Inhalable ultrathin nanosheet as well as preparation method and application thereof

Abstract

The invention provides an inhalable ultrathin nanosheet as well as a preparation method and application thereof, and belongs to the technical field of antitumor drugs. The inhalable ultrathin nanosheet disclosed by the invention comprises a MnO2 nanosheet and BSA (Bovine Serum Albumin) loaded on the surface of the MnO2 nanosheet, the particle size of the inhalable ultrathin nanosheet is 100 to 300nm, and the thickness of the inhalable ultrathin nanosheet is 1 to 5nm. Through the design of the ultrathin nanosheets, it is guaranteed that the nanosheets can be efficiently deposited in the lung and deeply permeate into a tumor area; the stability of the MnO2 nanosheet is enhanced through loading of BSA, local tumor treatment is carried out through a photothermal effect, ROS generated by immune reaction is eliminated, adverse immune reaction is relieved, and through combination of the functions, the inhalable ultrathin nanosheet can effectively treat tumors and improve the treatment effect, and can be used as a medicine for treating tumors. The irAEs problem in cancer immunotherapy is solved by combining photothermal therapy and anti-inflammatory action.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-tumor drugs, and particularly to an inhalable ultrathin nanosheet and its preparation method and application. Background Art

[0002] The treatment of lung cancer faces many challenges. In recent years, immunotherapy, especially the application of immune checkpoint inhibitors (such as anti-PD-1 antibodies), has brought new hope to many lung cancer patients. However, these treatment methods are often accompanied by severe immune-related adverse events (irAEs), especially pulmonary immune reactive diseases, such as immune pneumonia, which may lead to the interruption of treatment and the death of patients in severe cases. How to solve the irAEs problem in lung cancer treatment and further improve the treatment effect has become an important topic in the field of tumor treatment.

[0003] In related technologies, the treatment of irAEs mainly relies on the use of immunosuppressants, which can temporarily relieve symptoms, but may weaken the anti-tumor function of the immune system and reduce the treatment effect. In addition, existing immunotherapy regimens still face problems such as local targeted delivery and systemic toxicity. Many drugs need to be injected into the body intravenously. Although they can be effectively delivered throughout the body, it is difficult to achieve efficient targeted deposition at the tumor site, and at the same time, it also brings systemic side effects. The emergence of nanotechnology has provided new ideas for the innovation of drug delivery systems.

[0004] Currently, the application of nanomaterials in tumor treatment has received extensive attention, especially the realization of spatiotemporally controllable treatment through photothermal therapy (PTT) combined with drug delivery systems, and then achieving efficient targeted deposition at the tumor site. PTT uses nanomaterials to generate a local temperature increase under the irradiation of a laser with a specific wavelength, so as to achieve the effect of tumor thermal ablation. However, traditional nanomaterials usually have a relatively large particle size (greater than 500 nm), resulting in their inability to achieve ideal deep penetration in the lungs and effective targeted delivery. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide an inhalable ultrathin nanosheet and its preparation method and application. The inhalable ultrathin nanosheet provided by the present invention has a particle size of 100 - 300 nm and a thickness of 1 - 5 nm, ensuring its efficient deposition in the lungs and deep penetration into the tumor area, and enabling effective targeted delivery.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions:

[0007] The present invention provides an inhalable ultrathin nanosheet, comprising MnO 2 nanosheets and loaded on the MnO 2Bovine serum albumin (BSA) on the surface of the nanosheets, the inhalable ultrathin nanosheets having a particle size of 100 to 300 nm and a thickness of 1 to 5 nm.

[0008] Preferably, the inhalable ultrathin nanosheets have a particle size of 150 to 200 nm.

[0009] Preferably, in the inhalable ultrathin nanosheets, the mass content of MnO 2 nanosheets is 70 to 90%, and the mass content of bovine serum albumin is 10 to 30%.

[0010] Preferably, the MnO 2 nanosheets have a crystal form of β-MnO 2 .

[0011] The present invention also provides a method for preparing the inhalable ultrathin nanosheets described in the above technical solution, comprising the following steps:

[0012] Mix the dispersion of MnO 2 nanosheets and the bovine serum albumin solution and then let it stand to obtain the inhalable ultrathin nanosheets.

[0013] Preferably, the concentration of MnO 2 in the dispersion of MnO 2 nanosheets is 100 to 200 μg / mL.

[0014] Preferably, the concentration of the bovine serum albumin solution is 10 to 20 mg / mL.

