A lung-targeted nanocomposite, a preparation method and application thereof

Abstract

The application discloses a lung-targeted nanocomposite and a preparation method and application thereof, wherein the nanocomposite comprises carbon nitride two-dimensional nanosheets and AIE photosensitizers loaded on surfaces of the carbon nitride two-dimensional nanosheets. The nanocomposite has excellent aggregation-induced emission properties and active oxygen generation capacity, can play a diagnostic role of the photosensitizer, and can produce a better tumor elimination effect, so that the diagnosis and treatment of lung cancer can be simultaneously realized. In addition, the nanocomposite has a suitable size, can enhance the retention of drugs in the lung, prolong the treatment time, and reduce the administration frequency; the nanocomposite can also be administered through tracheal spray, has the advantages of rapid onset, high local drug concentration, small drug dosage, convenient use, less systemic adverse reactions, easy monitoring and adjustment of the dosage, and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a lung-targeting nanocomposite material, its preparation method, and its application. Background Technology

[0002] The metastatic, spreading, and fatal nature of lung cancer makes lung cancer research of great significance. However, traditional treatments, such as chemotherapy and radiotherapy, often fail to specifically target the lungs and have significant side effects, resulting in poor treatment outcomes. Traditional imaging techniques, such as CT and MRI, suffer from low resolution and poor accuracy, particularly in their inability to accurately identify subtle early-stage cancerous tissues, leading to delays in diagnosis. Furthermore, traditional diagnostic and treatment methods cannot be implemented simultaneously, which greatly limits the efficiency of lung cancer diagnosis and treatment.

[0003] Therefore, existing technologies still need to be improved and developed. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the purpose of this invention is to provide a lung-targeting nanocomposite material, its preparation method and application, in order to solve the problem that existing lung-targeting drugs cannot simultaneously achieve diagnosis and treatment.

[0005] The technical solution of the present invention is as follows:

[0006] In a first aspect, the present invention provides a lung-targeting nanocomposite material comprising two-dimensional carbon nitride nanosheets and an AIE photosensitizer loaded on the surface of the two-dimensional carbon nitride nanosheets.

[0007] Optionally, the mass ratio of the carbon nitride two-dimensional nanosheets to the AIE photosensitizer is 1-10:1.

[0008] Optionally, the planar dimensions of the carbon nitride two-dimensional nanosheets are 0.5-2 μm.

[0009] Optionally, the structure of the AIE photosensitizer is selected from... One of them.

[0010] Optionally, the carbon nitride two-dimensional nanosheets are proton-functionalized graphitic carbon nitride.

[0011] Optionally, the AIE photosensitizer is loaded onto the surface of the carbon nitride two-dimensional nanosheets via electrostatic force.

[0012] A second aspect of the present invention provides a method for preparing a nanocomposite material as described in the first aspect, comprising:

[0013] Carbon nitride two-dimensional nanosheets and AIE photosensitizer were dissolved in a mixed solution of organic and aqueous phases, and the mixture was stirred to obtain the nanocomposite material.

[0014] Optionally, the temperature of the stirring reaction is 10-35℃ and the time is 12-24h.

[0015] Optionally, after the stirring reaction, the process further includes washing with water and filtering.

[0016] A third aspect of the present invention provides the use of the nanocomposite material as described in the first aspect in the preparation of medicaments for treating and diagnosing lung cancer.

[0017] Beneficial Effects: This invention provides a lung-targeting nanocomposite material, its preparation method, and its application. The nanocomposite material comprises two-dimensional carbon nitride nanosheets and an AIE photosensitizer loaded on the surface of the carbon nitride nanosheets. The nanocomposite material of this invention possesses excellent aggregation-induced emission properties and reactive oxygen species (ROS) generation capabilities, enabling it to exert both the diagnostic function of a photosensitizer and a good tumor elimination effect, thus simultaneously achieving the diagnosis and treatment of lung cancer. Furthermore, the nanocomposite material has a suitable size, which enhances drug retention in the lungs, prolongs treatment time, and reduces the frequency of administration. It can also be administered via tracheal spray, offering advantages such as rapid onset of action, high local drug concentration, low dosage, ease of use, few systemic adverse reactions, and easy monitoring and dosage adjustment. Attached Figure Description

[0018] Figure 1 It is g-C3N4 in Embodiment 1 of the present invention + Scanning electron microscope image of nanosheets.

