A natural ingredient-based diagnosis and treatment integrated nano platform, a preparation method and application thereof

Abstract

The application discloses a kind of diagnosis and treatment integration nano platform based on natural ingredient and its preparation method and application, it is related to biomedical technical field.The method includes the natural melanin nanoparticles extracted from cuttlefish ink sac, obtains CINPs water dispersion;Curcumin is dissolved in organic solvent, dropwise added to CINPs water dispersion, obtains Cur@CINP nanoparticle;Cur@CINP nanoparticle is mixed with mannose modified polyetherimide and phenylboronic acid modified polyetherimide in buffer and incubated, by cross-linking reaction, mannose modified polyetherimide and phenylboronic acid modified polyetherimide are collectively modified to Cur@CINP nanoparticle surface, and product C@C-M nanoparticle is obtained.The application integrates the " diagnosis and treatment integration " nano platform of treatment and real-time monitoring function, to solve the problem of low drug delivery efficiency in ALI treatment, not accurate immune microenvironment regulation and the difficulty that curative effect is difficult to real-time evaluation in vivo.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a diagnostic and therapeutic nanoplatform based on natural ingredients, its preparation method, and its applications. Background Technology

[0002] Acute lung injury (ALI) and its severe form ARDS are common critical syndromes in clinical practice, characterized by uncontrolled lung inflammation, alveolar-capillary membrane damage, and refractory hypoxemia. The core pathological mechanism lies in the imbalance of the pulmonary immune microenvironment, in which the phenotypic polarization of alveolar macrophages plays a crucial regulatory role. In the early stages of injury, macrophages polarize into a pro-inflammatory, tissue-destructive M1 phenotype, releasing large amounts of pro-inflammatory factors such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), as well as reactive oxygen species (ROS), exacerbating the inflammatory cascade. During the repair phase, macrophages need to polarize into an anti-inflammatory, tissue-repairing M2 phenotype, secreting interleukin-10 (IL-10) and arginase-1 (Arg-1) to promote inflammation resolution and tissue healing. Therefore, reprogramming pulmonary macrophages from the M1 phenotype to the M2 phenotype is considered a promising new strategy for the treatment of ALI.

[0003] Currently, clinical treatment for ALI still lacks specific drugs that can directly regulate the immune microenvironment. Curcumin, a natural active molecule, possesses clear anti-inflammatory and antioxidant activities, but its extremely poor water solubility, rapid in vivo metabolism, low bioavailability, and lack of targeting severely limit its therapeutic efficacy. In recent years, the combination of nanotechnology and biomaterials has provided new ideas for overcoming these challenges. Among them, photothermal therapy (PTT), especially mild photothermal therapy (≈40-45℃), has attracted attention because it can non-invasively affect cell behavior and regulate the immune microenvironment. Studies have shown that moderate thermal stimulation in suitable functional nanobiomaterials can affect the metabolism and function of macrophages, with the potential to induce their polarization towards the M2 phenotype, providing a new avenue for the immunotherapy of ALI.

[0004] Meanwhile, precise diagnosis and treatment require real-time, non-invasive assessment of lesion status and treatment response. Traditional imaging methods, such as X-ray computed tomography (CT), involve radiation and cannot perform functional imaging; magnetic resonance imaging (MRI) is costly and time-consuming; and fluorescence imaging has limited penetration depth. Photoacoustic imaging (PAI), as an emerging hybrid modality imaging technology, combines the high contrast of optical imaging with the deep penetration of ultrasound imaging. It can non-invasively and label-free visualize biological tissue function and molecular information, providing unique advantages for dynamically monitoring lung inflammation and drug distribution. Summary of the Invention

[0005] To address the shortcomings of the aforementioned background technologies, this invention primarily solves the problems of low drug delivery efficiency, imprecise regulation of the immune microenvironment, and difficulty in real-time in vivo efficacy assessment in existing ALI treatments. This invention provides a therapeutic nanoplatform based on natural ingredients, its preparation method, and its applications.

[0006] The first objective of this invention is to provide a method for preparing a diagnostic and therapeutic nanoplatform based on natural ingredients, comprising the following steps: Natural melanin nanoparticles extracted from squid ink sacs were dispersed in an aqueous solution to obtain a CINPs aqueous dispersion. Curcumin was dissolved in ethanol and added dropwise to a CINPs aqueous dispersion. The mixture was stirred in the dark to allow curcumin to be adsorbed onto the surface of natural melanin nanoparticles through non-covalent interactions. After centrifugation and purification, Cur@CINP nanoparticles were obtained. Mannose-modified polyetherimide solution and phenylboronic acid-modified polyetherimide solution were synthesized respectively. Cur@CINP nanoparticles were mixed and incubated with mannose-modified polyetherimide solutions and phenylboronic acid-modified polyetherimide solutions. Through a cross-linking reaction, the mannose-modified polyetherimide solution and the phenylboronic acid-modified polyetherimide solution were co-modified onto the surface of the Cur@CINP nanoparticles. After purification, the product C@CM nanoparticles, namely the therapeutic nanoplatform, were obtained.

[0007] Preferably, the synthesis process of the mannose-modified polyetherimide solution includes: Mannose was dissolved in methanol to obtain a mannose solution; Polyetherimide was dissolved in methanol, preheated in an oil bath to 45-55°C, and then mannose solution was added. The mixture was stirred and reacted in an oil bath at 45-55°C for 12-24 hours to obtain mannose-modified polyetherimide. The obtained mannose-modified polyetherimide was uniformly dispersed in methanol to obtain a mannose-modified polyetherimide solution; The mass ratio of mannose to polyetherimide is 1:5~10.

