A type of Pt 2+ - Carbon dot@protoporphyrin sonodynamic drug delivery system, its preparation method, and applications

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a Pt 2+ - Carbon dot@protoporphyrin sonodynamic drug delivery system, its preparation method and application, by loading Pt onto the surface of carbon dots 2+ Modification layer, to obtain Pt 2+ After carbon dots are formed, protoporphyrin is loaded onto them to obtain a carbon dot core with Pt loaded on the carbon dot surface. 2+ Modification layer, in Pt 2+ A sonodynamic drug delivery system with a protoporphyrin-modified layer loaded on the outside of the modified layer can be used in anti-tumor drugs to screen liver cancer cells and normal cells. It has good biocompatibility and significant in vivo sonodynamic anti-tumor effects. It can promote the generation of reactive oxygen species, target and remain in the subcutaneous tumor site in vivo for a long time, increase the mortality rate of cancer cells, and inhibit tumor growth. In a 3D constructed in vitro hepatocellular tumor microenvironment model, this drug delivery system still has superior sonodynamic therapeutic effects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a Pt 2+ - Carbon dot@protoporphyrin sonodynamic drug delivery system, its preparation method, and its application. Background Technology

[0002] Sonodynamic therapy, which uses low-intensity ultrasound with a penetration depth (>10 cm) to stimulate the production of reactive oxygen species (ROS) in sonosensitizers, is a promising non-invasive approach to cancer treatment. Currently, protoporphyrins, an organic sonosensitizer, are widely used in sonodynamic therapy; while carbon dots serve as carriers for anticancer drugs, enabling targeted drug delivery to tumor sites. Therefore, using carbon dots as a carrier to deliver the sonosensitizer protoporphyrin into the cell nucleus can lead to the generation of ROS in the nucleus, thereby inducing apoptosis.

[0003] However, intracellular glutathione in the tumor microenvironment consumes a large amount of reactive oxygen species generated by sonodynamic therapy, conferring resistance to the therapy and leading to incomplete tumor regression and recurrence. Therefore, it is necessary to control the glutathione content, ensure the level of reactive oxygen species, and maximize drug release efficiency and target retention time to enhance the efficacy of sonodynamic therapy. Summary of the Invention

[0004] This invention provides a Pt 2+ - Carbon dot@protoporphyrin sonodynamic drug delivery system, its preparation method, and its application.

[0005] The technical solution of the present invention is as follows:

[0006] This invention provides a Pt 2+ - A carbon dot@protoporphyrin sonodynamic drug delivery system, with carbon dots as the core and Pt loaded on the carbon dot surface. 2+ Modification layer, in Pt 2+ A protoporphyrin-modified layer is loaded onto the modified layer.

[0007] The Pt of this invention 2+ - Carbon dots@protoporphyrin, with a particle size of 2.78±0.88 nm.

[0008] The Pt described in this invention 2+ The carbon dot@protoporphyrin sonodynamic drug delivery system was prepared through the following steps:

[0009] Pt loaded on carbon dot surface 2+ Modification layer, to obtain Pt 2+ - Carbon dots; Pt 2+ Carbon dots were added to a protoporphyrin solution and stirred in the dark to obtain Pt. 2+ - Carbon dot@protoporphyrin sonodynamic drug delivery system.

[0010] The present invention describes the obtaining of Pt 2+ - Carbon dots are obtained by adding K2PtCl6 solution to carbon dot solution, reacting, centrifuging and filtering.

[0011] The Pt of this invention 2+ The molar ratio of carbon to carbon is 0.2-1.8:1.

[0012] The Pt of this invention 2+ - The mass ratio of carbon dots to protoporphyrin is 0.5-3:1.

[0013] The protoporphyrin solution described in this invention is obtained by dissolving protoporphyrin, N,N'-dicyclohexylcarbodiimide, and 1-hydroxybenztriazole in dimethyl sulfoxide.

[0014] The stirring time described in this invention is 8-15 hours, and the temperature is 23-27℃.

[0015] The present invention also provides the aforementioned Pt 2+ - Application of carbon dot@protoporphyrin sonodynamic drug delivery system in antitumor drugs and 3D printed tumor microenvironment models.

