ZnO2 / ICG@ZIF-iRGD nanoparticles, their preparation methods, and applications

Abstract

This invention provides ZnO2 / ICG@ZIF-iRGD nanoparticles, their preparation method, and applications. The method for preparing the nanoparticles includes: S1, preparing matrix nanoparticles ZnO2 / ICG@ZIF loaded with ZnO2 and ICG; S2, preparing aminated matrix nanoparticles ZnO2 / ICG@ZIF and N-hydroxysuccinimide-modified iRGD; S3, mixing the modified iRGD with the aminated matrix nanoparticles ZnO2 / ICG@ZIF and reacting them to obtain ZnO2 / ICG@ZIF-iRGD nanoparticles. The nanoparticles provided by this invention possess suitable size, good structural stability, pH responsiveness, high drug loading capacity, and encapsulation efficiency, effectively inhibiting tumor growth and providing a powerful supplement to existing treatment methods for colorectal cancer peritoneal metastasis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a nanoparticle ZnO2 / ICG@ZIF-iRGD, its preparation method, and its application. Background Technology

[0002] Colorectal cancer (CRC) is one of the most common malignant tumors of the digestive system, with approximately 2 million new cases and 1 million deaths worldwide each year. The prognosis of CRC patients is closely related to the disease stage; the 5-year survival rate for early-stage patients can reach 91%, while the 5-year survival rate for locally advanced patients drops to 73%. Approximately 20% of CRC patients have distant metastases at the time of diagnosis, and the 5-year survival rate for these patients is only 14%.

[0003] Peritoneal metastasis of colorectal cancer (CRC) refers to secondary metastatic lesions formed on the peritoneal surface by tumor cells from the primary CRC lesion via hematogenous, lymphatic, or implantation pathways. Peritoneal metastasis is the third most common site of distant metastasis in CRC, after liver and lung metastasis, accounting for approximately 17% of metastatic CRC patients. The presence of peritoneal metastasis in colorectal cancer usually indicates advanced cancer progression, and the survival rate is significantly lower than that of patients with other types of distant metastasis; the median survival is typically only 6 to 9 months.

[0004] Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is currently the main treatment for peritoneal metastases of colorectal cancer. This combined treatment strategy first involves CRS surgery to remove the macroscopically visible primary or metastatic lesions, followed by HIPEC to further eliminate peritoneal micrometastases and free cancer cells, thereby controlling peritoneal metastasis progression and improving patient prognosis. Tumor burden assessment is a key factor in determining the feasibility and benefit of CRS surgery. Studies have shown that patients with lower tumor burdens are more likely to achieve complete resection, resulting in longer disease-free survival and overall survival. However, current imaging techniques have limited ability to detect small (<5mm) or diffusely disseminated peritoneal metastases, easily leading to missed diagnoses of some lesions preoperatively. Limitations in imaging examinations may result in some patients with actual high tumor burdens being incorrectly included in the indications for CRS surgery, increasing the risk of surgical failure and postoperative recurrence. Furthermore, HIPEC currently faces challenges such as uneven drug distribution, significant side effects, and short drug retention time. Therefore, exploring new diagnostic and treatment pathways is crucial for improving the accurate assessment of tumor burden and treatment outcomes in patients with peritoneal metastases from colorectal cancer. Summary of the Invention

[0005] This invention provides a ZnO2 / ICG@ZIF-iRGD nanoparticle, its preparation method, and its application.

[0006] In a first aspect, the present invention provides a method for preparing nanoparticles, comprising: S1. Preparation of matrix nanoparticles ZnO2 / ICG@ZIF loaded with ZnO2 and ICG: A first solution containing zinc salt was mixed with a second solution containing ZnO2, indocyanine green and 2-methylimidazole and reacted. After the reaction was completed, the matrix nanoparticles ZnO2 / ICG@ZIF were collected. S2. The matrix nanoparticles ZnO2 / ICG@ZIF are aminated to obtain aminated matrix nanoparticles ZnO2 / ICG@ZIF; iRGD, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are mixed and coupled to prepare modified iRGD. S3. The modified iRGD is mixed with the aminated matrix nanoparticles ZnO2 / ICG@ZIF and subjected to an amide reaction to obtain iRGD-loaded nanoparticles ZnO2 / ICG@ZIF-iRGD.

[0007] The ZnO2 / ICG@ZIF-iRGD nanoparticles provided in this invention utilize ZIF-8 as a nanocarrier, encapsulating ZnO2 and ICG as nanomedicines, with the outer surface modified using iRGD. iRGD can be recognized by αvβ3 / 5 integrin expressed on the surface of colorectal cancer peritoneal metastatic tumor cells, thereby increasing the tumor targeting of the nanoparticles, improving the aggregation efficiency of nano-ions in tumor tissues, and reducing damage to normal tissues. The ZIF-8 nanocarrier is pH sensitive and readily hydrolyzes to form Zn in the weakly acidic tumor microenvironment. 2+ The nanoparticles ZnO2 and ICG are released simultaneously with imidazole ions. ZnO2 is also sensitive to acidic pH and can further decompose to form H2O2 and Zn. 2+ Zn produced by the hydrolysis of ZIF-8 and ZnO2 2+ Both induce intracellular zinc ion overload, which, together with H2O2, further increases ROS, inducing apoptosis and immunogenic cell death. ICG, under NIR irradiation, can perform photodynamic therapy (PDT) and photothermal therapy (PTT) on the tumor site. At the same time, H2O2 can effectively generate O2 under the action of catalase, thereby improving the hypoxic state of the tumor site and enhancing the efficacy of PDT.

[0008] In the preparation method described above, the mass ratio of zinc salt, ZnO2, indocyanine green, and 2-methylimidazole is 150:20:5:2050.

[0009] The preparation method described above involves amination of the matrix nanoparticles ZnO2 / ICG@ZIF to obtain amination-treated matrix nanoparticles ZnO2 / ICG@ZIF, specifically including: The matrix nanoparticles ZnO2 / ICG@ZIF were dispersed in a mixed solution of ethanol and water, and 3-aminopropyltriethoxysilane was added to carry out an amination reaction. After the reaction was completed, the amination matrix nanoparticles ZnO2 / ICG@ZIF were obtained.

[0010] As described above, the volume of 3-aminopropyltriethoxysilane is 10 μL per 1 mg of the matrix nanoparticles ZnO2 / ICG@ZIF.

[0011] In the preparation method described above, the mass ratio of the modified iRGD to the aminated matrix nanoparticles ZnO2 / ICG@ZIF is 1:5.

[0012] In a second aspect, the present invention provides nanoparticles ZnO2 / ICG@ZIF-iRGD prepared according to any of the preparation methods described above.

[0013] As described above, the ZnO2&ICG@ZIF-iRGD nanoparticles have a particle size of approximately 188±12.34 nm, a Zeta potential of 17.7±1.30, and an aggregation index of 0.19±0.06. Scanning electron microscopy images show that ZnO2&ICG@ZIF-iRGD has a similar regular polyhedral shape to ZIF-8, and XRD patterns also indicate that ZnO2&ICG@ZIF-iRGD does not alter the typical crystal structure of ZIF-8. Energy dispersive spectroscopy analysis shows that the surface of ZnO2&ICG@ZIF-iRGD contains oxygen, carbon, nitrogen, and zinc elements. Ultraviolet absorption spectroscopy and Fourier transform infrared spectroscopy confirm that the iRGD peptide is modified on the surface of ZnO2&ICG@ZIF-iRGD, and ICG has been successfully encapsulated.