[0008] Preferably, the synthesis process of the phenylboronic acid-modified polyetherimide solution includes: Phenylated acid was dissolved in methanol to obtain a phenylboronic acid solution; Polyetherimide was dissolved in methanol, and after adding phenylboronic acid solution, it was stirred in an oil bath at 65-75°C for 12-24 hours. After purification with diethyl ether, phenylboronic acid-modified polyetherimide was obtained. The obtained phenylboronic acid-modified polyetherimide was uniformly dispersed in methanol to obtain a phenylboronic acid-modified polyetherimide solution; The mass ratio of phenylboronic acid to polyetherimide is 1:0.5~2.

[0009] Preferably, the Cur@CINP nanoparticles are mixed and incubated with a mannose-modified polyetherimide solution and a phenylboronic acid-modified polyetherimide solution, comprising: Curcumin was dissolved in ethanol and added dropwise to CINP aqueous dispersion under continuous stirring. After reacting for 12-24 hours, a phenylboronic acid-modified polyetherimide solution was added, and the reaction was continued for another 12-24 hours. The product was collected by centrifugation to obtain the precipitate. The precipitate was dispersed in water, and a mannose-modified polyetherimide solution was added with stirring. The reaction was carried out for 12 to 24 hours.

[0010] Preferably, the concentration of the mannose-modified polyetherimide solution is 0.5~2 mg / mL; the concentration of the phenylboronic acid-modified polyetherimide solution is 2~5 mg / mL.

[0011] Preferably, the organic solvent is ethanol.

[0012] Preferably, the concentration of the CINP aqueous dispersion is 0.5~2 mg / mL.

[0013] Preferably, the natural melanin nanoparticles extracted from the ink sac of squid include: Cuttlefish bile and ultrapure water were mixed evenly in a centrifuge tube and then centrifuged at 2000-4000 rpm for 3-6 minutes to remove large aggregates and insoluble impurities. The supernatant was collected and then centrifuged at 7000-9000 rpm for 8-12 minutes. The precipitate was retained and redispersed in water. The centrifugation at 8000 rpm for 8-12 minutes was repeated several times. The final precipitate was the purified melanin nanoparticles (CINPs).

[0014] The second objective of this invention is to provide a diagnostic and therapeutic nanoplatform based on natural ingredients.

[0015] The third objective of this invention is to provide an application of a natural component-based therapeutic nanoplatform in the preparation of photoacoustic imaging contrast agents for the treatment of acute lung injury, acute respiratory distress syndrome, or the imaging diagnosis and efficacy monitoring of acute lung injury.

[0016] This invention provides a diagnostic and therapeutic nanoplatform based on natural ingredients, its preparation method, and its application. Compared with existing technologies, it has the following advantages: Innovative from the source, safe and reliable: The core components CINPs and Cur are derived from natural edible substances, which fundamentally ensures the material's high biocompatibility, low immunogenicity and good in vivo safety.

[0017] Intelligent design, precise delivery: Through the dual functional design of "mannose targeting" and "ROS response", the active homing of nanomedicines to inflamed lung macrophages and intelligent controlled release within lesion cells are achieved.

[0018] Multiple synergies enhance efficacy: The innovative combination of mild photothermal physical stimulation with curcumin chemotherapy creates a cascade mechanism of "targeted accumulation - microenvironment-responsive drug release - photothermal / drug synergistic immune regulation", resulting in efficacy significantly superior to single drugs or single therapies.

[0019] Integrated diagnosis and treatment, dynamic visualization: cleverly utilizing the photothermal / photoacoustic properties of CINPs, combining the functions of therapeutic agents and contrast agents into one.

[0020] The preparation process is controllable and easy to scale up: the raw materials used are widely available and low in cost, and the preparation method is based on mature nano-assembly and surface modification technology, with stable process and good reproducibility. Attached Figure Description

[0021] Figure 1 Transmission electron microscopy (TEM) images of the prepared CINPs (A) and C@CM (B); Figure 2 The results of photothermal characterization and ultraviolet absorption spectrum of C@CM are shown in the figure; Figure 3 The graph shows the endocytosis efficiency of macrophages on Cur and C@CM. Figure 4 The figure shows the results of the antioxidant effect of C@CM combined with photothermal therapy on macrophages in vitro; Figure 5 and Figure 6 The result of C@CM combined with photothermal therapy regulating the polarization of M1 macrophages into M2 macrophages; Figure 7 The figure shows the results of dynamic monitoring of the distribution and metabolism of C@CM in the lungs of healthy and ALI mice using photoacoustic imaging. Figure 8 The image shows the pathological results of lung tissue sections in mice to demonstrate the therapeutic effect of C@CM combined with photothermal therapy on ALI. Figure 9 The image shows the ROS detection results of lung tissue in mice to illustrate the therapeutic effect of C@CM combined with photothermal therapy on ALI mice. Figure 10 The figure shows the immunomodulatory effect of C@CM combined with photothermal therapy on the lung tissue of ALI mice. Figure 11 This is a figure showing the safety evaluation results of C@CM on the major organs of ALI using histological analysis. Detailed Implementation

[0022] To enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the present invention.

[0023] The purpose of this invention is to provide a diagnostic and therapeutic nanoplatform based on natural ingredients, its preparation method and application. It is mainly a "diagnostic and therapeutic" nanoplatform with high biosafety, strong targeting and integrated treatment and real-time monitoring functions, in order to solve the problems of low drug delivery efficiency, imprecise regulation of immune microenvironment and difficulty in real-time in vivo efficacy assessment in ALI treatment.

