A size / charge double-converted nanoparticle-gelatin composite nanocluster, and a preparation method and application thereof

Abstract

The application discloses a size / charge double-converted nanoparticle-gelatin composite nanocluster and a preparation method and application thereof. The preparation method is as follows: first, the gelatin is modified by a targeting ligand and an acid anhydride compound or the gelatin is modified by the acid anhydride compound, so that the gelatin has a charge conversion characteristic from negative to positive under neutral to weak acid pH; a nanoparticle carrying a positive drug is prepared through a sol-gel method or a nano precipitation method; finally, the modified gelatin and the nanoparticle are electrostatically assembled to obtain the nanoparticle-gelatin composite nanocluster. The composite nanocluster utilizes the dual response of the modified gelatin to pH and enzymes in a disease microenvironment, can degrade and release the positive nanoparticle under the action of pH and enzymes, and realizes the double conversion of size and charge. The composite nanocluster prepared by the application has excellent penetration and internalization effects in a disease tissue, and can be widely applied in the fields of drug delivery of tumors, inflammation, bacterial biofilm and the like.

Description

Technical Field

[0001] This invention belongs to the field of responsive drug delivery materials and composite nanoclusters, specifically relating to a size / charge dual-conversion nanoparticle-gelatin composite nanocluster, its preparation method, and its application. Background Technology

[0002] Nanocarriers have attracted significant attention in recent years, promising to address the problems of poor water solubility, insufficient targeting ability, and high systemic toxicity in traditional drug delivery processes. However, the low penetration and internalization efficiency of nanocarriers in diseased tissues, especially deep tumors, greatly limits drug delivery efficiency and thus weakens therapeutic effects. Studies have shown that the size and surface charge of nanocarriers are important factors affecting in vivo drug delivery. Small-sized, positively charged nanocarriers are beneficial for improving tumor penetration and internalization, but this contradicts the morphology of nanocarriers (large size, negative charge) that facilitates circulation and accumulation. Therefore, designing nanocarriers with dual size / surface charge conversion has significant advantages in improving penetration and internalization. Compared to synthetic polymers, natural polymers have advantages such as good biocompatibility and low cost, and their molecular structures, such as hydroxyl, carboxyl, and amino groups, are easily functionalized, making them a promising candidate for developing drug delivery nanocarriers.

[0003] Gelatin can be degraded into low-molecular-weight peptides and amino acids by matrix metalloproteinases (MMP-2) highly expressed in tumor tissues. Organosilicon nanoparticles have advantages such as tunable size, the ability to be designed into hollow structures for efficient drug loading, good biocompatibility, and easy surface functionalization modification. Copper sulfide nanoparticles have photothermal and photodynamic antitumor effects and can also be designed into hollow structures for drug loading. Small-sized nanoparticles and polymers can be assembled through electrostatic, hydrogen bonding, and host-guest interactions to obtain nanoparticle-based composite nanoclusters, and the assembly forces can be removed under certain endogenous and exogenous stimuli, releasing the nanoparticles.

[0004] Existing literature reports an amphiphilic polymer bonded to an antitumor drug, which self-assembles into a clustered nanomedicine carrier of approximately 80 nm. This carrier helps prolong drug circulation in the blood and enrichment in tumor tissue, and rapidly disintegrates into particles smaller than 10 nm in the microacidic environment of the tumor, exhibiting strong tumor tissue penetration and accumulation capabilities, thereby delivering more drugs into tumor cells. However, the single structural characteristic transformation of the carrier cannot meet the requirements of each step in the delivery process. For example, nanoparticles that enhance tumor tissue penetration by reducing size are affected by surface PEG molecules or negative charges during cell internalization. Similarly, nanoparticles that promote cell internalization by surface charge conversion are limited in their tissue penetration due to their excessive size after circulating in the blood. (LI HJ,DU JZ,LIU J,et al.Smart Superstructures with Ultrahigh pH-Sensitivity for Targeting Acidic Tumor Microenvironment:Instantaneous Size Switching and Improved Tumor Penetration[J].ACS Nano,2016,10(7):6753-61.) Summary of the Invention

[0005] In order to overcome the limitations of existing technologies where the single-carrier structure cannot meet the requirements of each step in the delivery process, the purpose of this invention is to provide a size / charge dual-conversion nanoparticle-gelatin composite nanocluster, its preparation method, and its application.

[0006] This invention utilizes modified gelatin that is responsive to both tumor microacids and enzymes, and hollow mesoporous organosilicon or copper sulfide nanoparticles with high drug loading capacity, to construct nanoparticle-gelatin composite nanoclusters via electrostatic assembly. These nanoclusters can respond to microacid and enzyme stimuli in the tumor environment, achieving size and charge conversion, thereby enhancing tumor tissue penetration and anti-tumor therapeutic effects.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] This invention provides a method for preparing size / charge dual-conversion nanoparticle-gelatin composite nanoclusters, comprising the following steps:

[0009] (1) Reaction of gelatin with a targeting ligand, dialysis and freeze-drying, and then reaction with an acid anhydride compound under alkaline conditions, followed by dialysis and freeze-drying to obtain modified gelatin; or reaction of gelatin with an acid anhydride compound under alkaline conditions, followed by dialysis and freeze-drying to obtain modified gelatin.

[0010] (2) Prepare positively charged drug-loaded organosilicon nanoparticles by sol-gel method or prepare positively charged drug-loaded copper sulfide nanoparticles by nanoprecipitation method.

[0011] (3) The modified gelatin prepared in step (1) is prepared into a modified gelatin solution, and the nanoparticles prepared in step (2) are prepared into a nanoparticle dispersion. The modified gelatin solution and the nanoparticle dispersion are then mixed to obtain a size / charge double conversion nanoparticle-gelatin composite nanocluster through electrostatic interaction.

[0012] Preferably, the temperature at which the gelatin reacts with the targeting ligand in step (1) is 20°C to 80°C, and the reaction time is 2 to 24 hours.

[0013] Preferably, the reaction temperature of the gelatin and the acid anhydride compound in step (1) is 20℃~80℃, and the reaction time of the gelatin and the acid anhydride compound is 2-24h.

[0014] Preferably, the alkaline conditions described in step (1) are all pH 7.4 to 10.0.

[0015] Preferably, the targeting ligand in step (1) is one or more of folic acid and hyaluronic acid.

[0016] Preferably, the anhydride compound in step (1) is one or more of maleic anhydride, methylmaleic anhydride, and dimethylmaleic anhydride.

[0017] Preferably, the modified gelatin in step (1) is maleic anhydride modified gelatin, methyl maleic anhydride modified gelatin, dimethyl maleic anhydride modified gelatin, maleic anhydride folic acid modified gelatin, methyl maleic anhydride folic acid modified gelatin, dimethyl methyl maleic anhydride folic acid modified gelatin, maleic anhydride hyaluronic acid modified gelatin, methyl maleic anhydride hyaluronic acid modified gelatin, or dimethyl methyl maleic anhydride hyaluronic acid modified gelatin.

[0018] Preferably, in step (1), the mass ratio of the targeting ligand to gelatin is (1:15) to (1:1).

[0019] Preferably, in step (1), the mass ratio of the acid anhydride compound to gelatin is (1:25) to (1:9).

[0020] Preferably, the mass ratio of maleic anhydride, methylmaleic anhydride or dimethylmaleic anhydride to folic acid-modified gelatin is (1:25) to (1:9).

