Preparation and application of intelligent response nanoprobes based on manganese sulfide

Abstract

The application discloses preparation and application of intelligent response nanoprobes based on manganese sulfide, and the nanoprobes are water-soluble and size-controllable HAMnS nanoparticles coated / chelated with hyaluronic acid as a surface functional layer, and near-infrared fluorescent dyes are loaded on the surface of the nanoparticles through electrostatic interaction and coordination / amide coupling to construct pH / ROS double-lock response "OFF-ON" fluorescent photoacoustic magnetic resonance multi-modal nanoprobes. In the physiological neutral and low ROS circulation, the dyes are in a fluorescence quenching state due to the close distance / accumulation / electron transfer with the MnS surface, so that the background is extremely low; when the probes are enriched in the tumor / inflammatory / metastatic lymph node and other lesion microenvironments through EPR effect and HA-mediated CD44 targeting, weak acidity induces MnS degradation and combines with ROS oxidation / ligand replacement to release free dyes or eliminate quenching, so that strong near-infrared fluorescence is lighted up; the change of component density provides a photoacoustic tracking signal, so that intraoperative real-time navigation and accurate boundary judgment from the molecular level to anatomical positioning are realized.

Description

Technical Field

[0001] This invention relates to the fields of biomedical materials and molecular imaging technology, specifically to the preparation and application of a smart responsive nanoprobe based on manganese sulfide. Background Technology

[0002] The incidence of head and neck malignancies such as thyroid cancer is increasing year by year, with lymph node metastasis rates as high as 20%-50%. Currently, the core clinical challenge lies in the lack of effective methods to accurately identify small metastatic lesions in sentinel lymph nodes preoperatively and intraoperatively. While traditional prophylactic lymph node dissection can reduce recurrence rates, it often leads to serious complications such as recurrent laryngeal nerve injury and hypoparathyroidism, significantly impacting patients' quality of life. Therefore, developing an intraoperative navigation technology that can achieve "precise resection and function preservation," especially a highly specific identification technology for small metastatic lesions, is a major clinical need to be addressed in this field. To address these needs, small animal in vivo imaging and fluorescence imaging techniques have been widely studied due to their high sensitivity; however, their low spatial resolution makes it impossible to distinguish metastatic lesions smaller than 5 mm in diameter from normal lymph nodes. While photoacoustic imaging and magnetic resonance imaging can provide high-resolution anatomical information, existing commercial contrast agents (such as ICG, gadolinium, and ferrites) have significant limitations: Non-specific distribution: Traditional fluorescent dyes (such as ICG) have high background signals when circulating in vivo and lack a "smart response" mechanism, resulting in a low signal-to-noise ratio between target tissue and normal tissue, making it difficult to define tumor boundaries; Single-modal limitations: A single imaging mode cannot simultaneously achieve "molecular-level specificity" (fluorescence), "depth penetration" (photoacoustic) and "precise anatomical localization"; Stability and toxicity: Although inorganic quantum dots and other materials have excellent optical properties, their clinical translation is limited by the toxicity of heavy metals and the risk of long-term retention in the body. Manganese sulfide-based nanoprobes are degradable and release Mn under acidic conditions. 2+ The properties of MnS (for T2-weighted MRI) have attracted attention, but existing technologies are mostly limited to a single treatment or imaging function. For example, current technologies typically only utilize MnS as a drug carrier or chemokinetic therapeutic agent, failing to address the following key issues: Multimodal synergy not achieved: There is a lack of design that integrates "fluorescence illumination", "photoacoustic imaging" and "magnetic resonance enhancement" on the same manganese sulfide nanoplatform; Lack of effective background suppression strategies: Existing MnS probes have failed to achieve OFF-ON switching of "not lit during circulation, lit at lesion" by utilizing surface chemical regulation, resulting in severe background interference during intraoperative navigation; The preparation process is complex: it often uses high-temperature pyrolysis or complex surface ligand exchange, which makes it difficult to scale up production and leaves residual solvents that pose safety hazards. Therefore, developing a manganese sulfide nanoprobe with a mild preparation process, intelligent pH / ROS dual-lock response, and the ability to integrate the advantages of fluorescence / photoacoustic / MRI multimodal approaches is of decisive significance for promoting the clinical translation of intraoperative navigation technology. Therefore, we propose a method for preparing and applying a smart responsive nanoprobe based on manganese sulfide. Summary of the Invention

