Mitochondrial-targeting nanomaterials that release HNO, their preparation methods and applications

Abstract

This invention relates to the field of bionanomaterials technology, and particularly to mitochondrial-targeting nanomaterials capable of releasing HNO, their preparation methods, and applications. The preparation method of the mitochondrial-targeting nanomaterials capable of releasing HNO includes the following steps: synthesis of a silanized HNO donor: hydroxylamine hydrochloride is dissolved in a mixed solvent of anhydrous tetrahydrofuran and anhydrous pyridine, and a dichloromethane solution of 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane is added. The mixture is stirred at room temperature, and after post-treatment, a silanized HNO donor is obtained; synthesis of aminated carbon dots: chitosan, ethylenediamine, and mercaptosuccinic acid are dispersed in deionized water, and concentrated hydrochloric acid is added and stirred until dissolved. This preparation method can prepare mitochondrial-targeting nanomaterials capable of releasing HNO, achieving precise delivery, controlled release, and visual tracking of HNO to cardiac mitochondria, solving the technical problems of traditional HNO donors' inability to achieve stable loading on carriers and lack of mitochondrial targeting ability.

Description

Technical Field

[0001] This invention relates to the field of bionanomaterials technology, and in particular to mitochondrial-targeting nanomaterials that can release HNO, their preparation methods, and applications. Background Technology

[0002] Heart failure (HF) is a disease caused by impaired cardiac pumping function. Prolonged elevated cardiac filling pressure is a key cause of HF, as excessive mechanical stress disrupts intracellular and intercellular homeostasis. Mitochondrial dysfunction plays a crucial role in this process, often accompanied by metabolic disorders, excessive oxidative stress, and ion overload, and is involved in the development of various cardiovascular diseases (CVDs). Current clinical medications for HF, such as beta-blockers and angiotensin-converting enzyme inhibitors (ACEIs), primarily work by regulating the neuroendocrine system to slow disease progression, but they cannot directly target the mitochondria, a critical organelle, for precise pathological repair. Hydrogen nitrosyl (HNO), a signaling molecule with unique biological activity, has received widespread attention in cardiovascular protection in recent years. It can exert unique effects by regulating myocardial contraction and inhibiting oxidative stress. However, HNO is chemically unstable, and traditional HNO donors cannot achieve stable loading on carriers, resulting in an extremely short half-life and difficulty in maintaining effective therapeutic concentrations at the lesion site. Furthermore, traditional HNO donor molecules lack mitochondrial targeting capabilities, severely hindering their clinical translation. Summary of the Invention

[0003] In response to the problems raised in the background art, the purpose of this invention is to propose a method for preparing mitochondrial-targeting nanomaterials that can release HNO. This method can prepare mitochondrial-targeting nanomaterials that can release HNO, enabling precise delivery, controllable release, and visual tracking of HNO to myocardial mitochondria. It solves the technical problems that traditional HNO donors cannot achieve stable loading on a carrier and lack the ability to target mitochondria.

[0004] Another objective of this invention is to provide mitochondrial-targeting nanomaterials that can release HNO, prepared by the above-described method.

[0005] Another object of the present invention is to propose the application of the above-mentioned mitochondrial-targeting nanomaterials that can release HNO in the preparation of drugs for treating heart failure.

[0006] The above-mentioned technical objective of the present invention is achieved through the following technical solution: The preparation method of mitochondrial-targeting nanomaterials that can release HNO includes the following steps: Synthesis of silanized HNO donor: Hydroxylamine hydrochloride was dissolved in a mixed solvent of anhydrous tetrahydrofuran and anhydrous pyridine, and a dichloromethane solution of 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane was added. The reaction was stirred at room temperature, and the silanized HNO donor was obtained after post-treatment. Synthesis of aminated carbon dots: Chitosan, ethylenediamine and mercaptosuccinic acid were dispersed in deionized water, concentrated hydrochloric acid was added and stirred until dissolved, and the reaction was followed by post-treatment to obtain aminated carbon dots; Synthesis of HNO-releasing mitochondrial-targeting nanomaterials: Aminated carbon dots were dispersed in anhydrous toluene to obtain a carbon dot suspension; a silanized HNO donor was dissolved in anhydrous toluene to obtain a toluene solution of the silanized HNO donor; the toluene solution of the silanized HNO donor was added dropwise to the carbon dot suspension, reacted, and post-treated to obtain HNO-releasing mitochondrial-targeting nanomaterials.

[0007] To further clarify, in the synthesis step of the silanized HNO donor, the molar ratio of hydroxylamine hydrochloride to 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane is 5:1.

[0008] To further explain, in the synthesis step of the silanized HNO donor, the reaction time of the room temperature stirring reaction is 16-20 h, and the crude product is obtained after the reaction is completed.

[0009] To further explain, in the synthesis steps of the silanized HNO donor, the post-processing steps include: adding ethyl acetate to the crude product for extraction, washing the extracted organic phase with deionized water and saturated brine, drying it with anhydrous sodium sulfate, removing the solvent under reduced pressure, and drying it under vacuum to obtain the silanized HNO donor.

[0010] To further explain, in the synthesis step of the amination carbon dots, the reaction temperature is 180-190°C, the reaction time is 24-28 hours, and the reaction solution is obtained after the reaction is completed.

[0011] To further explain, in the synthesis step of the amination carbon dots, the post-processing step includes: filtering the reaction solution, dialyzing the filtrate, and then freeze-drying it to obtain the amination carbon dots.

[0012] To further explain, in the synthesis steps of the mitochondrial-targeting nanomaterial that can release HNO, the silanized HNO donor is dissolved in anhydrous toluene, and methanol is added dropwise to obtain a toluene solution of the silanized HNO donor.

[0013] To further explain, in the synthesis steps of the mitochondrial-targeting nanomaterials that can release HNO, the post-processing steps include: removing toluene solvent from the reactants by rotary evaporation under reduced pressure to obtain a solid crude product, washing the solid crude product with anhydrous ethanol, and obtaining the mitochondrial-targeting nanomaterials that can release HNO after vacuum drying.

[0014] A mitochondrial-targeting nanomaterial capable of releasing HNO was prepared using the aforementioned method for preparing mitochondrial-targeting nanomaterials capable of releasing HNO.

[0015] The application of the mitochondrial-targeting nanomaterials that release HNO in the preparation of drugs for treating heart failure.

[0016] Compared with the prior art, the embodiments of the present invention have the following beneficial effects: By preparing silanized HNO donors (Si-HNOD) and aminated carbon dots (CDs), Si-HNOD is covalently grafted onto the surface of CDs to form Si-OC bonds, constructing a core-shell structured nanosystem. This system integrates the targeting and fluorescence imaging advantages of CDs with the stable drug release characteristics of Si-HNOD, enabling precise delivery, controllable release, and visual tracking of HNO to myocardial mitochondria. The prepared mitochondrial-targeting nanomaterials that can release HNO can meet the needs of targeted therapy for heart failure. Attached Figure Description

[0017] Figure 1 This is a schematic diagram illustrating the preparation mechanism of the mitochondrial-targeting nanomaterials that can release HNO according to the present invention. Figure 1 (a) in the diagram is a schematic of the synthesis mechanism of silanized HNO donor (Si-HNOD). Figure 1 (b) is a schematic diagram of the mechanism of chemical grafting of hydroxyl groups on the surface of Si-HNOD and aminated carbon dots (CDs).

[0018] Figure 2 This is the 1H NMR spectrum of the silanized HNO donor (Si-HNOD) of Example 1 of the present invention.

[0019] Figure 3 This is the carbon NMR spectrum of the silanized HNO donor (Si-HNOD) of Example 1 of the present invention.

