Long afterglow nanocarrier, preparation method and application thereof

Abstract

The application discloses a long-afterglow nano carrier and a preparation method and application thereof, and belongs to the field of biomedical materials. The long-afterglow nano carrier comprises a near-infrared luminescent center, an organic mesoporous silica layer and an autophagy inhibitor; the organic mesoporous silica layer is coated on the outer layer of the near-infrared luminescent center, and the autophagy inhibitor is loaded in the mesopores of the organic mesoporous silica layer; the near-infrared luminescent center comprises a transition metal element; the organic mesoporous silica layer is prepared by co-condensation of an organic silane precursor and an inorganic silicon precursor, wherein the organic silane precursor is an organic silane containing a tetrasulfide bond, and the inorganic silicon precursor is a silicate compound. The carrier can specifically respond to excessively high concentration of hydrogen peroxide in a tumor microenvironment, and accurately release the loaded autophagy inhibitor; the release process can be observed by positioning with the aid of a high signal-to-noise ratio afterglow signal, thereby providing guidance for subsequent drug release, and finally realizing efficient radiotherapy sensitization.

Description

Technical Field

[0001] This application relates to a long afterglow nanocarrier, its preparation method and application, belonging to the field of biomedical materials. Background Technology

[0002] Radiotherapy primarily kills tumor cells by inducing DNA damage and generating reactive oxygen species (ROS), making it a core component of clinical cancer treatment. However, its efficacy is often limited by radiotherapy resistance developed in tumor cells. Among numerous mechanisms, autophagy, a highly conserved cellular self-degradation process, has been identified as a key driver of radiotherapy resistance. During radiotherapy, tumor cells can respond to this stress by activating protective autophagy. On one hand, autophagy clears damaged organelles and misfolded proteins accumulated due to radiation, reducing intracellular ROS buildup and genotoxicity, thereby maintaining cellular homeostasis and aiding cell survival. On the other hand, autophagy directly inhibits radiotherapy-induced apoptosis by degrading key proteins or activating survival signaling pathways. Therefore, autophagy is an important mechanism for radiotherapy resistance and self-protection in tumor cells.

[0003] The introduction of autophagy inhibitors is an important emerging strategy for reversing radiotherapy resistance. Their mechanism of action primarily focuses on blocking protective autophagic flux: by using inhibitors such as chloroquine and hydroxychloroquine, lysosomal function can be interfered with, preventing autophagosome degradation and causing the accumulation of damaged substances within cells, thereby enhancing radiation-induced metabolic stress and DNA damage. Studies have confirmed that the combination of radiotherapy and autophagy inhibition can significantly increase the sensitivity of tumor cells to radiation, promote apoptosis and necroptosis, and exhibit synergistic anti-tumor effects in various solid tumor models. However, currently reported administration methods for autophagy inhibitors are still mainly direct intravenous injection or single-factor stimulation, which suffer from problems such as non-targeted release and slow release rate. Summary of the Invention

[0004] To address practical needs and the shortcomings of existing technologies, this application provides a long-afterglow nanocarrier, its preparation method, and its application. The long-afterglow nanocarrier comprises a near-infrared luminescent center, an organic mesoporous silica layer, and an autophagy inhibitor. The organic mesoporous silica layer coats the outer layer of the near-infrared luminescent center, and the autophagy inhibitor is loaded within the mesopores of the organic mesoporous silica layer. The near-infrared luminescent center includes a transition metal element. The organic mesoporous silica layer is prepared by co-condensation of an organosilane precursor and an inorganic silicon precursor, wherein the organosilane precursor is a tetrasulfide-containing organosilane, and the inorganic silicon precursor is a silicate compound. This carrier can specifically respond to excessively high concentrations of hydrogen peroxide in the tumor microenvironment (TME) and precisely release the loaded autophagy inhibitor. The release process can be located and observed using a high signal-to-noise ratio afterglow signal, thereby providing guidance for subsequent drug release and ultimately achieving highly efficient radiosensitization.