[0021] Preferably, the specific steps of the sol-gel method in step (2) are as follows: Take 1-5g of hexadecyltrimethylammonium chloride (CTAC), add 10-40g of deionized water, then add 0.2-2g of an aqueous solution of triethanolamine (TEA) (the volume ratio of triethanolamine to water is 1:9), stir evenly at 60-120℃, then add 1-5mL of tetraethyl orthosilicate (TEOS), and react for 1-8h to obtain the mesoporous silica nanoparticle core (MSN). Continue to add a mixture of 1-5mL of TEOS and 0.1-5mL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTDS), react for 2-8h, centrifuge, and wash with deionized water and ethanol to obtain nanoparticles coated with mesoporous organosilicon (MON@MSN). CTAC was then removed by reflux in a mixed solution of ethanol and hydrochloric acid (volume ratio of 10:1) at 60–120 °C. The CTAC was redispersed in 100–800 mL of deionized water, and 2–10 mL of 25% ammonia solution (NH3·H2O) was added. After ultrasonic dispersion, the mixture was stirred at 60–120 °C for 2–8 h, centrifuged, washed with deionized water and ethanol, and freeze-dried to obtain hollow mesoporous organosilicon nanoparticles (HMON). 50–300 mg of HMON was dispersed in 20–200 mL of anhydrous ethanol, and 1–6 mL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed at 80 °C for 5–24 h and freeze-dried to obtain aminated hollow mesoporous organosilicon nanoparticles (HMON-NH2). After drug loading, drug-loaded aminated hollow mesoporous organosilicon nanoparticles were obtained.

[0022] Preferably, the specific steps of the sol-gel method in step (2) are as follows: Take 1-5g of hexadecyltrimethylammonium chloride (CTAC), add 10-40g of deionized water, then add 0.2-2g of an aqueous solution of triethanolamine (TEA) (the volume ratio of triethanolamine to water is 1:9), stir evenly at 60-120℃, then add 1-5mL of tetraethyl orthosilicate (TEOS), and react for 1-8h to obtain the mesoporous silica nanoparticle core (MSN). Continue to add 1-5mL of TEOS and 0.1-5mL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTDS), react for 2-8h, centrifuge, and wash with deionized water and ethanol to obtain nanoparticles coated with mesoporous organosilicon. Subsequently, CTAC is removed by reflux in a mixed solution of ethanol and hydrochloric acid (the volume ratio of ethanol to hydrochloric acid is 10:1) at 60-120℃, and freeze-dry to obtain mesoporous organosilicon nanoparticles MON. 50–300 mg of MON was dispersed in 20–200 mL of anhydrous ethanol, and 1–6 mL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed at 80 °C for 5–24 h and then freeze-dried to obtain aminated mesoporous organosilicon nanoparticles (MON-NH2). After drug loading, drug-loaded aminated mesoporous organosilicon nanoparticles were obtained.

[0023] Preferably, the specific steps of the nanoprecipitation method in step (2) are as follows: 0.2–5 mL of acetic acid is added to 10–50 mL of methanol under stirring at 200–1500 rpm, and the mixture is kept at 20–60 °C for 10–60 min. 1–5 mmol of copper acetate monohydrate is added under stirring, and after the copper acetate is completely dissolved, 1–5 mmol of thiourea is added. The mixture is stirred continuously at 20–60 °C for 8–48 h. The mixture is then transferred to a 25–100 mL polytetrafluoroethylene high-pressure reactor and kept at 80–200 °C for 2–12 h. After the reaction, the mixture is centrifuged, washed, and vacuum dried to obtain copper sulfide (CuS) nanoparticles. After drug loading, drug-loaded copper sulfide (CuS) nanoparticles are obtained.

[0024] Preferably, the organosilicon nanoparticles in step (2) are mesoporous organosilicon nanoparticles or hollow mesoporous organosilicon nanoparticles.

[0025] Preferably, in step (2), the size of the drug-loaded hollow mesoporous organosilicon nanoparticles is 10-100 nm, and the potential is +5 mV to +40 mV.

[0026] Preferably, in step (3), the mass percentage concentration of the modified gelatin solution is 2% to 20%, the mass percentage concentration of the nanoparticle dispersion is 2% to 20%, the pH of the prepared modified gelatin solution and the prepared nanoparticle dispersion before mixing is 7.4 to 10.0, and the volume ratio of the modified gelatin solution and the nanoparticle dispersion is (1:9) to (9:1).

[0027] This invention provides a size / charge dual-conversion nanoparticle-gelatin composite nanocluster prepared by any of the above preparation methods.

[0028] Preferably, the size of the size / charge dual-conversion nanoparticle-gelatin composite nanocluster is 40-400 nm, and the potential is -10 mV to -50 mV.

[0029] Preferably, the size / charge dual-conversion nanoparticle-gelatin composite nanoclusters undergo size conversion from large to small and charge conversion from negative to positive under the action of micro-acids and enzymes.

[0030] More preferably, the slightly acidic refers to pH = 6.5, and the enzyme is matrix metalloproteinase (MMP-2).

[0031] The present invention also provides the application of the size / charge dual-conversion nanoparticle-gelatin composite nanoclusters in drug delivery.

[0032] Preferably, the size / charge dual-conversion nanoparticle-gelatin composite nanoclusters serve as nanocarriers for drugs, promoting the penetration and internalization of drugs in tumors, arthritis, biofilms, etc.

[0033] This invention discloses a method for preparing a size / charge dual-conversion nanoparticle-gelatin composite nanocluster. First, gelatin undergoes a targeted ligand reaction, followed by modification with anhydride compounds, or by further modification with anhydride compounds to impart a charge-conversion characteristic from negative to positive at pH levels ranging from neutral to weakly acidic. Positively charged drug-loaded organosilicon nanoparticles are prepared via a sol-gel method, or positively charged drug-loaded copper sulfide nanoparticles are prepared via a nanoprecipitation method. Finally, the nanoparticle-gelatin composite nanocluster is obtained through electrostatic assembly. The specific steps include:

[0034] (1) Dissolve gelatin in deionized water at a mass percentage concentration of 1% by heating, add a targeting ligand and 1-ethyl-(3-dimethylaminopropyl), react at 20℃~80℃ for 2-24 hours, dialyze and freeze-dry to obtain targeted modified gelatin. Dissolve the targeted modified gelatin in deionized water, adjust the pH to 7.4~10.0, add an acid anhydride compound, react at 20℃~80℃ for 2-24 hours, dialyze and freeze-dry to obtain modified gelatin; or dissolve gelatin in deionized water at a mass percentage concentration of 1% by heating, adjust the pH to 7.4~10.0, add an acid anhydride compound, react at 20℃~80℃ for 2-24 hours, dialyze and freeze-dry to obtain modified gelatin.