[0016] The application described in this invention is the Pt 2+ - Application of carbon dot@protoporphyrin sonodynamic drug delivery system in anti-tumor drugs that target cancer cell nuclei, promote reactive oxygen species generation, prolong the residence time at the tumor site, increase cancer cell mortality, and inhibit tumor growth.

[0017] Beneficial effects

[0018] This invention successfully prepared a carbon dot-based material with Pt loaded on the carbon dot surface. 2+ Modification layer, in Pt 2+ Pt with protoporphyrin-modified layer loaded outside the modified layer 2+ - Carbon dot@protoporphyrin sonodynamic drug delivery system.

[0019] Pt prepared in this invention 2+ - Carbon dot@protoporphyrin sonodynamic drug delivery system, Pt 2+ It reacts with glutathione, thereby reducing glutathione levels, maintaining reactive oxygen species levels, and thus improving the efficacy of sonodynamic therapy.

[0020] Pt prepared in this invention 2+The carbon dot@protoporphyrin sonodynamic drug delivery system, when used in anti-tumor drugs, can screen liver cancer cells and normal cells. It has good biocompatibility and significant in vivo sonodynamic anti-tumor effects. It can target and remain in vivo subcutaneous tumor sites for a long time, effectively increasing cancer cell mortality and inhibiting tumor growth. This drug delivery system also has superior sonodynamic therapeutic effects in a 3D constructed in vitro hepatocellular tumor microenvironment model. Attached Figure Description

[0021] Figure 1 For CDs, Pt 2+ -CDs, Pt 2+ Transmission electron microscope images (a) and particle size distribution diagram (b) of CDs@PpIX.

[0022] Figure 2 For Pt 2+ -CDs@PpIX, CDs, Pt 2+ -CDs, PpIX and Pt 2+ Zeta potential (a) and hydration radius (b) of -CDs+GSH

[0023] Figure 3 This is a selective detection plot for GSH, where (a) shows different concentrations of Pt. 4+ The fluorescence intensity comparison diagram in CDs, (b) shows different concentrations of Pt. 4+ Fluorescence spectrum in CDs, (c) is Pt 2+ - Specific detection diagram of GSH by CDs, (d) shows the detection of different concentrations of GSH in Pt 2+ - Comparison of fluorescence intensity in CDs, (e) shows different concentrations of GSH in Pt 2+ - Fluorescence spectra of CDs, (f) GSH in Pt 2+ - Linear relationship between fluorescence intensity and GSH concentration in CDs

[0024] Figure 4 To use CDs(a), Pt 2+ -CDs(b) and Pt 2+ -CDs@PpIX(c) were used as fluorescent probes for in vitro fluorescence imaging of different cells.

[0025] Figure 5 The images show in vitro drug release detection, where (a) is fluorescence imaging of different drug delivery systems, and (b) is Pt at different mass ratios. 2+ -CDs and PpIX drug loading curves, (c) shows the drug loading rate of Pt at different pH values. 2+ -CDs, Pt 2+ -CDs@PpIX cumulative drug release curve over time.

[0026] Figure 6 The graph shows cell viability assays, where (a) represents different concentrations of Pt without sonication. 2+ -CDs@PpIX cell viability comparison chart, (b) shows the cell viability of different concentrations of Pt under ultrasound. 2+ Comparison of cell viability under CDs@PpIX, (c) is a comparison of cell viability under different treatments, (d) is a fluorescence image of SMMC-7721 hepatocellular carcinoma cells after different treatments stained with Calcein-AM / PI kit, (e) is a confocal image of SMMC-7721 hepatocellular carcinoma cells after different treatments stained with DCFH-DA / DAPI kit, and (f) is a fluorescence image of SMMC-7721 hepatocellular carcinoma cells after different treatments stained with Calcein-AM / PI kit and a confocal image of SMMC-7721 hepatocellular carcinoma cells stained with DCFH-DA / DAPI kit in a 3D tumor microenvironment model.

[0027] Figure 7 Pt labeled with indocyanine green 2+ Near-infrared fluorescence imaging was performed at different time points on the mouse abdomen (a) and right side of the body (b) after CDs@PpIX.

[0028] Figure 8 The images show a comparison of the effects of in vivo sonodynamic therapy with different treatment methods. (a) shows the total images of the 7 groups of mice after 21 days of treatment, (b) shows the images of the tumor tissue, (c) shows the weight of the tumor tissue, (d) shows the curve of treatment days versus tumor tissue volume, and (e) shows the curve of treatment days versus mouse body weight. Detailed Implementation

[0029] The following examples are intended to illustrate the present invention, and not to further limit the invention.