[0024] To achieve the above objectives, the first aspect of the present invention provides a method for preparing a diagnostic and therapeutic nanoplatform based on natural ingredients, comprising the following steps: Natural melanin nanoparticles extracted from squid ink sacs were dispersed in an aqueous solution to obtain a CINPs aqueous dispersion. Curcumin was dissolved in an organic solvent and added dropwise to a CINPs aqueous dispersion. The mixture was stirred in the dark to allow curcumin to be adsorbed onto the surface of natural melanin nanoparticles through non-covalent interactions. After centrifugation and purification, Cur@CINP nanoparticles were obtained. Mannose-modified polyetherimide solution and phenylboronic acid-modified polyetherimide solution were synthesized respectively. Cur@CINP nanoparticles were mixed with mannose-modified polyetherimide solutions and phenylboronic acid-modified polyetherimide solutions and incubated. Through cross-linking reaction, mannose-modified polyetherimide and phenylboronic acid-modified polyetherimide were co-modified onto the surface of Cur@CINP nanoparticles. After purification, the product C@CM nanoparticles, namely the therapeutic nanoplatform, were obtained.

[0025] The preparation mechanism of C@CM provided by this invention is as follows: Based on naturally derived curcumin (Cur) and squid ink melanin nanoparticles (CINPs), curcumin is loaded onto the surface of CINPs through electrostatic adsorption; further, polyetherimide (PEI) is used as a linker to modify the targeting molecule mannose (Man) and the reactive oxygen species (ROS) responsive group phenylboronic acid (BA), respectively, and grafted onto the surface of the nanoparticles. This process forms a multifunctional nanotherapeutic platform C@CM with active macrophage targeting, ROS-responsive drug release, and mild photothermal conversion capabilities.

[0026] The therapeutic nanoplatform provided by this invention, named C@CM, is a core-shell structured nanoparticle comprising: The core carrier is natural melanin nanoparticles (CINPs) extracted from the ink sac of cuttlefish (Sepia). These CINPs have a uniform particle size distribution, with an average hydrated particle size of 180-220 nm, and exhibit excellent photothermal conversion efficiency and antioxidant activity.

[0027] Therapeutic loading: Curcumin (Cur) loaded onto the core surface of the CINPs. The curcumin is loaded via physical adsorption, with a loading rate of not less than 8% (w / w).

[0028] The targeting functional molecule is mannose (Man) attached to the surface of the nanoparticles. Mannose is covalently modified on the surface of CINPs via a polycationic carrier (polyethyleneimine, PEI) to specifically recognize and bind to the mannose receptor (CD206) highly expressed on the surface of alveolar macrophages, thereby achieving active targeting.

[0029] Microenvironment responsive groups: Phenylboronic acid (BA) attached to the surface of nanoparticles acts as a reactive oxygen species (ROS) sensitive "switch", decomposing drugs in the high ROS environment of the inflammatory site.

[0030] The synthesis process of the mannose-modified polyetherimide solution includes: Mannose was dissolved in methanol to obtain a mannose solution; Polyetherimide was dissolved in methanol, preheated in an oil bath to 45-55°C, and then mannose solution was added. The mixture was stirred and reacted in an oil bath at 45-55°C for 12-24 hours to obtain mannose-modified polyetherimide. The obtained mannose-modified polyetherimide was uniformly dispersed in methanol to obtain a mannose-modified polyetherimide solution; The mass ratio of mannose to polyetherimide is 1:5~10.

[0031] The synthesis process of phenylboronic acid-modified polyetherimide solution includes: Phenylated acid was dissolved in methanol to obtain a phenylboronic acid solution; Polyetherimide was dissolved in methanol, and after adding phenylboronic acid solution, the mixture was stirred in an oil bath at 65-75°C for 12-24 hours. After purification with diethyl ether, phenylboronic acid-modified polyetherimide (PEI-BA) was obtained. The obtained phenylboronic acid-modified polyetherimide was uniformly dispersed in methanol to obtain a phenylboronic acid-modified polyetherimide solution; The mass ratio of phenylboronic acid to polyetherimide is 1:0.5~2.

[0032] In this invention, Cur@CINP nanoparticles are mixed and incubated with mannose-modified polyetherimide solution and phenylboronic acid-modified polyetherimide solution, including: Curcumin was dissolved in ethanol and added dropwise to CINP aqueous dispersion under continuous stirring. After reacting for 12-24 hours, a phenylboronic acid-modified polyetherimide solution was added, and the reaction was continued for another 12-24 hours. The product was collected by centrifugation to obtain the precipitate. The precipitate was dispersed in water, and a mannose-modified polyetherimide solution was added with stirring. The reaction was carried out for 12 to 24 hours.

[0033] The concentration of the mannose-modified polyetherimide (PEI-Man) solution is 0.5~2 mg / mL, preferably 1 mg / mL; the concentration of the phenylboronic acid-modified polyetherimide (PEI-BA) solution is 2~5 mg / mL, preferably 4 mg / mL. The concentration of CINP aqueous dispersion is 0.5~2 mg / mL, preferably 1 mg / mL.

[0034] The organic solvent used in this invention is ethanol.

[0035] In this invention, the natural melanin nanoparticles extracted from the ink sac of squid include: Cuttlefish bile and ultrapure water were mixed evenly in a centrifuge tube and then centrifuged at 2000-4000 rpm for 3-6 minutes to remove large aggregates and insoluble impurities. The supernatant was collected and then centrifuged at 7000-9000 rpm for 8-12 minutes. The precipitate was retained and redispersed in water. The centrifugation at 8000 rpm for 8-12 minutes was repeated several times. The final precipitate was the purified melanin nanoparticles (CINPs).

[0036] An exemplary method for preparing a therapeutic nanoplatform C@CM includes the following steps: S1. Extraction and purification of CINPs: Cuttlefish ink was centrifuged, washed, dialyzed, and filtered to obtain a CINPs aqueous dispersion with uniform particle size and good dispersibility.