[0035] (2) Dissolve triethanolamine, hexadecyltrimethylammonium chloride, and tetraethyl orthosilicate in deionized water and react at 60–120 °C for 1–8 h. Continue to add a mixture of tetraethyl orthosilicate and bis-[3-(triethoxysilane)propyl]-disulfide and react for another 2–8 h. Obtain hollow mesoporous organosilicon nanoparticles by extraction, etching, centrifugation, and drying. After drug loading, obtain drug-loaded hollow mesoporous organosilicon nanoparticles; or dissolve triethanolamine, hexadecyltrimethylammonium chloride, and tetraethyl orthosilicate in deionized water. The reaction mixture of tetraethyl orthosilicate and bis-[3-(triethoxysilane)propyl]-disulfide is added to water and reacted at 60–120 °C for 1–8 h. The reaction is continued for 2–8 h, followed by extraction, centrifugation, and drying to obtain mesoporous organosilicon nanoparticles. Drug-loaded mesoporous organosilicon nanoparticles are then obtained. Alternatively, copper acetate is added to a solution of acetic acid and methanol, followed by the addition of thiourea and stirring for 8–48 h. The mixture is then hydrothermally heated at 80–200 °C for 2–12 h, followed by centrifugation and washing to obtain hollow copper sulfide nanoparticles. Drug-loaded copper sulfide (CuS) nanoparticles are then obtained.

[0036] (3) The drug-loaded nanoparticles were ultrasonically dispersed in deionized water to prepare a dispersion with a mass percentage concentration of 2%-20%, and the pH was adjusted to 7.4-10.0. Modified gelatin was dissolved in deionized water at pH 7.4-10.0 to prepare a solution with a mass percentage concentration of 2%-20%. Under stirring conditions, the modified gelatin solution was slowly added to the drug-loaded nanoparticle dispersion. The mixture was stirred at room temperature for 24 hours, followed by dialyzing and freeze-drying to obtain nanoparticle-gelatin composite nanoclusters.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0038] (1) Gelatin has enzyme-degradable properties.

[0039] (2) The amino groups of gelatin are easy to modify and have the characteristic of changing from negative to positive charge at pH levels from neutral to weakly acidic.

[0040] (3) Small-sized hollow nanoparticles are easy to load with drugs, and after modification they become positively charged and can be assembled with negatively charged materials.

[0041] (4) The size / charge dual conversion nanoparticle-gelatin composite nanoclusters prepared in this invention utilize the dual responsiveness of modified gelatin to pH and enzymes in the disease microenvironment, and can release positively charged nanoparticles when pH and enzyme activity decrease, thus achieving dual conversion of size and charge.

[0042] (5) The size / charge dual-conversion nanoparticle-gelatin composite nanoclusters prepared by the present invention have excellent penetration and internalization effects in diseased tissues and can be widely used in drug delivery in the fields of tumors, inflammation, and bacterial biofilms. Attached Figure Description

[0043] Figure 1 In the figure, 'a' represents the preparation of a size / charge double-conversion nanoparticle-gelatin composite nanocluster (DOX-icluster) loaded with doxorubicin (DOX), the effects of tumor pH and MMP-2 on its size shrinkage and charge reversal, and a schematic diagram of glutathione (GSH) degradation and DOX release. Figure 1 In the diagram, b is a schematic diagram of the penetration, internalization, and DOX release behavior of the DOX-icluster into the tumor microenvironment.

[0044] Figure 2 The diagram shows the preparation of DOX-HMON-NH2 obtained by loading DOX onto HMON-NH2 prepared in Example 1, and the corresponding transmission electron microscope (TEM) image.

[0045] Figure 3 The zeta potential diagrams are for HMON in Example 1, HMON-NH2 in Example 1, and DOX-HMON-NH2 obtained by loading DOX on HMON-NH2 prepared in Example 1.

[0046] Figure 4 The image shows the DOX loading rate of HMON-NH2 prepared in Example 1.

[0047] Figure 5 This is a flowchart illustrating the preparation process of the modified gelatin in Example 1.

[0048] Figure 6 a and a1 are TEM images of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1; Figure 6 b and b1 in the image are TEM images of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 2; Figure 6 c and c1 in the image are TEM images of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 2.

[0049] Figure 7In the figure, 'a' represents the particle size distribution of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Examples 1-3. Figure 7 b in the figure represents the Zeta potential diagram of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Examples 1-3; Figure 7 In the figure, c represents the DOX loading rate of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Examples 1-3.

[0050] Figure 8 In Figure 'a', the image is a TEM image of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 in phosphate buffer at pH 7.4. Figure 8 In the image, b represents a TEM image of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 in a phosphate buffer solution at pH 6.5 containing 0.25 μg / mL MMP-2.

[0051] Figure 9 Size and potential stability of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 in phosphate buffer solution at pH 7.4 for 24 h.

[0052] Figure 10 The particle size distribution (DLS) of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 is shown in phosphate buffer at pH 7.4, phosphate buffer at pH 6.5 containing 0.25 μg / mL MMP-2, and acetate buffer at pH 5.0 containing 10 mM glutathione (GSH).

[0053] Figure 11 Zeta potential plot of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 at pH 7.4, pH 6.5 containing 0.25 μg / mL MMP-2, and pH 5.0 containing 10 mM glutathione (GSH).

[0054] Figure 12 In the figure, 'a' represents the drug release curves of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 under different conditions. Figure 12 In Example 2, b represents the drug release curves of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared under different conditions. Figure 12 In the figure, c represents the drug release curves under different conditions of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 3 after drug loading.

[0055] Figure 13 The survival rate of tumor cells on the first and third days after the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 were shown.

[0056] Figure 14 The size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 were subjected to different depth cross-sections of tumor spheres corresponding to tumor cells under the conditions of phosphate buffer (pH 7.4), acetate buffer (pH 6.5), and acetate buffer (pH 6.5) containing 0.25 μg / mL MMP-2.

[0057] Figure 15 The fluorescence intensity quantification results of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 at different depths of tumor spheres corresponding to tumor cells under the conditions of phosphate buffer (pH 7.4), acetate buffer (pH 6.5), and acetate buffer (pH 6.5) containing 0.25 μg / mL MMP-2.

[0058] Figure 16 Orthogonal graphs of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 acting on tumor spheres corresponding to tumor cells under the conditions of phosphate buffer at pH 7.4, phosphate buffer at pH 6.5, and phosphate buffer at pH 6.5 containing 0.25 μg / mL MMP-2.

[0059] Figure 17 The growth diagrams of 4T1 tumor spheres prepared in Example 1 with size / charge dual conversion organosilicon nanoparticles-gelatin composite nanoclusters under different conditions are shown.

[0060] Figure 18 The graph shows the relative volume changes of 4T1 tumor spheres prepared in Example 1 with size / charge dual conversion organosilicon nanoparticles-gelatin composite nanoclusters treated under different conditions.

[0061] Figure 19 A schematic diagram showing the collapse or shrinkage of 4T1 tumor spheres after treatment with size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 under the conditions of phosphate buffer at pH 7.4, phosphate buffer at pH 6.5, and phosphate buffer at pH 6.5 containing 0.25 μg / mL MMP-2.

[0062] Figure 20 This is a schematic diagram of the preparation of DOX-CuS obtained by loading DOX onto copper sulfide (CuS) nanoparticles prepared in Example 5.

[0063] Figure 21 Image a is a TEM image of copper sulfide (CuS) nanoparticles prepared in Example 5; Figure 21 In Figure b, DOX-CuS is a TEM image of copper sulfide (CuS) nanoparticles loaded with DOX prepared in Example 5.

[0064] Figure 22 Zeta potential maps of DOX-CuS were obtained for copper sulfide (CuS) nanoparticles prepared in Example 5 and copper sulfide (CuS) nanoparticles loaded with DOX prepared in Example 5.

[0065] Figure 23 The image shows the DOX loading rate of the copper sulfide (CuS) nanoparticles prepared in Example 5.