[0020] Figure 4 This is the time-dependent absorption spectrum of the silanized HNO donor (Si-HNOD) of Example 1 of the present invention at pH=7.40 and 37℃. Figure 4 The inset is a magnified view of a region that defines the characteristic absorption peak.

[0021] Figure 5 It is based on Figure 4The Si-HNOD degradation kinetics curve obtained by fitting the absorbance at the characteristic absorption peak of 235 nm over time.

[0022] Figure 6 This is a pH-responsive degradation behavior test diagram of the silanized HNO donor (Si-HNOD) of Example 1 of the present invention. Figure 6 (a) is the time-dependent absorption spectrum of the silanized HNO donor (Si-HNOD) of Example 1 of the present invention at pH=9 and 37°C. Figure 6 The inset in (a) is a Si-HNOD degradation kinetic curve obtained by fitting the absorbance at the characteristic absorption peak at 219 nm over time. Figure 6 (b) in the equation represents k. obs Trend graph of pH (7–14).

[0023] Figure 7 This is a graph showing the HNO release kinetics characterization results of the silanized HNO donor (Si-HNOD) of Example 1 of the present invention at pH=9.03, determined using the capture method. Figure 7 The inset is a kinetic curve obtained by fitting the absorbance at the 535nm characteristic absorption peak over time.

[0024] Figure 8 These are the HNO release kinetic characterization results for silanized HNO donors (Si-HNOD) of Example 1 of the present invention with different molar equivalents. Figure 8 The inset in the figure shows the results of a linear fitting analysis of the molar equivalent of Si-HNOD and the absorbance at 535 nm in the main figure.

[0025] Figure 9 This study uses a phosphorus capture method to quantitatively analyze the HNO release capacity of the silanized HNO donor Si-HNOD. 31 PNMR spectrum.

[0026] Figure 10 This is a transmission electron microscope (TEM) characterization image of the HNO-releasing mitochondrial-targeting nanomaterial (CDs-Si-HNOD) of Example 1 of the present invention. Figure 10 (a) in the figure shows the transmission electron microscopy (TEM) characterization results of CDs-Si-HNOD (scale bar is 20 nm). Figure 10 (b) in the middle is Figure 10 (a) TEM characterization results at magnification (scale bar 10 nm). Figure 10 (c) in the figure shows the transmission electron microscopy (TEM) characterization results of CDs-Si-HNOD (scale bar is 100 nm). Figure 10(d) in the middle is Figure 10 The top left corner of (c) shows the transmission electron microscope (TEM) characterization results (scale bar 10 nm). Figure 10 (e) in the figure is the transmission electron microscopy (TEM) characterization result of CDs-Si-HNOD (scale bar is 20 nm). Figure 10 (f) in the middle is Figure 10 The image shows the transmission electron microscopy (TEM) characterization results at magnification (e) (scale bar: 10 nm).

[0027] Figure 11 The Fourier transform infrared spectra of the HNO-releasing mitochondrial-targeting nanomaterials (CDs-Si-HNOD), silanized HNO donors (Si-HNOD), and aminated carbon dots (CDs) of Example 1 of the present invention are shown.

[0028] Figure 12 This is the UV-Vis absorption spectrum of the HNO-releasing mitochondrial-targeting nanomaterial (CDs-Si-HNOD) of Example 1 of the present invention. Figure 12 The illustrations in the image are magnified views of the defined areas.

[0029] Figure 13 This is a ZeTa potential characterization diagram of the HNO-releasing mitochondrial-targeting nanomaterial (CDs-Si-HNOD) and aminated carbon dots (CDs) of Example 1 of the present invention.

[0030] Figure 14 This is a three-dimensional fluorescence spectrum of the HNO-releasing mitochondrial-targeting nanomaterial (CDs-Si-HNOD) of Example 1 of the present invention.

[0031] Figure 15 This is an X-ray photoelectron spectroscopy (XPS) characterization result of the HNO-releasing mitochondrial-targeting nanomaterial (CDs-Si-HNOD) and amination carbon dots (CDs) of Example 1 of the present invention. Figure 15 In the image, (a), (b), (c), and (d) are the full spectrum scans of CDs-Si-HNOD, C, and C, respectively. 1s High-resolution XPS spectra, O 1s High-resolution XPS spectra and Si 2p High-resolution XPS spectra; Figure 15 (e) in the image is the full spectrum scan of the amination carbon dots (CDs).

[0032] Figure 16 This is a characterization diagram of HNO release from the mitochondrial-targeted nanomaterial (CDs-Si-HNOD) that releases HNO according to Example 1 of the present invention. Figure 16The inset is a kinetic curve obtained by fitting the absorbance at the 535nm characteristic absorption peak over time.

[0033] Figure 17 This is a characterization diagram of the loading rate of the HNO-releasing mitochondrial-targeting nanomaterial (CDs-Si-HNOD) of Example 1 of the present invention. Figure 17 (a) shows the UV-Vis absorption spectra of the total HNO released by CDs-Si-HNOD at different concentrations, determined using the hydroxycobalamin (HOCbl) capture method. Figure 17 (b) in the text is the adoption of 31 P NMR method was used to verify the nuclear magnetic resonance spectrum of HNO released by CDs-Si-HNOD.

[0034] Figure 18 The image shows the mitochondrial colocalization characterization test results of the HNO-releasing mitochondrial-targeting nanomaterial (CDs-Si-HNOD) of Example 1 of this invention, performed using laser scanning confocal microscopy (CLSM). Figure 18 (a1), (b1), (c1), and (d1) are images of the mitochondrial red fluorescence channel (scale bars are 40μm, 20μm, 10μm, and 10μm, respectively; c1 and d1 are magnified views of different locations). Figure 18 (a2), (b2), (c2), and (d2) are images of the green fluorescent channel of CDs-Si-HNOD (scale bars are 40μm, 20μm, 10μm, and 10μm, respectively; c2 and d2 are magnified views at different locations). Figure 18 In the image, (a3), (b3), (c3), and (d3) are images of the blue fluorescence channel in the cell nucleus (scale bars are 40μm, 20μm, 10μm, and 10μm, respectively; c3 and d3 are magnified views at different locations). Figure 18 (a4), (b4), (c4), and (d4) are three-channel fused images (scales of 40μm, 20μm, 10μm, and 10μm, respectively; c4 and d4 are magnified views at different locations).

[0035] Figure 19 It corresponds Figure 18 The fluorescence intensity line scan analysis plots of (a1), (b1), (c1), and (d1) are shown in the figure. Figure 19 (a), (b), (c), and (d) in the text correspond to... Figure 18 The (a1), (b1), (c1), and (d1) images were obtained by using ImageJ software to perform systematic quantitative colocalization analysis on multiple sets of parallel fluorescence images.

[0036] Figure 20This is a graph showing the experimental results of evaluating the toxicity of CDs-Si-HNOD to normal rat cardiomyocytes H9c2 using the CCK-8 assay.