[0014] The nanoparticles described above have the following drug loading and encapsulation efficiency: ZnO2 in ZnO2&ICG@ZIF-iRGD is 47±3.06, and ICG in ZIF-iRGD is 3.5±0.31; the drug loading and encapsulation efficiency are 53±2.08 and 4.4±0.31, respectively. ZnO2&ICG@ZIF-iRGD exhibits good stability in different solutions. In vitro drug release experiments show that the drug release rate of ZnO2&ICG@ZIF-iRGD is accelerated in a weakly acidic environment at pH 6.5.

[0015] Thirdly, the present invention provides a composition comprising the above-mentioned nanoparticles ZnO2 / ICG@ZIF-iRGD.

[0016] According to the above composition, the composition further includes a hydrogel. Further, the hydrogel is a thermosensitive hydrogel. Even further, the hydrogel is a PLEL hydrogel, which helps to increase the residence time of ZnO2 / ICG@ZIF-iRGD nanoparticles in the peritoneal cavity. The thermosensitivity of the PLEL hydrogel makes it more suitable for applications combining nanoparticles with HIPEC.

[0017] Fourthly, the present invention provides the use of the above-mentioned nanoparticles ZnO2 / ICG@ZIF-iRGD or the above-mentioned composition in the preparation of antitumor drugs.

[0018] In the application described above, the tumor is a peritoneal metastasis of colorectal cancer.

[0019] This invention provides a ZnO2&ICG@ZIF-iRGD nanoparticle, which possesses suitable size and excellent structural stability, pH responsiveness, high drug loading capacity, and encapsulation efficiency. Furthermore, it exhibits good tumor targeting in both in vitro and in vivo experiments. Intraperitoneal injection of ZnO2&ICG@ZIF-iRGD combined with fluorescence imaging can be used to locate tumors, providing a powerful supplement to existing diagnostic methods for colorectal cancer peritoneal metastasis. This nanoparticle effectively inhibits tumor growth by inducing apoptosis and immunogenic cell death. Further combination with other therapeutic strategies and intraperitoneal hyperthermic perfusion can produce a synergistic effect, significantly improving the treatment efficacy and prognosis in tumor-bearing mice with colorectal cancer peritoneal metastasis. Simultaneously, the ZnO2&ICG@ZIF-iRGD nanoparticle exhibits high biosafety, with no significant impact on normal human colonic epithelial cells or on liver, kidney function, and solid organs in tumor-bearing mice.

[0020] In vitro experiments showed that ZnO2&ICG@ZIF-iRGD effectively induced zinc ion overload in tumor cells, significantly increased intracellular reactive oxygen species (ROS) levels, and induced a decrease in mitochondrial membrane potential through ROS-mediated oxidative stress damage. Furthermore, ZnO2&ICG@ZIF-iRGD exhibited strong in vivo and in vitro photothermal efficacy under near-infrared light irradiation. Transcriptome sequencing analysis indicated that this nanoparticle drug delivery system primarily exerts its antitumor efficacy by regulating tumor cell apoptosis and immunogenic death. Flow cytometry analysis further confirmed that ZnO2&ICG@ZIF-iRGD effectively induced apoptosis in HCT-116 cells, and this effect was significantly enhanced under near-infrared light irradiation. In addition, this nanoparticle drug delivery system can also activate immunogenic death by promoting CAT+ eversion, inducing the release of high-mobility group box 1 (HMGB1), and increasing the proportion of activated dendritic cells. The ZnO2&ICG@ZIF-iRGD nanosystem exhibits a multi-modal synergistic mechanism that enables highly efficient anti-tumor effects under near-infrared light irradiation, providing a new strategy for the precision treatment of peritoneal metastases from colorectal cancer. Attached Figure Description

[0021] Figure 1 The image shows the morphology of ZnO2 & ICG@ZIF-iRGD nanoparticles as observed by transmission electron microscopy. Figure 2 The morphology of ZnO2&ICG@ZIF-iRGD nanoparticles observed by scanning electron microscopy; Figure 3 Crystal structure identification diagrams for ZIF-8 and ZnO2&ICG@ZIF-iRGD powders; Figure 4 The elemental composition of the surface of ZnO2 & ICG@ZIF-iRGD nanoparticles; Figure 5 Identification of the composition of ZnO2 & ICG@ZIF-iRGD nanoparticles; Figure 6 Identification of the composition of ZnO2 & ICG@ZIF-iRGD nanoparticles; Figure 7 The variation in hydrated particle size of ZnO2 & ICG@ZIF-iRGD nanoparticles; Figure 8 The pH sensitivity test results for ZnO2 & ICG@ZIF-iRGD nanoparticles are shown. Figure 9 The results of the temperature sensitivity test for PLEL hydrogel; Figure 10 The results of the temperature sensitivity test for PLEL hydrogel; Figure 11The appearance and morphology of ZnO2&ICG@ZIF-iRGD-PLEL are characterized; where A is a 500x magnification image and B is a 1000x magnification image. Figure 12 Cellular uptake of ZnO2&ICG@ZIF-iRGD, ZnO2&ICG / @ZIF, and ICG by HCT-116 cells; Figure 13 Cellular uptake assay of ZnO2 & ICG@ZIF-iRGD in NCM460 cells; Figure 14 Results of in vivo tumor targeting assay using ZnO2 & ICG@ZIF-iRGD-PLEL; Figure 15 Results of in vivo tumor retention effect assay for ZnO2 & ICG@ZIF-iRGD; Figure 16 Results of in vivo fluorescence intensity measurement of ZnO2 & ICG@ZIF-iRGD; Figure 17 Effects of ZnO2 & ICG@ZIF-iRGD on the viability of HCT-116 cells; Figure 18 Effects of ZnO2 & ICG@ZIF-iRGD on HCT-116 cell viability; Figure 19 Dynamic bioluminescence imaging range for different groups of tumor-bearing mice; Figure 20 The bioluminescence signal intensity changes of tumor-bearing mice in different groups; Figure 21 The curves showing the changes in PCI scores in mice of different groups; Figure 22 Photographs showing the distribution of abdominal tumors in mice from different treatment groups after dissection; Figure 23 Photographs showing the distribution of abdominal tumors in mice from different treatment groups after dissection; Figure 24 The curves showing the changes in body weight of tumor-bearing mice in different groups; Figure 25 Survival curves for mice bearing different tumors; Figure 26 Effects of ZnO2 & ICG@ZIF-iRGD on cell viability of NCM460 cells; Figure 27 Liver and kidney function were measured in mice in different groups. Figure 28 The effects of different groups on the major organs of mice. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0024] Example 1: Preparation and characterization of ZnO2 / ICG@ZIF-iRGD nanoparticles 1. Preparation of ZnO2 nanoparticles: 100 mg of zinc acetate and 100 mg of polyvinylpyrrolidone were dissolved in 5 mL of water. Hydrogen peroxide was rapidly added under ultrasonic conditions and the reaction was carried out for 24 hours. ZnO2 nanoparticles were collected by centrifugation (8000 rpm, 10 min).

[0025] 2. Preparation of ZnO2 / ICG@ZIF nanoparticles: A 0.5 mol / L zinc nitrate solution (solvent: methanol, zinc nitrate mass: 150 mg) was slowly added to a 2.5 mol / L 2-methylimidazole (CAS No. 693-98-1) solution containing 20 mg ZnO2 nanoparticles and 5 mg indocyanine green (ICG). After stirring for 30 min, the product was collected by centrifugation and washed three times with water to remove unreacted substances, thus obtaining ZnO2 / ICG@ZIF nanoparticles.