[0019] Figure 2 This is a graph showing the fluorescence intensity changes of C3N4@TDP at different proportions in Example 1 of the present invention.

[0020] Figure 3 This is a graph showing the test results of the reactive oxygen species generation capacity of C3N4, TDP, and C3N4@TDP in Example 1 of the present invention.

[0021] Figure 4 This is a graph showing the gas production capacity test results of C3N4, TDP, and C3N4@TDP in Embodiment 1 of the present invention.

[0022] Figure 5 This is a graph showing the results of cell dark toxicity and phototoxicity tests of C3N4, TDP, and C3N4@TDP in Example 2 of this invention.

[0023] Figure 6 This is a graph showing the results of the lung retention experiment of TDP and C3N4@TDP in Embodiment 3 of the present invention.

[0024] Figure 7 This is a graph showing the experimental results of C3N4@TDP in mouse lung cancer treatment in Example 4 of this invention. Detailed Implementation

[0025] This invention provides a lung-targeting nanocomposite material, its preparation method, and its application. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0026] Photosensitizers with aggregation-induced emission (AIE) properties are emerging and promising materials for integrated cancer diagnosis and treatment. In 2001, Professor Ben-Chung Tang, Chair Professor at the Hong Kong University of Science and Technology, proposed the concept of aggregation-induced emission and developed a series of compounds with this property. When well dispersed, upon excitation, the rotors rotate actively or the flexible parts vibrate, leading to non-radiative inactivation of the excited-state molecules. When aggregated, the tight arrangement of molecules restricts intramolecular motion, hindering non-radiative decay paths, thus allowing fluorescence emission in the aggregated state. Compared to traditional aggregation-induced quenching (ACQ) materials, AIE materials exhibit high luminescence efficiency and good photostability, showing great potential for visual tracking of metastatic lung cancer. Furthermore, some AIE photosensitizers with specific structures also possess phototherapy effects. Under excitation light stimulation, they generate cytotoxic reactive oxygen species (ROS) (photodynamic therapy, PDT) or heat (photothermal therapy, PTT), thereby killing tumors. These agents offer advantages such as non-invasiveness, spatiotemporal selectivity, tumor specificity, and high efficiency. The lungs are semi-transparent organs with abundant alveolar tissue, which greatly facilitates light penetration and internal scattering. AIE photosensitizers can effectively label and efficiently kill tumor tissue, achieving targeted treatment and providing a promising new approach for integrated diagnosis and treatment of lung cancer.

[0027] Two-dimensional nanosheets, with their anisotropy, rich surface properties, and unique functionality, have become the best candidate material for lung-targeting carriers of photosensitizers in acute lung injury (AIE). First, two-dimensional nanosheets show great potential for lung targeting. Second, their two-dimensional size and interlayer distance can be controlled through a simple ultrasonic exfoliation method, resulting in a large specific surface area and abundant functional groups and active sites on the surface. This allows for effective enhancement of drug loading through π-π interactions or hydrophilic-hydrophobic interactions, leading to excellent performance in drug accumulation. Finally, two-dimensional nanosheets are not only carrier materials but also possess many functionalities, such as photothermal properties, photocatalytic properties, and electrical conductivity, demonstrating unique performance in controlled drug release, radiotherapy, and immunotherapy. The combination of two-dimensional nanosheets and AIE photosensitizers has been reported before. For example, Tang Benzhong's team found that the surface of monolayer or few-layer transition metal sulfides has an aggregation effect on AIE molecules. The AIE aggregates (DDTA) interact with monolayer MoS2 nanosheets, resulting in fluorescence enhancement or quenching. In addition, the interlayer voids of multilayer α-ZrP nanosheets have a confinement effect on the embedded AIE photosensitizer. By matching the α-ZrP interlayer spacing and the AIE molecule size, the fluorescence intensity of the system can be effectively enhanced. Our team also found that PEG-modified AIE photosensitizer (PEG-TTPy) combined with black phosphorus through electrostatic force gives the system good biocompatibility and even mitochondrial targeting. The composite material combines the fluorescence imaging effect and reactive oxygen generation capacity of PEG-TTPy with the photothermal therapeutic effect of black phosphorus, thus achieving the effect of eliminating tumors.