[0037] Figure 21 This is a graph showing the results of an experiment evaluating the toxicity of CDs-Si-HNOD against four types of tumor cells (4T1, B16, HeLa, and HepG2) using the CCK-8 assay. Figure 21 (a) Figure 21 (b) Figure 21 (c) and Figure 21 The (d) in the text corresponds to four cell types: 4T1, B16, HeLa, and HepG2. Detailed Implementation

[0038] The preparation method of mitochondrial-targeting nanomaterials that can release HNO includes the following steps: Synthesis of silanized HNO donor (Si-HNOD): Hydroxylamine hydrochloride was dissolved in a mixed solvent of anhydrous tetrahydrofuran and anhydrous pyridine, and a dichloromethane solution of 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane was added. The reaction was stirred at room temperature, and the silanized HNO donor (Si-HNOD) was obtained after post-treatment. Synthesis of amminated carbon dots (CDs): Chitosan, ethylenediamine and mercaptosuccinic acid were dispersed in deionized water, concentrated hydrochloric acid was added and stirred until dissolved, and the reaction was carried out and post-treated to obtain amminated carbon dots (CDs). Synthesis of HNO-releasing mitochondrial-targeting nanomaterials (CDs-Si-HNOD): Aminated carbon dots were dispersed in anhydrous toluene to obtain a carbon dot suspension; a silanized HNO donor was dissolved in anhydrous toluene to obtain a toluene solution of the silanized HNO donor; the toluene solution of the silanized HNO donor was added dropwise to the carbon dot suspension, reacted, and post-treated to obtain HNO-releasing mitochondrial-targeting nanomaterials.

[0039] Addressing the core pathology of mitochondrial dysfunction in heart failure, and the clinical translational bottleneck caused by the unstable chemical properties and lack of targeted delivery capabilities of HNO, this invention focuses on the integrated design and synthesis of mitochondrial-targeting nanomaterials (CDs-Si-HNOD) capable of releasing HNO, centered around a "targeting-stabilizing-tracing" approach. The preparation mechanism of CDs-Si-HNOD in this invention is as follows: Figure 1 As shown: At the donor level, a silanized HNO donor, Si-HNOD, was designed. It achieves covalent grafting with carbon-based nanomaterials through a trimethoxysilyl group. The siloxane group of Si-HNOD undergoes a hydrolysis-condensation reaction with the hydroxyl groups on the surface of CDs: the siloxane group hydrolyzes to generate silanol groups, which then dehydrate and condense with the hydroxyl groups on the surface of CDs to form Si-OC bonds. This achieves stable loading of the HNO donor on the support, solving the problem that traditional donors cannot be stably loaded. Under physiological conditions, it slowly releases HNO at a 1:1 stoichiometric ratio (1 molecule of HNO is released per molecule of Si-HNOD degradation), with a half-life of 86 minutes, effectively avoiding burst release effects.

[0040] The carrier is an aminated carbon dot, utilizing its abundant hydroxyl groups to provide grafting sites. Protonated amino groups impart a positive potential of +31.55 mV, allowing for electrostatic targeting of mitochondria without the need for additional targeting ligand modification. Simultaneously, the wavelength-dependent fluorescence properties of the carbon dots enable real-time tracking of drug delivery. In the synthesis of CDs, chitosan, ethylenediamine, and mercaptosuccinic acid (mercaptosuccinic acid) serve as precursors. These three precursors undergo successive dehydration condensation, carbonization aromatization, and surface functionalization reactions. The active groups carried by the precursors cross-link to form covalent structures such as amide bonds and ether bonds, simultaneously constructing a sp-containing structure. 2 The hybrid carbon skeleton and N / S heteroatom-doped carbon core retain a large number of active functional groups on the carbon dot surface. Chitosan, as the main carbon source, provides hydroxyl and amino groups to participate in molecular cross-linking and carbon core construction, providing a positive charge basis and good water solubility for the carbon dots. Ethylenediamine provides a large number of amino groups, and N element doping regulates the fluorescence properties of the carbon dots, enhancing the positive charge density of the carbon dots. Mercaptosuccinic acid provides carboxyl and thiol groups, not only realizing S element doping and participating in cross-linking reactions, but also acting as a bridging molecule to induce the formation of delocalized aromatic structures of carbon dots. The synergistic effect of the groups of the three precursors enables the finally synthesized CDs to have both positive surface charge and delocalized structure, realizing the inherent mitochondrial targeting capability without additional targeting ligand modification.

[0041] Finally, through a silanization dehydration condensation reaction, Si-HNOD was covalently grafted onto the surface of CDs to form Si-OC bonds, constructing a core-shell structured nanosystem. This system integrates the targeting and fluorescence imaging advantages of CDs with the stable drug release characteristics of Si-HNOD, enabling precise delivery, controlled release, and visual tracking of HNO to myocardial mitochondria. The prepared mitochondrial-targeting nanomaterials that can release HNO can meet the needs of targeted therapy for heart failure.

[0042] It should be noted that, Figure 1The reaction mechanism of CDs-Si-HNOD in (b) is illustrated by taking only one hydroxyl group on CDs. In the actual reaction of CDs-Si-HNOD synthesis, the added Si-HNOD is in excess. Therefore, in the actual reaction, the other hydroxyl groups on CDs will also react with Si-HNOD.

[0043] Preferably, in the synthesis step of the silanized HNO donor, the molar ratio of hydroxylamine hydrochloride to 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane is 5:1.

[0044] Preferably, in the synthesis step of the silanized HNO donor, the reaction time of the stirring reaction at room temperature is 16-20 h, and the crude product is obtained after the reaction is completed.

[0045] Preferably, in the synthesis step of the silanized HNO donor, the post-processing step includes: adding ethyl acetate to the crude product for extraction, washing the extracted organic phase with deionized water and saturated brine, drying it with anhydrous sodium sulfate, removing the solvent under reduced pressure, and drying it under vacuum to obtain the silanized HNO donor (Si-HNO).

[0046] To further explain, in the synthesis step of the amination carbon dots, the reaction temperature is 180-190°C, the reaction time is 24-28 hours, and the reaction solution is obtained after the reaction is completed.

[0047] Preferably, in the synthesis step of the amination carbon dots, chitosan, ethylenediamine and mercaptosuccinic acid are dispersed in deionized water, concentrated hydrochloric acid is added and stirred until dissolved, and then transferred to a high-pressure reactor lined with polytetrafluoroethylene, and reacted at a constant temperature of 180°C for 24 hours.

[0048] Specifically, CDs use chitosan, ethylenediamine, and mercaptosuccinic acid (mercaptosuccinic acid) as precursors. In a high-temperature and high-pressure hydrothermal environment of 180℃ for 24 h, the three precursors undergo continuous dehydration condensation, carbonization and aromatization and surface functionalization reactions.

[0049] To further explain, in the synthesis step of the amination carbon dots, the post-processing step includes: filtering the reaction solution, dialyzing the filtrate, and then freeze-drying it to obtain the amination carbon dots.

[0050] By filtering the reaction solution, large particles and residues can be removed. The filtrate is dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 300 Da (with water changed every 6 hours), and then freeze-dried to obtain amination carbon dots.

[0051] To further explain, in the synthesis steps of the mitochondrial-targeting nanomaterial that can release HNO, the silanized HNO donor is dissolved in anhydrous toluene, and methanol is added dropwise to obtain a toluene solution of the silanized HNO donor.

[0052] When preparing a toluene solution of silanized HNO donor, adding methanol can increase the polarity of the solvent system (anhydrous toluene), allowing the silanized HNO donor to dissolve completely in anhydrous toluene.

[0053] To further explain, in the synthesis steps of the mitochondrial-targeting nanomaterials that can release HNO, the post-processing steps include: removing toluene solvent from the reactants by rotary evaporation under reduced pressure to obtain a solid crude product, washing the solid crude product with anhydrous ethanol, and obtaining the mitochondrial-targeting nanomaterials that can release HNO after vacuum drying.

[0054] Specifically, in the actual reaction for the synthesis of CDs-Si-HNOD, the added Si-HNOD is in excess, and the excess Si-HNOD can be removed by washing the crude solid product with anhydrous ethanol.

[0055] A mitochondrial-targeting nanomaterial capable of releasing HNO was prepared using the aforementioned method for preparing mitochondrial-targeting nanomaterials capable of releasing HNO.