[0026] 3. Preparation of ZnO2 / ICG@ZIF-iRGD nanoparticles: 50 mg of ZnO2 / ICG@ZIF nanoparticles were dispersed in an ethanol-water mixture, and 500 μL of 3-aminopropyltriethoxysilane was added and stirred for 1 h to obtain aminated ZnO2 / ICG@ZIF-8. Internalizing RGD (iRGD, amino acid sequence CRGDK / RGPD / EC, purchased from Xi'an Ruixi) was dissolved in water, and 20 mg of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (CAS No. 1892-57-5, purchased from Shanghai Covalent Chemical Technology Co., Ltd.) and 20 mg of N-hydroxysuccinimide (CAS No. 6066-82-6, purchased from Merck) were added and reacted for 3 h. This mixture was then combined with the aminated ZnO2 / ICG@ZIF-8 and reacted overnight. The precipitate was collected by centrifugation (13000 rpm, 10 min), washed with water, and redispersed to obtain the final product ZnO2 / ICG@ZIF-iRGD nanoparticles.

[0027] Comparative Example 1: Preparation of Zeolite imidazole framework-8 (ZIF-8) nanoparticles Zinc nitrate hexahydrate was dissolved in 5 mL of ultrapure water to prepare a zinc nitrate solution; 2-methylimidazole was dissolved in 10 mL of anhydrous methanol to prepare a 2-methylimidazole solution; the zinc nitrate solution and the 2-methylimidazole solution were sonicated for 10 min each. The zinc nitrate solution was rapidly added to the 2-methylimidazole solution under magnetic stirring, and the mixture was stirred for 15 min to form a milky white suspension. The product was collected by centrifugation (10000 rpm, 15 min), washed three times with methanol, and dried under vacuum at 50 °C for 24 h to obtain ZIF-8 nanoparticles.

[0028] Comparative Example 2: Preparation of ZnO2@ZIF Nanoparticles 1. Preparation of ZnO2 nanoparticles: 100 mg of zinc acetate and 100 mg of polyvinylpyrrolidone were dissolved in 5 mL of water. Hydrogen peroxide was rapidly added under ultrasonic conditions and the reaction was carried out for 24 hours. ZnO2 nanoparticles were collected by centrifugation (8000 rpm, 10 min).

[0029] 2. Preparation of ZnO2 / ICG@ZIF nanoparticles: A 0.5 mol / L zinc nitrate solution (solvent: methanol, zinc nitrate mass: 150 mg) was slowly added to a 2.5 mol / L 2-methylimidazole (CAS No. 693-98-1) solution containing 20 mg ZnO2 nanoparticles. After stirring for 30 min, the product was collected by centrifugation and washed three times with water to remove unreacted substances, thus obtaining ZnO2@ZIF nanoparticles.

[0030] (II) Characterization and detection of ZnO2 & ICG@ZIF-iRGD nano-drug delivery system 1. Particle size, aggregation index, and zeta potential of ZnO2 & ICG@ZIF-iRGD The particle size of nanoparticles in drug delivery systems significantly impacts their biodistribution, tumor penetration, and clearance of plasma and interstitial spaces. Currently, most approved anticancer nanoparticles range in size from 100 to 200 nm. Aggregation index is an important parameter for measuring the uniformity of particle size distribution in nanomaterials. It reflects the proportional distribution of particles of different sizes within the material by comparing the degree of variation in nanoparticle size. The aggregation index typically ranges from 0 to 1, with smaller values ​​indicating more uniform particle size distribution. A suitable sample was dispersed in water and diluted to an appropriate concentration. The sample was then sonicated in cold water for 15 min. The particle size and distribution of the sample were measured using a Malvern particle size analyzer. The sample was then transferred to a potentiometer to determine the Zeta potential. The test results are shown in Table 1. According to Table 1, the average hydrated particle size of ZnO2&ICG@ZIF-iRGD was 188±12.34 nm, the aggregation index was 0.19±0.06, and the Zeta potential was 20.78±0.87. This indicates that ZnO2&ICG@ZIF-iRGD has suitable and consistent dimensions as an antitumor nanodrug delivery system.

[0031] Table 1. Particle size, potential, and aggregation index of ZnO2 & ICG@ZIF-iRGD

[0032] In addition, the particle size, potential, and aggregation index of the synthesized ZIF-8 and ZnO2&ICG@ZIF were tested in this embodiment, and the test results are shown in Table 2-3. According to Table 2-3, the particle size of ZIF-8 is only 118±3.5 nm. After encapsulation with ZnO2 and ICG, the particle size of ZnO2&ICG@ZIF increases to 141±19.67 nm; after further modification with iRGD, the particle size of ZnO2&ICG@ZIF-iRGD increases to 188±12.34 nm. The Zeta potential of ZnO2&ICG@ZIF is 17.7±1.30. Since iRGD itself carries a positive charge, after modification with iRGD, the Zeta potential of ZnO2&ICG@ZIF-iRGD increases to 20.78±0.87.

[0033] Table 2. Particle size, potential, and aggregation index of ZnO2 & ICG@ZIF

[0034] Table 3. Particle size, potential, and aggregation index of ZIF-8

[0035] 2. Morphological characterization of ZnO2 & ICG@ZIF-iRGD 2.1 The morphology of ZnO2&ICG@ZIF-iRGD was observed using transmission electron microscopy. Specifically, the diluted ZnO2&ICG@ZIF-8-iRGD solution was dropped onto a copper grid using a pipette, stained negatively with 2% phosphatidylcholine, and dried. The morphology and size of the ZnO2&ICG@ZIF-8-iRGD were then observed using a transmission electron microscope. The observation results are as follows: Figure 1 As shown, ZnO2&ICG@ZIF-iRGD exhibits a regular polyhedral shape similar to the typical morphology of ZIF-8, indicating that the modification of iRGD and the successful encapsulation of ZnO2 and ICG did not alter the original structure of ZIF-8. The particle size of ZnO2&ICG@ZIF-iRGD can be estimated to be approximately 100 nm using the scale bar of the transmission electron microscope image. This particle size is smaller than the hydrated particle size measured by the Malvern particle size analyzer (188 ± 12.34), because the calculation of the hydrated particle size of ZnO2&ICG@ZIF-iRGD includes the diameter of the surface hydration layer.

[0036] 2.2. ZnO2 & ICG@ZIF-8-iRGD were dispersed in ethanol, and 5 μl was dropped onto a silicon wafer. After vacuum drying and 60 s gold sputtering, the mixture was observed using a scanning electron microscope. The observation results are as follows: Figure 2As shown, the particle size of ZnO2&ICG@ZIF-iRGD is also around 100nm, with relatively uniform particle size and regular polyhedral structure, and no obvious agglomeration between nanoparticles.

[0037] 3. Determination of the crystal structure of ZnO2 & ICG@ZIF-iRGD Two powder samples, ZIF-8 and ZnO2&ICG@ZIF-iRGD, were placed in the sample cassette of the X-ray diffractometer and pressed firmly. The 2θ range was measured from 5 to 50°. The X-ray diffraction patterns of ZIF-8 and ZnO2&ICG@ZIF-iRGD are shown below. Figure 3 As shown, the curves of the two are basically in agreement, indicating that ZnO2 & ICG@ZIF-iRGD did not change the original structure of ZIF-8, and both have good crystallinity.

[0038] 4. Identification of the composition of ZnO2 & ICG@ZIF-iRGD First, the elemental composition of the ZnO2 & ICG@ZIF-iRGD surface was analyzed using energy dispersive X-ray spectroscopy (EDS). The results are as follows: Figure 4 As shown, the elemental composition of ZnO2&ICG@ZIF-iRGD includes oxygen (O), nitrogen (N), zinc (Zn), and carbon (C). N and C elements mainly originate from the imidazole ligand of ZIF-8, the amino acid components of iRGD, and ICG; Zn elements mainly originate from ZIF-8 and ZnO2; O elements are present but less abundant, originating from ZnO2.

