Preparation of functionalized polyethyleneimine nanocomposites and their diagnosis and treatment applications

Abstract

The application discloses preparation of a functionalized polyethyleneimine nanocomposite material and diagnosis and treatment applications thereof. 131 Based on the unique physical and chemical properties of PEI.NH2, the SPECT imaging element (radioactive nuclide I), the CT imaging element (gold nanoparticle Au NPs) and the fluorescent imaging element (Cy7) are integrated by means of covalent bonding and physical wrapping, and the targeting molecule HA and the biocompatible molecule polyethylene glycol (PEG) are used for functional modification, so as to construct a nanosystem for in-vivo targeted tumor SPECT / CT / FL multimodal imaging. 131 The I-Au PHCNPs nanoparticle composite material can be well dispersed in a water-based solution, can specifically target tumors with high expression of CD44 receptors, and has good anticancer activity. 131 The I-Au PHCNPs nanoparticle composite material can realize tumor targeted SPECT / CT / FL multimodal imaging, has good application prospects for early and accurate diagnosis of cancer, and also lays a solid foundation for research and development of a new type of diagnosis and treatment reagent.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of functional nanomaterials, and particularly relates to a near-infrared fluorescent dye Cy7 modified functional polyethyleneimine coated gold nanoparticle composite material and application thereof. BACKGROUND

[0002] Statistical results of cancer show that the incidence and mortality of cancer in low-income countries and middle-income countries are still increasing year by year. Although there are various methods for treating cancer in the clinic, including surgical treatment, radionuclide treatment, chemotherapy and immunotherapy, these methods are only suitable for early-stage and treatment-sensitive cancer patients. At the same time, limited by the clinical diagnosis technology, most cancer patients are diagnosed as advanced cancer. Therefore, the above methods aim to maximize the elimination of cancer cells to prolong the survival of patients. Unfortunately, the effect of these methods will be weakened or even disappeared with the increase of drug resistance of cancer cells, and the practical results of achieving long-term survival of patients are not ideal. Therefore, based on the characteristics of early-stage cancer, such as strong concealment, poor treatment effect and poor prognosis, early accurate diagnosis is the key to reducing the mortality of cancer patients.

[0003] In the early diagnosis of cancer, the clinical application is more widely used in computed tomography (CT), magnetic resonance imaging (MR), positron emission tomography (PET), single photon emission computed tomography (SPECT) and so on. However, each single imaging method has its inherent limitations, and they cannot completely independently and comprehensively provide physiological and pathological information of tumors. Among them, nuclear medicine imaging (NMI) includes SPECT imaging and PET imaging, which can directly or indirectly detect the gamma rays produced by the decay of radionuclides injected into the body for imaging, with ultra-high sensitivity, no tissue penetration limit and other characteristics. However, the relatively low spatial resolution of NMI limits its diagnostic accuracy. CT imaging has the advantages of high spatial resolution, accurate anatomical information, and high cost performance. However, due to the low sensitivity of CT imaging itself, and the CT signal intensity is usually positively correlated with the concentration of contrast agent. Therefore, compared with SPECT, PET imaging, which only needs microgram level contrast agent for sensitive imaging, CT imaging needs to inject a large amount of contrast agent to have a better imaging effect. For fluorescence imaging (FL) which has not been clinically popularized, in addition to its high sensitivity, real-time visualization and other advantages, it also has the characteristics of clear imaging of lesion site boundary, which can guide the complete resection of tumors during surgery. However, due to the scattering and absorption of light in tissues, and the influence of light penetration depth on tissues, FL imaging is limited in biomedical applications. Therefore, based on the inherent characteristics of different diagnostic techniques, it is urgent to develop a multi-modal imaging technology that integrates different imaging modes to overcome the limitations of single imaging and obtain more comprehensive physiological information of tumor tissues.

