Nano immunologic adjuvant based on calcium-arginine coordination as well as preparation method and application of nano immunologic adjuvant

Abstract

The invention discloses a nano immunologic adjuvant for enhancing tumor cell immunogenic death based on calcium-arginine coordination as well as a preparation method and application of the nano immunologic adjuvant, and belongs to the technical field of biological medicines. Mixing the calcium chloride aqueous solution and the arginine aqueous solution; and adding a polyvinylpyrrolidone aqueous solution into the mixed system, adding hydrogen peroxide, mixing, adjusting the pH value, and stirring and reacting at room temperature to obtain the nano immunologic adjuvant (CaO2-Arg) based on calcium-arginine coordination. The calcium-arginine coordinated nano immunologic adjuvant for enhancing immunogenic death of tumor cells is disassembled and recombined under the triggering of acid, is converted into a hexahedral configuration from a spherical structure of an aggregate, and is still maintained in an assembled state, so that the diffusion of the nano immunologic adjuvant in the cells is retarded, and the residence time of the nano immunologic adjuvant in the tumor cells is prolonged. Meanwhile, the CaO2-Arg can also enable ROS and NO generated on the surface of the CaO2-Arg to effectively generate bimolecular collision, and high ONOO <-> concentration is locally realized, so that immune cell death is synergistically induced, and a new breakthrough is expected to be brought to the field of RONS-based tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a nano-immunoadjuvant for enhancing immunogenic cell death of tumor cells based on calcium-arginine coordination, and a preparation method and application thereof. Background Art

[0002] Cancer, a global public health problem, not only poses a serious threat to human health but also brings a huge burden to the medical system. Reactive oxygen and nitrogen species (RONS) have been proven to be potential effector molecules in cancer treatment and have become an important part of anti-cancer treatment regimens. However, RONS delivery or production strategies have limitations in achieving optimal RONS activation concentrations and kinetics and cannot effectively induce immunogenic cell death in the tumor microenvironment (TME). Therefore, improving the tumor delivery of RONS and in-situ generation and accumulation of high concentrations of RONS in tumors are expected to significantly improve the therapeutic effect.

[0003] Arginine (Arg), as an amino acid containing a guanidine group, can not only form stable coordination bonds with metal ions but also act as a donor of nitric oxide (NO) and participate in regulating the oxidative stress response in the TME. The metal-arginine coordination nano-immunoadjuvant can respond to the acidic environment of the TME and generate ROS and NO through the catalytic action of metal ions. These reactive substances show the potential of synergistic anti-cancer in tumor treatment. Heping Wang (Dual-Fuel Propelled Nanomotors with Two-Stage Permeation for Deep Bacterial Infection in the Treatment of Pulpitis, Adv. Sci. 2024, 11, 2305063) et al. developed dual-fuel propelled nanomotors (DPNM) by encapsulating L-arginine (L-Arg) and calcium peroxide (CaO2) in a metal-organic framework. L-Arg can react with reactive oxygen species (ROS) to release nitric oxide (NO), providing the driving force for movement. The consumed ROS can be replenished by the reaction between CaO2 and the abundant acid in the inflammatory microenvironment, effectively eliminating bacterial infection. Huan Liu (Tumor-Targeting CaO2-Based Nanoparticles for Cancer Therapy, ACS Appl. Nano Mater. 2024, 7, 26276−26286) encapsulated L-arginine (L-Arg) and calcium peroxide (CaO2) in a metal-organic framework and used folic acid modification for targeted and enhanced photodynamic / photothermal synergistic cancer treatment.

[0004] Although the metal-arginine coordination nano-immunoadjuvant shows promise in tumor treatment, current research still faces some challenges. The limited content of endogenous hydrogen peroxide (H2O2) in the TME restricts the generation and accumulation of ROS; the premature release of exogenous NO precursors may lead to a decrease in the biological activity of NO, affecting its role in tumor treatment; in terms of TME regulation, there is a lack of treatment regimens that can more accurately identify and locate tumor cells. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the present invention provides a calcium-arginine coordination-based nano-immunoadjuvant, its preparation method and application, which can disassemble and recombine in response to pH to synergistically and precisely release RONS, enhance the immunogenic death of tumor cells, and achieve in-situ anti-cancer.

[0006] The present invention is realized through the following technical solutions: A preparation method of a nano-immunoadjuvant based on calcium-arginine coordination, comprising the following steps: (1) Mix an aqueous calcium chloride solution and an aqueous arginine solution; (2) Add an aqueous polyvinylpyrrolidone solution to the mixed system of step (1), add hydrogen peroxide and mix, adjust the pH, and stir and react at room temperature to obtain a nano-immunoadjuvant based on calcium-arginine coordination.