[0039] The composition of ZnO2&ICG@ZIF-8-iRGD was then determined using a UV spectrophotometer and Fourier transform infrared chromatograph. ZnO2&ICG@ZIF-iRGD, ZnO2, ZIF-8, and iRGD were dispersed in methanol solutions, and their absorption curves in the 200-1000 nm wavelength range were measured using a UV spectrophotometer. The results are as follows: Figure 5As shown, ZIF-8 (blue curve), ZnO2&ICG@ZIF-8 (green curve), and ZnO2&ICG@ZIF-8-iRGD (dark blue curve) all exhibit a UV absorption peak at 230 nm, indicating that ZnO2&ICG@ZIF-8 and ZnO2&ICG@ZIF-8-iRGD both contain the ZIF-8 structure. The ICG (black curve) shows two UV absorption peaks, located near 210 nm and 780 nm, respectively. Similarly, ZnO2&ICG@ZIF-8 and ZnO2&ICG@ZIF-8-iRGD show ICG absorption peaks shifted to the right at 810 nm and 820 nm due to conjugation, indicating that ICG has been successfully encapsulated by ZnO2&ICG@ZIF-8 and ZnO2&ICG@ZIF-8-iRGD. No obvious UV absorption peaks were observed for ZnO2 (pink curve) and iRGD (red curve).

[0040] Fourier transform infrared chromatograph (FTIR) was used to characterize the nanoparticles using infrared spectroscopy. 1-2 mg of dried ICG, iRGD, ZnO2&ICG@ZIF-8, and ZnO2&ICG@ZIF-8-iRGD powders were weighed and ground uniformly with 200 mg of potassium bromide, then compressed into tablets. The spectra were scanned and recorded within a specific wavelength range. The results are as follows: Figure 6 As shown, it can be seen that ZnO2&ICG@ZIF-8 (blue curve), ZnO2&ICG@ZIF-8-iRGD (green curve), and ICG at approximately 1400 cm⁻¹ - The presence of the same absorption peak at ¹ indicates that the peak is formed by the deformation vibration of the CH bond in the indocyanine green methyl group, proving that ICG has been successfully encapsulated by ZnO2&ICG@ZIF-8-iRGD. ZnO2&ICG@ZIF-8-iRGD (green curve) and iRGD (red curve) show a peak at 1700 cm⁻¹. - The presence of the same absorption peak at position ¹ indicates that the iRGD in ZnO2&ICG@ZIF-8-iRGD was successfully modified by the stretching vibration of the C=O peptide bond in the peptide chain of iRGD.

[0041] 5. Determination of drug loading and encapsulation efficiency of ZnO2 & ICG@ZIF-iRGD Calculation of ZnO2 drug loading and encapsulation efficiency: ZnO2 was dissolved in hydrochloric acid to obtain ZnO2 solutions with concentrations of 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, 7.8125 μg / mL, 3.90625 μg / mL, and 0 μg / mL, respectively. The zinc ion concentration in these solutions was then determined using inductively coupled plasma-atomic emission spectrometry (ICP-AES), and a standard curve was plotted with ZnO2 concentration on the x-axis and zinc ion concentration on the y-axis. Equal masses of ZIF-8 and ZnO2&ICG@ZIF-8-iRGD samples were dissolved in 50 μL of hydrochloric acid, and the zinc ion concentration in the solutions was determined using ICP-AES. The difference in zinc ion concentration between the latter and the former represents the concentration of zinc ions released from the dissolution of ZnO2 in ZnO2&ICG@ZIF-8-iRGD. Substituting these results into the ZnO2 standard curve, the mass of ZnO2 in ZnO2&ICG@ZIF-8-iRGD is calculated. The drug loading and encapsulation efficiency are calculated using the following formulas: Drug Loading Efficiency (DLC%) = Mass of ZnO2 in ZnO2&ICG@ZIF-iRGD / Mass of ZnO2&ICG@ZIF-iRGD x 100%; Encapsulation Efficiency (EE%) = Mass of ZnO2 in ZnO2&ICG@ZIF-iRGD / Mass of ICG input x 100%.

[0042] Calculation of ICG loading and encapsulation efficiency: Weigh 100 mg of ICG into a 10 mL volumetric flask and dilute to volume with deionized water to obtain a 10 mg / mL ICG stock solution. Dilute the stock solution with deionized water to obtain solutions of ICG with concentrations of 62.5 μg / mL, 31.25 μg / mL, 15.625 μg / mL, 7.8125 μg / mL, 3.90625 μg / mL, and 0 μg / mL. Measure the absorbance of the solutions at 778 nm using a UV-Vis spectrophotometer and plot a standard curve with concentration on the x-axis and absorbance on the y-axis. Treat vacuum-dried ZnO2&ICG@ZIF-8-iRGD with 50 μL of hydrochloric acid and dilute with deionized water. Measure the absorbance of ZnO2&ICG@ZIF-iRGD using a UV-Vis spectrophotometer and substitute it into the standard curve for calculation. The drug loading and encapsulation efficiency of ICG are calculated using the following formulas: Drug Loading Efficiency (DLC%) = ICG mass / ZnO2 & ICG@ZIF-iRGD mass x 100%; Encapsulation Efficiency (EE%) = ICG mass / ICG input mass x 100%.

[0043] As shown in Table 4-5, the calculated drug loading and encapsulation efficiency of ZnO2 in ZnO2&ICG@ZIF-iRGD were 47±3.06 and 3.5±0.31, respectively, while those of ICG were 53±2.08 and 4.4±0.31. This suggests that ZIF-8, as a nanocarrier, has high drug loading and encapsulation efficiency, mainly due to its relatively regular pore structure, high pore volume, and large specific surface area.

[0044] Table 4. ZnO2 loading and encapsulation efficiency in ZnO2 & ICG@ZIF-iRGD

[0045] Table 5. Drug loading and encapsulation efficiency of ICG in ZnO2 & ICG@ZIF-iRGD

[0046] 6. Stability assessment of ZnO2 & ICG@ZIF-iRGD The prepared ZnO2&ICG@ZIF-iRGD was dispersed in PBS solution and cell basal medium (DMEM, RPMI 1640) and incubated in a shaker at 37°C. The hydrated particle size of ZnO2&ICG@ZIF-iRGD was measured at the same time every day for 7 days. The results are as follows: Figure 7As shown, the hydrated particle size of ZnO2&ICG@ZIF-iRGD in PBS did not change significantly. In DMEM and RPMI 1640 solutions, the particle size initially increased slightly, but there was no statistically significant difference compared to the hydrated particle size in PBS. These results indicate that ZnO2&ICG@ZIF-iRGD is stable in these solutions.

[0047] 7. pH sensitivity assessment of ZnO2 & ICG@ZIF-iRGD 10 mg of ZnO2 & ICG@ZIF-iRGD was weighed and dispersed in PBS solutions at pH 6.5 and pH 7.4. 1 mL of solution was taken out at intervals and 1 mL was added back. The OD value of the taken solutions was measured at 784 nm, and the ICG release was calculated based on the standard curve. Results are as follows: Figure 8 As shown, ZnO2&ICG@ZIF-iRGD exhibits pH sensitivity and slow release characteristics in ICG release. In a neutral PBS environment (pH 7.4) and a weakly acidic PBS environment (pH 6.5), the 24-hour average cumulative release rates of ICG from ZnO2&ICG@ZIF-iRGD were 20.57±2.08% and 42.73±2.83%, respectively. The ICG release curves of ZnO2&ICG@ZIF-iRGD in these two different environments showed statistically significant differences (p < 0.01).