[0004] The rapid development of nanomedicine has brought hope for multimodal imaging. Nanomaterials, with their nanoscale size, high surface-to-volume ratio, controllable drug release, and functionalizability, can be used to construct novel multifunctional nanocontrast agents. Among many nanomaterials, polyethyleneimine (PEI.NH2), based on its abundant amino groups on its surface and its internal cavity structure, can serve as an excellent nanocarrier. It can load various imaging or therapeutic elements through covalent bonding and physical encapsulation to construct novel nanocomposite systems for cancer diagnosis and treatment. For example, inspired by the nanotechnology of PEI.NH2 and the properties of alkoxyphenylsulfonamides (APAS), Zhu JY et al. (Zhu, JY; Shi, XY et al. Langmuir, 2019, 35, 13405-13412) designed a nanomaterial with pH-responsive charge-flipping properties and labeled with technetium-99m (… 99m Tc) nanoparticle APAS- 99m Tc-Au PENs were used for dual-modal imaging of cancer cells using enhanced SPECT / CT. Furthermore, Zhu J. Y. et al. (Zhu, JY; Zhao, JH et al. Biomater. Sci., 2020, 8, 3956-3965) used... 131 I labeled APAS-modified PEI.NH2 containing gold nanoparticles to obtain 131 I-APAS-Au PNPs are used for SPECT / CT dual-modal imaging of tumors and radionuclide therapy. Therefore, based on the unique properties of PEI.NH2, it can integrate multiple imaging elements used for cancer diagnosis and functionalize them with targeted reagents to achieve in vivo targeted multimodal tumor imaging.

[0005] Previous research on tumor targets has revealed that the Cluster of Differentiation 44 (CD44) molecule, as a multifunctional cell surface receptor, participates in various biological processes, including tumor metastasis and angiogenesis. Furthermore, compared to normal cells, the CD44 receptor is significantly expressed in cancer cells such as glioma, breast cancer, non-small cell lung cancer, and colon cancer. Further research has found that hyaluronic acid (HA), a natural biological polysaccharide, can specifically bind to the CD44 receptor on the surface of cancer cells. Therefore, based on the unique physicochemical properties of PEI.NH2, SPECT imaging elements (radioactive nuclides) are integrated through covalent bonding and physical encapsulation. 131I), CT imaging element (gold nanoparticles Au NPs) and fluorescence imaging element (Cy7) are integrated, and are modified by target molecules HA and biocompatible molecules polyethylene glycol (PEG) to construct a nano system for in vivo targeted tumor SPECT / CT / FL multi-modal imaging.

[0006] The retrieval of literatures and patents at home and abroad related to the construction of nano imaging contrast agent based on functionalized PEI.NH2 shows that the collection of fluorescent dye molecules Cy7, radionuclide 131 I and Au NPs multi-imaging elements are integrated and modified by HA for the study of targeted tumor SPECT / CT / FL three-mode imaging, and no related report has been seen. SUMMARY

[0007] The purpose of the present application is to provide a near-infrared fluorescent dye Cy7 modified functionalized polyethyleneimine coated gold nanoparticle composite material.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] A near-infrared fluorescent dye Cy7 modified functionalized polyethyleneimine coated gold nanoparticle composite material is prepared by the following steps:

[0010] Step 1, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to the NH2-PEG-COOH solution for activation, then polyethyleneimine PEI.NH2 solution is added, and stirring reaction is carried out to prepare PEI.NH2-PEG-NH2;

[0011] Step 2, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) are added to the hyaluronic acid HA solution for activation, then PEI.NH2-PEG-NH2 prepared in step 1 is added, and stirring reaction is carried out to prepare PEI.NH2-(PEG-HA);

[0012] Step 3, dimethyl sulfoxide solution of 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester HPAO is added to PEI.NH2-(PEG-HA) prepared in step 2, and stirring reaction is carried out to prepare PEI.NH2-(PEG-HA)-HPAO;

[0013] Step 4, sulfonic acid-based cyanine 7-N-hydroxysuccinimide ester Cy7 is added to PEI.NH2-(PEG-HA)-HPAO prepared in step 3, and stirring reaction is carried out in the dark to prepare PEI.NH2-(PEG-HA)-Cy7-HPAO;

[0014] Step 5, adding HAuCl4 solution to the PEI.NH2-(PEG-HA)-Cy7-HPAO prepared in step 4, stirring for 20-40 minutes, then adding NaBH4 solution to react, then adding N(C2H5)3, stirring for 20-40 minutes, then adding Ac2O, stirring to react, to prepare nanoparticle Au PHCNPs;

[0015] Step 6, adding chloramine T and Na 131 I to the nanoparticle Au PHCNPs prepared in step 5, stirring for 3 minutes, then adding Na2S2O5 and KI while stirring, reacting for 5 minutes, and finally separating and purifying the composite material through a desalting column.

[0016] Further, in step 1, the ratio of the sum of the amounts of substance of EDC and NHS to the amount of substance of NH2-PEG-COOH is 5:1, the ratio of the amounts of substance of EDC and NHS is 1:1, the ratio of the amounts of substance of NH2-PEG-COOH and PEI.NH2 is 20:1, and the stirring reaction time is 3 days.