[0015] Preferably, the standing time is 1 to 5 h and the temperature is 20 to 25 °C.

[0016] The present invention also provides the use of the inhalable ultrathin nanosheets described in the above technical solution or the inhalable ultrathin nanosheets prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs.

[0017] The present invention also provides the use of the inhalable ultrathin nanosheets described in the above technical solution or the inhalable ultrathin nanosheets prepared by the preparation method described in the above technical solution in the preparation of drugs for treating immune-related adverse events.

[0018] The present invention provides an inhalable ultrathin nanosheet, comprising MnO 2 nanosheets and bovine serum albumin loaded on the surface of the MnO 2 nanosheets, the inhalable ultrathin nanosheets having a particle size of 100 to 300 nm and a thickness of 1 to 5 nm.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] The present invention provides an inhalable ultrathin nanosheet (i.e., MnO 2 @BSA nanosheet hereinafter), which comprises a MnO 2 nanosheet and bovine serum albumin loaded on the surface of the MnO 2 nanosheet. The design of the MnO 2 @BSA nanosheet is based on the following considerations: First, through the design of ultrathin nanosheets (with a thickness of 1 - 5 nm) and a particle size of 100 - 300 nm, it is ensured that it can achieve efficient deposition in the lungs and deeply penetrate into the tumor area; Second, by loading BSA, the stability of the MnO 2 nanosheet is enhanced, preventing the aggregation of the MnO 2 nanosheet in the body, and it can be used for local tumor treatment through the photothermal effect, while scavenging reactive oxygen species (ROS) generated by the immune response and reducing immune adverse reactions. By combining these functions, the inhalable ultrathin nanosheet of the present invention can not only effectively treat tumors and improve the treatment effect, but also reduce systemic toxicity, and solve the irAEs problem in cancer immunotherapy by combining photothermal therapy and anti-inflammatory effects, such as alleviating lung damage caused by excessive immune response in immunotherapy.

[0021] The present invention also provides a preparation method of the inhalable ultrathin nanosheet described in the above technical solution. After mixing the dispersion of the MnO 2 nanosheet and the bovine serum albumin solution and then standing, the operation is simple and it is easy to realize industrial production. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 FIG. is the transmission electron microscope image of the inhalable ultrathin nanosheet at different magnifications, and the inset is the physical image of the inhalable ultrathin nanosheet solution;

[0023] Figure 2 FIG. is the particle size distribution diagram of the inhalable ultrathin nanosheet;

[0024] Figure 3 FIG. is the ultraviolet-visible spectrum diagram;

[0025] Figure 4 FIG. is the energy spectrum analysis diagram;

[0026] Figure 5 FIG. is the infrared thermal imaging diagram;

[0027] Figure 6 FIG. is the photothermal effect curve of the inhalable ultrathin nanosheet at different concentrations;

[0028] Figure 7 FIG. is the photothermal cycle curve;

[0029] Figure 8H clearance efficiency graphs at different time points; 2 O 2 ;

[0030] Figure 9 H clearance efficiency graphs at different concentrations; 2 O 2 ;

[0031] Figure 10 Schematic diagram of animal experiment;

[0032] Figure 11 Lung weight ratio graph;

[0033] Figure 12 HE staining section graphs of lung tissues of mice in each group;

[0034] Figure 13 Lung injury score graph;

[0035] Figure 14 Percentage graph of neutrophils;

[0036] Figure 15 Percentage graph of macrophages;

[0037] Figure 16 Percentage graph of dendritic cells;

[0038] Figure 17 Percentage graph of Th17 cells;

[0039] Figure 18 Bioluminescence imaging graph;

[0040] Figure 19 Bioluminescence imaging numerical statistical graph;

[0041] Figure 20 Tumor weight measurement result graph;

[0042] Figure 21 Body weight change graph;

[0043] Figure 22 HE staining graph and immunocyte marker (CD8 + T cells and DC cells) staining graph;

[0044] Figure 23 For CD8 + T cell infiltration graph;

[0045] Figure 24 DC cell infiltration graph;

[0046] Figure 25 In a, the stability of pure MnO 2 nanosheets in different solutions, and in b, MnO 2Stability of @BSA nanosheets in different solutions;

[0047] Figure 26 For inhalation of MnO in Example 1 2 @BSA nanosheets and BSA-MnO of the comparative example 2 After carbon dots, fluorescence intensity comparison diagram of mouse lungs. Detailed implementation manners

[0048] The present invention provides an inhalable ultrathin nanosheet, comprising MnO 2 nanosheets and bovine serum albumin loaded on the surface of the MnO 2 nanosheets. The particle size of the inhalable ultrathin nanosheet is 100 - 300 nm, and the thickness is 1 - 5 nm.