[0048] Example 2: Preparation and characterization of ZnO2 & ICG@ZIF-iRGD-PLEL hydrogel 1. Preparation of PLEL hydrogel Accurately weigh 9.0 g of PEG and place it in a vacuum reaction flask. Heat to 120 °C and evacuate for 0.5 h to remove water. After the reaction flask cools to room temperature, add 0.2% stannous octoate, followed by 21.0 g of lactide monomer. Evacuate to 70 Pa and react magnetically in an oil bath at 150 °C for 8 h. After the reaction, dissolve the product in dichloromethane, precipitate and purify in ethanol, repeating this process three times. Then, dry the product in a vacuum drying oven at 50 °C for 48 h to obtain a translucent solid PDLA-PEG-PDLA. Weigh a certain amount of PDLA-PEG-PDLA into a test tube, add a certain amount of deionized water to make the copolymer aqueous solution concentration 15%, and dissolve at approximately 2 °C for more than 12 h until the polymer is completely dissolved to obtain PLEL hydrogel.

[0049] 1 mg of powdered ZnO2 & ICG@ZIF-iRGD was added to 2 mL of PLEL solution, and then incubated in a water bath at 37 °C for 5 minutes to induce the formation of hydrogel, thus obtaining ZnO2 & ICG@ZIF-iRGD-PLEL hydrogel.

[0050] 2. Thermosensitivity determination of ZnO2 & ICG@ZIF-iRGD-PLEL hydrogel At room temperature, 2 ml of ZnO2 & ICG@ZIF-iRGD-PLEL hydrogel was added to a glass test tube. The test tube containing the hydrogel was placed in a 37°C water bath and heated. The test tube was then quickly inverted, and the state of the hydrogel was recorded by photograph. The results are as follows: Figure 9 As shown, the hydrogel has reached a gel state and does not flow when inverted.

[0051] The phase transition temperature of the ZnO2&ICG@ZIF-iRGD-PLEL hydrogel was determined using a rotational rheometer. 1 ml of the ZnO2&ICG@ZIF-iRGD-PLEL hydrogel (gel temperature 0℃) was added to the test pan of the rotational rheometer. The frequency was 1 Hz, the strain was 0.05%, the heating range was set to 0-70℃, and the heating rate was 1℃ / min. The elastic modulus G' (storage modulus) and viscous modulus G'' (dissipation modulus) were used as observation indicators. The temperature at which G' = G'' was considered the phase transition temperature. Figure 10 As shown, in the low-temperature region (<30°C), the storage modulus of the hydrogel is lower than its loss modulus, indicating that the hydrogel exhibits strong fluidity at this temperature. When the temperature rises to around 35°C, the storage modulus equals the loss modulus, indicating that the hydrogel has undergone a transition from a solution state to a gel state. With further increases in temperature, the ratio of storage modulus to loss modulus gradually increases, suggesting that the hydrogel may have entered a stable gel state.

[0052] 4. SEM morphology of ZnO2 & ICG@ZIF-iRGD-PLEL hydrogel 2 ml of ZnO2&ICG@ZIF-iRGD-PLEL was added to a 6-well plate, rapidly cooled with liquid nitrogen, and then freeze-dried for 24 hours. A suitable sample was taken and its morphology and structure were determined using a scanning electron microscope to observe the appearance of the ZnO2&ICG@ZIF-iRGD-PLEL hydrogel and its drug loading. Figure 11 As shown, low-magnification scanning electron microscopy images reveal that the hydrogel has large and irregular pores, which helps to increase the drug loading capacity and promote sustained drug release; high-magnification scanning electron microscopy images show that ZnO2&ICG@ZIF-iRGD is relatively uniformly distributed on the surface of the hydrogel, indicating that it has been successfully loaded into the hydrogel.

[0053] Example 3: Study on tumor targeting and anti-tumor efficacy of ZnO2 & ICG@ZIF-iRGD The human colon adenocarcinoma cell line HCT-116 and the colon adenocarcinoma cell line CT26 (from BALB / c mice) labeled with luciferase D (D-Luciferin) used in the following examples were both purchased from Peking Union Medical College Hospital Cell Resource Center.

[0054] The BALB / c mice used in the following examples were female, 6-8 weeks old, weighing 18-20 g, and were specific pathogen-free (SPF) grade, purchased from Vital River Laboratory Animal Technology Co., Ltd.

[0055] 1. In vitro tumor targeting assessment of ZnO2 & ICG@ZIF-iRGD The fluorescence intensity of ICG was detected using laser confocal microscopy to assess the uptake of ZnO2 & ICG@ZIF-iRGD by HCT-116 cells. HCT-116 cells were cultured at a density of 1×10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 cells / well in 24-well confocal culture plates. ZnO2&ICG@ZIF-iRGD was co-cultured with HCT-116 cells for 1 h and 12 h, respectively. Cells were then washed three times with PBS to remove cell debris or untaken samples. Cells were fixed with 4% paraformaldehyde for 20 min, then incubated with DAPI in the dark for 5 min, followed by three washes with PBS. Free ICG and ZnO2&ICG / @ZIF were treated in the same manner, and their in vitro tumor targeting strength was compared with that of ZnO2&ICG@ZIF-iRGD.

[0056] The results are as follows Figure 12 As shown, after co-culturing ZnO2&ICG@ZIF-iRGD with HCT-116 cells for 1 hour, a small amount of ICG red fluorescence appeared in the cells, proving that ZnO2&ICG@ZIF-iRGD can be rapidly taken up by tumor cells. After co-culturing with HCT-116 cells for 12 hours, the intensity of intracellular red fluorescence significantly increased, indicating that more ZnO2&ICG@ZIF-iRGD was taken up by the cells at this time. However, after co-culturing free ICG and ZnO2&ICG@ZIF with HCT-116 cells for 1 hour or 12 hours, the intracellular ICG fluorescence intensity was significantly lower than that of the corresponding ZnO2&ICG@ZIF-iRGD co-culture group. These results indicate that ZnO2&ICG@ZIF-iRGD is most easily taken up by tumor cells.

[0057] After co-culturing ZnO2 & ICG@ZIF-iRGD with normal human colonic epithelial NCM460 cells for 12 h, the intracellular ICG signal intensity was observed using confocal laser scanning microscopy. Results are as follows: Figure 13As shown, after co-culturing ZnO2&ICG@ZIF-iRGD with NCM460 for 12 years, no obvious ICG red fluorescence signal was observed in the cells, proving that ZnO2&ICG@ZIF-iRGD was rarely taken up by normal cells and has good tumor targeting.

[0058] 2. In vivo tumor targeting assessment of ZnO2 & ICG@ZIF-iRGD A mouse model of peritoneal metastasis of colon cancer was established. Mice were first held using the dorsal fixation method, with their heads slightly tilted back and abdomens facing upwards. The left hind limb was stabilized with the little finger. The injection site was disinfected with a 75% alcohol swab. The syringe needle was inserted into the skin, reaching the subcutaneous layer, and then advanced 2-3 mm subcutaneously, inserting the needle into the mouse's peritoneal cavity at a 45-degree angle to the skin. 200 μl of a 2x10⁻⁶ concentration was injected. 6 CT26 cells labeled with D-Luciferin were injected into mice at a concentration of 1 / ml. The cells were then kneaded in the abdomen of mice to distribute them as evenly as possible. After 5 days, a mouse model of peritoneal metastasis of colon cancer was induced.