[0005] According to a first aspect of this application, a long-afterglow nanocarrier is provided, which exhibits uniform particle size, good dispersibility, and high stability. Under near-infrared light excitation, hydrogen peroxide can specifically break tetrasulfide bonds in the silicon layer, thus achieving a specific response to hydrogen peroxide and enabling the controlled release of the loaded autophagy inhibitor, thereby enhancing radiosensitization. The long-afterglow nanocarrier, under near-infrared excitation, can achieve precise and highly sensitive localization of tumors without biological background interference, while simultaneously guiding subsequent drug release, providing a new method for enhancing the sensitization of tumor radiotherapy.

[0006] A long-afterglow nanocarrier, the long-afterglow nanocarrier comprising a near-infrared luminescent center, an organic mesoporous silica layer, and an autophagy inhibitor; The organic mesoporous silica layer covers the outer layer of the near-infrared luminescent center, and the autophagy inhibitor is loaded in the mesopores of the organic mesoporous silica layer. The near-infrared luminescent center includes a transition metal element; The organic mesoporous silica layer is prepared by co-condensation of an organosilane precursor and an inorganic silicon precursor, wherein the organosilane precursor is an organosilane containing a tetrasulfide bond, and the inorganic silicon precursor is a silicate ester compound.

[0007] The near-infrared luminescent center has near-infrared afterglow imaging capabilities, which can monitor its distribution and retention within the tumor through afterglow signals, guiding radiotherapy and the timing of autophagy inhibitor release; the organic mesoporous silica layer has porous adsorption properties, which can load autophagy inhibitors through electrostatic adsorption, and the tetrasulfide bonds it contains can undergo redox bond breaking reactions with hydrogen peroxide, thereby controlling the release of autophagy inhibitors.

[0008] Optionally, the particle size of the long afterglow nanocarrier is 250-350 nm.

[0009] Optionally, the transition metal element includes zinc, gallium, germanium, and chromium.

[0010] Optionally, the molar ratio of zinc, gallium, germanium and chromium is (0.8-1.4):(1.4-2.2):(0.05-0.15):(0.001-0.01).

[0011] Optionally, the organosilane containing a tetrasulfide bond is bis[3-(triethoxysilyl)propyl]tetrasulfide.

[0012] Optionally, the silicate compounds include orthosilicate compounds.

[0013] Optionally, the orthosilicate compound is ethyl orthosilicate.

[0014] Optionally, the volume ratio of the organosilane precursor to the inorganic silicon precursor is (6-4):(4-3).

[0015] Optionally, the autophagy inhibitor is chloroquine.

[0016] The controlled release mechanism of chloroquine utilizes the high concentration of hydrogen peroxide in the tumor microenvironment and the hydrogen peroxide generated by X-rays during radiotherapy. The hydrogen peroxide reacts with the tetrasulfide bonds in the silicon layer of the long-afterglow nanocarrier via a redox reaction, breaking these bonds. Upon excitation by near-infrared light, the luminescent centers release an enhanced afterglow signal. Detection of this enhanced afterglow signal through in vivo imaging guides subsequent drug release. This achieves precise response to hydrogen peroxide, high signal-to-noise ratio tumor localization, and subsequent drug release guidance, further enhancing the sensitization effect of tumor radiotherapy.

[0017] Optionally, in the long-afterglow nanocarrier, the mass ratio of the near-infrared luminescent center, the organic mesoporous silica layer, and the autophagy inhibitor is (0.5-1):(0.5-1):(5-10).

[0018] According to a second aspect of this application, a method for preparing the long afterglow nanocarrier described above is provided.

[0019] A method for preparing the above-described long afterglow nanocarrier, the method comprising the following steps: S1. Dissolve the transition metal source in water, adjust the pH value and then perform a hydrothermal reaction. Then, separate, wash and dry the samples in sequence to obtain the near-infrared luminescent center. S2. Dissolve the near-infrared luminescent center, template agent and alkaline catalyst in water, then add the mixed solution, react, and then perform separation, washing and drying treatment in sequence to obtain the near-infrared luminescent center with an outer organic mesoporous silica layer. The mixed solution includes an organosilane precursor, an inorganic silicon precursor and an organic solvent. S3. The near-infrared luminescent centers of the outer organic mesoporous silica layer and the autophagy inhibitor are dissolved in water for electrostatic adsorption, and then separated, washed and dried in sequence to obtain the long afterglow nanocarrier.

[0020] Optionally, in S1, the transition metal source includes metal ion salts corresponding to zinc, gallium, germanium, and chromium.