[0007] Further, in step (1), the molar ratio of calcium chloride to arginine is 20:1 to 1:1.

[0008] Further, in step (2), the mass ratio of polyvinylpyrrolidone to calcium chloride in step (1) is 1:1 to 1:10.

[0009] Further, in step (2), hydrogen peroxide is an aqueous solution with a mass concentration of 25-30%; the volume ratio of hydrogen peroxide to the mixed system of step (1) is 1:2 to 1:5.

[0010] Further, in step (2), add alkali to adjust the pH value to 9-10.

[0011] Further, the reactants in step (2) are washed with ethanol and freeze-dried to obtain a nano-immunoadjuvant based on calcium-arginine coordination.

[0012] Further, in step (1), the concentration of the aqueous calcium chloride solution is 200 mM, and the concentration of the aqueous arginine solution is 200 mM; in step (2), the concentration of the aqueous polyvinylpyrrolidone solution is 0.024 g / mL.

[0013] In the present invention, the nano-immunoadjuvant based on calcium-arginine coordination prepared by the above-mentioned preparation method.

[0014] Further, the nano-immunoadjuvant based on calcium-arginine coordination is a spherical particle.

[0015] In the present invention, the application of the above-mentioned nano-immunoadjuvant based on calcium-arginine coordination in the preparation of an anti-tumor preparation.

[0016] The beneficial effects obtained by the present invention are: In the present invention, the nano-immunoadjuvant based on calcium-arginine coordination rationally designs the CaO2 and L-Arg precursors, so that they are disassembled and recombined under acid triggering, changing from the spherical structure of the aggregate to the hexahedron configuration. Although the configuration changes, the nano-immunoadjuvant still maintains the assembled state, slows down its diffusion in cells, and prolongs its residence time in tumor cells. At the same time, CaO2-Arg can also effectively cause bimolecular collision of the ROS and NO generated on its surface, achieving high ONOO locally -Concentration to synergistically induce immunogenic cell death. This precisely designed CaO2-Arg can effectively generate ONOO at the subcellular level - , overcoming the limitations of traditional RONS delivery and production strategies, providing a new method for cancer treatment, and promising to bring new breakthroughs to the field of RONS-based cancer therapy. Description of the Drawings

[0017] Figure 1 Schematic diagram of the synthesis of calcium-arginine coordinated nano-immunoadjuvant; Figure 2 SEM images of calcium-arginine coordinated nano-immunoadjuvant, (a) CaO2-Arg, (b) after acidolysis for 0.5 h; Figure 3 Fourier transform infrared spectra of Arg, CaO2, CaO2-Arg, and CaO2-Arg after acidolysis for 0.5 h (CaO2-Arg + H + ); Figure 4 X-ray photoelectron spectra of calcium-arginine coordinated nano-immunoadjuvant before and after acidolysis (0.5 h), (a) CaO2-Arg, (b) CaO2-Arg after acidolysis for 0.5 h (CaO2-Arg + H + ); Figure 5 Ability diagrams of CaO2-Arg and CaO2 to generate •OH in acidic or neutral environments, (left) acidic environment, (right) neutral environment; Figure 6 Quantitative bar chart of NO generation by CaO2-Arg under acidic conditions; Figure 7 Confocal images of cell staining after treating 4T1 cells with the control group (DPBS), CaO2, and CaO2-Arg; Figure 8 TEM image of 4T1 cells treated with CaO2-Arg for 6 h; Figure 9 ROS, NO, and CRT immunofluorescence staining images of tumor tissues 3 days after intratumoral injection of the control group (DPBS), CaO2, and CaO2-Arg; Figure 10 Schematic diagram of the immune response of CaO2-Arg. Detailed Implementation Modes

[0018] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0019] Example 1 Preparation of nano-immunoadjuvant based on calcium-arginine coordination. The synthesis schematic diagram of the calcium-arginine coordination nano-immunoadjuvant is as Figure 1 shown: (1) Dissolve 0.3552 g of CaCl2 in 16 mL of water, add 2 mL of L-Arg (200 mM) aqueous solution, and mix; (2) Add polyvinylpyrrolidone (0.2367 g of PVP dissolved in 10 mL of water) to the mixed system in step (1), then add 6.4 mL of 30% hydrogen peroxide, and adjust the pH value to (9 - 10) with NaOH solution; stir and react at room temperature for 12 h, wash the precipitate of the reactant 3 times with absolute ethanol, and freeze-dry to obtain the calcium-arginine coordination nano-immunoadjuvant (CaO2-Arg).