[0059] A colon cancer peritoneal metastasis model was established in 12 mice, which were divided into two groups: an ICG group and a ZnO2&ICG@ZIF-iRGD group, with 6 mice in each group. 20 μl of ICG and ZnO2&ICG@ZIF-iRGD were injected intraperitoneally into the mice in the form of PLEL hydrogel. After anesthetizing all mice, 100 μl of luciferase substrate solution was injected into each mouse. Using the "bioluminescence imaging" and "fluorescence imaging" modes of a multimodal animal in vivo imaging system, the location of the tumor and the location of ICG emission were recorded at 0h, 1h, 6h, 24h, and 48h. The excitation filter for the fluorescence imaging mode was set to 745nm, and the emission filter was set to 820nm.

[0060] The results are as follows Figure 14 As shown, the fluorescence position of ZnO2&ICG@ZIF-iRGD is close to that of bioluminescence, indicating that the latter has good in vivo tumor targeting. Furthermore, at 48 hours post-intraperitoneal injection, the ZnO2&ICG@ZIF-iRGD group still showed strong red fluorescence, demonstrating the drug's good retention effect in mouse peritoneal tumor tissue. In contrast, the intraperitoneal fluorescence intensity of the ICG group was significantly reduced compared to 24 hours, indicating that most of the ICG was metabolized in the mice within 48 hours.

[0061] Half of the mice in each group were sacrificed and dissected. Tumors, liver, kidneys, spleen, lungs, and heart were collected. Bioluminescence imaging and fluorescence imaging of the tumors and major organs were used again to record bioluminescence and ICG fluorescence images, and the fluorescence intensity of tumors and major organs in different treatment groups was calculated. The results are as follows: Figure 15As shown, significant bioluminescence and fluorescence were observed in tumor tissues of the ZnO2 & ICG@ZIF-iRGD group, while only bioluminescence was observed in tumor tissues of the ICG group. These results indicate that ZnO2 & ICG@ZIF-iRGD has a higher tumor retention effect than ICG. Figure 16 As shown, there was a statistically significant difference in fluorescence intensity in tumor tissues between the ZnO2&ICG@ZIF-iRGD group and the ICG group (p < 0.01). The liver and kidneys were the organs with the highest fluorescence intensity besides tumors, suggesting that ZnO2&ICG@ZIF-iRGD is mainly metabolized by the liver and kidneys. Fluorescence intensity levels in the lungs, heart, and spleen were lower and showed no significant difference.

[0062] 3. In vitro antitumor efficacy of ZnO2 & ICG@ZIF-iRGD HCT-116 cells were used at a rate of 1 x 10 5 Cells were seeded at a density of 100 cells / well in two 96-well plates. ZnO2 & ICG@ZIF-iRGD solutions of different concentrations were prepared, and the drugs were diluted with DMEM medium containing 10% FBS to concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL, respectively. The medium containing different concentrations of ZnO2 & ICG@ZIF-iRGD was added to the two 96-well plates. The unseeded group served as the blank control, and untreated cells served as the control group. 100 μl of medium was added to each well in the blank, control, and drug-treated groups, with five replicates in each group. The plates were incubated for 24 h. After incubation, one of the 96-well plates was irradiated with near-infrared light at 808 nm for 5 min. 10 μl of CCK-8 reagent was added to each well, and the absorbance (optical density, OD) was measured at 450 nm using a microplate reader. Cell viability was determined by calculating absorbance values ​​using the following formula, and the IC50 value of ZnO2&ICG@ZIF-iRGD under irradiation with or near-infrared light was calculated.

[0063] Cell viability (%) = (OD of test OD of blank) / (OD of control OD of blank) × 100%; In the above formula, "OD of test" represents the absorbance value of the sample treatment group, "OD of control" represents the absorbance value of the untreated sample group, and "OD of blank" represents the absorbance value of the cell-free group.

[0064] The results are as follows Figure 17As shown, ZnO2&ICG@ZIF-iRGD exhibits dose-dependent cytotoxicity against HCT-116 cells, meaning that the cell viability of tumor cells gradually decreases with increasing drug concentration. In the absence of near-infrared light irradiation, the IC50 of ZnO2&ICG@ZIF-iRGD was 60 μg / ml. However, in the near-infrared light irradiation group, the anti-proliferative ability of ZnO2&ICG@ZIF-iRGD was further enhanced, with an IC50 of 40 μg / ml.

[0065] Then, the effects of ZIF-8, ZnO2 / @ZIF, ZnO2&ICG / @ZIF, and ZnO2&ICG / @ZIF-iRGD on the viability of HCT-116 cells were compared under external conditions with and without near-infrared light irradiation. HCT-116 cells were cultured at 1x10⁻⁶ cells per cell line. 5 Cells were seeded at a density of 10 cells / well in two 96-well plates. Based on the IC50 of the obtained ZnO2&ICG@ZIF-iRGD nanoparticles, ZIF-8, ZnO2 / @ZIF, ZnO2&ICG / @ZIF, and ZnO2&ICG / @ZIF-iRGD were prepared at the corresponding concentrations. Five replicates were set up for the blank control, control group, and different concentration groups. The plates were incubated in a cell culture incubator for 24 h. After incubation, one of the 96-well plates was irradiated with near-infrared light at 808 nm for 5 min. 10 μL of CCK-8 reagent was added to each well, and the cell viability of different groups was determined by measuring the OD value at 450 nm using a microplate reader.

[0066] The results are as follows Figure 18 As shown, compared with other components, ZnO2&ICG@ZIF-iRGD exhibited the strongest antitumor efficacy. At the same concentration, ZnO2&ICG@ZIF-iRGD and ZnO2&ICG@ZIF showed a statistically significant difference in their inhibitory effects on HCT-116 cell survival (p < 0.01). Furthermore, near-infrared light irradiation significantly affected the inhibitory effects on HCT-116 cell survival in both the ZnO2&ICG@ZIF-iRGD and ZnO2&ICG@ZIF treatment groups (p < 0.05).

[0067] 4. In vivo anti-tumor efficacy of ZnO2 & ICG@ZIF-iRGD Mice bearing colorectal cancer peritoneal metastases were established using the methods described above. A hyperthermic intraperitoneal perfusion system was then established, comprising: a constant-temperature water bath, 50ml centrifuge tubes, a peristaltic pump, and a thermometer. The constant-temperature water bath was heated to 43℃, and centrifuge tubes containing 50ml of distilled water were placed in the water bath for heating. Mice were anesthetized with isoflurane (5%, 10μL / g body weight), and their abdomens were disinfected with 75% alcohol. 0.5mm needles were inserted into the left upper abdomen and right lower abdomen of the mice, respectively, to serve as the inlet and outlet for the hyperthermic intraperitoneal perfusion. The outlet in the mouse abdomen was connected to the pump inlet of the peristaltic pump via tubing, and the outlet in the mouse abdomen was connected to the head of the centrifuge tube via tubing, and the other end of the centrifuge tube head was connected to the inlet of the peristaltic pump via tubing. Two temperature probes of the thermometer were inserted into the inlet and outlet tubing of the mice to monitor the temperature during the perfusion process. The peristaltic pump was adjusted to maintain the perfusion fluid flow rate at 5 ml / min, and intraperitoneal hyperthermic perfusion was performed twice on days 0 and 7. Finally, a mouse PCI scoring system was established: the mouse abdomen was divided into 9 equal regions, and a score was assigned based on the area occupied by the tumor in each region. The specific scoring criteria are as follows: 0 points - no tumor; 1 point - tumor area less than 50% of the region; 2 points - 50% < tumor area ≤ 80%; 3 points - 80% < tumor area. The final PCI score is the sum of the scores for each region.