[0003] To achieve the above objectives, the present invention provides the following technical solution: a smart-response nanoprobe based on manganese sulfide, comprising: Manganese sulfide core, composed of Mn 2+ Source and S 2- The source is formed by co-precipitation in the aqueous phase, with the chemical formula MnS. x Where 0.8≤x≤1.2, the crystal structure of the core is zincblende phase or wurtzite phase; Hyaluronic acid (HA) modified layer, through the carboxyl / hydroxyl groups in the HA molecule and Mn 2+ The coordination and physical adsorption of the manganese sulfide core surface form a core-shell structure. Near-infrared fluorescent dyes are loaded onto the HA modified layer and the core surface through electrostatic adsorption and / or hydrophobic interactions. pH / ROS dual-response switching mechanism: at pH 7.35-7.45 and reactive oxygen species concentration <10. -6 In a physiological environment with a concentration of mol / L, the fluorescent dye is in a fluorescence-quenched state due to electronic coupling or spatial aggregation with the surface of the manganese sulfide core, with a fluorescence quantum yield ≤0.02; in a physiological environment with a pH ≤6.8 and / or a reactive oxygen species concentration ≥10 mol / L, the fluorescence quantum yield is ≤0.02. -5 In a pathological microenvironment of mol / L, the manganese sulfide core undergoes acid degradation and oxidative corrosion, disrupting the electron coupling or spatial aggregation, thereby increasing the fluorescence quantum yield to ≥0.15. Physical parameters: The hydrated particle size of the nanoprobe is 30-150 nm, the polydispersity index (PDI) is ≤0.25, and the zeta potential is -20 mV to -45 mV.

[0004] Preferably, the molecular weight of the hyaluronic acid is 7kDa-1500kDa, more preferably 50kDa-500kDa; the modification density of the hyaluronic acid is 0.5-2.5 HA segments per square nanometer surface.

[0005] Preferably, the near-infrared fluorescent dye is selected from one or more of indocyanine green, Cy7, Cy7.5, IR780, and DiR; the mass ratio of the fluorescent dye to the manganese sulfide core is 1:20-1:2, preferably 1:10-1:4.

[0006] Preferably, the manganese sulfide core is in a phosphate buffer solution at pH 6.5 for 12 hours, where Mn 2+ The release rate is 20%-80%; the released Mn 2+ Used to provide relaxation enhancement for T2-weighted magnetic resonance imaging, with a longitudinal relaxation rate r2 ≥ 50 mM -1 s -1 .

[0007] Preferably, the photoacoustic signal intensity of the nanoprobe under 808nm laser irradiation is increased by 1.5-5 times under pH 6.5 conditions compared to pH 7.4 conditions, and is used for photoacoustic imaging-guided lesion depth localization.

[0008] A method for preparing a smart-responsive nanoprobe based on manganese sulfide includes the following steps: Preparation of S1 precursor: Hyaluronic acid is dissolved in water under an inert atmosphere, a sulfur source is added, and after stirring to dissolve, the pH is adjusted to 7.35-7.45 with NaOH to obtain HA-S 2- Mixed solutions; S2 coprecipitation reaction: Under an inert atmosphere and at 0-10℃, Mn 2+ The aqueous solution was added dropwise to the HA-S at a rate of 0.2-1.0 mL / min. 2- In the mixed solution, maintain stirring, and continue the reaction for 1-4 hours after the addition is complete to form a dispersion of HA-coated manganese sulfide nanoparticles. S3 Purification: The dispersion was subjected to gradient dialysis using a dialysis membrane with a molecular weight cutoff of 50-300 kDa to remove free ions and oligomers; S4 Dye Loading: The dispersion of HA-coated manganese sulfide nanoparticles is mixed with an organic solution of near-infrared fluorescent dye, wherein the solvent of the organic solution is DMSO or ethanol, and accounts for 0.1%-2% of the total volume of the mixture. The mixture is stirred at room temperature in the dark for 6-24 hours to obtain the crude product. S5 Post-processing: The crude product is dialyzed using a dialysis membrane with a molecular weight cutoff of 1-10 kDa to remove free dye, thus obtaining the intelligent responsive multimodal nanoprobe.

[0009] Preferably, in step S1, the sulfur source is one or more of sodium sulfide, potassium sulfide, and sodium hydrosulfide; the concentration of the hyaluronic acid is 2-10 mg / mL; and the S... 2- With Mn 2+The molar ratio is 1:1-2:1. Preferably, in step S2, the Mn 2+ The source is one or more of manganese chloride, manganese sulfate, manganese nitrate, and manganese acetate; the stirring speed is 250-500 rpm. Preferably, in step S3, the gradient dialysis is performed as follows: the dialysis fluid is changed every 20-40 minutes for the first 2 hours, and every hour for the next 4 hours. The dialysis fluid is ultrapure water. After step S5, a targeted modification step is also included: the carboxyl groups of the nanoprobe dispersion are activated by EDC / NHS, and then coupled with CD44 targeting peptide, anti-CD44 antibody, or fragment thereof. The reaction temperature is 4-25℃, and the time is 2-12 hours to obtain a targeted nanoprobe. An application of the nanoprobe according to any one of claims 1-5 in the preparation of a multimodal imaging contrast agent, wherein the multimodal imaging includes near-infrared fluorescence imaging, photoacoustic imaging, and magnetic resonance imaging; the application includes: The nanoprobe was introduced into a mammal via intravenous infusion or local injection. By utilizing the intelligent pH / ROS response characteristics of the nanoprobe, specific illumination of fluorescence signals can be achieved in tumor tissues, inflammatory lesions, or metastatic lymph nodes; By combining depth information provided by photoacoustic imaging and anatomical information provided by magnetic resonance imaging, a multimodal intraoperative navigation atlas is constructed; the local injection is either peritumoral injection or sentinel lymph node drainage area injection; the fluorescence signal reaches its peak fold 1-24 hours after injection, and the fluorescence intensity ratio (TNR) of the lesion to normal tissue is ≥ 5:1; the diseases targeted by the application include sentinel lymph node metastases of thyroid cancer, breast cancer, and melanoma, as well as the determination of surgical margins for solid tumors.