[0030] This invention provides a Pt 2+ - A carbon dot@protoporphyrin sonodynamic drug delivery system, with carbon dots as the core and Pt loaded on the carbon dot surface. 2+ Modification layer, in Pt 2+ A protoporphyrin-modified layer is loaded onto the modified layer.

[0031] This drug delivery system, using carbon dots as carriers, can deliver the sonosensitive agent protoporphyrin into the cell nucleus, while Pt 2+ The addition of [a specific ingredient] ensures that the reactive oxygen species generated by sonodynamic therapy are not consumed by glutathione.

[0032] Preferably, Pt 2+ In the carbon dot@protoporphyrin sonodynamic drug delivery system, ensuring a reasonable particle size distribution is crucial for achieving optimal therapeutic efficacy. The Pt... 2+The particle size of the carbon dots@protoporphyrin is 2.78±0.88 nm.

[0033] The Pt provided by this invention 2+ The carbon dot@protoporphyrin sonodynamic drug delivery system was prepared through the following steps:

[0034] Pt loaded on carbon dot surface 2+ Modification layer, to obtain Pt 2+ - Carbon dots; Pt 2+ Carbon dots were added to a protoporphyrin solution and stirred in the dark to obtain Pt. 2+ - Carbon dot@protoporphyrin sonodynamic drug delivery system.

[0035] Pt was loaded onto the carbon dot surface through a two-step modification process. 2+ Pt with synergistic effects was successfully prepared by modifying the layer and the protoporphyrin layer. 2+ - Carbon dot@protoporphyrin sonodynamic drug delivery system.

[0036] Specifically, the obtained Pt 2+ - Carbon dots, which are obtained by adding K2PtCl6 solution to carbon dot solution, Pt 4+ It is obtained by reacting with carbon dots in a redox reaction and then centrifuging and filtering.

[0037] Furthermore, to ensure Pt 2+ The thickness of the modified layer and the protoporphyrin modified layer, the Pt 2+ The molar ratio of Pt to carbon points is 0.2-1.8:1. 2+ - The mass ratio of carbon dots to protoporphyrin is 0.5-3:1.

[0038] In addition, protoporphyrin forms a protoporphyrin-modified layer through an amide reaction. Specifically, protoporphyrin, N,N'-dicyclohexylcarbodiimide, and 1-hydroxybenztriazole are dissolved in dimethyl sulfoxide to obtain a protoporphyrin solution.

[0039] Furthermore, in order to ensure Pt 2+ The carbon dots react fully in the protoporphyrin solution, with stirring for 8-15 hours at a temperature of 23-27°C.

[0040] The present invention also provides the aforementioned Pt 2+ - Application of carbon dot@protoporphyrin sonodynamic drug delivery system in antitumor drugs and 3D-printed tumor microenvironment models. Specifically, the Pt... 2+ - Application of carbon dot@protoporphyrin sonodynamic drug delivery system in anti-tumor drugs that target cancer cell nuclei, promote reactive oxygen species generation, prolong the residence time at the tumor site, increase the mortality rate of liver cancer cells, and inhibit tumor growth.

[0041] The Pt provided by this invention2+ The carbon dot@protoporphyrin sonodynamic drug delivery system can screen liver cancer cells and normal cells, has good biocompatibility, and exhibits significant in vivo sonodynamic anti-tumor effects. It can promote the generation of reactive oxygen species, target and remain in the subcutaneous tumor site in vivo for a long time, effectively kill cancer cells, increase cancer cell mortality, and inhibit tumor growth. This drug delivery system still has superior sonodynamic therapeutic effects in a 3D constructed in vitro liver cell tumor microenvironment model.

[0042] For the sake of brevity, the relevant terms are abbreviated in the following specific embodiments and test results, as shown in the table below:

[0043]

[0044]

[0045] Example 1

[0046] (1) Preparation of CDs: 0.3 g m-phenylenediamine and 0.675 g L-cysteine ​​were dissolved in 30 mL of deionized water and stirred. The stirred solution was placed in a reaction vessel and hydrothermally reacted at 160 °C for 10 h to obtain CDs.