[0066] Figure 24 The particle size distribution and TEM image of the size / charge dual-conversion CuS nanoparticle-gelatin composite nanoclusters prepared in Example 5 are shown.

[0067] Figure 25 In Figure a, CuS nanoparticles-gelatin composite nanoclusters with size / charge dual conversion prepared in Example 5 are used to treat tumor cells after adding hydrogen peroxide. Figure 25 Figure b shows the survival rate of tumor cells without hydrogen peroxide when the size / charge dual-conversion CuS nanoparticle-gelatin composite nanoclusters prepared in Example 5 are applied.

[0068] Figure 26 This is a comparison of cell viability between cells irradiated with 100 μM hydrogen peroxide (H2O2+NIR) using laser and the blank control group.

[0069] Figure 27 The images show the live / dead staining patterns of tumor cells with and without hydrogen peroxide after the CuS nanoparticle-gelatin composite nanoclusters with size / charge dual conversion prepared in Example 5 were applied. Detailed Implementation

[0070] The technical solution of the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings, but the embodiments and protection scope of the present invention are not limited thereto.

[0071] In the following examples, NaOH or HCl solution was used to adjust the pH.

[0072] Example 1

[0073] (1) Dissolve 1.0 g of gelatin in 100 mL of deionized water under heating at 50 °C to obtain a gelatin solution. Dissolve 0.067 g of folic acid (FA) in 10 mL of anhydrous dimethyl sulfoxide (DMSO) to obtain an FA solution. Add the FA solution dropwise to the gelatin solution at 20–60 drops / min under stirring. Activate with 1-ethyl-(3-dimethylaminopropyl) (EDC) and react at 20 °C for 2 h. Subsequently, after dialyzing with deionized water, folic acid-modified gelatin (FA-Gel) was obtained by freeze-drying. 1.0 g of FA-Gel was dissolved in 100 mL of deionized water under heating at 20 °C, the pH was adjusted to 7.4, 0.11 g of dimethyl maleic anhydride (DMA) was added, and the reaction was carried out at 20 °C for 2 h while maintaining the pH at 7.4. After dialyzing with deionized water at pH 7.4 (the pH of the deionized water was adjusted with NaOH / HCl), folic acid-modified dimethyl maleic anhydride modified gelatin (FA-GelDMA) was obtained by freeze-drying.

[0074] (2) Take 2g of hexadecyltrimethylammonium chloride (CTAC), add 18g of deionized water, then add 0.8g of an aqueous solution of triethanolamine (TEA) (the volume ratio of triethanolamine to water is 1:9), stir evenly at 95℃, and then add 1mL of tetraethyl orthosilicate (TEOS). After reacting for 1h, mesoporous silica nanoparticle cores (MSN) are obtained. Continue to add a mixture of 1mL of TEOS and 0.6mL of bis-[3-(triethoxysilyl)propyl]-disulfide (BTDS), react for 4h, centrifuge, and wash with deionized water and ethanol to obtain nanoparticles coated with mesoporous organosilicon (MON@MSN). CTAC was then removed by reflux in a mixed solution of ethanol and hydrochloric acid (volume ratio of 10:1) at 78°C. The CTAC was redispersed in 400 mL of deionized water, and 8 mL of 25% ammonia solution (NH3·H2O) was added. After ultrasonic dispersion, the mixture was stirred at 95°C for 3 h, centrifuged, washed with deionized water and ethanol, and freeze-dried to obtain hollow mesoporous organosilicon nanoparticles (HMON). 100 mg of HMON was dispersed in 80 mL of anhydrous ethanol, and 1.2 mL of 3-aminopropyltriethoxysilane (APTES) was added. The mixture was refluxed at 80°C for 12 h and freeze-dried to obtain aminated hollow mesoporous organosilicon nanoparticles (HMON-NH2). After drug loading, drug-loaded HMON-NH2 was obtained.

[0075] (3) Prepare 5 mL of a 2% (w / w) aqueous dispersion of drug-loaded HMON-NH2 and adjust the pH to 7.4. Prepare 5 mL of a 2% (w / w) aqueous solution of FA-GelDMA and adjust the pH to 7.4. Add the FA-GelDMA solution dropwise to the aqueous dispersion of drug-loaded HMON-NH2 under stirring at 800 rpm. Stir at room temperature for 24 h and dialyze to obtain size / charge double conversion organosilicon nanoparticle-gelatin composite nanoclusters (icluster).

[0076] Example 1: The preparation process of modified gelatin is as follows Figure 5 As shown.

[0077] Example 2

[0078] (1) 1.0 g of gelatin was dissolved in 100 mL of deionized water under heating at 50 °C to obtain a gelatin solution. 0.133 g of FA was dissolved in 10 mL of DMSO to obtain an FA solution. The FA solution was added dropwise to the gelatin solution at 20-60 drops / min under stirring. EDC was added for activation, and the reaction was carried out at 50 °C for 12 h. Subsequently, the mixture was dialyzed with deionized water and freeze-dried to obtain FA-Gel. 1.0 g of FA-Gel was dissolved in 100 mL of water under heating at 50 °C, and the pH was adjusted to 8.5. 0.08 g of methylmaleic anhydride (CA) was added, and the reaction was carried out at 50 °C for 12 h while maintaining the pH at 8.5. The mixture was dialyzed with deionized water at pH 8.5 (the pH of the deionized water was adjusted with NaOH / HCl), and freeze-dried to obtain folic acid-modified methylmaleic anhydride-modified gelatin (FA-GelCA).

[0079] (2) Take 2g of CTAC, add 18g of deionized water, then add 0.8g of an aqueous solution of triethanolamine (TEA) (the volume ratio of triethanolamine to water is 1:9), stir evenly at 95℃, and then add 1mL of TEOS at 20-60 drops / min. After reacting for 1h, mesoporous silica nanoparticle cores are obtained. Continue to add a mixture of 1mL of TEOS and 0.6mL of BTDS, react for 4h, centrifuge, and wash with deionized water and ethanol to obtain nanoparticles coated with mesoporous organosilicon. Disperse 100mg of mesoporous organosilicon nanoparticles in 80mL of anhydrous ethanol, add 1.2mL of APTES, reflux at 80℃ for 12h, and freeze-dry to obtain aminated mesoporous organosilicon nanoparticles (MON-NH2). After drug loading, drug-loaded MON-NH2 is obtained.

[0080] (3) Prepare 4.5 mL of a drug-loaded MON-NH2 aqueous dispersion with a mass percentage concentration of 20% and adjust the pH to 8.5. Prepare 0.5 mL of a FA-GelCA aqueous solution with a mass percentage concentration of 10% and adjust the pH to 8.5. Add the FA-GelCA solution dropwise to the drug-loaded MON-NH2 aqueous dispersion under stirring at 800 rpm. Stir at room temperature for 24 h and dialyze to obtain organosilicon nanoparticle-gelatin composite nanoclusters with size / charge dual conversion.

[0081] Example 3

[0082] (1) Dissolve 1.0 g of gelatin in 100 mL of deionized water under heating at 50 °C to obtain a gelatin solution. Dissolve 1 g of FA in 10 mL of DMSO to obtain an FA solution. Add the FA solution dropwise to the gelatin solution at 20-60 drops / min under stirring. Activate with EDC and react at 80 °C for 24 h. Then dialyze with deionized water and freeze-dry to obtain FA-Gel. Dissolve 1.0 g of FA-Gel in 100 mL of water under heating at 80 °C, adjust the pH to 10, add 0.04 g of maleic anhydride (MA), maintain pH 10 and react at 80 °C for 24 h. Dialyze with deionized water at pH 10 (adjust the pH of the deionized water with NaOH / HCl) and freeze-dry to obtain folic acid-modified maleic anhydride-modified gelatin (FA-GelMA).