[0049] In the present invention, the particle size range of 100 - 300 nm is an ideal particle size range for pulmonary delivery.

[0050] The effects of the inhalable ultrathin nanosheet of the present invention include the following aspects: 1. Having dual functional roles: The MnO 2 @BSA nanosheets of the present invention combine two functions of reactive oxygen species (ROS) scavenging and photothermal therapy (PTT). In the treatment of lung cancer, it can not only reduce inflammation and inhibit immune-related adverse events (irAEs), but also effectively ablate tumors. This dual role is the core advantage of the present invention, benefiting from the excellent ROS scavenging ability of MnO 2 and the stabilizing effect of BSA. As a surface complex material, BSA enhances the stability of MnO 2 nanosheets and promotes their effective deposition in the lungs; 2. Efficient pulmonary delivery and local treatment: By the inhalation administration method, MnO 2 @BSA nanosheets can effectively target the lungs and tumors, increase the local drug concentration, while reducing systemic toxicity. Smaller particles (particle size of 100 - 300 nm, thickness of 1 - 5 nm) are easy to penetrate deep into the lungs, suitable for deep pulmonary deposition, can reach the alveolar region, ensure effective local treatment effects, avoid the problem that particles larger than 5 μm are easily confined in the upper respiratory tract, and improve the pulmonary delivery efficiency; 3. Longer stability and controlled release: By loading BSA, it plays a protective role, not only improving the stability of MnO 2 nanosheets, but also reducing the reduction of treatment effects caused by oxidation or aggregation, making MnO 2 nanosheets show good dispersibility and long-term stability under physiological conditions. The MnO 2@BSA nanosheets have long-term stability in vitro and in vivo, can continuously exert their effects in the lungs, and have good stability at 48 h and 72 h, ensuring that the drug can maintain its efficacy for a long time during the treatment process; 4. Enhancement of the immune system: The MnO in the present invention 2 @BSA nanosheets can activate CD8 + T cells by inducing immunogenic cell death (ICD), enhancing the anti-tumor immune response. The mechanism is as follows: Triggering the immune system response through the combined action of photothermal effect and ROS scavenging, overcoming the limitations of single treatment methods. This advantage benefits from the combination of photothermal therapy (PTT) and anti-inflammatory mechanisms, which can effectively promote the release of tumor antigens and the activation of the immune system.

[0051] In the present invention, the particle size of the inhalable ultrathin nanosheets is preferably 150-200 nm, specifically 152.6 nm, which can achieve the best anti-tumor effect and solve the problem of irAEs in lung cancer immunotherapy.

[0052] In the present invention, the thickness of the inhalable ultrathin nanosheets can specifically be 1, 2, 3, 4 or 5 nm.

[0053] In the present invention, the mass content of MnO 2 nanosheets in the inhalable ultrathin nanosheets is preferably 70-90%, specifically 70%, 80% or 90%. The MnO 2 nanosheets, as the main active ingredient, are responsible for achieving reactive oxygen species scavenging and photothermal therapy during the treatment process, enabling the temperature of the inhalable ultrathin nanosheets to rise to 40 °C within 1-2 minutes and having a high photothermal conversion efficiency.

[0054] In the present invention, the crystal form of the MnO 2 nanosheets is preferably β-MnO 2 The β-MnO 2 has a high photothermal conversion efficiency and ROS scavenging ability, and can effectively scavenge H 2 O 2 The scavenging efficiency can reach 80%-90%.

[0055] In the present invention, the particle size of the MnO 2 nanosheets is preferably 100-300 nm, specifically 100, 150, 152.6 or 300 nm.

[0056] In the present invention, the thickness of the MnO 2 nanosheets is preferably 1-5 nm, specifically 1, 2, 3, 4 or 5 nm. The present invention has requirements for the MnO 2The source of the nanosheets is not particularly limited and can be obtained by methods well-known to those skilled in the art.

[0057] In the present invention, the mass content of bovine serum albumin in the inhalable ultrathin nanosheets is preferably 10-30%, specifically 10%, 20% or 30%. As a surface modifier, the BSA can improve the stability and dispersibility of the MnO 2 nanosheets, and at the same time help the effective delivery of the MnO 2 nanosheets in the organism and prevent the rapid clearance of the MnO 2 nanosheets in the body.