[0047] (2)Pt 2+ Preparation of -CDs: An equal volume of K2PtCl6 solution was slowly added to the carbon dot solution. After reacting for 30 min, the mixture was centrifuged at 10000 rpm for 5 min. This process was repeated three times to obtain Pt. 2+ -CDs. Where Pt 2+ The molar ratio of CDs is 0.2:1.

[0048] (3)Pt 2+ Preparation of -CDs@PpIX: First, dissolve 2 mg PpIX, 2.69 mg DCC, and 1.76 mg HOBt in 5 mL DMSO and react for 6 h to obtain a PpIX solution; then add Pt... 2+ -CDs were dissolved in 5 mL of DMSO to obtain Pt 2+ -CDs dispersion; PpIX solution and Pt 2+ The CDs dispersion was mixed and stirred at 25°C in the dark for 12 hours, then dialyzed at 3500 Da for 24 hours, and finally freeze-dried to obtain Pt. 2+ -CDs@PpIX. Wherein, Pt... 2+ The quality ratio of CDs to PpIX is 2:1.

[0049] Example 2

[0050] (1) Preparation of CDs: 0.3 g m-phenylenediamine and 0.675 g L-cysteine ​​were dissolved in 30 mL of deionized water and stirred. The stirred solution was placed in a reaction vessel and hydrothermally reacted at 160 °C for 10 h to obtain CDs.

[0051] (2)Pt 2+ Preparation of -CDs: An equal volume of K2PtCl6 solution was slowly added to the carbon dot solution. After reacting for 30 min, the mixture was centrifuged at 10000 rpm for 5 min. This process was repeated three times to obtain Pt. 2+ -CDs. Where Pt 2+ The molar ratio with CDs is 1:1.

[0052] (3)Pt 2+ Preparation of -CDs@PpIX: First, dissolve 2 mg PpIX, 2.69 mg DCC, and 1.76 mg HOBt in 5 mL DMSO and react for 6 h to obtain a PpIX solution; then add Pt... 2+ -CDs were dissolved in 5 mL of DMSO to obtain Pt 2+ -CDs dispersion; PpIX solution and Pt 2+ -CDs dispersion was mixed and stirred at 27°C in the dark for 8 hours, then dialyzed at 3500 Da for 24 hours, and finally freeze-dried to obtain Pt. 2+ -CDs@PpIX. Wherein, Pt... 2+ The quality ratio of CDs to PpIX is 0.5:1.

[0053] Example 3

[0054] (1) Preparation of CDs: 0.3 g m-phenylenediamine and 0.675 g L-cysteine ​​were dissolved in 30 mL of deionized water and stirred. The stirred solution was placed in a reaction vessel and hydrothermally reacted at 160 °C for 10 h to obtain CDs.

[0055] (2)Pt 2+ Preparation of -CDs: An equal volume of K2PtCl6 solution was slowly added to the carbon dot solution. After reacting for 30 min, the mixture was centrifuged at 10000 rpm for 5 min. This process was repeated three times to obtain Pt. 2+ -CDs. Where Pt 2+ The molar ratio of CDs is 1.8:1.

[0056] (3)Pt 2+ Preparation of -CDs@PpIX: First, dissolve 2 mg PpIX, 2.69 mg DCC, and 1.76 mg HOBt in 5 mL DMSO and react for 6 h to obtain a PpIX solution; then add Pt... 2+-CDs were dissolved in 5 mL of DMSO to obtain Pt 2+ -CDs dispersion; PpIX solution and Pt 2+ The CDs dispersion was mixed and stirred at 23°C in the dark for 15 h, then dialyzed at 3500 Da for 24 h, and finally freeze-dried to obtain Pt. 2+ -CDs@PpIX. Wherein, Pt... 2+ The quality ratio of CDs to PpIX is 3:1.

[0057] Comparative Example 1

[0058] Comparative Example 1 only provides samples without Pt 2+ The modified and PpIX-modified CDs were prepared without steps (2) and (3) compared with Example 1. The remaining operations were the same as in Example 1, and the same reaction conditions were used.

[0059] Comparative Example 2

[0060] Comparative Example 2 provides a solution without Pt 2+ Compared with Example 1, the modified CDs@PpIX drug delivery system has no step (2) in its preparation method. In step (3), the PpIX solution is mixed with the CDs dispersion, and the rest of the operation is the same as in Example 1, using the same reaction conditions as in Example 1.