[0017] Further, in step 2, the ratio of the sum of the amounts of substance of EDC and NHS to the amount of substance of HA is 5:1, the ratio of the amounts of substance of EDC and NHS is 1:1, the ratio of the amounts of substance of HA and PEI.NH2-PEG-NH2 is 30:1, and the stirring reaction time is 3 days.

[0018] Further, in step 3, the ratio of the amounts of substance of HPAO and PEI.NH2-(PEG-HA) is 10:1, and the stirring reaction time is 1-2 days.

[0019] Further, in step 4, the ratio of the amounts of substance of Cy7 and PEI.NH2-(PEG-HA)-HPAO is 16:1, and the stirring reaction time is 3 days.

[0020] Further, in step 5, the ratio of the amounts of substance of HAuCl4 and NaBH4 is 1:5, the ratio of the amounts of substance of HAuCl4 and PEI.NH2-(PEG-HA)-Cy7-HPAO is 100:1, the ratio of the amounts of substance of N(C2H5)3, Ac2O and PEI.NH2-(PEG-HA)-Cy7-HPAO is 120-660:100-550:1, and the stirring reaction time is 24h.

[0021] Further, in step 6, the ratio of the amounts of substance of Na 131 I is 185-365 MBq.

[0022] The composite material is applied to preparation of tumor diagnosis and treatment reagents.

[0023] The use of polyethylene glycol (PEG) in the application can reduce the uptake of the material by immune cells, improve the biocompatibility of the material, prolong the circulation time of the material in the body, and further prolong the imaging time and radionuclide treatment time in the body.

[0024] In the application, the targeting molecule HA is used to modify the surface of PEI.NH2, so as to endow the material with the function of targeting CD44 receptor overexpressing tumors in the body. Meanwhile, based on the good biocompatibility of HA, the modification of HA can also improve the biocompatibility of the material.

[0025] In the application, HPAO is used as a chelating agent for chelating radionuclides 131 I is used as a beam bridge to indirectly label the surface of PEI.NH2.

[0026] In the application, Sulfo-Cy7 NHS ester is used to directly react with the amino group on the surface of PEI.NH2 through its terminal NHS ester group, and is modified on the surface of PEI.NH2, so as to be used for in-vivo and in-vitro fluorescence imaging.

[0027] In the application, after the addition of HAuCl4 solution, the reaction is continuously and rapidly stirred for 20-40 minutes, AuCl4 - is used to enter the internal cavity of PEI.NH2 through the affinity with the amino group on PEI.NH2, and then is rapidly reduced by NaBH4, so as to obtain functionalized PEI.NH2 wrapped with gold nanoparticles, which can effectively prevent the aggregation of gold nanoparticles.

[0028] In the application, N(C2H5)3 and Ac2O are used to acetylate the remaining amino groups on the surface of PEI.NH2, so as to reduce the surface potential and improve the biocompatibility of the material. N(C2H5)3 is added to create an alkaline environment to ensure the smooth progress of acetylation.

[0029] In the application, Na 131 I is added to the formed nanoparticle Au PHCNPs solution, and is stirred to make 131 I fully react with HPAO on the surface of Au PHCNPs, and the unreacted Na 131 I is separated by PD-10 desalting chromatography column, and finally the pure 131 I-Au PHCNPs are collected.

[0030] In the application, the functionalized PEI.NH2 is prepared 131The I-labeled Au PHCNPs nanoparticles can provide high-resolution anatomical information and high-sensitivity biological information at the molecular level about the tumor at the same time, and are expected to realize targeted tumor SPECT / CT / FL multimodal imaging. 131 The I-labeled Au PHCNPs nanoparticles can emit β rays (0.606 MeV, 89.9%) during the decay process, 131 The I-Au PHCNPs nanoparticles can be used for tumor radionuclide therapy.

[0031] Beneficial effects

[0032] (1) The preparation process of the present application is simple, the reaction conditions are mild, and the preparation process can be used for the preparation of other fluorescent imaging reagents and radionuclide modified functionalized polyethyleneimine coated gold nanoparticles, and has good use value.