[0010] Compared with existing technologies, this invention provides a method for preparing and applying a manganese sulfide-based smart responsive nanoprobe, which has the following advantages: 1. The preparation and application of a manganese sulfide-based intelligent responsive nanoprobe: By utilizing the dynamic regulation capability of the manganese sulfide (MnS) surface on fluorescent dyes, a pH / ROS dual-lock response "OFF-ON" intelligent switching system was constructed, fundamentally solving the problem of high background noise in traditional fluorescent probes. During physiological cycles (pH 7.4, low ROS), dye molecules are in a deep "dormant" state (quantum yield ≤0.02) through aggregation-induced quenching and photoinduced electron transfer effects. Once the probe accumulates in the microenvironment of tumors or metastatic lymph nodes (pH ≤6.8, increased ROS), the MnS core undergoes acid-triggered degradation and oxidative corrosion, disrupting the electronic coupling between the dye and the core, releasing the dye in a free or spatially decoupled state, achieving a hundredfold activation of the fluorescence signal (ON state). This "zero background, high contrast" characteristic significantly improves the fluorescence intensity ratio between lesions and normal tissues, solving the industry pain point that existing technologies such as IVIS cannot distinguish between micrometastases and normal lymphoid tissues.

[0011] 2. The fabrication and application of this manganese sulfide-based intelligent response nanoprobe marks the first time that three modalities—near-infrared fluorescence, photoacoustic imaging, and magnetic resonance imaging—have been integrated onto a single MnS nanoplatform, achieving complementary advantages. Specifically: At the molecular level, NIRF provides a highly sensitive "light-up" signal for real-time identification of tumor margins and micrometastases smaller than millimeters. At the tissue depth level, PAI utilizes the photoacoustic conversion effect to overcome the depth limitations of optical imaging, providing three-dimensional distribution of lesions and blood perfusion information. At the anatomical level, Mn released from MnS degradation 2+ As a highly efficient T2 contrast agent, it provides high-resolution soft tissue anatomical maps and lymph node morphological information.

[0012] The "multimodal intraoperative navigation system" constructed by integrating the three can not only see "where the enemy is" (fluorescence) but also know "what the surrounding terrain is like", effectively avoiding miscutting or incomplete cleaning due to the complexity of anatomical structures.

[0013] 3. The preparation and application of this manganese sulfide-based intelligent response nanoprobe, compared to the cumbersome processes of traditional quantum dots or upconversion nanomaterials requiring high-temperature pyrolysis and ligand exchange, utilizes an all-aqueous synthesis route. Through the molecular template effect of hyaluronic acid, precise control of particle size (30-150 nm) and structure can be achieved at 4℃ and under normal pressure in an inert atmosphere. The process is simple, reproducible, and easy to scale up for production and freeze-drying. Furthermore, the use of HA as a natural targeting ligand not only enhances tumor enrichment efficiency through CD44 receptor mediation but also avoids the immunogenicity caused by synthetic polymers or exogenous proteins. In addition, MnS can degrade into the essential trace element Mn in an acidic environment.2+ It contains non-toxic metabolites, avoiding the risk of inorganic materials remaining in the body for a long time, and providing a solid safety guarantee for clinical translation and subsequent application in human critical illness.

[0014] The above overview is for illustrative purposes only and is not intended to be limiting in any way. In addition to the illustrative aspects, embodiments, and features described above, further aspects, embodiments, and features of the invention will become readily apparent from the accompanying drawings and the following detailed description. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This is a physical image of the nanoparticles of the present invention; Figure 2 This is a transmission electron microscope image of the nanoparticles of the present invention, using ICG fluorescent dye as an example. Figure 3 This is the ultraviolet spectrum of the nanoparticles of the present invention, using ICG fluorescent dye as an example; Figure 4 The fluorescence emission spectrum of the nanoparticles of this invention, using ICG fluorescent dye as an example, is shown below. Figure 5 This is a particle size distribution diagram of the nanoparticles of the present invention, using ICG fluorescent dye as an example. Figure 6 This is a zeta potential diagram of the nanoparticles using ICG fluorescent dye as an example in this invention. Detailed Implementation