[0068] Forty mice were randomly divided into four groups: Group I (control group), Group II (ZnO2 & ICG / @ZIF-iRGD intraperitoneal injection group), Group III (ZnO2 & ICG / @ZIF-iRGD intraperitoneal injection + near-infrared light irradiation group), and Group IV (ZnO2 & ICG / @ZIF-iRGD intraperitoneal injection + near-infrared light irradiation + intraperitoneal hyperthermic perfusion group, with ZnO2 & ICG / @ZIF-iRGD administered as a gel). Intraperitoneal hyperthermic perfusion was performed three times, once every two days. Strict disinfection and temperature control were maintained during the perfusion process. No complications related to intraperitoneal hyperthermic perfusion were observed in the treatment groups. Mouse weight and abdominal circumference were recorded at the same time every five days. Every ten days, the extent and fluorescence intensity of the tumor within the peritoneal cavity of tumor-bearing mice were detected using a small animal in vivo imaging system. PCI scores were calculated for different groups of mice based on the bioluminescence imaging range, and bioluminescence intensity change curves were calculated for different groups of mice based on the bioluminescence intensity. On day 17, half of the mice were euthanized, and the distribution and size of tumors within the abdominal cavity were observed and recorded after dissection. Survival curves were plotted after all remaining mice had died. Data were performed at least three times independently, and results are presented as mean ± standard deviation. GrapHpad Prism 9 and Adobe Illustrator CC 2022 software were used for statistical analysis and graphing. Student's test was used to analyze two or more groups of quantitative data, as applicable; Kaplan-Meier test and Logrank test were used for survival analysis. A p-value less than 0.05 was considered statistically significant.

[0069] The results are as follows Figure 19-20 As shown, the signal intensity of bioluminescence imaging in Group I gradually increased over time, indicating lesion enlargement or increased activity, reaching its highest level particularly on day 20. Groups II, III, and IV showed reduced luminescence range and intensity in bioimaging compared to the control group, indicating tumor shrinkage and necrosis after treatment. Group II showed some signal enhancement from day 10 to day 20, but slightly weaker than Group I, suggesting an anti-tumor effect of intraperitoneal injection of ZnO2&ICG@ZIF-8-iRGD+PLEL in tumor-bearing mice. Mice in Groups III and IV showed tumor shrinkage and reduced fluorescence intensity as early as day 10, with Group IV showing the most significant reduction, indicating a synergistic effect of ZnO2&ICG@ZIF-8-iRGD-PLEL combined with near-infrared light irradiation and intraperitoneal hyperthermic perfusion.

[0070] like Figure 21As shown, PCI scores were calculated by observing the bioluminescence imaging range of different treatment groups. In Group I (gray curve), the PCI score of tumor-bearing mice continuously increased over time, with the largest increase, indicating a continuous worsening of the condition. In Group II (blue curve), the PCI score of tumor-bearing mice did not change significantly in the early stages, but gradually increased in the later stages, indicating that the treatment strategy of intraperitoneal injection of ZnO2 & ICG@ZIF-8-iRGD + PLEL may have some effect in the short term, but the long-term effect is limited. In Group III (purple curve), the PCI score of tumor-bearing mice decreased in the early stages, then increased slightly, but the overall fluctuation was not significant, indicating that ZnO2 & ICG@ZIF-8-iRGD + PLEL combined with near-infrared light irradiation can inhibit the progression of colorectal cancer peritoneal metastasis to some extent. In Group IV (orange curve), the PCI score decreased the most and remained at a low level throughout the experiment, indicating that ZnO2 & ICG@ZIF-8-iRGD + PLEL combined with near-infrared light irradiation and intraperitoneal hyperthermic perfusion can effectively inhibit disease progression and even improve the condition.

[0071] Mice in each group were dissected after treatment to observe the size, number, and distribution of tumors in the abdominal cavity. Figure 22-23 As shown, a large number of scattered peritoneal tumors were observed in the abdominal cavity of the control group, while the number of tumors in group II was slightly less than that in the control group. Only a small number of scattered tumors were observed in the abdominal cavity of mice in groups III and IV. Abdominal tumors were collected and photographed from each group of mice. The results showed that the tumor volume and number in group II were reduced compared to the control group, indicating that ZnO2&ICG@ZIF-8-iRGD+PLEL still possesses certain anti-tumor efficacy in vivo. Based on this, the nano-hydrogel composite material combined with near-infrared light irradiation significantly enhanced the anti-tumor efficacy. The volume and number of tumors in the abdominal cavity of group III were significantly reduced compared to group II. Group IV mice received additional intraperitoneal hyperthermic perfusion therapy in addition to the treatment given to group III mice, further improving the therapeutic effect. This indicates that the nano-hydrogel composite material and intraperitoneal hyperthermic perfusion have a good synergistic effect.

[0072] like Figure 24 As shown in the curves, the weight change of tumor-bearing mice generally shows an increasing weight trend in all groups, but the specific trends differ. Group I (gray curve) saw a steady increase in weight with the largest increase, indicating rapid disease progression and a significant increase in ascites in this group. Groups II, III, and IV (blue, purple, and orange curves) experienced a slight decrease in weight in the early stages, possibly related to tumor cachexia; the gradual recovery and increase in weight in the later stages was mainly due to ascites formation induced by peritoneal metastasis. However, because groups II, III, and IV effectively controlled tumor progression, ascites formation was less than in the control group, and the rate of weight gain was slower. Figure 25As shown in the curves, the prognosis of mice in groups III and IV was significantly better than that in the control group. All mice in the control group died within 30 days, while some mice in groups III and IV still survived after 30 days.

[0073] In summary, the nanodrug delivery system provided by this invention, modified with iRGD, significantly enhances targeted uptake of tumor cells, exhibiting excellent in vitro tumor targeting. Furthermore, in vivo animal experiments show that ZnO2&ICG@ZIF-iRGD can efficiently accumulate at the tumor site and prolong its residence time in the tumor microenvironment through the sustained-release properties of ZIF-8, thereby improving therapeutic efficacy. In the antitumor efficacy study, the CCK-8 assay showed that ZnO2&ICG@ZIF-iRGD significantly inhibited the proliferation of HCT-116 cells, and further enhanced cytotoxicity under near-infrared light irradiation, demonstrating a synergistic effect of photothermal / photodynamic therapy. Animal experiments further confirmed that intraperitoneal injection of ZnO2&ICG@ZIF-iRGD combined with near-infrared light irradiation and intraperitoneal hyperthermic perfusion achieved effective in vivo tumor inhibition. Moreover, survival analysis results showed that the survival time of mice in the ZnO2&ICG@ZIF-iRGD+NIR+hyperthermic perfusion group was significantly prolonged, further validating the therapeutic advantages of this nanosystem. In summary, this study fully demonstrates the superiority of the ZnO2&ICG@ZIF-iRGD nanosystem in the treatment of peritoneal metastases from colorectal cancer, providing new ideas and potential clinical application value for efficient and targeted combination therapy strategies.

[0074] Example 4: Biosafety testing of the ZnO2&ICG@ZIF-iRGD nanoparticle drug delivery system 1. In vitro biosafety assessment of ZnO2 & ICG@ZIF-iRGD NCM460 cells (derived from the human normal colonic epithelial cell line NCM460, purchased from Peking Union Medical College Hospital Cell Resource Center) were used at a rate of 1x10⁻⁶. 5Cells were seeded at a density of 100 cells / well in two 96-well plates. ZnO2 & ICG@ZIF-iRGD solutions of different concentrations were prepared, and the drugs were diluted with DMEM medium containing 10% FBS to concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL. The medium containing different concentrations of ZnO2 & ICG@ZIF-iRGD was added to each of the two 96-well plates. 100 μl of medium was added to each well. The unseeded group served as the blank control, and the untreated cells served as the control group. Five replicates were set up for each of the blank, control, and drug-treated groups. The plates were incubated in a cell culture incubator for 24 h. After incubation, one of the 96-well plates was irradiated with near-infrared light at 808 nm for 5 min. 10 μl of CCK-8 reagent was added to each well, and the absorbance (optical density, OD) was measured at 450 nm using a microplate reader. Cell viability was determined by calculating absorbance values ​​using the following formula, and the IC50 value of ZnO2&ICG@ZIF-iRGD under irradiation with or near-infrared light was calculated.