[0056] The prepared mitochondrial-targeting nanomaterials that can release HNO can achieve precise delivery, controlled release, and visual tracking of HNO to myocardial mitochondria, solving the technical problems of traditional HNO donors being unable to achieve stable loading on carriers and lacking the ability to target mitochondria.

[0057] The application of the mitochondrial-targeting nanomaterials that release HNO in the preparation of drugs for treating heart failure.

[0058] To facilitate understanding of the present invention, a more complete description is provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0059] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0060] Example 1 The preparation method of mitochondrial-targeting nanomaterials that can release HNO includes the following steps: Step (1), Synthesis of silanized HNO donor (Si-HNOD): 107 mg of hydroxylamine hydrochloride (1.54 mmol) was dissolved in a mixed solvent of 15 mL of anhydrous tetrahydrofuran (THF) and 10 mL of anhydrous pyridine. 200 mg of a dichloromethane solution of 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane (2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane was dissolved in dichloromethane during raw material preparation, with a mass fraction of 50 wt%. The CAS number of 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane is 126519-89-9, brand: Aladdin) (0.308 mmol of 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane) was stirred at room temperature for 16 h. After the reaction was completed, the crude product was obtained. Post-processing: The crude product was extracted with 80 mL of ethyl acetate. The extracted organic phase was washed with deionized water (3 × 50 mL) and saturated brine, dried with anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The product was then dried under vacuum at 30 °C for 12 h to obtain a light green oily product, Si-HNOD (yield 76%).

[0061] Step (2), Synthesis of amination carbon dots (CDs): Weigh 1 g chitosan, 200 μL ethylenediamine and 500 mg mercaptosuccinic acid, disperse them in 29 mL deionized water, add 1 mL 12 M concentrated hydrochloric acid and stir until dissolved, transfer to a 50 mL polytetrafluoroethylene-lined high-pressure reactor, and react at 180 °C for 24 h. After the reaction is completed, the reaction solution is obtained. Post-processing: After the reactor cooled down, the reaction solution was filtered to remove large particles and residues. The filtrate was dialyzed for 24 hours using a dialysis bag with a molecular weight cutoff of 300 Da (the water was changed every 6 hours). After freeze drying, brownish-black CDs powder was obtained.

[0062] Step (3), Synthesis of mitochondrial-targeting nanomaterials that can release HNO (CDs-Si-HNOD): 50 mg of CDs powder was dispersed in 20 mL of anhydrous toluene and sonicated for 30 min to make it evenly dispersed and eliminate agglomeration, resulting in a carbon dot suspension; then 50 mg of Si-HNOD was dissolved in 2 mL of anhydrous toluene, and 1 drop of methanol was added to increase the polarity so that Si-HNOD could be completely dissolved, resulting in a toluene solution of silanized HNO donor. The toluene solution of silanized HNO donor was added dropwise to the carbon dot suspension, and the reaction was refluxed at 80 °C for 8 h under nitrogen protection. Post-processing: Toluene solvent in the reactants was removed by rotary evaporation under reduced pressure to obtain a crude solid product. The crude solid product was washed 3-5 times with 2 mL of anhydrous ethanol and dried in a vacuum drying oven at 25 °C for 12 h to obtain a black powdery solid CDs-Si-HNOD.

[0063] Performance testing and characterization: 1. Structural characterization of the silanized HNO donor (Si-HNOD) of Example 1.

[0064] (1) Analysis of the results of nuclear magnetic resonance hydrogen spectrum detection.

[0065] Analysis of the molecular structure of Si-HNOD reveals that the hydrogen atoms in the molecule can be classified into several types: aromatic hydrogens of the benzene ring, hydrogens of the N-hydroxysulfonamide functional group, methylene hydrogens of the ethyl chain, and methoxy hydrogens of the trimethoxysilyl group. Based on this, the structure of Si-HNOD can be further analyzed. 1 HNMR analysis was performed, such as Figure 2 As shown, the results were tested at 400 MHz in Chloroform-d solvent conditions. 1 In the 1H NMR spectrum, each signal peak can be clearly assigned: δ 8.69–8.64 (t, 2H) corresponds to the hydrogen atom adjacent to the sulfonyl group on the benzene sulfonyl ring; δ 7.80 (t, J = 7.7 Hz, 2H) is the meta-hydrogen on the benzene ring; δ 7.40 (t, 2H) is the para-hydrogen on the benzene ring; δ 2.02 (t, J = 15.1 Hz, 2H) belongs to the methylene hydrogen attached to the benzene sulfonyl group on the ethyl chain; δ 1.25 (s, 9H) is the hydrogen of the three equivalent methoxy groups in the trimethoxysilane group; and δ 0.88 (t, J = 6.8 Hz, 2H) corresponds to the methylene hydrogen attached to the silicon atom on the ethyl chain. Crucially, the characteristic peak at δ 8.64–8.69 directly corresponds to the hydrogen signal of the -NHOH functional group in the product. The appearance of this signal clearly proves that the sulfonyl chloride in the raw material has successfully reacted with hydroxylamine hydrochloride to form the target functional group. Combining the chemical shift, integral area and coupling constant of each peak, it can be clearly confirmed that the synthesized product is the target silanized HNO donor Si-HNOD, and its molecular structure is completely consistent with the design scheme.

[0066] (2) Analysis of the results of nuclear magnetic resonance carbon spectroscopy.

[0067] like Figure 3 As shown, 13 C NMR infers information about the carbon skeleton and substituents of compounds by measuring the nuclear magnetic resonance absorption signals of carbon atoms. Therefore, it was used to analyze Si-HNOD. 13C10 NMR spectroscopy was performed to further determine the structure and substituent positions of the product. The tests were conducted in DMSO-d6 solvent at 100 MHz. The results and analysis are as follows: The signal peaks at chemical shifts δ 148.07, 137.84, 128.50, 127.97, 124.85, and 124.63 ppm correspond to the six non-equivalent carbon atoms on the benzene ring in the benzenesulfonyl structure. The peaks at δ 148.07 and 137.84 ppm are attributed to the quaternary carbons of the benzene ring attached to the sulfonyl group and the ethyl group, while the remaining peaks are CH carbons on the benzene ring. The signals at δ 19.49, 13.09, and 11.27 ppm correspond to the carbons on the methoxy group (–OCH3) attached to silicon in the trimethoxysilyl group, consistent with the chemical shift characteristics of the CH3-O bond. The signals at δ 60.16 and 52.82 ppm... The peak at ppm is attributed to the two methylene carbons on the ethyl chain (–(CH2)2–), where δ 60.16 corresponds to the carbon corresponding to the methylene group attached to the quaternary carbon of the benzene ring, and δ 52.82 corresponds to the carbon corresponding to the methylene group attached to silicon. All signal peaks correspond one-to-one with the molecular structure of Si-HNOD (benzenesulfonyl hydroxylamine backbone + trimethoxysilyl ethyl side chain), with no obvious impurity peaks, indicating that the product has high purity and its structure is completely consistent with the designed silanized HNO donor.

[0068] 2. Ultraviolet kinetic characterization of the silanized HNO donor (Si-HNOD) of Example 1.