[0058] In the present invention, the BSA binds to the MnO 2 nanosheets through electrostatic interaction and hydrophobic interaction, enhancing its stability and reducing the agglomeration phenomenon, and at the same time improving the deposition efficiency in the lungs.

[0059] In the present invention, the inhalable ultrathin nanosheets are stable nanocomposites formed by loading BSA on the surface of the MnO 2 nanosheets. Taking the MnO 2 nanosheets as the core and BSA as the shell layer load, a composite material in the shape of 2D ultrathin nanosheets is formed.

[0060] In the present invention, the surface charge of the inhalable ultrathin nanosheets is preferably -16.5 to -20.9 mV, specifically -16.5, -18.7 or -20.9 mV, having good stability and being able to prevent aggregation in the body.

[0061] The present invention also provides a preparation method of the inhalable ultrathin nanosheets described in the above technical solution, including the following steps:

[0062] Mix the dispersion of the MnO 2 nanosheets and the bovine serum albumin solution and then let it stand to obtain the inhalable ultrathin nanosheets.

[0063] Preferably, the present invention disperses the MnO 2 nanosheets in a dispersion liquid to obtain a dispersion of the MnO 2 nanosheets; mixes bovine serum albumin with a buffer solution to obtain a bovine serum albumin solution; then mixes the dispersion of the MnO 2 nanosheets and the bovine serum albumin solution and then let it stand to obtain the inhalable ultrathin nanosheets.

[0064] The present invention disperses the MnO 2 nanosheets in a dispersion liquid to obtain a dispersion of the MnO 2 nanosheets.

[0065] In the present invention, the MnO2 MnO in the dispersion of nanosheets 2 The concentration of is preferably 100 - 200 μg / mL, and specifically can be 100, 150 or 200 μg / mL.

[0066] In the present invention, the dispersion is preferably phosphate buffer (PBS buffer).

[0067] The present invention preferably prepares the MnO 2 nanosheets by a solution method, and more preferably includes the following steps: Dissolve MnCl 2 in deionized water to make a MnCl solution with a concentration of 0.1 M. 2 Add hydrogen peroxide to the MnCl 2 solution, keep the temperature at room temperature for a reaction time of 4 h, and centrifuge the obtained product (the rotation speed is preferably 8000 rpm) for 10 min to remove the unreacted MnCl 2 to obtain the MnO 2 nanosheets.

[0068] The present invention mixes bovine serum albumin with a buffer solution to obtain a bovine serum albumin solution.

[0069] In the present invention, the concentration of the bovine serum albumin solution is preferably 10 - 20 mg / mL, and specifically can be 10, 15 or 20 mg / mL.

[0070] In the present invention, the buffer solution is preferably phosphate buffer (PBS buffer).

[0071] The present invention preferably adds the bovine serum albumin solution to the dispersion of the MnO 2 nanosheets.

[0072] In the present invention, the standing time is preferably 1 - 5 h, and specifically can be 1, 2, 3, 4 or 5 h, and the temperature is preferably 20 - 25 °C, and specifically can be room temperature; during the standing process, BSA binds to the surface of the MnO 2 nanosheets through electrostatic interaction and hydrophobic interaction.

[0073] After the standing is completed, the present invention preferably centrifuges the obtained material liquid to remove the unbound BSA, and then washes it with PBS buffer to thoroughly remove the unbound BSA to obtain the inhalable ultrathin nanosheets.

[0074] In the present invention, the rotation speed of the centrifugation is preferably 8000 rpm, and the time is preferably 10 min.

[0075] In the present invention, the number of washing times is preferably 3 - 5 times, and specifically can be 3, 4 or 5 times.

[0076] The present invention also provides the use of the inhalable ultrathin nanosheets described in the above technical solution or the inhalable ultrathin nanosheets prepared by the preparation method described in the above technical solution in the preparation of anti-tumor drugs. Through the inhalation administration method, MnO 2 @BSA nanosheets can effectively target the lungs and tumors, increase the local drug concentration, while reducing systemic toxicity, are suitable for deep lung deposition, can reach the alveolar region, and ensure an effective local treatment effect.

[0077] The present invention preferably uses the inhalable ultrathin nanosheets in the preparation of anti-lung cancer drugs.

[0078] The present invention also provides the use of the inhalable ultrathin nanosheets described in the above technical solution or the inhalable ultrathin nanosheets prepared by the preparation method described in the above technical solution in the preparation of drugs for treating immune-related adverse events.