[0033] (2) The I-Au PHCNPs nanoparticles prepared by the present application can be well dispersed in a water-based solution, can specifically target tumors with high expression of CD44 receptors, and exhibit good anticancer activity. 131 The I-Au PHCNPs nanoparticles can realize tumor targeted SPECT / CT / FL multimodal imaging, have good early precise diagnosis application prospect for cancer, and also lay a solid foundation for the research and development of new diagnosis and treatment reagents. 131 The I-Au PHCNPs nanoparticles can realize tumor targeted SPECT / CT / FL multimodal imaging, have good early precise diagnosis application prospect for cancer, and also lay a solid foundation for the research and development of new diagnosis and treatment reagents. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The I-Au PHCNPs and the non-targeting material without HA modification 131 The I-Au PHCNPs and the non-targeting material without HA modification 131 The synthesis path diagram of the I-Au PCNPs.

[0035] Figure 2 The I-Au PHCNPs and the non-targeting material without HA modification m The I-Au PHCNPs and the non-targeting material without HA modification m The nuclear magnetic resonance hydrogen spectrum of PEG and (e) PEI.NH2-

[0036] Figure 3 The Fourier infrared spectrum of Cy7, PEI.NH2-(PEG-HA)-HPAO and PEI.NH2-(PEG-HA)-Cy7-HPAO.

[0037] Figure 4Fluorescence spectra of Cy7 and PEI.NH2-(PEG-HA)-Cy7-HPAO dissolved in water.

[0038] Figure 5 (a) UV-Vis absorption spectra of PEI.NH2-(PEG-HA)-HPAO, Cy7 and PEI.NH2-(PEG-HA)-Cy7-HPAO. (b) UV-Vis absorption spectra of PEI.NH2-(PEG-HA)-Cy7-HPAO at different concentrations. (c) Standard curve of Cy7 at 763 nm. (d) UV-Vis absorption spectra of Au PHCNPs, inset is the photo of Au PHCNPs dispersed in water.

[0039] Figure 6 (a) TEM image, (b) histogram of particle size distribution, (c) high resolution TEM image, (d) SAED image and (e) EDS image of Au PHCNPs prepared in this invention.

[0040] Figure 7 UV-Vis absorption spectra of Au PHCNPs prepared in this invention at different (a) pH, (b) temperature conditions and (c) time points.

[0041] Figure 8 Hydrodynamic size distribution of Au PHCNPs dispersed in (a) PBS and (b) water at different time points determined by DLS method; and (c) average hydrodynamic size and (d) polydispersity index of Au PHCNPs at different time points.

[0042] Figure 9 CT imaging of (a) Au PHCNPs and (b) Optipak at different molar concentrations of radiodense elements (gold or iodine). (c) Plot of X-ray attenuation intensity (HU) of Au PHCNPs and Optipak as a function of molar concentration of radiodense elements (gold or iodine).

[0043] Figure 10 (a) UV-Vis absorption spectra of Au PHCNPs at different time points at room temperature, dissolved in PBS buffer 131 Radiochemical purity of I-Au PCNPs and 131 Radiochemical purity of I-Au PHCNPs at different time points at 37 °C, dissolved in FBS 131 Radiochemical purity of I-Au PCNPs and 131 Radiochemical purity of I-Au PHCNPs.

[0044] Figure 11Flow cytometry detection results after 4T1-HCD44 cells were incubated with (a) PBS, (b) Au PCNPs and (c) Au PHCNPs, 4T1-LCD44 cells were incubated with (d) Au PCNPs and (e) Au PHCNPs for 2 h, respectively; and (f) the average fluorescence intensity chart of 4T1-HCD44 cells and 4T1-LCD44 cells treated with PBS, Au PCNPs and Au PHCNPs for 2 h, respectively. The concentration of all materials was 5 μM.

[0045] Figure 12 Laser scanning confocal microscope images of 4T1-LCD44 and 4T1-HCD44 cells treated with PBS, Au PCNPs and Au PHCNPs for 2 h, respectively.

[0046] Figure 13 ICP-OES analysis chart of 4T1-LCD44 and 4T1-HCD44 cells incubated with Au PHCNPs with gold concentration of 0, 25, 50, 100 μM for 2 h, respectively.

[0047] Figure 14 (a) Au PCNPs and Au PHCNPs with different gold concentrations, and (b) I-Au PCNPs and I-Au PHCNPs with different radioactivity concentrations. 131 I-Au PCNPs, 131 I-Au PHCNPs incubated with 4T1 cells for 24 h, and the 4T1 cell viability detected by CCK-8 method.