[0069] To demonstrate that the Si-HNOD donor can degrade under physiological conditions, the decomposition of the Si-HNOD donor (50.0 μmol / L) in phosphate buffer (pH 7.40, 0.10 M) and methanol (95:5, v / v) at 37 °C was monitored using a UV-Vis spectrophotometer. The corresponding UV-Vis spectral changes are summarized as follows: Figure 4 As shown, a clear, regular decrease in absorbance is observed at 235 nm in the spectrum, and a clean isoabsorption point is clearly visible at 229 nm. This indicates that the donor compound underwent only a single degradation reaction under physiological conditions. Figure 5 As shown, the Si-HNOD degradation kinetics curve was obtained by monitoring the absorbance change at 235 nm. Based on the absorption data at 235 nm wavelength, the complete decomposition of the Si-HNOD donor takes approximately 86.8 minutes. The degradation process of the donor molecule under physiological conditions completely conforms to the first-order reaction (k... obs = 0.00799±4.27688×10 -5 s -1 , t 1 / 2 = 86.8 min). Specifically, under physiological conditions, the Si-HNOD donor can achieve slow HNO release during the donor degradation process.

[0070] Figure 6 The pH-responsive degradation behavior of Si-HNOD was demonstrated. Figure 6 (a) shows the time-dependent absorption spectrum at pH=9 and 37 ℃. It can be seen that the characteristic absorption peaks at 219 nm and 229 nm continuously increase with time. Based on the change in absorbance at 219 nm, we can obtain... Figure 7 The inset in (a) is used to obtain k through nonlinear fitting. obs = 0.0138±3.2861×10 -5 s -1 Half-life t 1 / 2 =50 min. Furthermore, a pure isoabsorbance point appeared at 229 nm in the figure, which demonstrates that the pH-responsive degradation behavior of the silanized HNO donor Si-HNOD follows first-order reaction kinetics. Figure 6 (b) in the middle presents k obs Trends with pH (7–14): In the weakly alkaline range of pH 7–10, k obs Growth is gradual, such as when pH=8, k obs = 0.0108±3.2526×10 -5 s -1 (t) 1 / 2 = 65 min); when pH > 11 enters the strongly alkaline range, k obs The pH value spikes dramatically, peaking at 13-14, demonstrating a high sensitivity to strongly alkaline environments. In summary, the degradation rate of Si-HNOD increases significantly with increasing pH, providing important evidence for its application in targeted delivery.

[0071] 3. Characterization of HNO release from the silanized HNO donor (Si-HNOD) in Example 1.

[0072] The hydroxycobalamin (HOCbl) specific capture method was employed, based on its efficient and specific reaction with HNO to generate nitrosocbalamin (NOCbl) accompanied by characteristic UV-Vis absorption spectral changes. This method was used to systematically characterize the HNO release performance, kinetics, and stoichiometry of Si-HNOD. The experiment was conducted under anaerobic conditions. Using a 0.1 M phosphate buffer solution (pH 9.03) and methanol (95:5 v / v) as the system, 100.0 μM Si-HNOD was added to a 50.0 μM HOCbl solution. The reaction process was monitored in real-time using time-resolved UV-Vis absorption spectroscopy. Figure 7The image shows the changes in the UV-Vis absorption spectra of the system after Si-HNOD was added to the HOCbl solution at different time points. As can be seen from the figure, three clear isoabsorbance points appear at 341 nm, 366 nm, and 496 nm, indicating that only two stable absorbing species, HOCbl and NOCbl, exist in the system, with no obvious side reactions, confirming the high specificity of the HNO-HOCbl reaction. Simultaneously, the absorbance of the maximum characteristic absorption peak of HOCbl at 352 nm decreases significantly, and the characteristic peak at 535 nm shows a significant blue shift. This spectral change is consistent with the specific reaction spectral behavior of HNO, directly confirming that Si-HNOD can effectively release HNO. Figure 7 The kinetic fit of the inset shows that the reaction follows a first-order kinetic model, with an apparent rate constant kobs of 0.0125 ± 6.16 × 10⁻⁶. -5 s -1 The half-life of 55 min is highly consistent with the degradation half-life of Si-HNOD at pH 9.03, indicating that HNO release is dominated by donor degradation, and the two rates are synchronized. This provides a key basis for the controllable and sustained release of HNO and the design of cell drug delivery time windows. To clarify the stoichiometry, the HOCb1 concentration was fixed, and 0–2.0 molar equivalents of Si-HNOD were added, such as… Figure 8 As shown, the spectrum after equilibrium shows that the absorbance at 535 nm decreases regularly with the molar equivalent of Si-HNOD. Linear fitting confirms that the amount of HNO generated is strictly related to the amount of Si-HNOD added in a 1:1 ratio, which clarifies that each molecule of Si-HNOD releases 1 molecule of HNO during degradation.

[0073] To further verify the HNO release capacity and stoichiometry of the silanized HNO donor Si-HNOD, a phosphorus trapping method was used for quantitative analysis. Given the susceptibility of HNO to side reactions with oxygen, the entire experiment was strictly conducted in an oxygen-free glove box to avoid oxidation interference. Commercially available 2-diphenylphosphine methyl terephthalate (CAS No.: 361154-31-6) was used as the specific trapping agent. A mixture of 2.25 mmol / L phosphine compound (2-diphenylphosphine methyl terephthalate) and 0.75 mmol / L Si-HNOD donor at a volume ratio of 3:1 was placed in an anaerobic carbonate buffer solution (pH 9.03) and deuterated acetonitrile (volume ratio 40:60). After incubation for 18 hours, samples were collected. 31 P NMR spectrum. For example, Figure 9The spectral results showed two clear characteristic phosphorus signal peaks in the system, with chemical shifts of δ 33.79 ppm and δ 34.45 ppm, corresponding to the two characteristic products generated in the reaction: phosphine oxide and phosphonamide. Their relative integral ratio was approximately 1:1, directly confirming at the NMR level that Si-HNOD can effectively decompose and release HNO under the specified conditions. Combined with previous experimental results using the hydrocobalamin (HOCbl) specific capture method, both independent detection methods clearly confirmed that the HNO release process from Si-HNOD follows a strict stoichiometric ratio of 1:1, meaning that each molecule of Si-HNOD releases one molecule of HNO. This conclusion provides a reliable basis for the accurate quantification of HNO release. By directly measuring the concentration of Si-HNOD, the HNO release amount can be rapidly calculated, laying a core foundation for subsequent performance evaluation of the nanodelivery system and optimization of drug delivery dosage.

[0074] 4. Structural and performance characterization of the HNO-releasing mitochondrial-targeting nanomaterial (CDs-Si-HNOD) of Example 1 and comparison with aminated carbon dot CDs.

[0075] (1) Transmission electron microscopy (TEM) characterization of CDs-Si-HNOD.

[0076] Transmission electron microscopy (TEM) was used to examine silanized grafted CDs. Si The microstructure, particle size, crystal structure, and core-shell structure of HNOD carbon-based hybrid nanomaterials were characterized. For example... Figure 10 As shown in the low-magnification TEM, the hybrid particles are uniformly quasi-spherical, well-dispersed, with no obvious agglomeration, and only a very small amount of weak aggregation, indicating that Si HNOD grafting can improve the dispersion stability of carbon dots by modulating steric hindrance and surface charge. Through particle size analysis, CDs... Si The average particle size of HNOD was 5–10 nm, slightly larger than that of pure carbon dots, confirming the successful coating of the surface with an organosilicon layer. High-resolution TEM (HRTEM) revealed its typical "carbon crystal nuclei". Amorphous organic shell core-shell structure: the particle center has clear and continuous lattice fringes, corresponding to well-crystallized sp. 2 The hybrid carbon nuclei, with lattice spacing consistent with graphitized carbon, demonstrate that the grafting reaction did not disrupt the intrinsic crystal structure of the carbon dots. The nucleus size is 3–8 nm, essentially identical to that of pure carbon dots. A uniform and intact amorphous shell, 1–2 nm thick, is visible on the periphery, corresponding to Si. HNOD organic functional layers directly confirm the morphological properties of Si. O The C-covalent grafting was successful, which is corroborated by the infrared spectroscopy results below. This uniform organosilicon shell can both inhibit aggregation and improve colloidal stability, and provide stable covalent loading sites for HNO3. While retaining the fluorescence properties of carbon dots, it enables controllable loading and delivery of HNO3, laying the structural foundation for its mitochondrial targeting, real-time fluorescence tracing, and heart failure-related biomedical applications.