[0061] Comparative Example 3

[0062] Comparative Example 3 provides a Pt without PpIX modification. 2+ -CDs, compared with Example 1, the preparation method has no step (3), the rest of the operation is the same as in Example 1, and the same reaction conditions as in Example 1 are used.

[0063] Experimental results

[0064] In the following test results, the Pt used in the experiment... 2+ -CDs@PpIX, CDs, CDs@PpIX, Pt 2+ -CDs were prepared in Example 1, Comparative Example 1, Comparative Example 2, and Comparative Example 3, respectively.

[0065] 1. Morphology and particle size analysis

[0066] The morphology and particle size of the carrier are important factors affecting its physical stability and in vivo distribution. Generally, the high-permeability long-retention effect (EPR effect) aggregates small-sized (<200nm) nanoparticles at the tumor site, achieving long-term therapeutic retention. Figure 1 (a) It can be seen that CDs, Pt 2+ -CDs and Pt 2+-CDs@PpIX are all spherical and well-dispersed, which is significant for the subsequent action of the system on the tumor site. Based on statistical data obtained from transmission electron microscopy images using ImageJ software, a particle size distribution map was obtained, as shown below. Figure 1 As shown in (b). CDs, Pt 2+ -CDs and Pt 2+ -CDs@PpIX have average particle sizes of 1.12±0.03nm, 2.14±0.61nm, and 2.78±0.88nm, respectively. The particle size values ​​are lower than the diameter of the nuclear pore complex (9-15nm), which is beneficial for passing through the nuclear membrane and reaching the nucleolus region.

[0067] 2. Characterization of Zeta potential and hydration radius

[0068] For Pt 2+ -CDs@PpIX, CDs, Pt 2+ -CDs, PpIX and Pt 2+ Characterization of zeta potential and hydration radius using -CDs+GSH, such as Figure 2 As shown. Compared to CDs, CDs have a load Pt 2+ Subsequently, the potential changed from negative to positive, and the hydration radius increased from 78.8 d·nm to 164.4 d·nm, indicating that Pt 2+ Successfully loaded onto CDs. (With Pt) 2+ Compared to -CDs, when GSH is present, Pt 2 + The hydrodynamic size of CDs decreased from 164.4 nm to 107.0 d·nm, and the Zeta potential decreased from 6.56 ± 2.1 mV to 2.34 ± 1.6 mV, indicating that GSH induces Pt 2+ A reduction reaction occurred. With Pt 2+ Compared to -CDs, Pt modified with PpIX 2+ -CDs@PpIX, with a positive Zeta potential, are conducive to reacting with the negatively charged surface of cancer cells.

[0069] 3. Selective detection of GSH

[0070] from Figure 3 From (a) and (b) in the diagram, we can see that Pt is added to CDs. 4+ The higher the concentration, the weaker the fluorescence intensity, especially when Pt 4+ At a concentration of 1.8 μM, the fluorescence of CDs was almost completely quenched. Pt at a concentration of 1.8 μM was then added. 4+ Pt after quenching 2+-CDs were placed under different conditions (Blank, AA, BSA, PBS, GSH, NaCl, KCl, MgCl2, L-Thr, Arg, Asp) to selectively detect GSH, such as Figure 3 As shown in (c), it can be seen that Pt after quenching... 2+ -CDs and GSH showed the strongest fluorescence intensity, indicating that Pt after quenching 2+ -CDs are specific for GSH. In Pt 2+ Different concentrations of GSH were added to CDs, resulting in... Figure 3 As shown in (d) and (e), the higher the concentration of GSH added, the stronger the fluorescence intensity, indicating that Pt 2+ -CDs exhibit high sensitivity to GSH. Further processing revealed a linear relationship between GSH concentration and fluorescence intensity, such as... Figure 3 As shown in (f), Pt 2+ -CDs have a wide detection range (0-8 mM) and a low fluorescence intensity detection limit (1.8 μM) for GSH.