[0083] (2) Take 2g of CTAC, add 18g of deionized water, then add 0.8g of an aqueous solution of triethanolamine (TEA) (the volume ratio of triethanolamine to water is 1:9), stir evenly at 95℃, and slowly add 1mL of TEOS. After reacting for 1h, mesoporous silica nanoparticle cores (MSN) are obtained. Continue to add a mixture of 1mL of TEOS and 0.6mL of BTDS, react for 4h, centrifuge, and wash with water and ethanol to obtain nanoparticles coated with mesoporous organosilicon (MON@MSN). Then, CTAC is removed by reflux in a mixed solution of ethanol and hydrochloric acid (volume ratio of ethanol to hydrochloric acid is 10:1) at 78℃, redispersed in 400mL of water, and 8mL of 25% ammonia water (NH3·H2O) is added. After ultrasonic dispersion, stir at 95℃ for 3h, centrifuge, wash with water and ethanol, and freeze-dry to obtain hollow mesoporous organosilicon nanoparticles HMON. 100 mg of HMON was dispersed in 80 mL of anhydrous ethanol, and 6 mL of APTES was added. The mixture was refluxed at 80 °C for 12 h and then freeze-dried to obtain aminated hollow mesoporous organosilicon nanoparticles (HMON-NH2). After drug loading, drug-loaded HMON-NH2 was obtained.

[0084] (3) Prepare 0.5 mL of a 10% HMON-NH2 aqueous dispersion and adjust the pH to 10.0. Prepare 4.5 mL of a 20% GelMA aqueous dispersion and adjust the pH to 10.0. Add the GelMA solution dropwise to the HMON-NH2 aqueous dispersion under stirring at 800 rpm and stir at room temperature for 24 h. Dialyze to obtain size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters.

[0085] Example 4

[0086] (1) Dissolve 1.0 g of gelatin in 100 mL of deionized water under heating at 50 °C to obtain a gelatin solution. Dissolve 0.1 g of hyaluronic acid (HA) in 40 mL of phosphate buffered saline (PBS) to obtain an HA solution. Add the HA solution dropwise to the gelatin solution at a rate of 20–60 drops / min under stirring. Activate with 1-ethyl-(3-dimethylaminopropyl)(EDC)N-hydroxysuccinimide (NHS) and react at 50 °C for 24 h. Subsequently, hyaluronic acid-modified gelatin (HA-Gel) was obtained by dialyzing with deionized water and freeze-drying. 1.0 g of HA-Gel was dissolved in 100 mL of deionized water under heating at 50 °C, the pH was adjusted to 8.5, 0.11 g of dimethyl maleic anhydride (DMA) was added, and the reaction was carried out at 50 °C for 24 h while maintaining the pH at 8.5. After dialyzing with deionized water at pH 8.5 (the pH of the deionized water was adjusted with NaOH / HCl), the gelatin was obtained by freeze-drying.

[0087] (2) 0.8 mL of acetic acid was added to 30 mL of methanol under stirring at 800 rpm, and the mixture was stirred continuously at 30 °C for 30 min. 1.24 mmol of copper acetate monohydrate was added under stirring at 800 rpm, and after the copper acetate was completely dissolved, 1.24 mmol of thiourea was added. The mixture was stirred continuously at 25 °C for 24 h. The mixture was then transferred to a 50 mL polytetrafluoroethylene high-pressure reactor and reacted at 100 °C for 6 h. After the reaction, the mixture was centrifuged, washed, and vacuum dried to obtain copper sulfide (CuS) nanoparticles. After drug loading, drug-loaded CuS nanoparticles were obtained.

[0088] (3) Prepare 5 mL of a 2% (w / w) aqueous dispersion of drug-loaded CuS nanoparticles and adjust the pH to 8.5. Prepare 5 mL of a 2% (w / w) aqueous solution of HA-GelDMA and adjust the pH to 8.5. Add the HA-GelDMA solution dropwise to the aqueous dispersion of drug-loaded CuS nanoparticles under stirring at 800 rpm. Stir at room temperature for 24 h and dialyze to obtain CuS nanoparticle-gelatin composite nanoclusters with size / charge double conversion.

[0089] Example 5

[0090] (1) Dissolve 1.0 g of gelatin in 100 mL of deionized water under heating at 50 °C to obtain a gelatin solution. Dissolve 0.33 g of HA in 40 mL of phosphate buffered saline (PBS) to obtain an HA solution. Add the HA solution dropwise to the gelatin solution at 20–60 drops / min under stirring. Activate with 1-ethyl-(3-dimethylaminopropyl)(EDC)N-hydroxysuccinimide (NHS) and react at 50 °C for 24 h. Subsequently, hyaluronic acid-modified gelatin (HA-Gel) was obtained by dialyzing with deionized water and freeze-drying. 1.0 g of HA-Gel was dissolved in 100 mL of deionized water under heating at 50 °C, the pH was adjusted to 8.5, 0.08 g of CA was added, and the reaction was carried out at 50 °C for 24 h while maintaining the pH at 8.5. After dialyzing with deionized water at pH 8.5 (the pH of the deionized water was adjusted with NaOH / HCl), the gelatin was obtained by freeze-drying and hyaluronic acid-modified methyl maleic anhydride gelatin (HA-GelCA).

[0091] (2) 0.8 mL of acetic acid was added to 30 mL of methanol under stirring at 800 rpm, and the mixture was stirred continuously at 30 °C for 30 min. 1.24 mmol of copper acetate monohydrate was added under stirring at 800 rpm, and after the copper acetate was completely dissolved, 1.24 mmol of thiourea was added. The mixture was stirred continuously at 25 °C for 24 h. The mixture was then transferred to a 50 mL polytetrafluoroethylene high-pressure reactor and reacted at 100 °C for 8 h. After the reaction, the mixture was centrifuged, washed, and vacuum dried to obtain CuS nanoparticles. After drug loading, drug-loaded CuS nanoparticles were obtained.

[0092] (3) Prepare 4.5 mL of an aqueous dispersion of drug-loaded CuS nanoparticles with a mass percentage concentration of 20% and adjust the pH to 7.4. Prepare 0.5 mL of an aqueous solution of HA-GelCA with a mass percentage concentration of 10% and adjust the pH to 7.4. Add the HA-GelCA solution dropwise to the aqueous dispersion of drug-loaded CuS nanoparticles under stirring at 800 rpm. Stir at room temperature for 24 h and dialyze to obtain CuS nanoparticle-gelatin composite nanoclusters with size / charge double conversion.

[0093] Example 6

[0094] (1) Dissolve 1.0 g of gelatin in 100 mL of deionized water under heating at 50 °C, adjust the pH to 8.5, add 0.04 g of MA, maintain pH 8.5 and react at 50 °C for 24 h, dialyze with deionized water at pH 8.5 (adjust the pH of deionized water with NaOH / HCl), and freeze dry to obtain maleic anhydride modified gelatin (GelMA).