[0037] Preparation of S2.Cur@CINP: Curcumin was dissolved in an organic solvent and added dropwise to the CINP dispersion. The mixture was stirred in the dark to allow curcumin to be adsorbed onto the surface of CINPs through non-covalent interactions. After centrifugation and purification, Cur@CINP was obtained.

[0038] S3. Synthesis of functionalized polymers: Mannose-modified polyetherimide (PEI-Man) and phenylboronic acid-modified polyetherimide (PEI-BA) were synthesized respectively.

[0039] Construction of S4.C@CM: The Cur@CINP nanoparticles obtained in step S2 were mixed and incubated with PEI-Man and PEI-PBA obtained in step S3. Through cross-linking reaction, PEI-Man and PEI-PBA were co-modified onto the surface of Cur@CINP. After purification, the final product C@CM nanoparticles were obtained.

[0040] A second aspect of this invention provides a diagnostic and therapeutic nanoplatform based on natural ingredients.

[0041] The third aspect of this invention provides the application of a natural component-based therapeutic nanoplatform in the preparation of photoacoustic imaging contrast agents for the treatment of acute lung injury, acute respiratory distress syndrome, or the imaging diagnosis and efficacy monitoring of acute lung injury (ALI).

[0042] For example, the application of the theologous nanoplatform C@CM in the preparation of drugs for treating acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) involves the following cascade process as a synergistic therapeutic mechanism: (1) Active lung targeting and accumulation: Mannose-mediated specific enrichment of nanoparticles in macrophages at sites of lung inflammation.

[0043] (2) ROS-responsive drug release: In the high ROS microenvironment within M1 macrophages, phenylboronic acid bonds break, enabling the site-specific release of curcumin.

[0044] (3) Photothermal-drug synergistic immune reprogramming: Under near-infrared light irradiation, CINPs generate mild photothermal (≈40℃), which works synergistically with the released curcumin to promote macrophage polarization from M1 phenotype to M2 phenotype, while efficiently clearing ROS and reshaping the anti-inflammatory repair microenvironment.

[0045] For example, the application of the theotherapy nanoplatform C@CM in the preparation of photoacoustic imaging contrast agents for the imaging diagnosis and efficacy monitoring of acute lung injury (ALI) demonstrates that the inherent strong light absorption properties of CINPs enable them to generate strong photoacoustic signals under near-infrared light excitation. This allows for non-invasive, real-time, and dynamic visualization monitoring of the distribution, accumulation, and metabolic processes of nanoparticles in the lungs via photoacoustic tomography (PAT), thereby enabling quantitative assessment of inflammation development and treatment response.

[0046] It should be noted that, unless otherwise specified, the experimental methods used in this invention are all conventional methods; and the reagents and materials used, unless otherwise specified, are all commercially available.

[0047] Example 1 A method for preparing a therapeutic nanoplatform based on natural ingredients includes the following steps: 1) Mix 15g of cuttlefish bile with ultrapure water in a centrifuge tube and disperse evenly on a magnetic stirring plate. Then, sonicate the mixture in an ultrasonic bath for 10 minutes to achieve uniform dispersion. After equilibration, centrifuge the sample at 3000 rpm for 5 minutes to remove large aggregates and insoluble impurities. Collect the supernatant and further centrifuge at 8000 rpm for 10 minutes. Retain the precipitate, redisperse it in water, and repeat the 8000 rpm centrifugation step several times. The final precipitate obtained is the purified melanin nanoparticles (CINPs), which are collected for subsequent experiments. CINP nanoparticles were dispersed in an aqueous solution to obtain a CINP aqueous dispersion. 2) Phenylboronic acid (507.7 mg, 0.282 mmol, 214.85 g / mol, CAS: 68162-47-0) was dissolved in 7 mL of methanol. This solution was slowly added dropwise to a solution of polyetherimide (PEI, 507.7 mg, 1.175 mmol, 1800 g / mol, CAS: 9002-98-6) dissolved in 7 mL of methanol. The reaction mixture was stirred in an oil bath at 70 °C for 24 hours to obtain the crude product. The product was then purified as follows: The crude product was transferred to a 50 mL centrifuge tube, and approximately 2 mL of diethyl ether was slowly added dropwise while gently shaking. Methanol was added to approximately half the volume of the centrifuge tube. After equilibration, the tube was centrifuged at 3000 rpm for 3 minutes. The supernatant was discarded, the precipitate was collected and redissolved with a small amount of methanol, and diethyl ether was added again to precipitate the product. The tube was then centrifuged at 3000 rpm for 3 minutes. This washing step, involving ether precipitation and centrifugation, was repeated three times to ensure complete purification. The final precipitate was collected and dried under vacuum to obtain phenylboronic acid-modified polyetherimide (PEI-BA). PEI-BA was uniformly dispersed in methanol to obtain a PEI-BA solution with a concentration of 4 mg / mL. The purified conjugate (5 mg) was dissolved in 100 µL of methanol for ¹H nuclear magnetic resonance (NMR) spectroscopy analysis.