[0071] 4. In vitro fluorescence imaging of different cells

[0072] Human normal hepatocyte line LO2, human umbilical vein endothelial cell line HUVEC, and human hepatocellular carcinoma lines SMMC-7721 and HepG2 were selected as cell models, and CDs and Pt were used. 2+ -CDs and Pt 2+ -CDs@PpIX were used as fluorescent probes to treat cell models and then fluorescence imaging was performed. Figure 4 As shown. From Figure 4 (a) It can be seen that when CDs are used as fluorescent probes and incubated with four types of cells, CDs are internalized into the cytoplasm of all cells and there is no obvious distinction in the fluorescence imaging of the four types of cells. Figure 4 In (b), Pt is used. 2+ When -CDs are used as fluorescent probes, even after 24 hours of incubation, Pt 2+ -CDs-labeled LO2 and HUVEC cells were "negative" (with almost no fluorescence contrast). In contrast, SMMC-7721 and HepG2 cells showed significant fluorescence contrast (i.e., "positive") after 6 hours of incubation. As incubation time increased, the fluorescence intensity in SMMC-7721 and HepG2 cells gradually stabilized due to a slow decrease in high levels of GSH in the cytoplasm. Figure 4 (c) indicates that Pt 2+ -CDs loaded with the drug PpIX still interact with Pt 2+ -CDs have the same GSH detection capability. The above results indicate that Pt 2+The -CDs@PpIX drug delivery system can screen liver cancer cells and normal cells.

[0073] 5. In vitro drug release

[0074] Commercial nuclear dye DAPI was used to react with Pt 2+ -CDs@PpIX, CDs@PpIX, CDs, and PpIX were used to co-stain SMMC-7721 liver cancer cells, and the effects of PpIX, CDs@PpIX, and Pt were investigated. 2+ The internalization and localization of -CDs@PpIX within cells. For example... Figure 5 As shown in (a), after 1 hour of incubation with SMMC-7721 hepatocellular carcinoma cells, Pt 2+ -CDs@PpIX rapidly and massively endocytoingest cells and release PpIX, resulting in red fluorescence in the cytoplasm and nucleus. In contrast, free PpIX only exhibits red fluorescence in the cytoplasm, indicating that Pt... 2+ -CDs carry PpIX into the cell nucleus, achieving nuclear targeting; compared to free PpIX, Pt 2+ -CDs@PpIX exhibit green fluorescence, indicating that Pt 2+ -CDs can carry PpIX into the cell nucleus. Furthermore, compared with the staining results of SMMC-7721 hepatocellular carcinoma cells using only the commercially available nuclear dye DAPI, it can be seen that Pt... 2+ The red fluorescence of -CDs@PpIX coincides with the blue fluorescence of DAPI, which further illustrates the significance of Pt. 2+ -CDs@PpIX exhibit nuclear targeting functionality. Compared to the PpIX red fluorescence of CDs@PpIX, Pt... 2+ The red fluorescence intensity of -CDs@PpIX was significantly weaker in the cytoplasm, indicating that Pt 2+ The modification makes Pt 2+ The CDs@PpIX drug delivery system exhibits sustained-release capability. These results demonstrate that, compared to free PpIX, CDs@PpIX and Pt... 2+ -CDs@PpIX exhibit significantly increased cellular uptake and nuclear delivery capabilities, demonstrating excellent nuclear targeting capabilities.

[0075] Using Pt with different mass ratios 2+ -CDs and PpIX are linked to optimize drug loading to obtain the optimal drug loading system, such as Figure 5 As shown in (b), the loading rate of PpIX reached its maximum at a mass ratio of 1:2, with a drug loading rate of 88.4%. Further investigation was conducted on Pt... 2+ -In vitro drug release from the CDs@PpIX drug delivery system, such as Figure 5As shown in (c), it can be seen that at pH = 5.0 and pH = 6.2, CDs@PpIX and Pt 2+ The cumulative release of PpIX in the CDs@PpIX drug delivery system was significantly higher than that at pH 7.4, with the highest cumulative release at pH 5.0. This indicates that both drug delivery systems can achieve pH-responsive drug release, and that acidic conditions facilitate the release of PpIX. 2+ PpIX release in -CDs@PpIX.