[0048] Figure 15 Inverted biological microscope images of 4T1 cells treated with (a) PBS, (b) Au PCNPs with gold concentration of 20 μM, (c) Au PHCNPs with gold concentration of 20 μM, (d) Au PCNPs with gold concentration of 100 μM, (e) Au PHCNPs with gold concentration of 100 μM, (f) I-Au PCNPs with radioactivity concentration of 50 μCi / mL, (g) I-Au PHCNPs with radioactivity concentration of 50 μCi / mL, (h) I-Au PCNPs with radioactivity concentration of 200 μCi / mL, (i) I-Au PHCNPs with radioactivity concentration of 200 μCi / mL for 24 h, respectively. 131 I-Au PCNPs and (g) 131 I-Au PHCNPs, (h) 131 I-Au PCNPs and (i) 131 I-Au PHCNPs for 24 h, respectively.

[0049] Figure 16 (a) CT cross-sectional imaging chart of 4T1 tumor-bearing mice at different time points after treated with Au PCNPs and Au PHCNPs, respectively, and (b) CT value of the corresponding tumor site. The white "*" in the chart indicates the tumor position.

[0050] Figure 17 (a) Fluorescence imaging of 4T1 tumor-bearing mice at different time points after injection of Au PCNPs and Au PHCNPs, and (b) fluorescence intensity values ​​of the corresponding tumor sites. (c) The ratio of tumor to muscle fluorescence intensity in 4T1 tumor-bearing mice at 4 h and 6 h after injection of Au PCNPs and Au PHCNPs, respectively. (d) Fluorescence imaging of 4T1 tumor-bearing mice ex vivo from tumors and major organs at 24 h after injection of Au PCNPs and Au PHCNPs, and (e) the corresponding fluorescence intensity.

[0051] Figure 18 To be injected separately 131 I-Au PCNPs and 131 (a) In vivo SPECT images of 4T1 tumor-bearing mice at different time points after I-Au PHCNPs and (b) TBR values ​​of the corresponding SPECT signal intensity. 4T1 tumor-bearing mice were injected with... 131 I-AuPCNPs and 131 (c) Radiation intensity of different organs after 24 h of I-Au PHCNPs, (d) SPECT imaging of the ex vivo tumor, and (e) corresponding SPECT signal intensity. Circles indicate tumor location.

[0052] Figure 19 The curves show the changes in (a) relative tumor volume and (b) relative body weight of tumor-bearing mice in different treatment groups.

[0053] Figure 20 H&E staining and TUNEL staining images of tumors in tumor-bearing mice in different treatment groups (scale bar in the figure is 100 μm).

[0054] Figure 21 The tumor cell apoptosis rate corresponding to the TUNEL staining images of tumors in tumor-bearing mice in different treatment groups.

[0055] Figure 22 The gold content in major organs (heart, liver, spleen, lung, kidney) and tumors at different time points (0, 24, 48, 72 h) after Au PHCNP injection. Detailed Implementation

[0056] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and substance of the invention are within the scope of the present invention. Experimental methods and reagents not specifically described in the embodiments are performed according to conventional conditions in the art.

[0057] Example 1

[0058] (1) Dissolve 32 mg of NH2-PEG-COOH (Mw = 2000) in 10 mL of distilled water, and add 15.34 mg of EDC and 9.20 mg of NHS dissolved in 4 mL of distilled water successively dropwise while stirring, wherein the ratio of the sum of the amounts of substances of EDC and NHS to the amount of substance of NH2-PEG-COOH is 5:1. After stirring for 3 hours, add 20 mg of PEI.NH2 (Mw = 25000) dissolved in 10 mL of distilled water to the solution, wherein the ratio of the amount of substance of NH2-PEG-COOH to the amount of substance of PEI.NH2 is 20:1. After stirring for 3 days, the resulting solution is dialyzed for 1 day in a phosphate buffer using a cellulose dialysis membrane (MWCO = 8000-14000), and then dialyzed for 2 days in distilled water, and the solution used for dialysis is changed 3 times a day during the dialysis, and finally freeze-dried to obtain PEI.NH2-PEG-NH2.

[0059] (2) Dissolve 182.28 mg of HA (Mw = 10000) in 18 mL of distilled water, and add 17.47 mg of EDC and 10.48 mg of NHS dissolved in 2 mL of distilled water successively dropwise while stirring, wherein the ratio of the sum of the amounts of substances of EDC and NHS to the amount of substance of HA is 5:1. After stirring for 3 hours, add 35 mg of PEI.NH2-PEG-NH2 dissolved in 12 mL of distilled water to the solution, wherein the ratio of the amount of substance of HA to the amount of substance of PEI.NH2-PEG-NH2 is 30:1. After stirring for 3 days, the resulting solution is dialyzed for 1 day in a phosphate buffer using a cellulose dialysis membrane (MWCO = 8000-14000), and then dialyzed for 2 days in distilled water, and the solution used for dialysis is changed 3 times a day during the dialysis, and finally freeze-dried to obtain PEI.NH2-(PEG-HA).