[0048] 3) Weigh 260.7 mg of polyetherimide (PEI) into a reaction flask, add 7 mL of methanol to dissolve it completely, obtaining a PEI solution. Then add a magnetic stir bar to the flask and place it in an oil bath preheated to 50°C. Separately, dissolve 33.4 mg of mannose in about 2 mL of methanol and stir until completely dissolved, obtaining a mannose solution. Slowly add this mannose solution dropwise to the PEI solution using a dropper. After the addition is complete, continue stirring at 50°C for 24 hours to obtain mannose-modified polyetherimide (PEI-Man). Disperse PEI-Man uniformly in methanol to obtain a PEI-Man solution with a concentration of 1 mg / mL. 4) Dissolve curcumin (10 mg) in ethanol and slowly add the solution dropwise to 50 mL of CINP aqueous dispersion (1 mg / mL) while stirring continuously (400 rpm). React for 24 hours to allow curcumin to gradually precipitate and adsorb onto the CINP surface through electrostatic interaction, thus obtaining a mixture. Subsequently, 1 mL of 4 mg / mL PEI-BA solution was slowly added dropwise to the mixture in step 4) above, and stirring was continued at 1200 rpm for 12 hours. The product was collected by centrifugation, and the precipitate was retained. The precipitate was redispersed in water, and 10 µL of a pre-prepared 1 mg / mL PEI-Man solution was slowly added dropwise to the dispersion while stirring (1200 rpm). The reaction was continued for 12 hours to obtain C@CM nanoparticles, a therapeutic nanoplatform based on natural ingredients. The nanoparticles obtained from the reaction were washed three times by centrifugation at 10,000 rpm with ultrapure water and redispersed in water for subsequent use.

[0049] 5) CINP and C@CM nanoparticles were diluted 10-fold with ultrapure water and sonicated for 1 minute. The particle size distribution, PDI, and Zeta potential were measured using a dynamic light scattering instrument. The sample was dropped onto a copper grid of a carbon support film and dried at 37°C for 24 hours. The morphology was then observed using a transmission electron microscope.

[0050] See Figure 1 As shown, transmission electron microscopy (TEM) imaging reveals that both CINP and C@CM exhibit a uniform spherical morphology with a diameter of 100–150 nanometers.

[0051] 6) Record the absorption spectra of 200-1000 nm using a UV-Vis-NIR spectrometer. Monitor the temperature changes of samples under different power densities and concentration gradients under 808 nm laser irradiation to evaluate photothermal performance and stability.

[0052] See Figure 2As shown in Figure (A), under irradiation at different power densities, the temperature of the C@CM dispersion system increases with increasing concentration, with higher power densities leading to faster temperature rise: at a power density of 2 W / cm², the solution temperature rises to above 40°C within 2 minutes and exceeds 70°C after 10 minutes; while at a power density of 1 W / cm², the temperature also rises to approximately 40°C within 2 minutes and stabilizes between 40-45°C over the following 10 minutes, demonstrating controllable and sustained photothermal response characteristics suitable for mild thermotherapy applications. Based on these results, a power density of 1 W / cm² was selected for subsequent experiments. It is worth noting that... (See also...) Figure 2 As shown in Figure (B), even after successive dilutions of 2, 4, 8, and 16 times, C@CM maintains a stable photothermal conversion efficiency under 1 W / cm² irradiation, indicating its robust and concentration-independent photothermal efficiency. Furthermore, see [reference needed]. Figure 2 As shown in (C), the photothermal stability of C@CM was further verified by performing three consecutive heating-cooling cycles (10 minutes of irradiation followed by natural cooling) under 1 W / cm² NIR irradiation. See also Figure 2 As shown in Figure (D), CINP and C@CM exhibit strong absorption characteristics in a wide wavelength range of 200–600 nm, demonstrating excellent photothermal conversion performance.

[0053] Example 2 This embodiment is basically the same as Embodiment 1, except that... In step 4), adjust the order and dosage of PEI-BA and PEI-Man. The specific steps are as follows: Step 4): After loading curcumin (10 mg) onto the surface of CINPs, dual modification was performed sequentially: First, 2 mL of PEI-BA solution (2 mg / mL) was slowly added dropwise to the above mixture, and the mixture was stirred at 1200 rpm for 6 hours. The precipitate was then collected by centrifugation. The precipitate was then redispersed in water, and 20 µL of PEI-Man solution (0.5 mg / mL) was slowly added dropwise to the dispersion. The mixture was stirred at 1200 rpm for 6 hours.

[0054] The remaining steps are the same as in Example 1.

[0055] Expected Results: By adjusting the modification order and ratio of PEI-BA and PEI-Man, the density and spatial distribution of the two functional groups on the surface of nanoparticles can be controlled. The C@CM obtained in this embodiment has a higher PEI-BA grafting density and a more moderate PEI-Man density, making it suitable for therapeutic scenarios requiring strong ROS-responsive drug release while maintaining targeting capability.

[0056] Example 3 This example demonstrates small-scale preparation, suitable for rapid screening and optimization in the laboratory.

[0057] Step 1): Take 5 grams of cuttlefish bile, reduce the amount of other reagents proportionally, and follow the same steps as in Example 1 to obtain an aqueous dispersion of CINPs.

[0058] Step 2): Dissolve phenylboronic acid (169.0 mg, 0.094 mmol) in 2.5 mL of methanol, and slowly add it dropwise to a PEI (169.0 mg, 0.39 mmol) solution dissolved in 2.5 mL of methanol. Stir at 70°C for 24 hours, and reduce the purification steps in the same proportion.

[0059] Step 3): Weigh 87.0 mg of PEI and dissolve it in 2.5 mL of methanol. Separately, dissolve 11.1 mg of mannose in 1 mL of methanol and slowly add it dropwise to the PEI solution. Stir at 50°C for 24 hours.

[0060] Step 4): Curcumin (3.3 mg) was loaded into 17 mL of CINP aqueous dispersion (1 mg / mL), and then 0.33 mL of PEI-BA solution (4 mg / mL) and 3.3 µL of PEI-Man solution (1 mg / mL) were added sequentially. The reaction time and stirring speed were the same as in Example 1.

[0061] Expected results: Obtain small batches of C@CM nanoparticles with the same physicochemical properties (total yield is about 1 / 3 of that in Example 1), suitable for preliminary condition optimization or cell experiment pre-testing.