[0017] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0018] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0019] Example An Example of the Preparation and Application of a Smart Response Nanoprobe Based on Manganese Sulfide A smart-response nanoprobe based on manganese sulfide, comprising: Manganese sulfide core, composed of Mn 2+ Source and S2- The source is formed by co-precipitation in the aqueous phase, with the chemical formula MnS. x Where 0.8≤x≤1.2, the crystal structure of the core is zincblende phase or wurtzite phase; Hyaluronic acid (HA) modified layer, through the carboxyl / hydroxyl groups in the HA molecule and Mn 2+ The coordination and physical adsorption of the manganese sulfide core surface form a core-shell structure. Near-infrared fluorescent dyes are loaded onto the HA-modified layer and the core surface through electrostatic adsorption and / or hydrophobic interactions. pH / ROS dual-response switching mechanism: at pH 7.35-7.45 and reactive oxygen species concentration <10. -6 In a physiological environment with a concentration of mol / L, the fluorescent dye is in a fluorescence-quenched state due to electronic coupling or spatial aggregation with the surface of the manganese sulfide core, with a fluorescence quantum yield ≤0.02; in a physiological environment with a pH ≤6.8 and / or a reactive oxygen species concentration ≥10 mol / L, the fluorescence quantum yield is ≤0.02. -5 In a pathological microenvironment of mol / L, the manganese sulfide core undergoes acid degradation and oxidative corrosion, disrupting electron coupling or spatial aggregation, thereby increasing the fluorescence quantum yield to ≥0.15. Physical parameters: The hydrated particle size of the nanoprobe is 30-150 nm, the polydispersity index (PDI) is ≤0.25, and the zeta potential is -20 mV to -45 mV.

[0020] Specifically, the molecular weight of hyaluronic acid is 7kDa-1500kDa, preferably 50kDa-500kDa; the modification density of hyaluronic acid is 0.5-2.5 HA segments per square nanometer surface.

[0021] Specifically, the near-infrared fluorescent dye is selected from one or more of indocyanine green, Cy7, Cy7.5, IR780, and DiR; the mass ratio of the fluorescent dye to the manganese sulfide core is 1:20-1:2, preferably 1:10-1:4.

[0022] Specifically, the manganese sulfide core in phosphate buffer at pH 6.5 showed a Mn content of [missing information] over 12 hours. 2+ The release rate is 20%-80%; the released Mn 2+ Used to provide relaxation enhancement for T2-weighted magnetic resonance imaging, with a longitudinal relaxation rate r2 ≥ 50 mM -1 s -1 .

[0023] Specifically, the photoacoustic signal intensity of the nanoprobe under 808nm laser irradiation is 1.5-5 times higher at pH 6.5 than at pH 7.4, which can be used for photoacoustic imaging-guided lesion depth localization.

[0024] A method for preparing a smart-responsive nanoprobe based on manganese sulfide includes the following steps: Preparation of S1 precursor: Hyaluronic acid is dissolved in water under an inert atmosphere, a sulfur source is added, and after stirring to dissolve, the pH is adjusted to 7.35-7.45 with NaOH to obtain HA-S 2- Mixed solutions; S2 coprecipitation reaction: Under an inert atmosphere and at 0-10℃, Mn 2+ The aqueous solution was added dropwise to HA-S at a rate of 0.2-1.0 mL / min. 2- In the mixed solution, maintain stirring, and continue the reaction for 1-4 hours after the addition is complete to form a dispersion of HA-coated manganese sulfide nanoparticles. S3 purification: The dispersion is subjected to gradient dialysis using a dialysis membrane with a molecular weight cutoff of 50-300kDa to remove free ions and oligomers; S4 dye loading: HA-coated manganese sulfide nanoparticle dispersion was mixed with an organic solution of near-infrared fluorescent dye, wherein the solvent of the organic solution was DMSO or ethanol, and the organic solution accounted for 0.1%-2% of the total volume of the mixture. The mixture was stirred at room temperature in the dark for 6-24 hours to obtain the crude product. S5 post-processing: The crude product is dialyzed using a dialysis membrane with a molecular weight cutoff of 1-10 kDa to remove free dye, thus obtaining the intelligent responsive multimodal nanoprobe.

[0025] Specifically, in step S1, the sulfur source is one or more of sodium sulfide, potassium sulfide, and sodium hydrosulfide; the concentration of hyaluronic acid is 2-10 mg / mL; S 2- With Mn 2+ The molar ratio is 1:1-2:1. Specifically, in step S2, Mn 2+ The source is one or more of manganese chloride, manganese sulfate, manganese nitrate, and manganese acetate; the stirring speed is 250-500 rpm. Specifically, in step S3, the gradient dialysis operation is as follows: the dialysate is changed every 20-40 minutes for the first 2 hours, and every hour for the next 4 hours. The dialysate is ultrapure water. After step S5, a targeted modification step is also included: the carboxyl groups of the nanoprobe dispersion are activated by EDC / NHS, and then coupled with CD44 targeting peptide, anti-CD44 antibody, or fragments thereof. The reaction temperature is 4-25℃, and the time is 2-12 hours to obtain targeted nanoprobes. An application of the nanoprobe of any one of claims 1-5 in the preparation of multimodal imaging contrast agents, where multimodal imaging includes near-infrared fluorescence imaging, photoacoustic imaging, and magnetic resonance imaging; the application includes: Nanoprobes are introduced into mammals via intravenous infusion or local injection. By utilizing the intelligent pH / ROS response characteristics of nanoprobes, specific illumination of fluorescence signals can be achieved in tumor tissues, inflammatory lesions, or metastatic lymph nodes; By combining depth information provided by photoacoustic imaging with anatomical information provided by magnetic resonance imaging, a multimodal intraoperative navigation atlas is constructed; local injection is performed peritumoral or sentinel lymph node drainage area; the fluorescence signal brightness factor reaches its peak 1-24 hours after injection, and the fluorescence intensity ratio (TNR) of the lesion to normal tissue is ≥ 5:1; the application targets diseases including sentinel lymph node metastases of thyroid cancer, breast cancer, and melanoma, as well as the determination of surgical margins for solid tumors.