[0060] (3) 12 mL of distilled water was used to dissolve 120 mg of PEI.NH2-(PEG-HA) with a dry weight, to obtain an aqueous solution of PEI.NH2-(PEG-HA). 12 mL of DMSO was used to dissolve 1.51 mg of HPAO with a dry weight, to obtain a DMSO solution of HPAO. Under stirring conditions, the aqueous solution of PEI.NH2-(PEG-HA) and the DMSO solution of HPAO were added alternately and sequentially, and the two solutions were mixed in equal volumes, wherein the molar ratio of HPAO to PEI.NH2-(PEG-HA) was 10:1. After stirring for 24 h, the obtained solution was dialyzed in a cellulose dialysis membrane (MWCO = 8000-14000) in a phosphate buffer for 1 day, and then in distilled water for 2 days, and the dialysis solution was changed 3 times a day during dialysis. Finally, the freeze-drying treatment was performed to obtain PEI.NH2-(PEG-HA)-HPAO.

[0061] (4) 8 mL of distilled water was used to dissolve 80 mg of PEI.NH2-(PEG-HA)-HPAO with a dry weight, and 4.73 mg of Cy7 with a dry weight was added dropwise in 10 mL of distilled water under stirring and light shielding conditions, wherein the molar ratio of Cy7 to PEI.NH2-(PEG-HA)-HPAO was 16:1. After stirring for 3 days under light shielding conditions, the obtained solution was dialyzed in a cellulose dialysis membrane (MWCO = 8000-14000) in a phosphate buffer for 1 day, and then in distilled water for 2 days, and the dialysis solution was changed 3 times a day during dialysis. Finally, the freeze-drying treatment was performed to obtain PEI.NH2-(PEG-HA)-Cy7-HPAO.

[0062] (5) 6 mL of distilled water was used to dissolve 60 mg of PEI.NH2-(PEG-HA)-Cy7-HPAO with dry weight, and light protection was performed. 381.04 μL of chloroauric acid solution (30 mg / mL) was added dropwise while stirring, and the molar ratio of chloroauric acid to PEI.NH2-(PEG-HA)-Cy7-HPAO was 100:1. After stirring for 30 min, 5.25 mg of NaBH4 dissolved in 2 mL of distilled water was quickly added dropwise, and the solution turned wine red instantly, and the molar ratio of chloroauric acid to NaBH4 was 1:5. Subsequently, 7.72 μL of N(C2H5)3 was added to the reaction solution under light protection at room temperature after stirring for 2 h, and 5.21 μL of Ac2O was added after stirring for 30 min, and the reaction was stirred for 24 h under light protection at room temperature. Finally, the obtained solution was dialyzed with a cellulose dialysis membrane (MWCO = 8000-14000) in a phosphate buffer for 1 day, and then dialyzed in distilled water for 2 days, and the dialysis solution was replaced every day during dialysis. After dialysis, the solution was freeze-dried to obtain [(Au 0 ) 100 -PEI.NHAc-(PEG-HA)-Cy7-HPAO] NPs nanoparticles (Au PHCNPs).

[0063] (6) The obtained Au PHCNPs nanoparticles (150 μg) were dissolved in 250 μL of PBS, and then 150 μg of chloramine T and radioactive 370 MBq of Na 131 I (500 μL) were added, and the reaction was stirred for 3 min. Then, Na2S2O5 (150 μg) and KI (100 μg) were added while stirring, and the reaction was performed for 5 min. Finally, the radioactive activity liquid was separated and purified by a PD-10 desalting column, and the Au PHCNPs labeled with 131 I were obtained by collecting the separated radioactive activity liquid with PBS (pH = 7.0-7.4) as the mobile phase. 131 I-Au PHCNPs). The reaction process is shown in the attached Figure 1 .