[0062] Example 4 Same as in Example 1, except that the concentration of PEI-Man solution is 2 mg / mL; the concentration of PEI-BA solution is 5 mg / mL.

[0063] To illustrate the performance of the integrated diagnostic and therapeutic nanoplatform based on natural ingredients provided by the present invention, the product provided in Example 1 will be used as an example.

[0064] (I) Functional verification at the in vitro cellular level 1) RAW264.7 cells were loaded at a rate of 2 × 10⁻⁶. 4 Cells were seeded per well in 12-well plates and cultured overnight. LPS stimulation was used for 24 hours to induce M1 phenotype polarization. Cells were then incubated for another 4 hours in serum-free medium containing Curcumin (dissolved in 1% DMSO) and C@CM (10 μM curcumin). Cells were washed and collected, and mean fluorescence intensity (MFI) of curcumin was measured by flow cytometry.

[0065] 2) RAW264.7 cells were loaded at a rate of 2 × 10⁻⁶.5 Cells were seeded per confocal culture dish and incubated overnight in complete medium. After polarization was induced by LPS for 24 hours under the same conditions, the medium was replaced with serum-free medium containing Cur or C@CM (10 μM curcumin) and incubated for 4 hours. After washing, cell fluorescence was observed using a laser confocal microscope.

[0066] 3) RAW264.7 cells were seeded at 2×10³ cells / well in 96-well plates and cultured to about 80% confluence. Then, medium containing 1 μg / mL LPS was added for 24 hours to induce M1 polarization. The LPS-containing medium was discarded, the cells were washed once with PBS, and then replaced with fresh medium containing different concentrations of Cur, CINP or C@CM (the final curcumin concentration was 0-20 μM, and the CINP concentration was matched accordingly) and cultured for another 24 hours.

[0067] 4) Discard the culture medium, add 100 μL of serum-free culture medium containing 10% CCK-8 reagent to each well, incubate at 37°C for 2 hours, measure the absorbance at 450 nm wavelength, and calculate the cell viability with the untreated group as a reference.

[0068] 5) RAW264.7 cells (3×10⁻⁶) 4 (Numbers / well) were seeded into 24-well plates pre-placed with coverslips and allowed to adhere overnight. After inducing M1 polarization with LPS (1 μg / mL) for 24 hours, the cells were treated with Cur (10 μM), CINP (10 μM), or C@CM (10 μM curcumin equivalent concentration) for 10–12 hours, respectively. The photothermal groups (CINP+PTT, C@C-M+PTT) were simultaneously subjected to light treatment.

[0069] After treatment, cells were fixed with 4% paraformaldehyde for 20 minutes, permeabilized with 0.5% Triton X-100 for 20-25 minutes, and blocked with 5% goat serum for 1 hour (37℃). Then, primary antibodies against CD86 (M1 marker) or CD206 (M2 marker) (both purchased from Guangzhou Beyotime) were added and incubated overnight at 4℃. After washing, cells were incubated with Alexa Fluor 488 or Cy3-labeled goat anti-rabbit IgG secondary antibody (Shanghai Yisheng, 1:100) at 37℃ for 1 hour, followed by nucleus staining with DAPI (Soleb, 1:500). Finally, the cells were mounted and observed and images acquired under a laser confocal microscope.

[0070] (II) Construction of ALI mouse model and in vivo treatment study 1) Immediately weigh the wet weight of the lung tissue, then dry it at 80°C for 72 hours to obtain the dry weight, and calculate the wet-to-dry weight ratio to assess the degree of pulmonary edema.

[0071] 2) The lungs were irrigated with ice-cold PBS to collect bronchoalveolar lavage fluid. The supernatant was centrifuged and the levels of pro-inflammatory factors (TNF-α, IL-1β, IL-6) and anti-inflammatory markers (Arg-1, IL-10, CD206) were detected using an ELISA kit.

[0072] 3) Take a portion of lung tissue, fix it with 4% paraformaldehyde, embed it in paraffin and section it (4-5 μm thick). After H&E staining, observe the inflammatory cell infiltration, alveolar structure and tissue integrity under a light microscope.

[0073] 4) After dewaxing, hydration and antigen retrieval of paraffin sections, block with 5% BSA for 30 minutes, incubate overnight at 4°C with DHE fluorescent probe (1:400) or anti-ROS antibody (1:500), anti-TNF-α antibody (1:500, M1 marker) and anti-IL-10 antibody (1:500, M2 marker), respectively. After washing, incubate with the corresponding secondary antibody (FITC or Cy3 labeled, 1:200) at room temperature for 1 hour. Counterstain cell nuclei with DAPI, mount with anti-quenching, and observe under a laser confocal microscope.

[0074] (III) In vivo dynamic monitoring based on photoacoustic imaging A mouse model of ALI was first induced by intraperitoneal injection of lipopolysaccharide (LPS). In vivo photoacoustic baseline images of the lungs were acquired before treatment. After intratracheal administration of C@CM, longitudinal lung imaging was performed at 0, 3, 6, 12, 24, and 48 hours post-treatment. Healthy mice receiving C@CM served as a control group, and the same imaging protocol was used. Furthermore, to track systemic clearance of C@CM, dynamic photoacoustic imaging of the liver and kidneys was performed in all experimental animals.

[0075] (iv) System biosafety evaluation Twenty-four hours after drug administration, mice in each group were sacrificed, and vital organs such as the heart, liver, spleen, and kidneys were quickly harvested and immediately fixed in 4% paraformaldehyde for 24 hours. The fixed tissues were then embedded in paraffin, sectioned (4-5 μm thick), and stained with hematoxylin and eosin (H&E) according to standard procedures. Finally, the tissue morphology was observed under an optical microscope to assess for any toxic damage or structural abnormalities.