[0079] MnO of the present invention 2 @BSA nanosheets trigger the immune system response through the combined action of photothermal effect and ROS scavenging, overcome the limitations of single treatment means, combine photothermal therapy with anti-inflammatory mechanisms, can effectively promote the release of tumor antigens and the activation of the immune system, and activate CD8 through inducing immunogenic cell death (ICD) + T cells, enhancing the anti-tumor immune response.

[0080] Next, the technical solutions in the present invention will be clearly and completely described in conjunction with the examples in the present invention. Obviously, the described examples are only a part of the examples of the present invention, rather than all the examples. All other examples obtained by those of ordinary skill in the art based on the examples in the present invention without making creative efforts belong to the scope of protection of the present invention.

[0081] Raw materials:

[0082] MnCl 2 (Manganese chloride), used to prepare MnO 2 Nanosheets, sourced from Sigma-Aldrich.

[0083] H 2 O 2 (30 wt% hydrogen peroxide), used as an oxidant, sourced from Sigma-Aldrich.

[0084] BSA, sourced from Sigma-Aldrich, with a molecular weight of 66 kDa.

[0085] PBS buffer, sourced from Sigma-Aldrich.

[0086] Example 1

[0087] MnO 2 Synthesis of Nanosheets

[0088] 1. Take MnCl 2 Dissolve it in deionized water to prepare a solution with a concentration of 0.1 M.

[0089] 2. Slowly add 30 wt% hydrogen peroxide and react at room temperature for 4 h to form MnO 2 nanosheets.

[0090] 3. After the reaction, obtain MnO 2 nanosheets by centrifugation (8000 rpm, 10 min).

[0091] BSA-Loaded MnO 2 Nanosheets

[0092] 1. Disperse the synthesized MnO 2 nanosheets in PBS buffer at a concentration of 100 μg / mL.

[0093] 2. Dissolve BSA in PBS solution at a concentration of 10 mg / mL and add it to the MnO 2 dispersion. Let it stand and react at room temperature for 2 h, then centrifuge (8000 rpm, 10 min), and wash the obtained solid with PBS buffer 3 times to obtain the inhalable ultrathin nanosheets. The mass content of MnO 2 nanosheets in the inhalable ultrathin nanosheets is 70%, and the mass content of bovine serum albumin is 30%.

[0094] Product Characterization:

[0095] Transmission electron microscope images of the inhalable ultrathin nanosheets at different magnifications are as Figure 1 shown. It can be seen that the inhalable ultrathin nanosheets are in a flaky structure with a thickness of 1 - 5 nm. The inset in it is a physical picture of the inhalable ultrathin nanosheet solution.

[0096] Particle Size and Dispersibility:

[0097] The particle size of MnO 2 @BSA nanosheets is measured by dynamic light scattering (DLS) to be 152.6 nm, the PDI is 0.175, and the surface charge is -18.7 mV.

[0098] Figure 2 The particle size distribution diagram of the inhalable ultrathin nanosheets shows the uniform distribution of the nanosheets, ensuring their good dispersibility.

[0099] UV-Vis Absorption Spectrum:

[0100] Through ultraviolet-visible spectroscopy ( Figure 3 ), the inhalable ultrathin nanosheets exhibited a wide range of absorption, especially in the range of 200 - 1200 nm, indicating their potential application in photothermal therapy.

[0101] Energy-dispersive X-ray spectroscopy (EDS):

[0102] Through energy-dispersive X-ray spectroscopy ( Figure 4 ), it was found that the elements Mn, O, C, and N were uniformly distributed in the inhalable ultrathin nanosheets, verifying the successful loading of MnO 2 nanosheets.

[0103] The inhalable ultrathin nanosheets were dissolved in pure water to obtain an inhalable ultrathin nanosheet solution for subsequent performance testing.

[0104] Photothermal performance:

[0105] Infrared thermal imaging:

[0106] Through infrared thermal imaging ( Figure 5 ), it was shown that the temperature of the 200 μg / mL inhalable ultrathin nanosheets increased rapidly under 1064 nm laser irradiation, showing a good photothermal effect. Compared with the water control group, the temperature of the inhalable ultrathin nanosheet solution increased to 60 °C, showing a significant photothermal effect.

[0107] Figure 6 For the photothermal effect curves of inhalable ultrathin nanosheet solutions with different concentrations, it can be seen that the inhalable ultrathin nanosheet solutions with different concentrations (25 - 200 μg / mL) showed a concentration-dependent photothermal effect under 1064 nm laser irradiation. The concentration of 200 μg / mL increased the temperature to over 60 °C within 10 min, proving its strong photothermal conversion efficiency.