[0026] Through the above technical solution, this invention utilizes the dynamic regulation capability of manganese sulfide (MnS) surface for fluorescent dyes to construct a pH / ROS dual-lock response "OFF-ON" intelligent switching system, fundamentally solving the problem of high background noise in traditional fluorescent probes. During physiological cycles (pH 7.4, low ROS), dye molecules are in a deep "dormant" state (quantum yield ≤0.02) through aggregation-induced quenching and photoinduced electron transfer effects. Once the probe accumulates in the microenvironment of tumors or metastatic lymph nodes (pH ≤6.8, increased ROS), the MnS core undergoes acid-triggered degradation and oxidative corrosion, disrupting the electronic coupling between the dye and the core, releasing the dye in a free or spatially decoupled state, achieving a hundredfold activation of the fluorescence signal (ON state). This "zero background, high contrast" characteristic significantly improves the fluorescence intensity ratio between lesions and normal tissues, solving the industry pain point that existing technologies such as IVIS cannot distinguish between micrometastases and normal lymphoid tissues. For the first time, it integrates near-infrared fluorescence, photoacoustic imaging, and magnetic resonance imaging modalities onto a single MnS nanoplatform, achieving complementary advantages. Specifically: At the molecular level, NIRF provides a highly sensitive "light-up" signal for real-time identification of tumor margins and micrometastases smaller than millimeters. At the tissue depth level, PAI utilizes the photoacoustic conversion effect to overcome the depth limitations of optical imaging, providing three-dimensional distribution of lesions and blood perfusion information. At the anatomical level, Mn released from MnS degradation 2+ As a highly efficient T2 contrast agent, it provides high-resolution soft tissue anatomical maps and lymph node morphological information.

[0027] The "multimodal intraoperative navigation system" constructed by integrating the three can not only see "where the enemy is" (fluorescence) but also know "what the surrounding terrain is like", effectively avoiding miscutting or incomplete cleaning due to the complexity of anatomical structures.

[0028] Compared to traditional quantum dots or upconversion nanomaterials, which require cumbersome processes such as high-temperature pyrolysis and ligand exchange, this invention employs an all-aqueous synthesis route. Through the molecular template effect of hyaluronic acid, precise control of particle size (30-150 nm) and structure can be achieved at 4°C and under an inert atmosphere at normal pressure. The process is simple, reproducible, and easy to scale up for production and freeze-drying. Furthermore, the use of hyaluronic acid (HA) as a natural targeting ligand not only enhances tumor accumulation efficiency through CD44 receptor mediation but also avoids the immunogenicity caused by synthetic polymers or exogenous proteins. In addition, MnS can degrade into the essential trace element Mn in an acidic environment. 2+ It contains non-toxic metabolites, avoiding the risk of inorganic materials remaining in the body for a long time, and providing a solid safety guarantee for clinical translation and subsequent application in human critical illness.

[0029] Example 1: Preparation of manganese sulfide nanoprobes (HA-MnS@ICG) based on 90 kDa hyaluronic acid 1. Preparation of raw materials and instruments Sodium hyaluronate (HA-Na, Mw=90 kDa, pharmaceutical grade, Sigma-Aldrich).

[0030] Sodium sulfide nonahydrate (Na2S·9H2O, ≥98%), manganese chloride tetrahydrate (MnCl2·4H2O, ≥99%).

[0031] Indocyanine green (ICG, purity ≥98%).

[0032] Dialysis bags (MWCO 100 kDa and 3 kDa, Spectra / Por).

[0033] Nitrogen cylinders, thermostatic magnetic stirrers, pH meters, and freeze dryers.

[0034] 2. Preparation steps Step S1: Synthesis of HA-MnS nanonuclei Weigh 100 mg of sodium hyaluronate, add it to 24 mL of ultrapure water, and place it in a 100 mL three-necked flask.

[0035] High-purity nitrogen (N2) gas is bubbled through the system for 15 minutes to remove dissolved oxygen and prevent Mn from entering the reaction. 2+ Oxidize in advance.