[0076] Figure 3 The graph shows the endocytosis efficiency of macrophages on Cur and C@CM.

[0077] like Figure 3As shown, cells treated with C@CM exhibited significantly stronger green fluorescence than cells treated with free Cur, indicating enhanced macrophage uptake capacity. In the diagram, Controll represents M1 macrophages from the control group, while Cur and C@CM represent M1 macrophages treated with Cur and C@CM, respectively. BF is a bright-field image showing cell outlines; DAPI (blue) is used to label cell nuclei; FITC (green) represents the green fluorescence emitted by Cur and C@CM; Merge is a superimposed image of the bright-field, DAPI, and FITC channels, with a scale bar of 20 micrometers.

[0078] Figure 4 This figure shows the results of the antioxidant effect of C@CM combined with photothermal therapy on macrophages in vitro.

[0079] like Figure 4 As shown, LPS stimulation significantly increased ROS levels compared to the untreated control group. Treatment with Cur or CINP alone did not significantly reduce ROS, but a more significant decrease was observed in the C@CM and C@C-M+PTT groups. Notably, the C@C-M+PTT group achieved the most significant ROS scavenging effect, highlighting the synergistic effect of targeted nanodelivery and mild PTT in alleviating oxidative stress. In the data, Controll represents unstimulated M0 macrophages, LPS represents LPS-treated M1 macrophages, and Cur, C@CM, and C@C-M+PTT represent cells from each treatment group; BF is a bright-field image showing cell outlines; DAPI (blue) is used to label cell nuclei; ROS represents intracellular reactive oxygen species levels (green); Merge is a superimposed image of the above bright-field, DAPI, and green fluorescent channels, with a scale bar of 20 micrometers.

[0080] Figure 5 and Figure 6 This image shows the results of C@CM combined with photothermal therapy regulating the polarization of M1 macrophages into M2 macrophages.

[0081] like Figure 5 and 6 As shown, LPS stimulation significantly upregulated the expression level of the M1 marker CD86 (red); all treatments inhibited its expression to varying degrees, with the C@C-M+PTT group showing the most significant inhibition. Conversely, the expression of the M2 marker CD206 (green) showed the opposite trend: its expression was significantly enhanced after treatment, a phenomenon particularly evident in the C@C-M+PTT group. (Where DAPI (blue) is used to label the cell nucleus; CD86 represents the intracellular expression level of CD86 (red); CD206 represents the intracellular expression level of CD206 (green); Merge is a superimposed image of the above bright field, DAPI, and green fluorescence channels, scale bar 20 micrometers).

[0082] Figure 7 This figure shows the results of dynamic monitoring of the distribution and metabolism of C@CM in the lungs of healthy and ALI mice using photoacoustic imaging.

[0083] like Figure 7 As shown, in healthy mice, a transient photoacoustic signal was detected in the lungs shortly after intratracheal administration of C@CM. This signal gradually weakened and became almost undetectable within 6 hours, indicating rapid systemic clearance of the drug in the absence of inflammation. In contrast, in mice with acute lung injury (ALI), C@CM exhibited specific targeting and persistent accumulation in inflamed lung tissue. The corresponding photoacoustic signal continuously enhanced after administration, peaking within 12 hours, reflecting sufficient drug accumulation at the lesion site. Subsequently, the signal gradually weakened after 24 hours and was essentially cleared within 48 hours. Here, Control represents healthy mice, ALI represents mice with acute lung injury, and PA Intensity represents the photoacoustic signal intensity.

[0084] Figure 8 The image shows the pathological results of lung tissue sections in mice to demonstrate the therapeutic effect of C@CM combined with photothermal therapy on ALI.

[0085] like Figure 8 As shown, histopathological analysis revealed significant inflammatory cell infiltration, interstitial congestion, alveolar edema, and increased secretions and hemorrhage within the bronchioles in the ALI model group mice. While free-form Cur treatment moderately alleviated the inflammatory characteristics, the overall damage remained severe. Compared to Cur alone, the C@CM group showed superior therapeutic efficacy. Notably, mice receiving the C@C-M+PTT combination therapy exhibited significantly reduced inflammatory cell aggregation, alveolar exudate, and tissue congestion, with lung tissue structure essentially restored to near-normal morphology.

[0086] Figure 9 The image shows the ROS detection results of lung tissue in mice with ALI, indicating the therapeutic effect of C@CM combined with photothermal therapy. Figure 9 It was found that the ROS level in ALI mice was significantly higher than that in the healthy control group. All treatments led to a decrease in ROS signal, with the decrease being more significant in the C@CM group than in the free Cur group. The most significant reduction in ROS level was observed in the C@CM + PTT combined treatment group, highlighting the synergistic antioxidant effect of this combined regimen. In the diagram, DAPI (blue) was used to label cell nuclei in mouse lung tissue pathological sections; ROS represents the level of reactive oxygen species in lung tissue (red); Merge is a superimposed image of DAPI and the red fluorescent channel, with a scale bar of 50 micrometers.

[0087] Figure 10The figure shows the immunomodulatory effect of C@CM combined with photothermal therapy in the lung tissue of ALI mice.

[0088] from Figure 10 Compared with the healthy control group, the levels of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) associated with M1 macrophages were significantly increased in the ALI model. Therapeutic interventions reduced the levels of these cytokines, with C@CM treatment showing a greater reduction than free Cur; the C@CM combined with PTT (C@C-M+PTT) group achieved the most significant inhibitory effect. Conversely, after treatment, the expression of anti-inflammatory cytokines (Arg-1, CD206, IL-10) was enhanced, with the upregulation induced by C@CM being stronger than that induced by Cur alone. Notably, the C@C-M+PTT group showed the most significant increase among all detected anti-inflammatory cytokines. Overall, these results indicate that C@CM—especially when combined with photothermal stimulation—can promote a shift from a pro-inflammatory to an anti-inflammatory state in the lung microenvironment.