[0075] Cell viability (%) = (OD of test OD of blank) / (OD of control OD of blank) × 100% In the above formula, "OD of test" represents the absorbance value of the sample treatment group, "OD of control" represents the absorbance value of the untreated sample group, and "OD of blank" represents the absorbance value of the cell-free group.

[0076] The results are as follows Figure 26 As shown, even under near-infrared light irradiation, different concentrations of ZnO2&ICG@ZIF-iRGD did not exhibit significant cytotoxicity against NCM460 cells. These results are primarily due to the modification of iRGD and the encapsulation of ZIF-8. The modification of iRGD increased the targeting specificity of ZnO2&ICG@ZIF-iRGD, while ZIF-8 enhanced the drug's stability, preventing its easy degradation and cellular uptake.

[0077] 2. In vivo biosafety assessment of ZnO2 & ICG@ZIF-iRGD 2.1 Six- to eight-week-old female BALB / c mice were randomly divided into four groups: Group I was the PLEL blank control group; Group II was the ZnO2&ICG / @ZIF-iRGD+PLEL group; Group III was the ZnO2&ICG / @ZIF-iRGD+PLEL+Near-infrared light group; and Group IV was the ZnO2&ICG / @ZIF-iRGD+PLEL+Near-infrared light+peritoneal hyperthermic perfusion group. Mice were euthanized on day 17, and samples were collected for the following tests.

[0078] First, the liver and kidney function of mice in different groups was measured. Mice were immobilized with the left hand, and the eyeballs were compressed to protrude as much as possible. After trimming the whiskers, the eyeballs were quickly removed using curved forceps. Blood was dripped into a 1.5ml EP tube containing an anticoagulant; blood flow could be accelerated by pressing the mouse's heart. After the blood had drained, the mice were euthanized by dislocation. The collected blood was allowed to stand at 4℃ for 2 hours, then centrifuged at 3000rpm for 20 minutes. The supernatant was the serum, and excess serum was stored at -80℃. The liver and kidney function of mice was assessed using a kit from Nanjing Jiancheng Biotechnology Institute. The assays included alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (Urea), and creatinine (CRE).

[0079] The results are as follows Figure 27 As shown, there were no significant differences in ALT, AST, CRE, and UREA levels between the control group and different treatment groups, and all were within the normal range.

[0080] 2.2. Hearts, livers, spleens, lungs, and kidneys of mice from each group were collected, washed with PBS, fixed with 4% paraformaldehyde, and then sectioned in paraffin and stained with hematoxylin-eosin (HE). The specific steps for paraffin embedding and sectioning are as follows: The samples were fixed in 4% paraformaldehyde for 48 h, washed with PBS, cut into pieces, and placed in an embedding cassette. A gradient of ethanol was used for dehydration at concentrations of 70%, 80%, 90%, 95%, and 100%. The samples were cleared with xylene and then immersed in hot-melt paraffin. Liquid paraffin was poured into the embedding cassette to soak the tissue pieces. After solidification, the embedding cassette was removed. The tissue pieces were cut into 5 μm thin sections using a paraffin microtome, flattened, placed in a slide tray, and allowed to adhere to glass slides. After slightly drying, the slides were baked in a slide baking machine. The specific steps for H&E staining are as follows: Paraffin sections are immersed sequentially in xylene solutions I, II, and III for 10 min each, followed by dewaxing in anhydrous ethanol I, II, 90%, 80%, 70%, and 50% ethanol for 5 min each. Next, they are stained with hematoxylin for 1 min, rinsed with water, differentiated with 1% hydrochloric acid ethanol for 5 s, rinsed again with water, and then blued with 1% ammonia solution for 1 min. After rinsing under running water and slightly drying, they are stained with eosin for 30 s and rinsed again under running water. The stained paraffin sections are then immersed sequentially in 75% and 85% ethanol, and anhydrous ethanol I and II for 5 min each, and finally cleared in xylene for 5 min. After the slides are dried, they are mounted with neutral resin. After these steps are completed, the sections are observed and photographed using a panoramic digital section scanning microscope, where the cell nuclei appear blue and the cytoplasm appears red.

[0081] The results are as follows Figure 28 As shown, no obvious organ damage was observed in the heart, liver, spleen, lungs, and kidneys of mice in each group, and the morphology of each organ tissue was normal. These results indicate that the nanohydrogel composite material has good biocompatibility in vivo. This is closely related to the good tumor targeting and biocompatibility of the nanohydrogel composite material, as well as the ability to release drugs sustainably, reducing the exposure to instantaneous high concentrations, thus laying the foundation for its application in tumor treatment.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for the preparation of nanoparticles, characterized in that, include: S1. Preparation of matrix nanoparticles ZnO2 / ICG@ZIF loaded with ZnO2 and ICG: A first solution containing zinc salt was mixed with a second solution containing ZnO2, indocyanine green and 2-methylimidazole and reacted. After the reaction was completed, the matrix nanoparticles ZnO2 / ICG@ZIF were collected. S2. The matrix nanoparticles ZnO2 / ICG@ZIF are aminated to obtain aminated matrix nanoparticles ZnO2 / ICG@ZIF; iRGD, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide are mixed and coupled to prepare modified iRGD. S3. The modified iRGD is mixed with the aminated matrix nanoparticles ZnO2 / ICG@ZIF and subjected to an amide reaction to obtain iRGD-loaded nanoparticles ZnO2 / ICG@ZIF-iRGD.

2. The preparation method according to claim 1, characterized in that, The mass ratio of zinc salt, ZnO2, indocyanine green and 2-methylimidazole is 150:20:5:2050.

3. The preparation method according to claim 1, characterized in that, The matrix nanoparticles ZnO2 / ICG@ZIF are amination-treated to obtain amination-treated matrix nanoparticles ZnO2 / ICG@ZIF, specifically including: The matrix nanoparticles ZnO2 / ICG@ZIF were dispersed in a mixed solution of ethanol and water, and 3-aminopropyltriethoxysilane was added to carry out an amination reaction. After the reaction was completed, the amination matrix nanoparticles ZnO2 / ICG@ZIF were obtained.

4. The preparation method according to claim 3, characterized in that, Based on each 1 mg of the matrix nanoparticles ZnO2 / ICG@ZIF, the volume of the 3-aminopropyltriethoxysilane is 10 μL.

5. The preparation method according to claim 1, characterized in that, The mass ratio of the modified iRGD to the aminated matrix nanoparticles ZnO2 / ICG@ZIF is 1:

5.

6. The ZnO2 / ICG@ZIF-iRGD nanoparticles prepared by the preparation method according to any one of claims 1-5.

7. The ZnO2 / ICG@ZIF-iRGD nanoparticles according to claim 6, characterized in that, The drug loading and encapsulation efficiency of ZnO2 in the ZnO2&ICG@ZIF-iRGD nanoparticles were 47±3.06 and 3.5±0.31, respectively, and the drug loading and encapsulation efficiency of ICG were 53±2.08 and 4.4±0.31, respectively.

8. A composition, characterized in that, Including the ZnO2 / ICG@ZIF-iRGD nanoparticles as described in claim 6 or 7.

9. The composition according to claim 8, characterized in that, It also includes hydrogels.

10. The use of the nanoparticles ZnO2 / ICG@ZIF-iRGD according to claim 6 or 7, or the composition according to any one of claims 8-9, in the preparation of antitumor drugs.

Images