLs); Figure 7 The middle C represents representative flow cytometry plots of M1 and M2 macrophages. The results show that FCDP-NPs+NIR can effectively promote CD4+. + and CD8 + T cell activation and an increase in the M1 / M2 TAM ratio demonstrate their advantage in enhancing systemic immune responses.

[0074] Experimental Example 8

[0075] The preparations obtained in Examples 1 and 2 of this invention were subjected to immune memory analysis and anti-tumor lung metastasis tests.

[0076] Experimental protocol as follows Figure 8 As shown in Figure A, on day 36 post-treatment, cured mice in the FCP-NPs+NIR and FCDP-NPs+NIR groups were injected via the tail vein with 4T1 cells (1×10⁻⁶).5 (Each mouse was tested). Untreated mice of the same age and sex served as controls. On day 2 post-injection, spleens were collected from each group of mice, prepared into single-cell suspensions, centrifuged at 450g for 5 min, and incubated at 4℃ for 30 min with antibodies against CD3e, CD4, CD8a, CD44, and CD62L. The proportion of effector memory T cells was determined using flow cytometry. Furthermore, lung tissue was collected from each group 20 days after injection, and tumor nodules were observed by photography.

[0077] Figure 8 The BE study showed that in the FCDP-NPs+NIR group, effector memory T cells accounted for a significant proportion of CD8+ cells. + and CD4 + The proportion of T cells was significantly higher in the FCP-NPs+NIR group and the control group. The control group had numerous and large tumor metastases in its lung tissue. Figure 8 In the FCP-NPs+NIR group, the number of tumor nodules was relatively small, while in the FCDP-NPs+NIR group, only one mouse had a tumor metastasis in its lung tissue, and the lung tissue maintained good integrity. This may be because a large number of effector memory T cells effectively prevented the lung metastasis of the tumor.

[0078] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.

Claims

1. A method for preparing a multifunctional carrier-free self-assembled nanoadjuvant, characterized in that, Includes the following steps: (a) After mixing the aqueous solution of ferrous chloride tetrahydrate, the aqueous solution of CpG and deionized water, the reaction was carried out. After the reaction was completed, the reaction solution was centrifuged and the precipitate was washed to obtain precipitate 1. (b) Resuspend precipitate 1, add DOTAP aqueous solution and shake to react. After the reaction is complete, centrifuge and wash the reaction solution to obtain precipitate 2. (c) Resuspend precipitate 2, add dopamine hydrochloride Tris-HCl solution and shake to react. After the reaction is complete, centrifuge the reaction solution, wash and collect the precipitate to obtain the multifunctional carrier-free self-assembled nano-adjuvant. In step (a), the reaction temperature is 90~100℃ and the reaction time is 2.5~4h; The resuspension solvent for precipitate 1 is deionized water, and the resuspension solvent for precipitate 2 is Tris-HCl buffer solution.

2. The preparation method according to claim 1, characterized in that, In step (a), the concentration of the ferrous chloride tetrahydrate aqueous solution is 3~5 mg / mL; the concentration of the CpG aqueous solution is 8~12 μL / OD; The volume ratio of ferrous chloride tetrahydrate aqueous solution, CpG aqueous solution and deionized water is 3: (6~10): (45~55).

3. The preparation method according to claim 1, characterized in that, In step (b), the volume ratio of the DOTAP aqueous solution to the deionized water in step (a) is 1:(0.8~1.2); the concentration of the DOTAP aqueous solution is 3~5 mg / mL.

4. The preparation method according to claim 1, characterized in that, In step (b), the shaking reaction time is 1.5~2.5h; the reaction temperature is room temperature.

5. The preparation method according to claim 1, characterized in that, In step (c), the volume ratio of the solvent used for resuspension to the deionized water in step (a) is 1:(0.8~1.2), and the solvent is a 10 mM Tris-HCl buffer solution with pH 8.0~8.

5.

6. The preparation method according to claim 1, characterized in that, In step (c), the volume ratio of dopamine hydrochloride Tris-HCl solution to deionized water in step (a) is 1:(0.8~1.2); the concentration of dopamine hydrochloride Tris-HCl solution is 3~5 mg / mL. The shaking reaction time is 6-8 hours; the reaction temperature is room temperature.

7. The multifunctional carrier-free self-assembled nanoadjuvant prepared by any of the preparation methods described in claims 1 to 6.

8. The application of the multifunctional carrier-free self-assembled nanoadjuvant prepared by any one of claims 1 to 6 in the preparation of products for treating breast cancer tumors.

9. The application of the multifunctional carrier-free self-assembled nanoadjuvant prepared by any one of claims 1 to 6 in the preparation of products that inhibit breast cancer tumor metastasis.

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