[0089] Figure 11 This is a figure showing the safety evaluation results of C@CM on the major organs of ALI using histological analysis.

[0090] from Figure 11 As can be seen, compared with the healthy mouse control group, no significant tissue damage or morphological abnormalities were observed in the major organs of any of the treatment groups. These results indicate that C@CM and its constituent compounds Cur and CINP have good biocompatibility both in vitro and in vivo, supporting their potential for safe therapeutic application. Here, Heart represents a pathological section of the heart, Liver represents a pathological section of the liver, Spleen represents a pathological section of the spleen, and Kidney represents a pathological section of the kidney.

[0091] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a diagnostic and therapeutic nanoplatform based on natural ingredients, characterized in that, Includes the following steps: Natural melanin nanoparticles extracted from squid ink sacs were dispersed in an aqueous solution to obtain a CINPs aqueous dispersion. Curcumin was dissolved in ethanol and added dropwise to a CINPs aqueous dispersion. The mixture was stirred in the dark to allow curcumin to be adsorbed onto the surface of natural melanin nanoparticles through non-covalent interactions. After centrifugation and purification, Cur@CINP nanoparticles were obtained. Mannose-modified polyetherimide solution and phenylboronic acid-modified polyetherimide solution were synthesized respectively. Cur@CINP nanoparticles were mixed and incubated with mannose-modified polyetherimide solutions and phenylboronic acid-modified polyetherimide solutions. Through a cross-linking reaction, the mannose-modified polyetherimide solution and the phenylboronic acid-modified polyetherimide solution were co-modified onto the surface of the Cur@CINP nanoparticles. After purification, the product C@CM nanoparticles, namely the therapeutic nanoplatform, were obtained.

2. The preparation method of the diagnostic and therapeutic integrated nanoplatform based on natural ingredients according to claim 1, characterized in that, The synthesis process of mannose-modified polyetherimide solution includes: Mannose was dissolved in methanol to obtain a mannose solution; Polyetherimide was dissolved in methanol, preheated in an oil bath to 45-55°C, and then mannose solution was added. The mixture was stirred and reacted in an oil bath at 45-55°C for 12-24 hours to obtain mannose-modified polyetherimide. The obtained mannose-modified polyetherimide was uniformly dispersed in methanol to obtain a mannose-modified polyetherimide solution; The mass ratio of mannose to polyetherimide is 1:5~10.

3. The method for preparing the integrated diagnostic and therapeutic nanoplatform based on natural ingredients according to claim 1, characterized in that, The synthesis process of phenylboronic acid-modified polyetherimide solution includes: Phenylated acid was dissolved in methanol to obtain a phenylboronic acid solution; Polyetherimide was dissolved in methanol, and after adding phenylboronic acid solution, it was stirred in an oil bath at 65-75°C for 12-24 hours. After purification with diethyl ether, phenylboronic acid-modified polyetherimide was obtained. The obtained phenylboronic acid-modified polyetherimide was uniformly dispersed in methanol to obtain a phenylboronic acid-modified polyetherimide solution; The mass ratio of phenylboronic acid to polyetherimide is 1:0.5~2.

4. The method for preparing the integrated diagnostic and therapeutic nanoplatform based on natural ingredients according to claim 1, characterized in that, Cur@CINP nanoparticles were mixed and incubated with mannose-modified polyetherimide solutions and phenylboronic acid-modified polyetherimide solutions, including: Curcumin was dissolved in ethanol and added dropwise to CINP aqueous dispersion under continuous stirring. After reacting for 12-24 hours, a phenylboronic acid-modified polyetherimide solution was added, and the reaction was continued for another 12-24 hours. The product was collected by centrifugation to obtain the precipitate. The precipitate was dispersed in water, and a mannose-modified polyetherimide solution was added with stirring. The reaction was carried out for 12 to 24 hours.

5. The preparation method of the integrated diagnostic and therapeutic nanoplatform based on natural ingredients according to claim 4, characterized in that, The concentration of mannose-modified polyetherimide solution was 0.5~2 mg / mL; the concentration of phenylboronic acid-modified polyetherimide solution was 2~5 mg / mL.

6. The method for preparing the integrated diagnostic and therapeutic nanoplatform based on natural ingredients according to claim 1, characterized in that, The organic solvent is ethanol.

7. The method for preparing the integrated diagnostic and therapeutic nanoplatform based on natural ingredients according to claim 1, characterized in that, The concentration of CINP aqueous dispersion is 0.5~2 mg / mL.

8. The method for preparing the integrated diagnostic and therapeutic nanoplatform based on natural ingredients according to claim 1, characterized in that, Natural melanin nanoparticles extracted from squid ink sacs include: Cuttlefish bile and ultrapure water were mixed evenly in a centrifuge tube and then centrifuged at 2000-4000 rpm for 3-6 minutes to remove large aggregates and insoluble impurities. The supernatant was collected and then centrifuged at 7000-9000 rpm for 8-12 minutes. The precipitate was retained and redispersed in water. The centrifugation at 8000 rpm for 8-12 minutes was repeated several times. The final precipitate was the purified melanin nanoparticles (CINPs).

9. A therapeutic nanoplatform based on natural ingredients prepared by the method of any one of claims 1 to 8.

10. The application of the natural ingredient-based therapeutic nanoplatform of claim 9 in the preparation of photoacoustic imaging contrast agents for the treatment of acute lung injury, acute respiratory distress syndrome, or imaging diagnosis and efficacy monitoring of acute lung injury.

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