[0028] Therefore, it is evident that AIE photosensitizers and two-dimensional nanosheets are the "best partners," but there are currently no reports on their application in the field of lung-targeted integrated diagnosis and treatment.

[0029] Based on this, embodiments of the present invention provide a lung-targeting nanocomposite material, comprising two-dimensional carbon nitride nanosheets and an AIE photosensitizer loaded on the surface of the two-dimensional carbon nitride nanosheets.

[0030] In one embodiment, the mass ratio of the carbon nitride two-dimensional nanosheets to the AIE photosensitizer is 1-10:1; for example, it can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc.; in a preferred embodiment, the mass ratio of the carbon nitride two-dimensional nanosheets to the AIE photosensitizer is 5:1.

[0031] In one embodiment, the planar size of the carbon nitride two-dimensional nanosheet is 0.5-2 μm; for example, it can be 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, 1.0 μm, 1.1 μm, 1.3 μm, 1.5 μm, 1.7 μm, 2.0 μm, etc.; in a preferred embodiment, the planar size of the carbon nitride two-dimensional nanosheet is 1.0 μm.

[0032] In one embodiment, the structure of the AIE photosensitizer is selected from... One of the following, but not limited to, can be replaced with other types of AIE photosensitizers; in a preferred embodiment, the structure of the AIE photosensitizer is as follows:

[0033]

[0034] In one embodiment, the carbon nitride two-dimensional nanosheets are proton-functionalized graphitic carbon nitride.

[0035] In one embodiment, when the carbon nitride two-dimensional nanosheet is proton-functionalized graphitic carbon nitride, the AIE photosensitizer is loaded onto the surface of the carbon nitride two-dimensional nanosheet by electrostatic force.

[0036] This invention also provides a method for preparing the nanocomposite material as described above, comprising:

[0037] Carbon nitride two-dimensional nanosheets and AIE photosensitizer were dissolved in a mixed solution of organic and aqueous phases, and the mixture was stirred to obtain the nanocomposite material.

[0038] In one embodiment, the temperature of the stirring reaction is 10-35°C; for example, it can be 10°C, 12°C, 15°C, 18°C, 20°C, 22°C, 25°C, 27°C, 30°C, 32°C, 35°C, etc.

[0039] In one embodiment, the stirring reaction time is 12-24 hours; for example, it can be 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, etc.

[0040] In one embodiment, the reaction is followed by washing with water and filtration.

[0041] In one embodiment, the organic phase is selected from one or more of methanol, ethanol, tetrahydrofuran (THF), and dimethyl sulfoxide (DMSO), but is not limited thereto; in a preferred embodiment, the organic phase is DMSO.

[0042] In one embodiment, the ratio of the organic phase to the aqueous phase in the mixed solution is 1:9 to 9:1; for example, it can be 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, etc.; in a preferred embodiment, the ratio of the organic phase to the aqueous phase is 5:5.

[0043] In one embodiment, the carbon nitride two-dimensional nanosheets and AIE photosensitizer are dissolved in a mixed solution of an organic phase and an aqueous phase, and the concentration of the carbon nitride two-dimensional nanosheets in the mixed solution is 10-100 μg / mL; for example, it can be 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL.