[0021] Optionally, in S1, the pH value is 6-7.

[0022] Optionally, in S1, the hydrothermal reaction is carried out at a temperature of 180-200°C for a time of 10-12 hours.

[0023] Optionally, in S2, the template agent is selected from hexadecyltrimethylammonium bromide.

[0024] Optionally, in S2, the alkaline catalyst is selected from triethanolamine.

[0025] Optionally, in S2, the organic solvent is selected from cyclohexane.

[0026] Optionally, in S2, the reaction temperature is 55-60°C and the time is 12-24 hours.

[0027] Optionally, in S2, the washing is an alternating water / alcohol wash.

[0028] Optionally, the separation is performed by centrifugation, with a centrifugation speed of 10,000-12,000 rpm and a time of 8-10 minutes.

[0029] Optionally, the drying is performed using vacuum drying, with the temperature being 20-30°C and the time being 12-16 hours.

[0030] According to a third aspect of this application, the use of the aforementioned long-afterglow nanocarrier in the preparation of radiosensitizing drugs for tumor radiotherapy is provided.

[0031] Optionally, the tumor includes hypopharyngeal cancer.

[0032] The application of long-afterglow nanocarriers in tumor radiosensitization includes the following steps: Step 1: Disperse 10 mg of long afterglow nanocarrier in 1 mL of phosphate buffer solution and mix by sonication at room temperature for 10 seconds.

[0033] Step 2: Take the appropriate mass of the mixture obtained in Step 1 according to the weight of the tumor-bearing nude mice, and inject the material into the tumor for 6-8 hours via intratumoral injection. The concentration of the long afterglow nanocarrier is 50 mg / kg.

[0034] Step 3: After the tumor-bearing nude mice that have been injected into the tumor have been placed for the appropriate time, they are excited under 659nm red light for 5 minutes. Immediately afterwards, the afterglow signal is acquired on the imaging system with an exposure time of 10 seconds to ensure the enrichment of materials and to guide the subsequent drug release.

[0035] Step 4: Based on the results of afterglow imaging in Step 3, administer radiotherapy to the tumor at the corresponding time. The treatment cycle is 9 days, with one treatment every 3 days. Each radiotherapy session is 6 Gy, for a total of 18 Gy.

[0036] The beneficial effects that this application can produce include: The long-persistence nanocarrier involved in this application has an excitation wavelength in the near-infrared region, which can improve the penetration depth in biological systems, thereby enabling the observation of tumor sites under in vivo imaging and guiding subsequent drug release. Furthermore, the long-persistence nanocarrier can be detected without an excitation light source, achieving accurate and highly sensitive detection without background fluorescence. The high concentration of hydrogen peroxide in the tumor microenvironment compared to normal tissue, as well as hydrogen peroxide produced under X-ray irradiation, are selected as good stimuli-responsive molecules for the controlled release of the autophagy inhibitor chloroquine, giving it a good radiosensitizing effect. Moreover, this nanoprobe has high stability, good dispersibility, and uniform particle size distribution. Through long-persistence imaging technology, it provides a new strategy for the controlled release of the autophagy inhibitor chloroquine, thus enhancing the sensitization of tumor radiotherapy. Attached Figure Description

[0037] Figure 1 This is an EDS elemental distribution diagram of the near-infrared emitting centers (Z@M) of the outer organic mesoporous silica layer in Example 1 of this application.

[0038] Figure 2 This is a transmission electron microscope (TEM) image of the long afterglow nanocarrier (Z@M-CQ) in Example 1 of this application.

[0039] Figure 3 This is a diagram showing the pore size distribution of the near-infrared luminescent center (Z@M) (right) and the long-afterglow nanocarrier (Z@M-CQ) (left) in Example 1 of this application, which are coated with an organic mesoporous silica layer.

[0040] Figure 4 The long afterglow nanocarrier (Z@M-CQ) in Example 1 of this application was used to simulate the release of CQ in the TME in PBS solution and after radiotherapy.

[0041] Figure 5 This is a curve showing the ratio of afterglow emission intensity at the tumor site to that at normal tissue in Example 1 of this application for the long afterglow nanocarrier (Z@M-CQ).