[0095] (2) 0.8 mL of acetic acid was added to 30 mL of methanol under stirring at 800 rpm, and the mixture was stirred continuously at 30 °C for 30 min. 1.24 mmol of copper acetate monohydrate was added under stirring at 800 rpm, and after the copper acetate was completely dissolved, 1.24 mmol of thiourea was added. The mixture was stirred continuously at 25 °C for 24 h. The mixture was then transferred to a 50 mL polytetrafluoroethylene high-pressure reactor and reacted at 100 °C for 6 h. After the reaction, the mixture was centrifuged, washed, and vacuum dried to obtain CuS nanoparticles. After drug loading, drug-loaded CuS nanoparticles were obtained.

[0096] (3) Prepare 5 mL of an aqueous dispersion of drug-loaded CuS nanoparticles with a mass percentage concentration of 10%, adjust the pH to 10.0, prepare 0.5 mL of an aqueous solution of GelCA with a mass percentage concentration of 20%, and adjust the pH to 10.0; under stirring conditions of 800 rpm, add GelMA solution dropwise to the aqueous dispersion of drug-loaded CuS nanoparticles, stir at room temperature for 24 h, and dialyze to obtain CuS nanoparticle-gelatin composite nanoclusters with size / charge double conversion.

[0097] Effect evaluation

[0098] I. Drug Loading Experiment

[0099] The aminated hollow mesoporous organosilicon nanoparticles (HMON-NH2) obtained in step (2) of Examples 1 and 3 were dispersed in 5 mL of PBS solution (pH 7.4), and DOX (doxorubicin) aqueous solution (concentration 1 mg / mL) was added at 20-60 drops / min to obtain a series of solutions with different mass ratios of doxorubicin to HMON-NH2. The solutions were stirred in the dark for 24 h, centrifuged, and freeze-dried to obtain drug-loaded nanoparticles (DOX-HMON-NH2). Figure 2 TEM images of MSN, MSN@MON, HMON, HMON-NH2, and DOX-HMON-NH2 show that approximately 20 nm MSN was first synthesized, then a mesoporous organosilicon layer of approximately 20 nm (named MSN@MON) was coated onto the MSN, and the MSN core was selectively etched to obtain hollow mesoporous organosilicon nanoparticles (HMON) of approximately 40 nm. After amino modification, the morphology of HMON-NH2 remained unchanged, but the size increased to approximately 45 nm. After DOX loading, the surface of the nanoparticles became smooth, indicating that DOX was successfully loaded into HMON-NH2. Figure 3 The Zeta potential diagrams are for HMON in Example 1, HMON-NH2 in Example 1, and DOX-HMON-NH2 obtained by loading DOX onto HMON-NH2 prepared in Example 1 (i.e., the drug-loaded HMON-NH2 described in step (2) of Example 1). Figure 3The average Zeta potentials of HMON, HMON-NH2, and DOX-HMON-NH2 are -41.8±7.6, -3.3±3.1, and +30.2±1.0 mV, respectively. This is because the amino group modification can offset part of the negative charge of HMON, and the subsequent DOX loading further enhances the positive charge. Figure 4 As can be seen, the drug loading rate of the prepared nanoparticles can reach 32.6% when m(DOX):m(HMON-NH2) = 90%.

[0100] The preparation method of the drug-loaded nanoparticles in step (2) of Example 2 is the same as above.

[0101] Figure 1 This diagram illustrates the preparation of a DOX-icluster, a composite nanoparticle-gelatin nanoparticle structure with size / charge dual conversion, formed by electrostatic attraction between modified gelatin and drug-loaded hollow mesoporous organosilicon nanoparticles. The example uses folic acid-modified dimethylmaleic anhydride-modified gelatin (FA-GelDMA) as the modified gelatin and doxorubicin (DOX) as the drug loading. In the tumor microenvironment, the 2,3-dimethylmaleic anhydride amide bond (DMA bond) of the modified gelatin breaks, and the gelatin is degraded by MMP-2, releasing small-sized, positively charged drug-loaded hollow mesoporous organosilicon nanoparticles that facilitate penetration and internalization. Figure 1 i in b and Figure 1 (ii) In section b of the text, drug-loaded hollow mesoporous organosilica nanoparticles internalized into tumor cells can be degraded by GSH highly expressed in the cells, thereby releasing DOX (…). Figure 1 (iii in b).

[0102] Figure 6 TEM images of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Examples 1-3. Figure 6 a and a1 are TEM images of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1; Figure 6 b and b1 are TEM images of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 2; Figure 6 C and C1 are TEM images of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 3. Figure 6 The particle sizes of the obtained size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters are 213.7±18.6, 269.2±40.5, and 199.1±30.0 nm, respectively.

[0103] Figure 7The particle size distribution, zeta potential, and DOX loading rate of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Examples 1-3 are shown. Figure 7 The size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Examples 1-3 have potentials of -29.7±1.3mV, -39.4±1.8mV, and -36.7±0.6mV, respectively. The DOX loading rate of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 3 is higher than that in Examples 2 and 1. When the mass ratio of DOX to HMON-NH2 and MON-NH2 reaches 90%, the drug loading rate can reach 21.9±5.1%.

[0104] The preparation method of DOX-loaded copper sulfide (CuS) nanoparticles is the same as that of the DOX-HMON-NH2 preparation method described above, except that HMON-NH2 in the DOX-HMON-NH2 preparation method is replaced with the copper sulfide (CuS) nanoparticles obtained in step (2) of Examples 4-6. The copper sulfide (CuS) nanoparticles obtained in step (2) of Examples 4-6 were used to prepare corresponding copper sulfide (CuS) nanoparticles loaded with DOX (i.e., drug-loaded CuS nanoparticles).

[0105] Figure 21 TEM images of DOX-CuS nanoparticles prepared in Example 5 and DOX-loaded copper sulfide (CuS) nanoparticles prepared in Example 5 were obtained. Figure 21 It is known that copper sulfide (CuS) nanoparticles loaded with DOX have a darker color, and their internal hollow structure is filled with DOX.

[0106] Figure 22 The zeta potentials of DOX-CuS were obtained by comparing the copper sulfide (CuS) nanoparticles prepared in Example 5 and the copper sulfide (CuS) nanoparticles loaded with DOX prepared in Example 5. Figure 22 It is known that copper sulfide (CuS) nanoparticles are negatively charged (-52.3mV), but their potential becomes positively charged (14.2mV) after being loaded with DOX.

[0107] Figure 23 This is a DOX loading diagram of the copper sulfide (CuS) nanoparticles prepared in Example 5. Figure 23 It can be seen that as the mass ratio of DOX to copper sulfide (CuS) nanoparticles increases, the loading rate of DOX gradually increases, and the maximum loading rate is 70% when the mass ratio reaches 220%.

[0108] Figure 20 This is a schematic diagram of the preparation of DOX-CuS obtained by loading DOX onto copper sulfide (CuS) nanoparticles prepared in Example 5.

[0109] Figure 24 The particle size distribution and TEM image of the size / charge dual-conversion CuS nanoparticle-gelatin composite nanoclusters prepared in Example 5 are shown. Figure 24 The size of CuS is further increased by coating it with a layer of high-molecular-weight gelatin.