[0044] In one embodiment, the carbon nitride two-dimensional nanosheets and AIE photosensitizer are dissolved in a mixed solution of an organic phase and an aqueous phase, and the concentration of AIE photosensitizer in the mixed solution is 1-10 μg / mL; for example, it can be 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, 7 μg / mL, 8 μg / mL, 9 μg / mL, or 10 μg / mL.

[0045] In one specific technical solution of the present invention, the following steps are included:

[0046] Graphitic carbon nitride (g-C3N4) was prepared by calcining dicyandiamide at 550℃ under an inert atmosphere at a heating rate of 2.3℃ / min, and then held at 550℃ for 4-5 hours to obtain bulk g-C3N4 material. At room temperature, 1-2 g of bulk g-C3N4 was protonated by vigorous stirring in 20-50 mL of 8-10 M HCl for 1-2 hours. The solution was filtered and repeatedly washed with water to remove the ultra-concentrated HCl. Then, 20-50 mg of protonated g-C3N4 was dispersed in 100-200 mL of deionized water and sonicated for 1-3 hours. The sonicated mixture was centrifuged at 3000 rpm to remove unexfoliated aggregates, leaving a uniform aqueous dispersion of proton-functionalized g-C3N4 nanosheets.

[0047] An aqueous solution of the above proton-functionalized g-C3N4 nanosheets was continuously sonicated for 1-3 hours to obtain g-C3N4 nanosheets with a size of approximately 0.5-2 μm. + Nanosheets;

[0048] Take 1-20 mL of g-C3N4 + The nanosheet aqueous solution (10-100 μg / mL) and 1-20 mL of TDP aqueous solution (1-10 μg / mL) were added to 1-50 mL of DMSO and stirred at room temperature for 12-24 h. The mixture was then repeatedly washed with water and filtered to obtain the nanocomposite material.

[0049] This invention provides an application of the nanocomposite material described above in the preparation of drugs for treating and diagnosing lung cancer.

[0050] The present invention will be further described below through specific embodiments.

[0051] Example 1

[0052] This embodiment provides a lung-targeting nanocomposite material, and evaluates its spectroscopic properties, photo-induced reactive oxygen species (ROS) generation capacity, and gas production capacity. Details are as follows:

[0053] (1) Preparation of nanocomposite materials

[0054] ①Preparation of AIE photosensitizer

[0055] The preparation method of the AIE photosensitizer is described in patent CN 115490846 A. In this embodiment, the structural formula of the AIE photosensitizer (TDP) is shown below:

[0056]

[0057] ② Preparation of carbon nitride two-dimensional nanosheets

[0058] Graphitic carbon nitride (g-C3N4) was prepared by calcining dicyandiamide at 550 °C under an inert atmosphere at a heating rate of 2.3 °C / min, and then held at 550 °C for 4 hours to obtain bulk g-C3N4 material. At room temperature, 1 g of bulk g-C3N4 was protonated by vigorous stirring in 25 mL of 10 M HCl for 1 hour. The solution was filtered and repeatedly washed with water to remove the ultra-concentrated HCl. Then, 50 mg of protonated g-C3N4 was dispersed in 100 mL of deionized water and sonicated for 2 hours. The sonicated mixture was centrifuged at 3000 rpm to remove unexfoliated aggregates, leaving a uniform aqueous dispersion of proton-functionalized g-C3N4 nanosheets.

[0059] An aqueous solution of the above proton-functionalized g-C3N4 nanosheets was continuously sonicated for about 2 hours to obtain g-C3N4 nanosheets with a size of about 1 μm. + Nanosheets, such as Figure 1 As shown.

[0060] ③ Preparation of nanocomposite materials

[0061] Take 10 mL of g-C3N4 at different concentrations + Nanosheet aqueous solutions (10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL) and 10 mL TDP aqueous solution (10 μg / mL) were added to 20 mL DMSO and stirred at room temperature for 24 h. After repeated washing with water and filtration, C3N4@TDP in different proportions were obtained, which are nanocomposites.