[0036] Add a pre-prepared 2 mg / mL sodium sulfide solution (containing S) 2- (Approximately 26.1 mmol / L), turn on magnetic stirring (350 rpm).

[0037] The pH of the reaction solution was precisely adjusted to 7.40 ± 0.05 using 0.1 M NaOH solution. This pH value was intended to ensure that the HA molecular chains fully extended while avoiding the formation of Mn. 2+ Premature hydrolysis and precipitation.

[0038] The reaction system was kept at a constant temperature of 4°C in an ice bath, maintaining an N2 atmosphere. 5 mL of 6 mg / mL MnCl2 aqueous solution (Mn...) was added... 2 + The solution (at a concentration of approximately 48.2 mmol / L) was added dropwise through a constant-pressure dropping funnel at a rate of 0.5 mL / min.

[0039] After the addition was complete, the reaction was continued to be stirred for 2 hours at 4℃ under N2 protection. At this time, the system showed a uniform light brown opalescence, indicating the formation of HA-MnS nanonuclei.

[0040] Step S2: Gradient dialysis purification The reaction solution was transferred to a MWCO 100 kDa dialysis bag and placed in a 4°C freezer for gradient dialysis.

[0041] Dialysis procedure: Change the external dialysate (ultrapure water) every 30 minutes for the first 2 hours; change it every hour for the next 4 hours.

[0042] This step aims to remove free Na. + Cl - Unreacted S 2- and small molecule oligomers.

[0043] Step S3: Fluorescent dye loading (ICG) Take the HA-MnS dispersion after dialysis (solid content approximately 1.2 mg / mL, calculated as MnS) and adjust the pH to 7.2 with 0.1 M NaOH.

[0044] Weigh an appropriate amount of ICG and dissolve it in a small amount of anhydrous DMSO to prepare a stock solution of 1 mg / mL.

[0045] ICG-DMSO solution was slowly added dropwise to HA-MnS dispersion at a mass ratio of ICG : HA-MnS = 1 : 5 (w / w). The volume fraction of DMSO in the total system was strictly controlled not to exceed 0.5% to prevent damage to the HA shell and subsequent nanoparticle aggregation.

[0046] The reaction was carried out under light-protected conditions with magnetic stirring (200 rpm) for 12 hours. During this process, positively charged ICG molecules adsorbed onto the negatively charged HA-MnS surface through electrostatic attraction and hydrophobic interaction, and the high concentration aggregation led to fluorescence quenching (OFF state).

[0047] Step S4: Remove free dye and freeze-dry Transfer the reaction solution to a MWCO 3 kDa dialysis bag and dialyze for 6 hours in the dark (changing the solution twice) to completely remove free ICG.

[0048] Samples were taken for physicochemical characterization (see subsequent verification).

[0049] The remaining product was packaged and pre-frozen at -80°C for 4 hours, then transferred to a freeze dryer (vacuum degree <10 Pa) and dried for 24 hours to obtain a loose and porous black powder (HA-MnS@ICG), which was dried and stored at 4°C in the dark.

[0050] 3. Product Validation Morphology and particle size: Transmission electron microscopy (TEM) showed that the particles were spherical and well dispersed, with an average particle size of about 45 nm; dynamic light scattering (DLS) measured the hydrated particle size to be 65.8 ± 3.2 nm, the PDI to be 0.182, and the Zeta potential to be -32.5 ± 2.1 mV.

[0051] Spectral characteristics: The UV-Vis absorption spectrum shows a characteristic ICG peak at 780 nm, but the peak shape is slightly broadened; the fluorescence spectrum shows that in pH 7.4 PBS, the fluorescence intensity of the probe is only 1.8% of that of the same concentration of free ICG, confirming that the OFF state is effective.

[0052] Example 2: Validation of the pH / ROS dual-lock intelligent response mechanism 1. Experimental Design Four control groups were set up to verify the fluorescence recovery of the probe under different environments: Group A: pH 7.4 (physiological environment) Group B: pH 6.5 (tumor acidic environment) Group C: pH 7.4 + 100 μM H2O2 (inflammation / ROS environment) Group D: pH 6.5 + 100 μM H2O2 (tumor / metastatic lesion microenvironment) 2. Testing Process The HA-MnS@ICG probe prepared in Example 1 was diluted with the above four buffer solutions to the same Mn concentration (0.1 mg / mL).

[0053] Incubate at 37℃ for 30 minutes.

[0054] Fluorescence intensity was measured using a fluorescence spectrophotometer (excitation wavelength 780 nm, emission wavelength 820 nm).

[0055] 3. Results and Mechanism Analysis Group A had extremely low fluorescence intensity (set as baseline 1.0); Group B and Group C had fluorescence intensities that increased to 4.5 times and 3.8 times that of Group A, respectively; Group D had fluorescence intensity that surged to 21.3 times that of Group A.