[0076] 6. Biocompatibility

[0077] Pt was tested using the CCK-8 experiment. 2+ The biocompatibility of the CDs@PpIX drug delivery system was investigated, and the efficacy of in vitro sonodynamic therapy was evaluated by incubating SMMC-7721 hepatocellular carcinoma cells. The Pt drug delivery system prepared in Example 1 was selected as the target drug. 2+ Experiments were conducted using the -CDs@PpIX drug delivery system. Figure 6 (a) It can be seen that, without US, even if Pt 2+ When the concentration of -CDs@PpIX reached 100 μg / mL, the survival rate of SMMC-7721 liver cancer cells was higher than 85%, indicating that Pt 2+ -CDs@PpIX exhibits very low cytotoxicity and good biocompatibility. For example... Figure 6 As shown in (b), when using US(1.5W / cm) 2 After 10 minutes of action, Pt 2+ - The survival rate of SMMC-7721 hepatocellular carcinoma cells incubated with CDs@PpIX increased with Pt 2+ -CDs@PpIX concentration decreased rapidly with increasing concentration, indicating that Pt 2+ The CDs@PpIX sonodynamic drug delivery system has good sonodynamic therapeutic effects.

[0078] Specifically, with Pt 2+ -CDs@PpIX, CDs@PpIX, Pt 2+ A comparative experiment was conducted using CDs and PpIX, with group 1 being the control group (SMMC-7721 liver cancer cells only), group 2 being US only, and group 3 being Pt. 2+ -CDs@PpIX, Group 4 is Pt 2+ -CDs+US, Group 5 is PpIX+US, Group 6 is CDs@PpIX+US, Group 7 is Pt 2+ -CDs@PpIX+US; as shown Figure 6 As shown in (c), without ultrasound treatment, only Pt in group 3... 2+-CDs@PpIX had a relatively small impact on cell viability. In group 4, sonication was used. 2+ -CDs had almost no effect on cell survival; under the same US conditions, after 2 hours of incubation of SMMC-7721 hepatocellular carcinoma cells, PpIX, CDs@PpIX, and Pt... 2+ The cell viability rates of SMMC-7721 cells treated with CDs@PpIX were 66%, 28%, and 10%, respectively. These results further indicate that Pt... 2+ The CDs@PpIX sonodynamic drug delivery system has good sonodynamic therapeutic effects.

[0079] Pt was detected using a live-dead co-staining assay. 2+ -CDs@PpIX activity levels in US-radiated cells. SMMC-7721 hepatocellular carcinoma cells treated in groups 1-7 were stained with a Calcein-AM / PI kit to observe cell death; live cells showed green fluorescence, and dead cells showed red fluorescence. Figure 6 As shown in (d), the green fluorescence area in groups 5-7 is significantly smaller than that in groups 1-4, demonstrating that ultrasound helps the drug delivery system exert its therapeutic effect and promotes cell death. SMMC-7721 hepatocellular carcinoma cells treated with groups 1-7 were co-stained with DAPI and 2',7'-dichlorofluorescein diacetic acid (DCFH-DA) to observe the generation of reactive oxygen species within the cells. Figure 6 As shown in (e), it can be seen that the control group 1 with only SMMC-7721 liver cancer cells, the group 2 with only US cells, and the Pt group... 2+ No obvious ROS fluorescence signal was observed in group 3 of -CDs@PpIX, while in Pt 2+ In group 7 of -CDs@PpIX+US, bright ROS fluorescence signals were observed, demonstrating that Pt under ultrasound conditions... 2+ The -CDs@PpIX drug delivery system helps generate intracellular ROS.

[0080] After processing the 3D-printed in vitro liver cancer tumor model using methods 1-7, co-staining of live and dead cells was performed, such as... Figure 6 As shown in (f), the red fluorescence area is the largest in group 7, proving that Pt 2+ -CDs@PpIX+US still exhibited significant cell-killing activity in a 3D-printed in vitro liver cancer tumor model, and Pt 2+ -CDs@PpIX+US group 7 showed obvious ROS fluorescence signals, and the fluorescence signal intensity was higher than that of other groups.

[0081] 7. In vivo imaging experiments

[0082] Pt labeled with the near-infrared fluorescent dye indocyanine green (ICG) 2+ The -CDs@PpIX drug delivery system was intravenously injected into mice with SMMC-7721 tumors for in vivo imaging experiments. Figure 7 As shown, Pt 2+ -CDs@PpIX effectively accumulated in tumor tissue, demonstrating excellent tumor targeting ability. The strongest fluorescence signal was detected at the tumor site 24 hours after injection; and the fluorescence signal was still observed at the tumor site after 264 hours, indicating that Pt... 2+ The -CDs@PpIX drug delivery system can achieve long-term retention at the tumor site.