[0042] Figure 6 The image shows a nude mouse model of hypopharyngeal cancer tumor after treatment, as described in Example 1 of this application. Detailed Implementation

[0043] The present application is described in detail below with reference to the embodiments, but the present application is not limited to these embodiments.

[0044] Unless otherwise specified, all raw materials used in the embodiments of this application were purchased through commercial channels.

[0045] ZGGC refers to the near-infrared luminescent center of zinc gallium germanium chromium; Z@M refers to the near-infrared luminescent center of the outer layer coated with organic mesoporous silica; CQ refers to chloroquine; Z@M-CQ refers to long afterglow nanocarrier (ZMC is its abbreviation); CTAB refers to hexadecyltrimethylammonium bromide; TEA refers to triethanolamine; TEOS refers to tetraethyl orthosilicate; BTES refers to bis[3-(triethoxysilyl)propyl]tetrasulfide; NH4NO3 refers to ammonium nitrate; PBS refers to phosphate buffer solution; TME refers to tumor microenvironment.

[0046] Unless otherwise specified, all test methods are standard and all instrument settings are those recommended by the manufacturer.

[0047] (1) A dispersion of long-afterglow nanocarrier (Z@M-CQ) was dropwise added to the surface of a copper mesh and allowed to dry naturally at room temperature to prepare the observation sample. Subsequently, the overall morphology and dispersion state of the material were preliminarily observed using a transmission electron microscope. Representative fields of view of the above process were imaged and saved.

[0048] (2) Measure the specific surface area, pore volume and pore diameter of the near-infrared luminescent center (Z@M) and the long afterglow nanocarrier (Z@M-CQ) of the outer organic mesoporous silica layer.

[0049] (3) A 10 mg / kg Z@M-CQ solution was placed in PBS and a 500 μM hydrogen peroxide solution, respectively, and thoroughly mixed. The mixture was then reacted under shaking conditions for 5 min. Subsequently, the solution mixed with hydrogen peroxide solution was irradiated with X-ray for 5 min. After standing for 1 h, the two mixtures were immediately centrifuged at 10,000 rpm / min for 5 min to terminate the reaction. The supernatant was collected, and the characteristic absorption intensity of CQ was measured by UV-Vis spectrophotometry. The CQ release of Z@M-CQ in PBS solution and in simulated TME after radiotherapy was then compared.

[0050] (4) 20 mg / kg of Z@M-CQ solution was injected into the tumor interior and the subcutaneous area of ​​adjacent normal tissue in tumor-bearing mice, and dynamic observations were conducted at time points of 5 min, 30 min, and 60 min. Before each imaging, the mice were gas-anesthetized, and then the injection area was excited with red light at a wavelength of 659 nm for 5 min. Five seconds after the light exposure stopped, the afterglow signal was immediately acquired using a small animal in vivo imaging system with an exposure time of 10 s to compare the afterglow intensity ratio of ZMC in tumor tissue and normal tissue and to observe the enhancement difference.

[0051] (5) Inject 20 mg / kg of Z@M-CQ solution into the tumor of tumor-bearing mice, and irradiate with X-ray (6 Gy each time) 6 hours later, once every three days, for a total of 18 Gy, for a treatment cycle of 9 days.

[0052] Example 1 First, ZGGC was synthesized via a hydrothermal method: 2.2 mL of 1 mol / L zinc nitrate, 7.2 mL of 0.5 mol / L gallium nitrate, 0.5 mL of 20 mol / L sodium germanate, and 0.5 mL of 20 mol / L chromium nitrate were sequentially added to 49.6 mL of deionized water. The pH was adjusted to 6.5 with ammonia while stirring vigorously. After stirring at room temperature for 2 h, the mixture was hydrothermally reacted at 200 °C for 12 h. The resulting product was centrifuged, washed with water, and dried to obtain ZGGC powder.

[0053] Subsequently, 50 mg ZGGC and 0.75 g CTAB were dispersed in 30 mL of ultrapure water, sonicated, and then 0.375 mL of LTEA (25% w / v) was added. After stirring at 60 °C for 1 h, a mixed solution of 600 μL LTEOS and 600 μL BTES dissolved in 8 mL of cyclohexane was added dropwise, and stirring was continued for 24 h. The Z@M sample collected by centrifugation was washed alternately with water and ethanol, and then the CTAB template was removed by treatment three times with 0.6% NH4NO3 / ethanol solution, finally obtaining the purified nanocomposite.