[0108] Figure 7 For the photothermal cycling curve, it can be seen that the inhalable ultrathin nanosheets of the present invention can stably last for more than 5 cycles, indicating that the inhalable ultrathin nanosheets of the present invention have a stable photothermal effect and can be recycled repeatedly.

[0109] H 2 O 2 Scavenging ability:

[0110] The H 2 O 2 scavenging efficiency at different time points is shown in Figure 8 , and it can be seen that the H 2 O 2 scavenging efficiencies of the 100 μg / mL inhalable ultrathin nanosheets at 0, 1, and 2 h treatment times were 85%, 90%, and 92% respectively, showing their excellent ROS scavenging ability.

[0111] H at different concentrations 2 O 2 The scavenging efficiency is shown in Figure 9 , and it can be seen that in the 1-hour reaction, the inhalable ultrathin nanosheet solution with a concentration of 25 μg / mL to 100 μg / mL exhibits a concentration-dependent H 2 O 2 scavenging effect, among which the scavenging efficiency at a concentration of 100 μg / mL is close to 95%, significantly better than other concentration groups.

[0112] The anti-inflammatory effect of inhalable ultrathin nanosheets

[0113] Raw materials:

[0114] Inhalable ultrathin nanosheet solution with a concentration of 200 μg / mL; mice (C57BL / 6J, body weight about 20 g); anti-PD-1 antibody (α-PD1); LPS (lipopolysaccharide): used to induce immune response; PBS: used for drug administration in the control group

[0115] Experimental procedures:

[0116] Establishment of mouse model:

[0117] Inject Lewis Lung Carcinoma (LLC) cells (1×10 6 cells) into the lungs of mice to establish a lung cancer model.

[0118] On day 0, inject LPS + α-PD1 to induce immune response and initiate anti-PD-1 treatment.

[0119] Starting from day 2, administer drugs to different treatment groups according to the experimental design (intravenous injection or intratracheal administration).

[0120] Drug administration method:

[0121] Intravenous administration group (i.v.): Inject 50 μL of 5 mg / kg MnO 2 @BSA solution.

[0122] Intratracheal administration group (i.t.): Administer 50 μL of 5 mg / kg MnO 2 @BSA solution through intratracheal administration.

[0123] PBS control group: Similarly, inject PBS solution through intravenous injection or intratracheal injection.

[0124] Treatment cycle:

[0125] Drug administration lasts for 5 days, and then the mice are sacrificed and lung tissues are collected for analysis.

[0126] Experimental results:

[0127] The schematic diagram of the animal experiment is as shown in Figure 10 .

[0128] The lung-to-body weight ratio, as shown in Figure 11 , shows that the i.t. MnO 2 @BSA group significantly reduced the lung weight. Compared with the PBS control group, it indicates that the inhalable ultrathin nanosheets have the effect of reducing lung inflammation.

[0129] Histological analysis

[0130] Figure 12 showed the HE-stained sections of the lung tissues of mice in each group. The lung tissue structure of the control group (Control group) was intact; while the lungs of the i.v. MnO 2 @BSA and i.t. MnO 2 @BSA groups showed less infiltration of inflammatory cells and relatively normal alveolar structures; the i.v. PBS group and i.t. PBS group showed severe inflammatory reactions and cell infiltration, indicating that the inhalable ultrathin nanosheets have anti-inflammatory effects.

[0131] The lung injury scores are shown in Figure 13 . It can be seen that the score of i.t. MnO 2 @BSA was significantly lower than that of other treatment groups, proving that the inhalable ultrathin nanosheets can effectively reduce the lung injury caused by immunotherapy.

[0132] Flow cytometry was used to evaluate inflammatory cells

[0133] Mouse lung tissues were collected

[0134] Flow cytometry detection (FACS): To evaluate the proportions of CD45 + immune cells, neutrophils (CD11b+Ly6G+), macrophages (CD11b+F4 / 80+), dendritic cells (CD11c+), and Th17 cells (CD4+IL-17a+).

[0135] Figure 14 This is the proportion diagram of neutrophils. It can be seen that compared with the control group (Control group) and the PBS group, the proportion of neutrophils in the lung tissues of mice in the i.t. MnO 2 @BSA group decreased significantly (p<0.0001). The i.v. MnO 2 @BSA group also reduced the proportion of neutrophils, but the effect was less than that of intratracheal administration.