[0077] (2) Fourier transform infrared (FT-IR) characterization of CDs-Si-HNOD.

[0078] By comparing pure carbon dots (CDs) and Si HNOD and CDs Si HNOD's FT IR spectroscopy confirmed the molecular bonding level of Si. Successful covalent grafting of HNOD onto carbon dot surfaces. For example... Figure 11 As shown, CDs Si HNOD at 1039 cm -1 Si appears at the location O The C-asymmetric stretching vibration peak proves that the hydroxyl groups on the CDs surface interact with Si. The silanol groups after HNOD hydrolysis undergo dehydration condensation to form stable covalent bonds, providing direct evidence for the construction of hybrid interfaces. Simultaneously, at 563 cm⁻¹... -1 802 cm -1 The location appears with Si The consistent characteristic peaks of HNOD indicate that the silane group and HNO donor structure are completely preserved after grafting, providing a structural basis for subsequent HNO delivery function.

[0079] CDs-Si-HNOD in 3000~3700 cm -1 O at the location H stretching vibration peak and 1500 cm -1 The significantly weakened bending vibration peak of nearby hydroxyl groups indicates that a large number of hydroxyl groups on the carbon dot surface participated in the reaction with Si. O The C bond formation mechanism is consistent. And 2337 cm -1 The intrinsic characteristic peaks of the carbon dots remained largely unchanged, indicating that the grafting modification did not damage the core structure of the carbon dots, and their inherent fluorescence properties were preserved. In summary, FT... IR results confirm that CDs and Si HNOD via Si O The stable covalent bonding of C and C elucidates the chemical structure and interfacial reaction mechanism of the hybrid material, providing key structural evidence for the realization of the material's optical properties, dispersion stability, and mitochondrial-targeted biological functions.

[0080] (3) Ultraviolet-visible spectral characterization of CDs-Si-HNOD.

[0081] Comparing the UV-Vis absorption spectra of CDs-Si-HNOD and pure carbon dots (CDs), it can be seen that both types of samples exhibit intrinsic carbon dot absorption in the 200–400 nm range. Figure 12 As shown, pure CDs have a strong absorption peak at 200 nm, with an absorbance of 2.8, which originates from sp. 2 The carbon core undergoes π-π transitions and surface functional groups undergo n-π transitions, which then decrease rapidly with increasing wavelength, approaching zero after 400 nm, consistent with typical carbon dot absorption patterns. After Si-HNOD grafting, the absorption peak of CDs-Si-HNOD at 200 nm is enhanced to 3.1, and it exhibits a significant broad absorption range from 220 to 350 nm, which is significantly different from pure CDs. The absorbance at 220 nm remains above 0.5, and continuous characteristic absorption appears from 250 to 280 nm, corresponding to the electronic transitions between the silanized group and Si-HNOD, directly proving the successful covalent modification of the organic functional molecule. After 300 nm, the absorption trends of both are similar, decreasing rapidly, indicating that the modification did not change the intrinsic long-wavelength behavior of the carbon dot. Overall, the results show that CDs-Si-HNOD retains the inherent optical properties of the carbon dot core while introducing the characteristic absorption of Si-HNOD, optically confirming the success of the grafting. Its wide absorption characteristics in the ultraviolet region provide an important optical basis for biomedical applications such as fluorescence imaging, controlled delivery of HNO, and intracellular tracing.

[0082] (4) Zeta potential characterization of CDs-Si-HNOD.

[0083] like Figure 13Zeta potential measurements of CDs-Si-HNOD showed that the pure CDs had a Zeta potential of +31.55 mV. This strong positive charge originates from the protonated nitrogen-containing functional groups and cationic active sites on its surface. While this ensures dispersion in the aqueous phase, the excessively high positive potential can easily lead to non-specific protein adsorption and biotoxicity, which does not meet the requirements for biomedical applications. After Si-HNOD grafting modification, the Zeta potential of CDs-Si-HNOD decreased to +7.88 mV. The positive charge was controllably reduced and remained stable, confirming the successful grafting at the surface charge level, which is consistent with the FT-IR and UV-Vis characterization results. The reduction in potential is due to two synergistic effects: first, the electronegative Si-HNOD grafting covers the positively charged active sites on the CDs surface, reducing the charge density; second, the grafting reaction consumes the positively charged active groups on the CDs surface, changing the interfacial charge distribution, consistent with the functional group changes observed in infrared spectroscopy. From an application perspective, the suitable potential of +7.88 mV has dual advantages: it inhibits aggregation and improves dispersion and storage stability through electrostatic repulsion, while avoiding the biotoxicity of excessively high positive potentials and optimizing biocompatibility; at the same time, the retained positive charge can target the negative transmembrane potential of mitochondria, providing support for mitochondrial targeted delivery. Combined with its fluorescence properties, it can meet the dual needs of drug delivery and fluorescence tracing, laying the foundation for subsequent biological applications related to the treatment of heart failure.

[0084] (5) Fluorescence spectral characterization of CDs-Si-HNOD.

[0085] like Figure 14 The three-dimensional fluorescence spectroscopy of the CDs-Si-HNOD nanomaterials reveals that the CDs-Si-HNOD hybrid nanomaterials exhibit wavelength-dependent fluorescence emission characteristics, consistent with typical optical features of carbon dots, demonstrating the synergistic regulatory effect of the hybrid structure on optical performance. When the excitation wavelength increases from 260 nm to 580 nm, its fluorescence emission peak redshifts from 300–500 nm to 500–800 nm, with the emission intensity first increasing and then decreasing, reaching a peak value (≥40000 au) under excitation at 360–460 nm. Excellent fluorescence response is still observed in the visible light region, and the wide-range tunable excitation-emission characteristics provide a foundation for bioimaging. In terms of the luminescence mechanism, under short-wavelength excitation (260–320 nm), fluorescence originates from exciton recombination in the carbon dot core, surface defect states, and electronic transitions in the Si-HNOD organic chromophores. Under near-ultraviolet-visible excitation (350–500 nm), the conjugated carbon domains of the carbon dots and the surface hybrid structure are the main luminescence centers, and the redshift of the emission peak originates from the interfacial electronic coupling effect of the Si-OC covalent bond, confirming the success of covalent grafting. In summary, CDs-Si-HNOD retains the excellent fluorescence performance of carbon dots and achieves wide-range tunable fluorescence emission. Its broad spectral characteristics are suitable for multi-channel imaging, providing support for the dual needs of real-time tracking of intracellular drug delivery, mitochondrial targeted delivery, and fluorescence imaging.

[0086] (6) X-ray photoelectron spectroscopy (XPS) characterization of CDs-Si-HNOD.

[0087] XPS was used to further verify the successful synthesis of CDs-Si-HNOD from the perspectives of elemental composition and chemical valence state, and the results are as follows: Figure 15 As shown in the XPS display, CDs-Si-HNOD mainly contains four elements: C, O, N, and Si. Si is a characteristic element of Si-HNOD. Figure 15 As can be seen from (e), pure CDs mainly contain three elements: C, N, and O, and there is no Si element signal, which is consistent with the composition of the raw material. This result directly proves that Si-HNOD was successfully grafted onto the surface of CDs.