[0110] II. Particle size and potential characterization

[0111] Dynamic light scattering (DLS) and zeta potential were measured using a Malvern Zetasizer Nano ZSU3200, with three measurements taken at 25 °C and the average value taken. Morphology was determined using a transmission electron microscope (TEM) JEM-2100 with an accelerating voltage of 200 kV. Figure 2 and Figure 18 The average sizes of drug-loaded hollow mesoporous organosilicon nanoparticles (DOX-HMON-NH2 nanoparticles) and drug-loaded CuS nanoparticles (DOX-CuS nanoparticles) were 47.5 nm and 175.3 nm, respectively. Figure 6 It can be seen that the average sizes of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters in Examples 1 to 3 are 213.7 nm, 269.2 nm, and 199.1 nm, respectively. Figure 24 It can be seen that the average size of the CuS nanoparticle-gelatin composite nanoclusters with size / charge dual conversion in Example 5 is 227.7 nm.

[0112] III. Size and Charge Conversion

[0113] 100 μL of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in Example 1 were dispersed in 5 mL of acetate buffer solution containing MMP-2 (pH 6.5, 0.25 μg / mL MMP-2) and stirred for 24 h. Samples were then taken for TEM analysis. Figure 8 As shown in the figure, the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters prepared in this invention can undergo responsive degradation under weakly acidic and enzymatic environments, releasing small-sized nanoparticles. The stability of the size / charge dual-conversion nanoparticle-gelatin composite nanoclusters was determined by monitoring their size and potential after immersion in PBS (pH 7.4) for 24 hours. Figure 9As shown, in acetate buffer solution (pH 7.4), the size and zeta potential of the size / charge double-conversion organosilicon nanoparticle-gelatin composite nanoclusters remained essentially unchanged over 24 h, indicating good stability and favorable for long-term in vivo circulation. The size / charge double-conversion organosilicon nanoparticle-gelatin composite nanoclusters of Example 1 were dispersed in 5 mL of an aqueous solution containing MMP-2 (pH adjusted to 6.5 with NaOH / hydrochloric acid, 0.25 μg / mL MMP-2) and stirred for 24 h, after which the size and zeta potential were measured. Figure 10 It is known that the size shrinks to 73.3 ± 15.2 nm under weakly acidic and enzymatic conditions. From Figure 11 It can be seen that the charge of the composite nanoclusters changes from negative to positive.

[0114] IV. In vitro drug release behavior of composite nanoclusters

[0115] Take 1.6 mL of the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanocluster (DOX-icluster) prepared in Examples 1-3 and place it in a dialysis bag (MWCO = 14000 Da). Dialyze it in 8 mL of different pH buffer solutions (phosphate buffer solution with pH 7.4, phosphate buffer solution with pH 6.5, acetate buffer solution with pH 5.0, phosphate buffer solution with pH 6.5 containing 0.25 μg / mL MMP-2, and acetate buffer solution with pH 5.0 containing 10 mM glutathione GSH) at 37 °C and 100 r / min on a shaker. 0.4 mL dialysis buffer samples were collected at different time points (1, 2, 4, 8, 12, 24, 36, 48, 72, 96, 120, 144, 168 h) and replaced with the corresponding 0.4 mL buffer. The drug content in the buffer samples was measured using a fluorescence microplate reader to calculate the drug release process. Figure 12 It is known that the weakly acidic conditions of tumor tissue and the environment rich in matrix metalloproteinases and glutathione are conducive to drug release.

[0116] V. Toxicity of composite nanoclusters loaded with drugs against mouse breast cancer (4T1) tumor cells

[0117] 4T1 cells were fed at a dose of 1×10 4Cells were seeded at a density of 10 cells / well in 96-well plates and in 150 μL of culture medium. After incubation at 37°C and 5% CO2 for 24 h, once cells were fully adherent, the old culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). Then, 150 μL of the size / charge double-conversion organosilicon nanoparticle-gelatin composite nanoclusters (DOX-icluster) corresponding to Example 1 (DOX concentrations of 0.01, 0.05, 0.1, 0.5, 1, and 5 μg / mL) was added. Control group 1 used the empty size / charge double-conversion organosilicon nanoparticle-gelatin composite nanoclusters (icluster) obtained in Example 1, and control group 2 used DOX aqueous solution (DOX concentrations of 0.01, 0.05, 0.1, 0.5, 1, and 5 μg / mL). Cells were incubated in fresh culture medium for 24 h and 72 h, and cell viability was tested using Presto blue. The results are shown below. Figure 13 ( Figure 13 In the graph, 'a' represents the survival rate after 24 hours of incubation. Figure 13 (Figure b shows the survival rate after 72 hours of incubation). Figure 13 It was found that after incubation for 24 h and 72 h, the size / charge dual-conversion organosilicon nanoparticle-gelatin composite nanoclusters inhibited 4T1 cells by 24.9% and 51.4%, respectively, with a half-inhibitory concentration (IC50) of 100%. 50 The values ​​were 14.18±2.64 μg / mL and 4.66±0.79 μg / mL, respectively.

[0118] 4T1 cells were fed at a dose of 1×10 4 Cells were seeded at a density of 10 cells / well in 96-well plates and in 150 μL of culture medium. The plates were cultured at 37°C and 5% CO2 for 24 h. After complete cell adhesion, the old culture medium was removed, and the plates were washed twice with phosphate-buffered saline (PBS). 150 μL of the size / charge double-conversion CuS nanoparticle-gelatin composite nanoclusters (DOX-icluster) corresponding to Example 5 (DOX concentrations of 0.01, 0.05, 0.1, 0.5, 1, and 5 μg / mL) was added. Control group 1 used the empty size / charge double-conversion CuS nanoparticle-gelatin composite nanoclusters (icluster) obtained in Example 5, and control group 2 used DOX aqueous solution (DOX concentrations of 0.01, 0.05, 0.1, 0.5, 1, and 5 μg / mL). The plates were incubated in fresh culture medium for 24 h. The effect of adding H2O2 was also compared. 10 μL of H2O2 was added to 10 mL of culture medium, and 100 μL of this solution was further diluted to 10 mL with more culture medium to prepare a 100 μM H2O2-containing medium. The group without hydrogen peroxide served as a control. Cell viability was tested using Presto blue, and the results are as follows: Figure 25 ( Figure 25 In this context, 'a' represents the addition of hydrogen peroxide (H2O2). Figure 26 In the figure, b represents the absence of hydrogen peroxide (H2O2). As shown in the figure, after 24 hours of incubation, the size / charge double-conversion CuS nanoparticle-gelatin composite nanoclusters (DOX-icluster) exhibited inhibition rates of 51.7% and 89.2% on 4T1 cells with and without H2O2 at a DOX concentration of 1 μg / mL. The effect of laser irradiation was also compared (laser power 2.0 W / cm²). 2 After 1 minute, the control group was not used. Cell viability was tested using Presto blue, and the results were as follows: Figure 25 As shown, the size / charge dual-conversion CuS nanoparticle-gelatin composite nanoclusters (DOX-icluster) exhibit good chemotherapeutic / photothermal / photodynamic effects.

[0119] 4T1 cells were fed at a dose of 1×10 4 Cells were seeded at a density of 100 cells / well in 96-well plates and in 150 μL of culture medium. The cells were cultured at 37°C and 5% CO2 for 24 h. After complete cell adhesion, the old culture medium was removed, and the cells were washed twice with phosphate-buffered saline (PBS). Then, 150 μL of fresh culture medium containing 100 μM H2O2 was added, and the cells were incubated for 2 h. Following this, the cells were irradiated with laser light (NIR) for 1 min and cultured for another 22 h. Cell viability was assessed using Presto blue assay. Figure 26 This is a comparison of cell viability between H2O2+NIR and the blank control group. Figure 26 It can be seen that the H2O2+NIR group alone has little effect on cell growth.