[0064] During the synthesis process, the obtained intermediate products were characterized by nuclear magnetic resonance hydrogen spectrum. According to the integral calculation of each peak, it can be known that 16.3 molecules of PEG (characteristic peak at 3.5-3.75 ppm), 15.2 molecules of HA (characteristic peak at 1.8-2.0 ppm), and 6.3 molecules of HPAO (characteristic peak at 6.6-6.8, 6.9-7.1 ppm) were modified on each molecule of PEI.NH2. The attached Figure 2The intermediate product PEI.NH2-(PEG-HA)-Cy7-HPAO was characterized by FTIR, fluorescence, and UV-Vis spectroscopy. FTIR spectroscopy results: After the reaction of Sulfo-Cy7 NHS ester with PEI.NH2-(PEG-HA)-HPAO, the fluorescence intensity was significantly higher in the 1700-1750 cm⁻¹ range. -1 The absorption peak of the C=O double bond stretching vibration of the NHS group attributed to Sulfo-Cy7 NHS ester disappeared. Simultaneously, the absorption peaks of the product PEI.NH2-(PEG-HA)-Cy7-HPAO at 1516 and 1206 cm⁻¹ disappeared. -1 The absorption peaks are attributed to the conjugated C=C double bonds and -SO3 in the Cy7 molecule, respectively. - The absorption peak is generated by the stretching vibration of S=O. All the above FTIR characteristics indicate that Cy7 was successfully modified on the PEI.NH2 surface. See the appendix of the instruction manual. Figure 3 Fluorescence spectroscopy characterization results: Under 750 nm excitation light, the PEI.NH2-(PEG-HA)-Cy7-HPAO solution and the Cy7 solution showed almost identical fluorescence emission peaks, proving that the Cy7 modification was successful. (See the instruction manual appendix.) Figure 4 UV-Vis spectral characterization results: The PEI.NH2-(PEG-HA)-Cy7-HPAO prepared in this invention has a strong absorption peak at 763 nm, which is almost identical to the absorption peak of Cy7, proving that Cy7 was successfully modified on the PEI.NH2 surface. Furthermore, UV quantitative analysis shows that one molecule of PEI.NH2 is modified with 6.1 molecules of Cy7. (See attached specification). Figure 5 Meanwhile, the UV-Vis spectra of the prepared Au PHCNPs nanoparticles showed strong absorption peaks at 534 nm and 763 nm, respectively, which are attributed to the surface plasmon resonance (SPR) peak unique to Au NPs and the characteristic UV absorption peak of Cy7, indicating that PEI.NH2-(PEG-HA)-Cy7-HPAO successfully encapsulated the gold nanoparticles. (See the attached instruction manual.) Figure 5 d.

[0065] Comparative Example 1

[0066] (1) Dissolve 32 mg of the product in 10 mL of distilled water. m PEG-COOH (Mw = 2000) was stirred while simultaneously adding dropwise 15.34 mg (dry weight) of EDC dissolved in 4 mL of distilled water and 9.20 mg (dry weight) of NHS dissolved in 4 mL of distilled water. The sum of the moles of EDC and NHS was... mThe molar ratio of PEG-COOH to PEI.NH2was 20:1. After stirring for 3 days, the resulting solution was dialyzed against phosphate buffer using a cellulose dialysis membrane (MWCO = 8000-14000) for 1 day, and then against distilled water for 2 days, with the dialysis solution being changed 3 times per day during dialysis, and finally freeze-dried to obtain PEI.NH2- m The molar ratio of PEG-COOH to PEI.NH2was 20:1. After stirring for 3 days, the resulting solution was dialyzed against phosphate buffer using a cellulose dialysis membrane (MWCO = 8000-14000) for 1 day, and then against distilled water for 2 days, with the dialysis solution being changed 3 times per day during dialysis, and finally freeze-dried to obtain PEI.NH2- m PEG.

[0067] (2) 30 mg of PEI.NH2- m PEG was dissolved in 10 mL of distilled water to obtain an aqueous solution of PEI.NH2- m PEG. 1.38 mg of HPAO was dissolved in 10 mL of DMSO to obtain a DMSO solution of HPAO. Under stirring, the aqueous solution of PEI.NH2- m PEG and the DMSO solution of HPAO were added alternately and sequentially in equal volumes to mix the two solutions, with the molar ratio of HPAO to PEI.NH2- m PEG being 10:1. After stirring for 24 h, the resulting solution was dialyzed against phosphate buffer using a cellulose dialysis membrane (MWCO = 8000-14000) for 1 day, and then against distilled water for 2 days, with the dialysis solution being changed 3 times per day during dialysis, and finally freeze-dried to obtain PEI.NH2- m PEG-HPAO.

[0068] (3) 20 mg of PEI.NH2- m PEG-HPAO was dissolved in 5 mL of distilled water, and 4.73 mg of Cy7 was added dropwise to the solution while stirring in the dark, with the molar ratio of Cy7 to PEI.NH2- m PEG-HPAO being 16:1. After stirring for 3 days in the dark, the resulting solution was dialyzed against phosphate buffer using a cellulose dialysis membrane (MWCO = 8000-14000) for 1 day, and then against distilled water for 2 days, with the dialysis solution being changed 3 times per day during dialysis, and finally freeze-dried to obtain PEI.NH2- m PEG-Cy7-HPAO.