[0088] Peak fitting analysis was performed on the high-resolution energy spectrum of CDs-Si-HNOD: C 1s The spectrum can be divided into three characteristic peaks, corresponding to the CC / C=C bond at 284.5 eV, the CO / CN bond at 283.5 eV, and the C=O bond at 280.5 eV, which are consistent with the carbon skeleton structure of CDs; O 1s The high-resolution energy dispersive spectroscopy (EDS) spectrum can be divided into two characteristic peaks, corresponding to the C=O bond at 529.5 eV and the C-OH / COC bond at 530.5 eV (where the COC peak corresponds to adsorbed impurities), consistent with the carbon framework structure of CDs-Si-HNOD; Si 2p The spectrum can be divided into two characteristic peaks, corresponding to the Si-O bond at 98.0 eV and the Si-C bond at 97.0 eV, respectively. These two peaks are characteristic bonding peaks of Si-HNOD, which further verify the chemical state of Si and are consistent with the results of FTIR.

[0089] (7) Characterization of HNO release from CDs-Si-HNOD.

[0090] The formation of HNO3 was verified using the hydroxycobalamin (HOCbl) capture method. Under anaerobic conditions, after mixing CDs-Si-HNOD with HOCbl, the UV-Vis spectrum of the system changed significantly with reaction time, such as... Figure 16 As shown in the figure (the curves in the figure show the changes in absorbance over time): the characteristic absorption peak intensity of HOCbl at 352 nm continuously decreases, the absorption peak at 525 nm shows a significant red shift, and clear isoabsorption points appear at 341 nm, 366 nm, and 496 nm. These spectral changes are characteristic evidence of the reaction of HOCbl with HNO to form nitrocobalamin (NOCbl); Figure 16 As shown in the illustration, the kinetic fitting results indicate that the reaction conforms to a first-order reaction kinetic model, with an apparent rate constant k.obs =(1.25±0.06)×10 -2 s -1 Half-life t 1 / 2 =55 min, proving that the reaction rate is determined by the HNO release step, and that covalent grafting did not change the HNO release mechanism and stoichiometry of Si-HNOD.

[0091] (8) Calculation of the loading rate of CDs-Si-HNOD.

[0092] Based on HNO and VB 12 Hydroxycobalamin (HOCbl) exhibits a 1:1 stoichiometric specific reaction. The drug loading efficiency of Si-HNOD in CDs-Si-HNOD was indirectly quantified using UV-Vis spectrophotometry. 31 The P NMR method is used to cross-validate the measurement results to ensure the accuracy and reliability of the load rate data.

[0093] 150 μL of CDs-Si-HNOD at different concentrations (0.111, 0.167, 0.238, 0.267, 0.333, 0.667 mg / mL) was reacted with 50 μL of 50 μmol / L HOCb1 under anaerobic conditions at pH 9.03 and 37 ℃. The UV-Vis absorption spectra results are as follows: Figure 17 a) The results show that as the concentration of CDs-Si-HNOD decreased from 0.267 mg / mL to 0.238 mg / mL, the characteristic UV absorption peak at 535 nm gradually increased from its lowest point, proving that the HNO3 released by CDs-Si-HNOD at this concentration was insufficient to react with 50 μL of 50 μmol / L VB. 12 The reaction is complete, according to HNO and VB. 12 The molar amount released in a 1:1 stoichiometric reaction can be calculated as: n HNO =50 μL × 50 μmol / L = 2.5 × 10 -9 mol, meaning the molar amount of Si-HNOD supported is 2.5 × 10⁻⁶. -9 If mol, then the mass of the Si-HNOD loaded is: m Si-HNOD =321.0 g / mol × 2.5 × 10 -9 mol = 0.0008025 mg. The mass of CDs-Si-HNOD contained in 150 μL of 0.238 mg / mL CDs-Si-HNOD solution is: m CDs-Si-HNOD=150 μL × 0.238 mg / mL = 0.0357 mg. The mass of CDs-Si-HNOD contained in 150 μL of 0.267 mg / mL CDs-Si-HNOD solution is: m CDs-Si-HNOD =150 μL × 0.267 mg / mL = 0.0401 mg. Therefore, according to the drug loading calculation formula... Calculations show that the drug loading rate of Si-HNOD in CDs-Si-HNOD is 2.00%~2.24%.

[0094] To further verify the accuracy of the loading rate measurement results and to conduct further quantitative research on HNO release from CDs-Si-HNOD, the specific capture of HNO by organophosphorus compounds was employed. 31 The P NMR method was used to verify this. In the experiment, the concentration of CDs-Si-HNOD obtained from UV-Vis spectrophotometry at 0.238 mg / mL was approximately 50 μmol / L. 0.75 mmol / L represents the concentration of CDs-Si-HNOD required for the specific HNO3 capture experiment using phosphine compounds, according to the formula... Calculation x The concentration is 3.57 mg / mL. Since 100 μL of CDs-Si-HNOD solution only accounts for 1 / 10 of the total volume in a J-Young NMR tube, the final required concentration of CDs-Si-HNOD is... x 2 = 10 × x =1 = 35.7 mg / mL. Therefore, 35.7 mg of CDs-Si-HNOD was dispersed and dissolved in 1 mL of deuterated acetonitrile. In a J-Young NMR tube, 400 μL of pH 9.03 anaerobic carbonate buffer, 300 μL of organophosphorus compound stock solution (2.25 mmol), 200 μL of deuterated acetonitrile, and 100 μL of CDs-Si-HNOD solution were added sequentially. After incubation at 37 °C for 18 h under anaerobic conditions, the reaction system was collected. 31 p NMR spectrum ( Figure 17 (b) The results showed that two characteristic peaks appeared in the spectrum at δ=-34.54 ppm and δ=-33.99 ppm, corresponding to the phosphine oxide product and phosphoramide product generated by the reaction of HNO with organophosphine compounds, respectively. The integral area ratio of the two was approximately 1.00:1.06, which was completely consistent with the characteristic results of the HNO specific capture reaction. This directly confirmed that CDs-Si-HNOD can stably and quantitatively release HNO, and the accuracy and reliability of the loading rate measurement results were further verified.

[0095] The above results collectively demonstrate that after Si-HNOD is covalently grafted onto the surface of CDs, its HNO release function is fully preserved, and it can still quantitatively and continuously release HNO. Its loading efficiency can fully meet the dosage requirements of subsequent cellular and animal pharmacodynamic experiments.

[0096] (9) Characterization of mitochondrial colocalization of CDs-Si-HNOD.

[0097] Cellular colocalization experiments were conducted in rat H9c2 cardiomyocytes using laser scanning confocal microscopy (CLSM) to verify the mitochondrial targeting ability of CDs-Si-HNOD. Mitochondria were labeled with MitoTracker Deep Red FM (mitochondrial deep red fluorescent probe), the distribution of CDs-Si-HNOD was characterized by intrinsic green fluorescence, and the cell nucleus was labeled with DAPI (4',6-diamidindole-2-phenylindole). Imaging results from multiple parallel samples were consistent. Figure 18 CLSM imaging showed that the green fluorescence of CDs-Si-HNOD highly overlapped with the red fluorescence distribution in mitochondria, and the fused image presented a clear yellow co-localization region. No fluorescence diffusion in the cytoplasm or abnormal aggregation in the nucleus was observed, demonstrating that it can efficiently target mitochondria after endocytosis. Figure 19 Fluorescence intensity line scan analysis also verified the precise co-localization of the two. ImageJ software was used to perform co-localization analysis on images acquired by CLSM. Quantitative characterization was achieved by calculating the Pearson correlation coefficient between green fluorescence (CDs-Si-HNOD) and red fluorescence (MitoTracker). ImageJ quantitative analysis showed a Pearson correlation coefficient of 0.93±0.03, confirming an extremely high degree of co-localization, fully demonstrating that CDs-Si-HNOD possesses excellent and stable mitochondrial targeting ability in H9c2 cardiomyocytes. Its targeting ability stems from: firstly, the positive surface charge targets the negative transmembrane potential of mitochondria through electrostatic interaction, requiring no additional ligands; secondly, the uniform nanoscale size of the carbon dots promotes endocytosis and avoids lysosomal degradation. Furthermore, DAPI staining showed intact nucleus morphology and no apoptosis abnormalities, preliminarily verifying the material's good cell safety, consistent with the following cytotoxicity experiments. These results indicate that CDs-Si-HNOD overcomes the traditional HNO donor targeting bottleneck and can achieve precise delivery of HNO to cardiomyocyte mitochondria.