[0054] Finally, add 0.5 mL of chloroquine solution (20 mg / mL). -1 Add to Z@M solution (1 mg·mL) -1 The mixture was stirred at room temperature in the dark for 24 hours. The resulting ZMC complex was then centrifuged at 10,000 rpm for 10 min and washed three times with deionized water to remove unbound free CQ. The supernatant was collected to calculate the drug loading rate. The precipitate was collected and dried to obtain the long afterglow nanocarrier (Z@M-CQ).

[0055] The long-afterglow nanocarrier comprises a near-infrared luminescent center, an organic mesoporous silica layer, and an autophagy inhibitor. The organic mesoporous silica layer coats the outer layer of the near-infrared luminescent center, and the autophagy inhibitor is loaded in the mesopores of the organic mesoporous silica layer. The near-infrared luminescent center includes a transition metal element. The organic mesoporous silica layer is prepared by co-condensation of an organosilane precursor and an inorganic silicon precursor, wherein the organosilane precursor is an organosilane containing a tetrasulfide bond, and the inorganic silicon precursor is a silicate ester compound. The particle size of the long-afterglow nanocarrier is 320 nm.

[0056] Example 2 The specific operation is the same as in Example 1, except that the concentration of the chloroquine solution is 5 mg / mL. -1 The long afterglow nanocarrier has a particle size of 250 nm.

[0057] Example 3 The specific operation is the same as in Example 1, except that the concentration of the chloroquine solution is 10 mg / mL. -1The long afterglow nanocarrier has a particle size of 280 nm.

[0058] Example 4 The specific operation is the same as in Example 1, except that the concentration of the chloroquine solution is 15 mg / mL. -1 The long afterglow nanocarrier has a particle size of 300 nm.

[0059] Example 5 The specific operation is the same as in Example 1, except that the concentration of the chloroquine solution is 25 mg / mL. -1 The long afterglow nanocarrier has a particle size of 350 nm.

[0060] Characterization and testing The long afterglow nanocarrier prepared in Example 1 is taken as an example: Figure 1 This is an EDS elemental distribution map of the near-infrared emitting centers (Z@M) of the outer organic mesoporous silica layer in Example 1 of this application. Figure 2 This is a transmission electron microscope (TEM) image of the long-afterglow nanocarrier (Z@M-CQ) in Example 1 of this application. Figure 1 and Figure 2 As can be seen, Zn, Ga, Ge, and Cr elements are uniformly distributed in the central position, while Si, O, and S elements are uniformly distributed in the organosilicon-doped shell. Furthermore, the dispersibility and stability are improved by coating with a mesoporous organosilicon layer.

[0061] Figure 3 The diagram shows the pore size distribution of the near-infrared luminescent center (Z@M) (right) and the long-afterglow nanocarrier (Z@M-CQ) (left) in Example 1 of this application, indicating that Z@M has a rich mesoporous structure, which can meet the requirements of drug loading and achieve efficient drug loading.

[0062] Figure 4 The long afterglow nanocarrier (Z@M-CQ) in Example 1 of this application was used to simulate CQ release in TME after radiotherapy in PBS solution and after radiotherapy. Compared with the PBS environment, Z@M-CQ can significantly enhance CQ release in TME after radiotherapy.

[0063] Figure 5 The curves showing the ratio of afterglow emission intensity at the tumor site to that at normal tissue in Example 1 of this application indicate that the afterglow signal in the tumor region gradually increases over time, while the afterglow signal at the subcutaneous injection site does not change significantly.

[0064] Figure 6The images shown are of nude mice with hypopharyngeal carcinoma tumors after treatment in Example 1 of this application. It can be observed that the tumor volume in group (4) is the smallest, indicating that the anti-tumor effect of ZMC combined with radiotherapy is significantly improved compared with radiotherapy alone.

[0065] The applicant declares that the above embodiments illustrate a long-afterglow nanocarrier, its preparation method, and its application, but this application is not limited to the above embodiments, i.e., it does not mean that this application must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to this application, equivalent substitutions of the raw materials for the product, addition of auxiliary components, and selection of specific methods, etc., all fall within the protection scope and disclosure scope of this application.