[0020] Example 2 Preparation of CaO2 Dissolve 0.3552 g of CaCl2 in 16 mL of water, add polyvinylpyrrolidone (0.2367 g of PVP dissolved in 10 mL of water), then add 6.4 mL of 30% hydrogen peroxide, and adjust the pH value to 9 with NaOH solution; stir and react at room temperature for 12 h, wash the precipitate of the reactant 3 times with absolute ethanol, and freeze-dry to obtain CaO2.

[0021] The SEM image of the calcium-arginine coordination nano-immunoadjuvant prepared in Example 1 is as Figure 2 shown in (a). It can be seen that CaO2-Arg is irregular spherical particles, and the single particle diameter is about 100 nm, tending to an aggregated state.

[0022] The calcium-arginine coordination nano-immunoadjuvant prepared in Example 1 was reacted under acidic conditions (pH = 5.5) for 0.5 h, and its SEM image is as Figure 2 shown in (b). It can be seen that the spherical structure of CaO2-Arg has undergone a huge transformation into a regular hexahedron shape under acidic conditions.

[0023] The Fourier transform infrared spectra of CaO2-Arg prepared in Example 1, L-Arg (Arg), CaO2 prepared in Example 2, and CaO2-Arg after acidolysis (pH = 5.5) for 0.5 h (CaO2-Arg + H + ) are as Figure 3 shown, further verifying the successful synthesis of CaO2-Arg. And it is worth noting that the disassembly and reassembly of the nano-immunoadjuvant triggered by the acidic environment not only change the external morphology, but also maximize the retention of the original surface functional groups and residues.

[0024] X-ray photoelectron spectroscopy spectra of the calcium-arginine coordinated nano-immunoadjuvant prepared in Example 1 before and after acidolysis (0.5 h) are as follows Figure 4 shown, and it can be seen from Figure 4 the calculation that the percentage of peroxy bridge bonds decreased significantly after 0.5 h of acidolysis, from 42.4% before acidolysis to 22.6% after acidolysis, which is attributed to the release of H2O2 caused by the partial cleavage of peroxy bridge bonds after acidolysis.

[0025] Example 3 Detection of ROS production catalyzed by calcium-arginine coordinated nano-immunoadjuvant (CaO2-Arg): The ability of CaO2-Arg to generate reactive oxygen species was studied by using the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) in neutral or weak acidic environments.

[0026] Prepare 2 μM horseradish peroxidase (HRP), 2 mM ABTS, and 200 mg / mL test solutions (CaO2-Arg and CaO2, respectively); Adjust the pH to 5.5. Add 400 μL of CaO2 and CaO2-Arg to centrifuge tubes respectively, then add 20 μL of HRP solution, and finally add 200 μL of ABTS solution. Incubate at 37 °C for 15 min, then centrifuge and take the supernatant. Measure the ultraviolet absorbance of the supernatant with a UV-visible spectrophotometer; Adjust the pH to 7.4 and simulate the ability of CaO2 and CaO2-Arg to generate ROS in a neutral environment through similar operations above; The ability diagrams of CaO2 and CaO2-Arg to generate •OH in acidic (left) or neutral (right) environments are as follows Figure 5 shown. The intermediate •OH can oxidize ABTS from colorless to green, with an obvious characteristic absorption peak at about 415 nm. Compared with the neutral environment, CaO2-Arg has a stronger ability to generate ROS by acidolysis under acidic conditions.

[0027] Example 4 Detection of NO production catalyzed by CaO2-Arg: Prepare solutions of different concentrations of CaO2-Arg (0.5, 1, 2, 5 mg / mL). Incubate different concentrations of CaO2-Arg in a solution environment with pH = 5.5 at 37 °C for 15 min. After incubation, detect the production of NO with Griess reagent. The quantitative histogram of NO production is as follows Figure 6 shown; and it can be seen from Figure 6It can be seen that when the concentration of CaO2-Arg reached 5 mg / mL, 1.3 μM of NO was produced, indicating that CaO2-Arg has good NO generation performance.

[0028] Example 5 In vitro anti-tumor efficacy evaluation: Mouse breast cancer cells (4T1) were cultured in a 96-well plate (1×10 4 cells per well) and cultured with complete medium RPMI 1640. The cell growth state was observed. When the cell density reached 80%, the cells were then treated with different concentrations of CaO2 and CaO2-Arg and incubated at 37 °C in a sterile incubator for 24 h. The cell viability was measured by the CCK-8 method, and the absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. The confocal images of cell staining after treating 4T1 cells with the control group (DPBS), CaO2, and CaO2-Arg are as Figure 7 shown. The large-scale death of cells in the CaO2-Arg treatment group indicates that the nano-immunoadjuvant CaO2-Arg has superior anti-cancer efficacy. The transmission electron microscopy (TEM) image and enlarged image of 4T1 cells treated with CaO2-Arg for 6 h are as Figure 8 shown. It can be seen that CaO2-Arg can still stably exist in 4T1 cells in the form of hexahedrons 6 h after treating 4T1 cells.