[0098] (10) Cytotoxicity test of CDs-Si-HNOD.

[0099] The CCK-8 assay was used to evaluate the effects of CDs-Si-HNOD on the proliferation of normal H9c2 rat cardiomyocytes and four types of tumor cells (4T1, B16, HeLa, and HepG2), and to clarify its cytotoxicity, biocompatibility, and safe dosing window.

[0100] like Figure 20 The results showed no significant differences in the toxic response of different cell lines to this nanomaterial. Within the entire experimental concentration range of 16–8000 μg / mL, the relative survival rate of H9c2 cells remained stable at over 90% after 24 h of incubation, with no significant proliferation inhibition even at the highest concentration. No dose-dependent toxicity was observed, demonstrating excellent biocompatibility and safety. Figure 21 As shown, the relative survival rate of the four tumor cell types was over 95% within the concentration range of 60–2000 μg / mL, with no concentration-dependent toxicity, further validating the material's low toxicity stability and universality. Its low toxicity stems from two aspects: first, the carbon dot core itself has excellent biocompatibility, and silanization modification shields potential active sites, reducing non-specific interactions; second, the material has a stable intracellular structure, does not degrade to produce toxic byproducts, and does not induce abnormal increases in reactive oxygen species. Based on this, its safe dosage concentration range was determined to be 0–8000 μg / mL. Within this range, effective release of HNO is ensured while exhibiting no significant toxicity to H9c2 cells, providing a reliable safety basis for its in vitro and in vivo application in the treatment of heart failure.

[0101] In summary, CDs-Si-HNOD overcomes the technical limitations of traditional HNO donors. The synthesized silanized donor Si-HNOD solves the problem of unstable loading of traditional donors, stably releasing HNO at a 1:1 stoichiometric ratio under physiological conditions with a half-life of 86 minutes, balancing efficacy and controllability. Constructed using carbon dots as a carrier, CDs-Si-HNOD achieves mitochondrial targeting without additional modification due to the positive surface potential (+7.88 mV) of the carbon dots, with a Pearson colocalization coefficient of 0.93±0.03, successfully delivering HNO to the core target site of mitochondrial heart failure. Simultaneously, the fluorescence properties of the carbon dots endow the system with real-time tracking capabilities, forming an integrated "delivery-tracking" function. This system also solves key issues of biocompatibility and stability, maintaining over 90% survival rate in H9c2 cardiomyocytes even at a high concentration of 8000 μg / mL, and covalent grafting prevents premature HNO leakage. The drug loading capacity of CDs-Si-HNOD can be precisely controlled from 2.0% to 2.24%. CDs-Si-HNOD integrates the pharmacological advantages of HNO, the targeting and imaging properties of carbon dots, and the stable grafting advantages of silanization technology, effectively breaking through the core bottleneck of HNO clinical translation. It provides a novel nanomaterial for mitochondrial targeted precision treatment of heart failure and also provides an important reference for the targeted delivery of reactive nitrogen species.

[0102] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for preparing a mitochondria-targeting nanomaterial that can release HNO, characterized in that, Includes the following steps: Synthesis of silanized HNO donor: Hydroxylamine hydrochloride was dissolved in a mixed solvent of anhydrous tetrahydrofuran and anhydrous pyridine, and a dichloromethane solution of 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane was added. The reaction was stirred at room temperature, and the silanized HNO donor was obtained after post-treatment. Synthesis of aminated carbon dots: Chitosan, ethylenediamine and mercaptosuccinic acid were dispersed in deionized water, concentrated hydrochloric acid was added and stirred until dissolved, and the reaction was followed by post-treatment to obtain aminated carbon dots; Synthesis of HNO-releasing mitochondrial-targeting nanomaterials: Aminated carbon dots were dispersed in anhydrous toluene to obtain a carbon dot suspension; a silanized HNO donor was dissolved in anhydrous toluene to obtain a toluene solution of the silanized HNO donor; the toluene solution of the silanized HNO donor was added dropwise to the carbon dot suspension, reacted, and post-treated to obtain HNO-releasing mitochondrial-targeting nanomaterials.

2. The method for preparing mitochondrial-targeting nanomaterials capable of releasing HNO according to claim 1, characterized in that, In the synthesis step of the silanized HNO donor, the molar ratio of hydroxylamine hydrochloride to 2-(4-chlorosulfonylphenyl)ethyltrimethoxysilane is 5:

1.

3. The method for preparing mitochondrial-targeting nanomaterials capable of releasing HNO according to claim 1, characterized in that, In the synthesis step of the silanized HNO donor, the reaction time of the stirred reaction at room temperature is 16-20 h, and the crude product is obtained after the reaction is completed.

4. The method of claim 3, wherein the mitochondrial-targeting nanomaterial releasable HNO is prepared by, In the synthesis steps of the silanized HNO donor, the post-processing steps include: adding ethyl acetate to the crude product for extraction, washing the extracted organic phase with deionized water and saturated brine, drying it with anhydrous sodium sulfate, removing the solvent under reduced pressure, and drying it under vacuum to obtain the silanized HNO donor.

5. The method for preparing mitochondrial-targeting nanomaterials capable of releasing HNO according to claim 1, characterized in that, In the synthesis step of the amination carbon dots, the reaction temperature is 180-190℃, the reaction time is 24-28h, and the reaction solution is obtained after the reaction is completed.

6. The method of claim 5, wherein the mitochondrial-targeted nanomaterial releasable HNO is prepared by, In the synthesis step of the amination carbon dots, the post-processing step includes: filtering the reaction solution, dialyzing the filtrate, and then freeze-drying it to obtain the amination carbon dots.

7. The method for preparing mitochondrial-targeting nanomaterials capable of releasing HNO according to claim 1, characterized in that, In the synthesis step of the mitochondrial-targeting nanomaterial that can release HNO, the silanized HNO donor is dissolved in anhydrous toluene, and methanol is added dropwise to obtain a toluene solution of the silanized HNO donor.

8. The method for preparing mitochondrial-targeting nanomaterials capable of releasing HNO according to claim 1, characterized in that, In the synthesis steps of the mitochondrial-targeting nanomaterials that can release HNO, the post-processing steps include: removing toluene solvent from the reactants by rotary evaporation under reduced pressure to obtain a solid crude product, washing the solid crude product with anhydrous ethanol, and drying it under vacuum to obtain the mitochondrial-targeting nanomaterials that can release HNO.

9. A mitochondrial-targeting nanomaterial capable of releasing HNO, characterized in that, It was prepared using the method for preparing mitochondrial-targeting nanomaterials that can release HNO as described in any one of claims 1 to 8.

10. The use of the mitochondrial-targeting nanomaterial capable of releasing HNO as described in claim 9 in the preparation of a medicament for treating heart failure.

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