[0066] The preferred embodiments of this application have been described in detail above. However, this application is not limited to the specific details of the above embodiments. Within the scope of the technical concept of this application, various simple modifications can be made to the technical solution of this application, and these simple modifications all fall within the protection scope of this application.

[0067] It should also be noted that the various specific technical features described in the above embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this application will not describe the various possible combinations separately.

Claims

1. A long afterglow nanocarrier, characterized in that, The long afterglow nanocarrier includes a near-infrared luminescent center, an organic mesoporous silica layer, and an autophagy inhibitor; The organic mesoporous silica layer covers the outer layer of the near-infrared luminescent center, and the autophagy inhibitor is loaded in the mesopores of the organic mesoporous silica layer. The near-infrared luminescent center includes a transition metal element; The organic mesoporous silica layer is prepared by co-condensation of an organosilane precursor and an inorganic silicon precursor, wherein the organosilane precursor is an organosilane containing a tetrasulfide bond, and the inorganic silicon precursor is a silicate ester compound.

2. The long afterglow nanocarrier according to claim 1, characterized in that, The particle size of the long afterglow nanocarrier is 250-350 nm.

3. The long afterglow nanocarrier according to claim 1, characterized in that, The transition metal elements include zinc, gallium, germanium, and chromium; Preferably, the molar ratio of zinc, gallium, germanium and chromium is (0.8-1.4):(1.4-2.2):(0.05-0.15):(0.001-0.01).

4. The long afterglow nanocarrier according to claim 1, characterized in that, The organosilane containing a tetrasulfide bond is bis[3-(triethoxysilyl)propyl]tetrasulfide; Preferably, the silicate ester compound includes orthosilicate compounds; Preferably, the orthosilicate compound is ethyl orthosilicate; Preferably, the volume ratio of the organosilane precursor to the inorganic silicon precursor is (6-4):(4-3).

5. The long afterglow nanocarrier according to claim 1, characterized in that, The autophagy inhibitor is chloroquine; Preferably, in the long afterglow nanocarrier, the mass ratio of the near-infrared luminescent center, the organic mesoporous silica layer, and the autophagy inhibitor is (0.5-1):(0.5-1):(5-10).

6. A method for preparing a long afterglow nanocarrier according to any one of claims 1 to 5, characterized in that, The preparation method includes the following steps: S1. Dissolve the transition metal source in water, adjust the pH value and then perform a hydrothermal reaction. Then, separate, wash and dry the samples in sequence to obtain the near-infrared luminescent center. S2. Dissolve the near-infrared luminescent center, template agent and alkaline catalyst in water, then add the mixed solution, react, and then perform separation, washing and drying treatment in sequence to obtain the near-infrared luminescent center with an outer organic mesoporous silica layer. The mixed solution includes an organosilane precursor, an inorganic silicon precursor and an organic solvent. S3. The near-infrared luminescent centers of the outer organic mesoporous silica layer and the autophagy inhibitor are dissolved in water for electrostatic adsorption, and then separated, washed and dried in sequence to obtain the long afterglow nanocarrier.

7. The preparation method according to claim 6, characterized in that, In S1, the transition metal source includes metal ion salts corresponding to zinc, gallium, germanium, and chromium; Preferably, in S1, the pH value is 6-7; Preferably, in S1, the hydrothermal reaction is carried out at a temperature of 180-200°C for 10-12 hours.

8. The preparation method according to claim 6, characterized in that, In S2, the template agent is selected from hexadecyltrimethylammonium bromide; Preferably, in S2, the alkaline catalyst is selected from triethanolamine; Preferably, in S2, the organic solvent is selected from cyclohexane; Preferably, in step S2, the reaction temperature is 55-60°C and the time is 12-24 hours. Preferably, in S2, the washing is an alternating water / alcohol wash; Preferably, the separation is performed by centrifugation, with a centrifugation speed of 10,000-12,000 rpm and a time of 8-10 minutes; Preferably, the drying is performed using vacuum drying, with a temperature of 20-30°C and a time of 12-16 hours.

9. The use of the long afterglow nanocarrier according to any one of claims 1 to 5 in the preparation of a radiosensitizing drug for tumor radiotherapy.

10. The application according to claim 9, characterized in that, The tumors include hypopharyngeal cancer.

Images