[0136] Figure 15 This is the proportion diagram of macrophages. It can be seen that i.t. MnO 2The proportion of macrophages in the BSA group of mice was significantly lower than that in the i.t. PBS group (p<0.05), indicating that inhalable ultrathin nanosheets administered intratracheally could inhibit the infiltration of inflammation-related macrophages, and the inhibitory effect of the intravenous administration group on macrophages was not obvious.

[0137] Figure 16 It is the proportion diagram of dendritic cells. It can be seen that compared with the control group, the proportion of dendritic cells in the i.t. MnO 2 @BSA group decreased significantly (p<0.01), indicating that inhalable ultrathin nanosheets may inhibit the inflammatory response by reducing the recruitment of antigen-presenting cells.

[0138] Figure 17 It is the proportion diagram of Th17 cells. It can be seen that Th17 cells play a key role in the pulmonary inflammatory response, and their proportion decreased significantly in the i.t. MnO 2 @BSA group (p<0.001), indicating that MnO 2 @BSA may reduce the recruitment of Th17 cells by reducing the pro-inflammatory environment, thereby reducing the immune inflammatory response.

[0139] Photothermal therapy and immune enhancement effect

[0140] Raw materials:

[0141] Inhalable ultrathin nanosheet aqueous solution with a concentration of 200 μg / mL; LLC (Lewis Lung Carcinoma) mouse model; anti-PD-1 antibody (α-PD1); PBS: control group.

[0142] Experimental steps:

[0143] Establishment of tumor model:

[0144] LLC cells (1×10 6 cells) were inoculated subcutaneously into the lungs of C57BL / 6 mice (weighing 20 g) to form a lung cancer model.

[0145] On day 0, mice were injected with LPS+α-PD1 to initiate anti-PD-1 immunotherapy.

[0146] Starting from day 2, different groups were treated, including intravenous injection (i.v.), intratracheal administration (i.t.) and PBS control group.

[0147] Treatment plan:

[0148] Intravenous injection group (i.v.): 5 mg / kg of MnO 2 @BSA nanosheet solution 50 μL was administered by intravenous injection.

[0149] Intratracheal administration group (i.t.): 5 mg / kg MnO was administered intratracheally 2 @BSA solution 50 μL.

[0150] PBS control group: PBS solution was also injected intravenously or intratracheally.

[0151] Treatment period:

[0152] The administration lasted for 7 days, and the tumor progression, body weight changes, and immune responses of the mice were observed every 2 days.

[0153] Experimental results:

[0154] Tumor growth:

[0155] Bioluminescence imaging showed Figure 18 , it can be seen that after photothermal therapy (i.t. MnO 2 @BSA group), the tumors of the mice were significantly inhibited, especially on the 5th and 7th days, and the bioluminescence intensity was significantly lower than that of the control group and the i.v. MnO 2 @BSA group.

[0156] The statistical values of bioluminescence imaging are shown in Figure 19 , it can be seen that after photothermal therapy, the tumors of the mice in the i.t. MnO 2 @BSA group were significantly inhibited.

[0157] The results of tumor weight measurement are shown in Figure 20 , it can be seen that the tumor weight of the i.t. MnO 2 @BSA group was significantly smaller than that of the i.v. MnO 2 @BSA group and the control group, demonstrating that photothermal therapy has a significant effect on tumor inhibition.

[0158] Body weight changes are shown in Figure 21 , it can be seen that the body weight changes of the mice in different groups were relatively small, indicating that no significant toxic reactions were observed during the treatment.

[0159] Histological analysis:

[0160] HE staining images and immune cell markers (CD8 + T cells and DC cells) are shown in Figure 22 , it can be seen that the tumor tissues of the i.t. MnO 2 @BSA group were relatively normal, and the number of tumor cells was significantly reduced, with a milder inflammatory response. In contrast, the control group had a larger number of tumor cells and more severe inflammatory cell infiltration; in the tumor tissues of the i.t. MnO 2 @BSA group, the number of CD8 + T cells (red) and dendritic cells (green) increased significantly.

[0161] Immune response:

[0162] CD8 + T cell infiltration was observed Figure 23 , it can be seen that i.t.MnO 2 In the tumor tissues of mice in the @BSA group, the number of CD8 + T cells increased significantly, indicating that photothermal therapy effectively activated the anti-tumor immune response.