[0120] Figure 27 for Figure 25 The corresponding live / dead staining diagram is derived from... Figure 27 It is known that CuS nanoparticles-gelatin composite nanoclusters with size / charge dual conversion have good chemotherapy / photothermal / photodynamic effects.

[0121] VI. Penetration of Three-Dimensional Tumor Spheres by Composite Nanoclusters

[0122] 4T1 cells were dropped into 10 μL of different culture media (pH 7.4, pH 6.5, and pH 6.5 containing 0.25 μg / mL MMP-2) at a density of 2000 cells / 10 μL onto the cap of a 48-well plate. 200 μL of PBS (pH 7.4) was added to each well. The plates were then incubated in a hanging drop incubator at 37°C and 5% CO2 for 4 days to establish and select a tumor spheroid model with regular and dense morphology. Each tumor spheroid model was treated with 1 μL of a 100 μg / mL DOX solution and 1 μL of an aqueous dispersion of the size / charge dual-conversion organosilicon nanoparticle-gelatin nanoclusters (DOX-icluster) prepared in Example 1, and cultured under different conditions (pH 7.4, pH 6.5, and pH 6.5 containing 0.25 μg / mL MMP-2, with a DOX concentration of 10 μg / mL) at 37°C for 3 h. The tumor spheroid sections at different depths were scanned using laser confocal microscopy to observe drug uptake and acquire images. Figure 14 , Figure 15 and Figure 16 As can be seen, the weakly acidic conditions of tumor tissue and the environment rich in matrix metalloproteinases enable the size / charge dual-conversion organosilicon nanoparticles-gelatin nanoclusters (DOX-icluster) to have a better penetration effect on three-dimensional tumor spheres.

[0123] VII. Growth Inhibition Effect of Nanoclusters on Three-Dimensional Tumor Spheres

[0124] 4T1 tumor spheres with regular morphology and appropriate size were cultured. One group was treated with 1 μL of a 100 μg / mL DOX solution, while the other group was treated with 1 μL of a dispersion of size / charge double-conversion organosilicon nanoparticles-gelatin nanoclusters (DOX-icluster) prepared in Example 1 (DOX concentration 100 μg / mL). The final solutions contained 10 μg / mL DOX in both cases. The spheres were cultured under different conditions (pH 7.4, pH 6.5, and pH 6.5 containing 0.25 μg / mL LMP-2). The volume change of the tumor spheres was measured using an inverted microscope. Figure 17 As can be seen, the weakly acidic conditions and MMP-2-rich environment of tumor tissue enable organosilicon nanoparticles-gelatin nanoclusters with size / charge double conversion to have a better inhibitory effect on three-dimensional tumor spheres. Figure 18 The results showed that in a culture medium containing 0.25 μg / mL MMP-2 at pH 6.5, the growth of tumor spheroids significantly decreased to 15.1% of their original volume, accompanied by cell detachment. Based on these results, the hypothesized mechanism of tumor spheroid growth inhibition is as follows: Figure 19 As shown, after the tumor spheres are treated with organosilicon nanoparticles-gelatin nanoclusters with size / charge dual conversion, they release drug-loaded hollow mesoporous organosilicon nanoparticles in response to pH and MMP-2 and penetrate into the interior of the tumor spheres, causing the peripheral cells to gradually collapse and undergo apoptosis, resulting in a reduction in volume.

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A method for preparing size / charge dual-conversion nanoparticle-gelatin composite nanoclusters, characterized in that, Includes the following steps: (1) Reaction of gelatin with a targeting ligand, dialysis and freeze-drying, and then reaction with anhydride compounds under alkaline conditions, followed by dialysis and freeze-drying to obtain modified gelatin; or reaction of gelatin with anhydride compounds under alkaline conditions, followed by dialysis and freeze-drying to obtain modified gelatin. (2) Positively charged drug-loaded copper sulfide nanoparticles were prepared by nanoprecipitation method. The specific steps of the nanoprecipitation method are as follows: 0.2-5 mL of acetic acid was added to 10-50 mL of methanol under stirring at 200-1500 rpm, and the mixture was kept at 20-60℃ for 10-60 min; 1-5 mmol of copper acetate monohydrate was added under stirring, and after the copper acetate was completely dissolved, 1-5 mmol of thiourea was added, and the mixture was stirred continuously at 20-60℃ for 8-48 h; then the mixture was transferred to a 25-100 mL polytetrafluoroethylene high-pressure reactor and kept at 80-200℃ for 2-12 h; after the reaction was completed, the mixture was centrifuged, washed and vacuum dried to obtain copper sulfide nanoparticles; after drug loading, drug-loaded copper sulfide nanoparticles were obtained. (3) Prepare the modified gelatin obtained in step (1) into a modified gelatin solution, prepare the drug-loaded copper sulfide nanoparticles obtained in step (2) into a nanoparticle dispersion, and then mix the modified gelatin solution and the nanoparticle dispersion to obtain a size / charge double conversion nanoparticle-gelatin composite nanocluster through electrostatic interaction; wherein, the mass percentage concentration of the modified gelatin solution is 2% to 20%, the mass percentage concentration of the nanoparticle dispersion is 2% to 20%, the pH of the prepared modified gelatin solution and the prepared nanoparticle dispersion before mixing is 7.4 to 10.0, and the volume ratio of the modified gelatin solution and the nanoparticle dispersion is (1:9) to (9:1).

2. The method for preparing a size / charge dual-conversion nanoparticle-gelatin composite nanocluster according to claim 1, characterized in that, The targeting ligand in step (1) is one or more of folic acid and hyaluronic acid; the reaction temperature of gelatin and the targeting ligand in step (1) is 20℃~80℃, the reaction time of gelatin and the targeting ligand is 2-24h, and the alkaline condition is pH7.4~10.0; the reaction temperature of gelatin and the acid anhydride compound in step (1) is 20℃~80℃, the reaction time of gelatin and the acid anhydride compound is 2-24h; the alkaline condition in step (1) is pH7.4~10.0; the acid anhydride compound in step (1) is one or more of maleic anhydride, methylmaleic anhydride, and dimethylmaleic anhydride.

3. The method for preparing a size / charge dual-conversion nanoparticle-gelatin composite nanocluster according to claim 1, characterized in that, In step (1), the mass ratio of the targeting ligand to gelatin is (1:15) to (1:1).

4. The method for preparing a size / charge dual-conversion nanoparticle-gelatin composite nanocluster according to claim 1, characterized in that, In step (1), the mass ratio of the acid anhydride compound to gelatin is (1:25) to (1:9).

5. A size / charge dual-conversion nanoparticle-gelatin composite nanocluster prepared by the preparation method according to any one of claims 1-4.

6. The size / charge dual-conversion nanoparticle-gelatin composite nanocluster according to claim 5, characterized in that, The size of the size / charge dual-conversion nanoparticle-gelatin composite nanocluster is 40-400 nm, and the potential is -10 mV to -50 mV.

7. The size / charge dual-conversion nanoparticle-gelatin composite nanocluster according to claim 5, characterized in that... The composite nanoclusters decrease in size and change in charge from negative to positive under the action of pH=6.5 and enzymes.

8. The application of the size / charge dual-conversion nanoparticle-gelatin composite nanocluster as described in claim 5 in the preparation of drug nanocarriers.

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