[0056] Conclusion: Acidic stimulation or ROS stimulation alone is insufficient for complete unlocking; the synergistic effect of both (group D) produced the strongest fluorescence recovery. This demonstrates a "dual-lock mechanism": an acidic environment promotes the degradation of the MnS backbone, while ROS oxidizes S... 2- Accelerated disintegration, together with the other two factors, disrupts the interaction between the dye and the core, causing the ICG to become free and regain fluorescence (ON state).

[0057] Example 3: Regulation of Particle Size and Targeting (Variable Experiment) 1. Particle size control (changing the molecular weight of HA) Repeat the steps of Example 1, except that the molecular weight of hyaluronic acid is replaced with 7 kDa, 200 kDa, or 1000 kDa, while keeping other conditions unchanged.

[0058] Results: As the molecular weight of HA increases, steric hindrance increases, and the nanoparticle size gradually decreases (approximately 110 nm for the 7 kDa group, approximately 66 nm for the 90 kDa group, and approximately 38 nm for the 1000 kDa group). This indicates that by selecting the molecular weight of HA, the particle size can be precisely controlled within the range of 30-150 nm to meet the permeation requirements of different sites (such as EPR effect or lymphatic targeting).

[0059] 2. Targeted modification (CD44 receptor targeting) After step S3 in Example 1, a targeted modification step is added: Take HA-MnS@ICG dispersion, add EDC / NHS to activate the carboxyl group of HA (molar ratio COOH:EDC:NHS = 1:1.2:1.2), and activate at room temperature for 15 minutes.

[0060] Add CD44 targeting peptide (sequence: CNYRARGC, containing free thiol group) and react at 4°C in the dark for 4 hours.

[0061] The HA-MnS@ICG-Target variant was purified using a MWCO 100 kDa dialysis bag. This variant showed enhanced uptake of CD44-overexpressing thyroid cancer cells, such as FTC-133, in cell experiments.

[0062] Example 4: Multimodal Imaging Application Example (Intraoperative Navigation Simulation) 1. Animal models and drug administration Establish a BALB / c nude mouse model of thyroid cancer lymph node metastasis.

[0063] The HA-MnS@ICG probe prepared in Example 1 was injected subcutaneously around the tumor (dosage: 10 mg / kg, calculated as Mn).

[0064] 2. Multimodal imaging acquisition Photoacoustic imaging: Two hours after injection, the axillary lymph node region was scanned using the PA system. The results showed that the photoacoustic signal in the metastatic lesion area was approximately 3.2 times enhanced compared to the contralateral normal tissue, providing depth information of the lesion.

[0065] Magnetic resonance imaging: T2-weighted imaging was performed using a 7.0 T small animal MRI scanner. Four hours post-injection, the metastatic lymph node areas showed a markedly low signal (darkened), indicating Mn. 2+ Release and enrich.

[0066] Fluorescence imaging: Six hours after injection, mice were sacrificed and lymph nodes were removed. Using an in vivo imaging system, the metastatic lymph nodes emitted strong fluorescence, while the normal lymph nodes showed only a weak background signal, with a TNR (target / background ratio) of 12.4 ± 1.8.

[0067] 3. Intraoperative navigation effectiveness In the simulated surgery, a handheld fluorescent probe was used to guide the surgeon to precisely remove luminescent metastatic lymph nodes, avoiding blood vessels and nerves, whereas these tiny nodules are difficult to identify under traditional visual observation. Pathological sections (H&E staining) confirmed the metastatic nature of the removed tissue.

[0068] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A smart-response nanoprobe based on manganese sulfide, characterized in that, include: Manganese sulfide core, composed of Mn 2+ Source and S 2- The source is formed by co-precipitation in the aqueous phase, with the chemical formula MnS. x Where 0.8≤x≤1.2, the crystal structure of the core is zincblende phase or wurtzite phase; Hyaluronic acid (HA) modified layer, through the carboxyl / hydroxyl groups in the HA molecule and Mn 2+ The coordination and physical adsorption of the manganese sulfide core surface form a core-shell structure. Near-infrared fluorescent dyes are loaded onto the HA modified layer and the core surface through electrostatic adsorption and / or hydrophobic interactions. pH / ROS dual-response switching mechanism: at pH 7.35-7.45 and reactive oxygen species concentration <10. -6 In a physiological environment with a concentration of mol / L, the fluorescent dye is in a fluorescence-quenched state due to electronic coupling or spatial aggregation with the surface of the manganese sulfide core, with a fluorescence quantum yield ≤0.02; in a physiological environment with a pH ≤6.8 and / or a reactive oxygen species concentration ≥10 mol / L, the fluorescence quantum yield is ≤0.

02. -5 In a pathological microenvironment of mol / L, the manganese sulfide core undergoes acid degradation and oxidative corrosion, disrupting the electron coupling or spatial aggregation, thereby increasing the fluorescence quantum yield to ≥0.

15. Physical parameters: The hydrated particle size of the nanoprobe is 30-150 nm, the polydispersity index (PDI) is ≤0.25, and the zeta potential is -20 mV to -45 mV.