[0062] (2) Spectral property testing

[0063] The aggregation-induced emission properties of TDP (10 μM) in C3N4@TDP with different proportions were evaluated, and their fluorescence spectra are shown below. Figure 2 As shown. By Figure 2 It can be seen that the aggregation-induced emission properties of TDP in different proportions of C3N4@TDP are linearly related to the proportion.

[0064] Subsequent experiments were conducted using C3N4@TDP with a ratio of C3N4:TDP = 5:1.

[0065] (3) Assessment of photo-induced reactive oxygen species generation capacity

[0066] The reactive oxygen species (ROS) generation capacity of C3N4, TDP, and C3N4@TDP was evaluated using DCFH-DA as an indicator. 0.5 mL of DCFH-DA (0.001 M) ethanol solution was added to 2 mL of NaOH (0.01 M) solution for activation to obtain DCFH. The pH of the solution was adjusted by adding 10 mL of PBS (pH 7.4), and the solution was stored in the dark. The ROS generation capacity of C3N4, TDP, and C3N4@TDP (0.2 μM) was evaluated using DCFH (5 μM) in PBS (pH 7.4). Irradiation was performed using a 660 nm laser, and the fluorescence intensity at 525 nm at different time points under 488 nm excitation was recorded using a fluorescence spectrophotometer to obtain the fluorescence enhancement factor. Experimental results are as follows: Figure 3 As shown. By Figure 3 It can be seen that C3N4@TDP has a strong ROS generation capability under 660nm laser irradiation.

[0067] (3) Assessment of gas production capacity

[0068] The ability of C3N4, TDP, and C3N4@TDP to produce CO, CH4, H2, and O2 was determined by gas chromatography. The experimental results are as follows: Figure 4 As shown. By Figure 4 It can be seen that the gas production capacity of C3N4@TDP is significantly enhanced, and further enhanced under CO2 supply conditions.

[0069] Example 2

[0070] This embodiment provides an experiment demonstrating the anti-proliferative effect of phototherapy on 4T1 breast cancer cells using C3N4@TDP (5:1) as described in Example 1. Details are as follows:

[0071] The test cells in this embodiment were 4T1 breast cancer cells; the test drugs were C3N4, TDP, and C3N4@TDP; and the light source used was a 660nm laser.

[0072] Cells in the logarithmic growth phase were digested with trypsin, resuspended in complete culture medium to form a cell suspension, and then subjected to a 5 × 10⁻⁶ ppm incubator.3 Seeds were planted at a density of cells / well in 96-well plates and incubated at 37°C in a 5% CO2 incubator. After 24 h, different concentrations of C3N4, TDP, and C3N4@TDP were added to achieve final concentrations of 0.5, 1, 2, 3, 4, and 5 μM, respectively. The plates were incubated for 12 h, followed by illumination (0.3 W / cm²). 2 The cells were exposed to a 660nm laser for 5 minutes. Simultaneously, a control group under the same experimental conditions but without light exposure underwent dark toxicity studies. After culturing for another 12 hours, the cells were washed three times with PBS solution, then cultured in fresh medium containing 10% CCK-8 without FBS for 2 hours in the dark. The absorbance (OD) value at 450nm was then measured using a microplate reader. The corresponding cell viability was calculated using the following formula: Cell viability (%) = (OD sample - OD background) / (OD control - OD background) × 100%. The experimental results are as follows: Figure 5 As shown. By Figure 5 It was found that at a drug concentration of 100 μg / mL, the cell survival rate in the control group not exposed to laser irradiation exceeded 95%, demonstrating that the compound has low dark toxicity and good biocompatibility. In the C3N4@TDP group, the cell survival rate was almost zero after laser irradiation, proving that C3N4@TDP has a significant phototherapy-induced anti-proliferative effect on 4T1 breast cancer cells.