[0083] 8. Internal sonodynamic therapy

[0084] Pt was detected by xenografting SMMC-7721 tumors into a mouse model. 2+ -In vivo antitumor effects of CDs@PpIX. Tumor-bearing mice were randomly divided into 7 groups of 3 mice each. Tumors were allowed to grow until the tumor volume reached 80-100 mm. 3 Different treatments were then administered. Specifically, group 1 was the control group, group 2 received +US, and group 3 received Pt. 2+ -CDs@PpIX, Group 4:Pt 2+ -CDs+US, Group 5:PpIX+US, Group 6:CDs@PpIX+US, Group 7:Pt 2+ -CDs@PpIX+US.

[0085] Figure 8 (a) Total images of the seven groups of mice after 21 days of treatment. Macroscopic observation showed that the tumor volume of mice in groups 5-7 was smaller than that in groups 1-4. Images of the tumor tissue and measurements of its weight and volume are shown below. Figure 8 As shown in (b), (c), and (d), it can be seen that the tumors in the control group grew faster, showed no inhibition trend, and had larger tumor volumes; groups 5, 6, and 7, namely PpIX+US, CDs@PpIX+US, and Pt... 2+ The -CDs@PpIX+US group showed some inhibitory effect on tumor growth and smaller tumor volume; among them, Pt 2+ The -CDs@PpIX+US group had the smallest tumor volume and the most significant inhibitory effect on tumor growth.

[0086] In addition, Pt was detected by monitoring changes in mouse body weight every two days during the treatment period. 2+ -Systemic toxicity of CDs@PpIX in mice. For example... Figure 8 As shown in (e), compared with the control group, no significant weight fluctuations were observed in the other six groups. In conclusion, Pt 2+-CDs@PpIX showed good sonodynamic therapeutic effects in mice and exhibited good biocompatibility.

Claims

1. A type of Pt 2+ - A carbon dot@protoporphyrin sonodynamic drug delivery system, characterized in that... Carbon dots as core, Pt loaded on the surface of carbon dots 2+ Modification layer, Pt loaded outside the modification layer 2+ Modification layer, protoporphyrin loaded outside the modification layer 2. The Pt according to claim 1 2+ - A carbon dot@protoporphyrin sonodynamic drug delivery system, characterized in that... The Pt 2+ Carbon dots@protoporphyrin with a particle size of 2.78 ± 0.88 nm.

3. The Pt according to claim 1 2+ - A carbon dot@protoporphyrin sonodynamic drug delivery system, characterized in that... It is prepared through the following steps: Pt loaded on carbon dot surface 2+ Modification layer, to obtain Pt 2+ - Carbon dots; Pt 2+ Carbon dots were added to a protoporphyrin solution and stirred in the dark to obtain Pt. 2+ - Carbon dot@protoporphyrin sonodynamic drug delivery system.

4. The Pt according to claim 3 2+ - A carbon dot@protoporphyrin sonodynamic drug delivery system, characterized in that... The obtained Pt 2 + - Carbon dots are obtained by adding K2PtCl6 solution to carbon dot solution, reacting, centrifuging and filtering.

5. The Pt according to claim 3 2+ - A carbon dot@protoporphyrin sonodynamic drug delivery system, characterized in that... The Pt 2+ The molar ratio of carbon to carbon is 0.2-1.8:

1.

6. The Pt according to claim 3 2+ - A carbon dot@protoporphyrin sonodynamic drug delivery system, characterized in that... The Pt 2+ - The mass ratio of carbon dots to protoporphyrin is 0.5-3:

1.

7. The Pt according to claim 3 2+ - A carbon dot@protoporphyrin sonodynamic drug delivery system, characterized in that... The protoporphyrin solution is obtained by dissolving protoporphyrin, N,N'-dicyclohexylcarbodiimide, and 1-hydroxybenztriazole in dimethyl sulfoxide.

8. The Pt according to claim 3 2+ - A carbon dot@protoporphyrin sonodynamic drug delivery system, characterized in that... The stirring time is 8-15 hours, and the temperature is 23-27℃.

9. A Pt according to any one of claims 1-8 2+ - Application of carbon dot@protoporphyrin sonodynamic drug delivery system in the preparation of antitumor drugs.

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