2. The manganese sulfide-based smart-response nanoprobe according to claim 1, characterized in that: The hyaluronic acid has a molecular weight of 7kDa-1500kDa, preferably 50kDa-500kDa; the modification density of the hyaluronic acid is 0.5-2.5 HA segments per square nanometer surface.

3. The manganese sulfide-based smart-response nanoprobe according to claim 1, characterized in that: The near-infrared fluorescent dye is selected from one or more of indocyanine green, Cy7, Cy7.5, IR780, and DiR; the mass ratio of the fluorescent dye to the manganese sulfide core is 1:20-1:2, preferably 1:10-1:

4.

4. The manganese sulfide-based smart-response nanoprobe according to claim 1, characterized in that: The manganese sulfide core was in a phosphate buffer solution at pH 6.5 for 12 hours, with Mn 2+ The release rate is 20%-80%; the released Mn 2+ Used to provide relaxation enhancement for T2-weighted magnetic resonance imaging, with a longitudinal relaxation rate r2 ≥ 50 mM -1 s -1 .

5. The manganese sulfide-based smart-response nanoprobe according to claim 1, characterized in that: The photoacoustic signal intensity of the nanoprobe under 808nm laser irradiation is increased by 1.5-5 times under pH 6.5 conditions compared to pH 7.4 conditions, and can be used for photoacoustic imaging-guided lesion depth localization.

6. A method for preparing a manganese sulfide-based smart-response nanoprobe, characterized in that: Includes the following steps: Preparation of S1 precursor: Hyaluronic acid was dissolved in water under an inert atmosphere, a sulfur source was added, and after stirring to dissolve, the pH was adjusted to 7.35-7.45 with NaOH to obtain HA-S. 2- Mixed solutions; S2 coprecipitation reaction: Under an inert atmosphere and at 0-10℃, Mn 2+ The aqueous solution was added dropwise to the HA-S at a rate of 0.2-1.0 mL / min. 2- In the mixed solution, maintain stirring, and continue the reaction for 1-4 hours after the addition is complete to form a dispersion of HA-coated manganese sulfide nanoparticles. S3 Purification: The dispersion was subjected to gradient dialysis using a dialysis membrane with a molecular weight cutoff of 50-300 kDa to remove free ions and oligomers; S4 Dye Loading: The dispersion of HA-coated manganese sulfide nanoparticles is mixed with an organic solution of near-infrared fluorescent dye, wherein the solvent of the organic solution is DMSO or ethanol, and accounts for 0.1%-2% of the total volume of the mixture. The mixture is stirred at room temperature in the dark for 6-24 hours to obtain the crude product. S5 Post-processing: The crude product is dialyzed using a dialysis membrane with a molecular weight cutoff of 1-10 kDa to remove free dye, thus obtaining the intelligent responsive multimodal nanoprobe.

7. The method for preparing a manganese sulfide-based smart-response nanoprobe according to claim 6, characterized in that: In step S1, the sulfur source is one or more of sodium sulfide, potassium sulfide, and sodium hydrosulfide; the concentration of the hyaluronic acid is 2-10 mg / mL; the S... 2- With Mn 2+ The molar ratio is 1:1-2:

1.

8. The method for preparing a manganese sulfide-based smart-response nanoprobe according to claim 6, characterized in that: In step S2, the Mn 2+ The source is one or more of manganese chloride, manganese sulfate, manganese nitrate, and manganese acetate; the stirring speed is 250-500 rpm.

9. The method for preparing a manganese sulfide-based smart-response nanoprobe according to claim 6, characterized in that: In step S3, the specific operation of the gradient dialysis is as follows: the dialysate is changed every 20-40 minutes for the first 2 hours, and every hour for the next 4 hours. The dialysate is ultrapure water. After step S5, a targeted modification step is also included: the carboxyl group of the nanoprobe dispersion is activated by EDC / NHS and then coupled with CD44 targeting peptide, anti-CD44 antibody or its fragment. The reaction temperature is 4-25℃ and the time is 2-12 hours to obtain the targeted nanoprobe.

10. The application of the nanoprobe according to any one of claims 1-5 in the preparation of a multimodal imaging contrast agent, characterized in that: The multimodal imaging includes near-infrared fluorescence imaging, photoacoustic imaging, and magnetic resonance imaging; the applications include: The nanoprobe was introduced into a mammal via intravenous infusion or local injection. By utilizing the intelligent pH / ROS response characteristics of the nanoprobe, specific illumination of fluorescence signals can be achieved in tumor tissues, inflammatory lesions, or metastatic lymph nodes; By combining depth information provided by photoacoustic imaging and anatomical information provided by magnetic resonance imaging, a multimodal intraoperative navigation atlas is constructed; the local injection is either peritumoral injection or sentinel lymph node drainage area injection; the fluorescence signal reaches its peak fold 1-24 hours after injection, and the fluorescence intensity ratio (TNR) of the lesion to normal tissue is ≥ 5:1; the diseases targeted by the application include sentinel lymph node metastases of thyroid cancer, breast cancer, and melanoma, as well as the determination of surgical margins for solid tumors.

Images