[0073] Example 3

[0074] This embodiment provides a retention experiment of C3N4@TDP(5:1) in the lungs as described in Example 1. Details are as follows:

[0075] TDP or C3N4@TDP in PBS solution (1 mg / mL, 25 μL) was injected into the lungs of mice with lung cancer via intratracheal spray. Mice were dissected at different time points, and fluorescence imaging of residual molecules in the mouse lungs was performed using a small animal imaging system. Experimental results are as follows: Figure 6 As shown. By Figure 6 It is known that, compared to TDP molecules, C3N4@TDP exhibits better retention in lung cancer tissue, which is beneficial for prolonging the therapeutic window and allowing for gradual elimination through metabolism within 72 hours. This is because the larger diameter of the two-dimensional nanosheets can hinder the passage of small molecules through lung epithelial cells, thereby extending the metabolic time.

[0076] Example 3

[0077] This embodiment provides an evaluation of the therapeutic effect of C3N4@TDP(5:1) on lung cancer in Example 1. Details are as follows:

[0078] C3N4@TDP in PBS solution (1 mg / mL, 25 μL) was injected into the lungs of mice with lung cancer via intratracheal spray, followed by laser irradiation of the lungs (660 nm laser, 0.3 W / cm²). 2 (5 min), and then bioluminescent imaging of lung cancer tissue was performed using a small animal imaging system. It was found that after treatment with C3N4@TDP, the lung cancer tissue in mice gradually disappeared, and there was no recurrence within 15 days, proving that C3N4@TDP has a good tumor elimination effect.

[0079] In summary, this invention provides a lung-targeted nanocomposite material, its preparation method, and its applications. The nanocomposite material comprises two-dimensional carbon nitride nanosheets and an AIE photosensitizer loaded on the surface of the carbon nitride nanosheets. The nanocomposite material of this invention possesses excellent aggregation-induced emission properties and reactive oxygen species (ROS) generation capabilities, enabling it to exert both the diagnostic function of a photosensitizer and a good tumor elimination effect, thus simultaneously achieving the diagnosis and treatment of lung cancer. Furthermore, the nanocomposite material has a suitable size, which enhances drug retention in the lungs, prolongs treatment time, and reduces the frequency of administration. It can also be administered via tracheal spray, offering advantages such as rapid onset of action, high local drug concentration, low dosage, ease of use, few systemic adverse reactions, and ease of monitoring and dosage adjustment.

[0080] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A nanocomposite material for lung targeting via endotracheal spray administration, characterized in that, It includes two-dimensional carbon nitride nanosheets and an AIE photosensitizer loaded on the surface of the two-dimensional carbon nitride nanosheets; The mass ratio of the carbon nitride two-dimensional nanosheets to the AIE photosensitizer is 1-10:1; The carbon nitride two-dimensional nanosheets are proton-functionalized graphitic carbon nitride. The AIE photosensitizer is loaded onto the surface of the carbon nitride two-dimensional nanosheets by electrostatic force; The planar dimensions of the carbon nitride two-dimensional nanosheets are 0.5-2 μm; The structure of the AIE photosensitizer is selected from... , , , and One of them.

2. A method for preparing the nanocomposite material as described in claim 1, characterized in that, include: Carbon nitride two-dimensional nanosheets and AIE photosensitizer were dissolved in a mixed solution of organic and aqueous phases, and the mixture was stirred to obtain the nanocomposite material.

3. The method for preparing the nanocomposite material according to claim 2, characterized in that, The stirring reaction is carried out at a temperature of 10-35℃ for 12-24 hours.

4. The method for preparing the nanocomposite material according to claim 3, characterized in that, The reaction process, including stirring, also includes washing with water and filtration.

5. The use of the nanocomposite material as described in claim 1 in the preparation of medicaments for the treatment and diagnosis of lung cancer.

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