[0163] DC cell infiltration was observed Figure 24 , it can be seen that in i.t.MnO 2 @BSA group, dendritic cells (DC cells) increased significantly, indicating that photothermal therapy can not only activate T cells, but also promote antigen presentation and enhance the immune response.

[0164] Stability test

[0165] Figure 25 In a, pure MnO 2 The stability of nanosheets in different solutions was observed. It can be seen that in PBS and DMEM solutions, MnO 2 nanosheets aggregated and precipitated and could not remain stable. In b, the stability of MnO 2 @BSA nanosheets in different solutions was observed. It can be known that the MnO 2 @BSA prepared in the present invention remained stable for a long time.

[0166] Comparative example

[0167] Protein-coated manganese dioxide nanoprobes (BSA-MnO 2 carbon dots) were synthesized in one step by a simple redox reaction using bovine serum albumin and potassium permanganate. The mass content of manganese dioxide in BSA-MnO 2 carbon dots was 70%, and the mass content of bovine serum albumin was 30%.

[0168] Raw materials:

[0169] The aqueous solution of Cy5-labeled MnO 2 @BSA nanosheets (mass concentration 200 μg / mL) in Example 1 and the aqueous solution of Cy5-labeled BSA-MnO 2 carbon dots (comparative example, mass concentration 200 μg / mL); the experimental animals were healthy adult C57BL / 6J mice.

[0170] Administration method:

[0171] Intratracheal administration of 5 mg / kg of MnO 2 @BSA nanosheet aqueous solution or BSA-MnO 2Aqueous solution of carbon dots.

[0172] Inhale the MnO of Example 1 2 @BSA nanosheet aqueous solution and BSA-MnO of the comparative example 2 After the aqueous solution of carbon dots, the comparison of the fluorescence intensity in the lungs of mice is as Figure 26 shown, indicating that MnO 2 The fluorescence intensity of @BSA nanosheets is significantly higher than that of BSA-MnO 2 carbon dots (p<0.0001). This indicates that MnO 2 @BSA nanosheets are more efficiently deposited in the lungs of mice and have stronger bioavailability. In contrast, BSA-MnO 2 The fluorescence signal of carbon dots is low, indicating that they may be easily excreted due to their too small size and cannot effectively remain in the lungs. This result supports the conclusion of MnO 2 @BSA nanosheets in the advantage of pulmonary drug delivery, especially in enhancing the therapeutic effect of the lungs.

[0173] The above is only the preferred embodiment of the present invention and does not impose any form of limitation on the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches should also be regarded as the protection scope of the present invention.

Claims

1. An inhalable ultrathin nanosheet, characterized in that: The invention comprises a MnO2 nanosheet and bovine serum albumin loaded on the surface of the MnO2 nanosheet. The particle size of the inhalable ultra-thin nanosheet is 100-300nm and the thickness is 1-5nm.

2. The inhalable ultrathin nanosheet according to claim 1, characterized in that The particle size of the inhalable ultra-thin nanosheet is 150-200 nm.

3. The inhalable ultrathin nanosheet according to claim 1, characterized in that The mass content of the MnO2 nanosheets in the inhalable ultra-thin nanosheets is 70-90%, and the mass content of the bovine serum albumin is 10-30%.

4. The inhalable ultrathin nanosheet according to claim 1, characterized in that The crystal form of the MnO2 nanosheets is β-MnO2.

5. The method for preparing the inhalable ultrathin nanosheets according to any one of claims 1 to 4, characterized in that: The following steps are involved: The dispersion of MnO2 nanosheets and the bovine serum albumin solution are mixed and then allowed to stand to obtain the inhalable ultrathin nanosheets.

6. The preparation method according to claim 5, characterized in that: The concentration of MnO2 in the dispersion of the MnO2 nanosheets is 100-200 μg / mL.

7. The preparation method according to claim 5, characterized in that: The concentration of the bovine serum albumin solution is 10-20 mg / mL.

8. The preparation method according to claim 5, characterized in that: The standing time is 1 to 5 hours, and the temperature is 20 to 25°C.

9. Use of the inhalable ultrathin nanosheet according to any one of claims 1 to 4 or the inhalable ultrathin nanosheet prepared by the preparation method according to any one of claims 5 to 8 in the preparation of anti-tumor drugs.

10. Use of the inhalable ultrathin nanosheet according to any one of claims 1 to 4 or the inhalable ultrathin nanosheet prepared by the preparation method according to any one of claims 5 to 8 in the preparation of drugs for treating immune-related adverse events.

Images