[0029] Example 6 In vivo detection of ROS, NO, and CRT: Different therapeutic drugs (DPBS (10 μL), CaO2 (10 μL, 10 mg / mL), CaO2-Arg (10 μL, 10 mg / mL)) were injected into the tumors of BALB / c mice bearing 4T1 xenograft tumors; 3 days later, the mice were euthanized and the tumors were dissected and separated; the tumor tissues were fixed with paraformaldehyde, embedded in paraffin, and sectioned. The sections were stained with DCFH-DA and DAF-FM DA probes respectively, and the production of ROS and NO in the stained tumor sections was observed under a confocal microscope respectively.

[0030] For the evaluation of immunogenic cell death (ICD), the sections were incubated with CRT primary antibody for 1.5 h, and then incubated with FITC-conjugated secondary antibody at room temperature for 2 h. The expression of CRT in the stained tumor sections was observed using a confocal microscope. The immunofluorescence staining images of ROS, NO, and CRT in tumor tissues 3 days after intratumoral injection of different drugs (control group (DPBS), CaO2, CaO2-Arg) are as Figure 9As shown, immunofluorescence staining revealed that there was still a certain concentration of ROS and NO accumulation in the CaO2-Arg treatment group after 3 days. Importantly, different from the CaO2 group that only showed an increase in ROS exposure, the CaO2-Arg treatment group specifically produced CRT (ICD marker). These results verified the excellent ability of CaO2-Arg to induce ICD by co-producing ROS / RNS.

[0031] In summary, the ROS / RNS produced by the calcium-arginine coordinated nano-immunoadjuvant (CaO2-Arg) prepared in the present invention play a synergistic role to promote the release of DAMPs from tumor cells and stimulate dendritic cells (DCs), thereby activating T cells and initiating an immune response. The schematic diagram of the immune response is as Figure 10 shown.

Claims

1. A preparation method of a nano-immunoadjuvant based on calcium-arginine coordination, characterized in that, It includes the following steps: (1) Mix an aqueous calcium chloride solution and an aqueous arginine solution; (2) Add an aqueous polyvinylpyrrolidone solution to the mixed system in step (1), add hydrogen peroxide and mix, adjust the pH, and stir and react at room temperature to obtain a nano-immunoadjuvant based on calcium-arginine coordination.

2. The preparation method of the nano-immunoadjuvant based on calcium-arginine coordination according to claim 1, characterized in that In step (1), the molar ratio of calcium chloride to arginine is 20:1 to 1:

1.

3. The preparation method of the nano-immunoadjuvant based on calcium-arginine coordination according to claim 1, wherein In step (2), the mass ratio of polyvinylpyrrolidone to calcium chloride in step (1) is 1:1 to 1:

10.

4. The preparation method of the nano-immunoadjuvant based on calcium-arginine coordination according to claim 1, wherein, In step (2), hydrogen peroxide is an aqueous solution with a mass concentration of 25-30%; the volume ratio of hydrogen peroxide to the mixed system in step (1) is 1:2 to 1:

5.

5. The preparation method of the nano-immunoadjuvant based on calcium-arginine coordination according to claim 1, wherein In step (2), add alkali to adjust the pH value to 9-10.

6. The preparation method of the nano-immunoadjuvant based on calcium-arginine coordination according to claim 1, characterized in that The reactants in step (2) are washed with ethanol and freeze-dried to obtain a nano-immunoadjuvant based on calcium-arginine coordination.

7. The preparation method of the nano-immunoadjuvant based on calcium-arginine coordination according to claim 1, characterized in that In step (1), the concentration of the aqueous calcium chloride solution is 200 mM, and the concentration of the aqueous arginine solution is 200 mM; in step (2), the concentration of the aqueous polyvinylpyrrolidone solution is 0.024 g / mL.

8. A nano-immunoadjuvant based on calcium-arginine coordination prepared by the preparation method according to any one of claims 1 to 7.

9. The nano-immunoadjuvant based on calcium-arginine coordination according to claim 8, characterized in that, The nano-immunoadjuvant based on calcium-arginine coordination is spherical particles.

10. Use of the nano-immunoadjuvant based on calcium-arginine coordination according to claim 8 or 9 in the preparation of an anti-tumor preparation.

Images