[0069] (4) 12 mg of PEI.NH2- mPEG-Cy7-HPAO was prepared and protected from light. 259.18 μL of chloroauric acid solution (30 mg / mL) was added dropwise while stirring. The chloroauric acid reacts with PEI·NH2-. m The molar ratio of PEG-Cy7-HPAO was 100:1. After stirring for 30 min, 3.57 mg (dry weight) of NaBH4 dissolved in 2 mL of distilled water was rapidly added dropwise, and the solution instantly turned wine-red. The molar ratio of chloroauric acid to NaBH4 was 1:5. After stirring for 2 h in the dark at room temperature, 5.25 μL of N(C2H5)3 was added to the reaction solution. After stirring for 30 min in the dark at room temperature, 3.55 μL of Ac2O was added, and the reaction was carried out for 24 h in the dark at room temperature. Finally, the resulting solution was dialyzed against a cellulose dialysis membrane (MWCO = 8000-14000) in phosphate buffer for 1 day, and then against distilled water for 2 days. The dialysis solution was changed 3 times a day during the dialysis period. Finally, the solution was freeze-dried to obtain [(Au 0 ) 100 -PEI.NHAc- m PEG-Cy7-HPAO]NPs nanoparticles (Au PCNPs).

[0070] (5) Dissolve the obtained Au PCNPs nanoparticles (150 μg) in 250 μL of PBS, then add 150 μg of chloramine T and radioactive 370 MBq of Na. 131 I (500 μL), after stirring for 3 minutes, Na2S2O5 (150 μg) and KI (100 μg) were added while stirring, and the reaction was continued for 5 minutes. Finally, the mixture was separated and purified by a PD-10 desalting column using PBS (pH = 7.0-7.4) as the mobile phase. The separated radioactive liquid was collected to obtain... 131 Au PCNPs with I-label ( 131 I-Au PCNPs). See the instruction manual for the reaction procedure. Figure 1 .

[0071] During the synthesis process, the comparative support material was characterized using 1H NMR spectroscopy. Integral calculations of each peak revealed that one molecule of PEI.NH2 was modified with 16.1 molecules of... m PEG and 6.1 molecule HPAO. And in the labeling 131 After I, the test results were formed 131 The radiochemical purity of I-Au PCNPs is above 94%, indicating successful labeling. 131 I.

[0072] This invention uses nuclear magnetic resonance hydrogen spectroscopy (NMR 1H spectroscopy) 1H NMR), Fourier transform infrared spectroscopy (FTIR), fluorescence spectroscopy, ultraviolet-visible spectroscopy (UV-Vis), transmission electron microscopy (TEM), X-ray energy dispersive spectroscopy (EDS), dynamic light scattering (DLS), X-ray attenuation test, and instant thin layer chromatography (ITLC) on the prepared 131 I-Au PHCNPs nanoparticles were subjected to basic characterization; the prepared nanoparticles were evaluated for their targeted tumor imaging performance, in vivo targeted tumor radionuclide treatment effect, and biological safety. Specifically as follows: 131 I-Au PHCNPs nanoparticles were subjected to basic characterization; the prepared nanoparticles were evaluated for their targeted tumor imaging performance, in vivo targeted tumor radionuclide treatment effect, and biological safety. Specifically as follows:

[0073] 1. The Au PHCNPs nanoparticles prepared in Example 1 were dissolved in water, and their ultraviolet absorption in the 400-900 nm waveband was determined by ultraviolet spectrophotometry. The results showed that the prepared Au PHCNPs particles had a characteristic absorption peak at 534 nm, which was attributed to the surface plasmon resonance (SPR) of the gold nanoparticles, proving that gold nanoparticles had been successfully prepared. See the accompanying drawings Figure 5 d. The TEM image showed that the gold nanoparticles had a spherical morphology, a diameter of 3.65 ± 0.84 nm, a narrow size distribution, and good dispersibility. The SAED test results proved the face-centered cubic crystal structure, and the EDS further proved the presence of gold elements in the product. These test results showed that Au PHCNPs nanoparticles had been successfully prepared. See the accompanying drawings Figure 6 .

[0074] 2. The stability of the material was analyzed by ultraviolet-visible absorption spectroscopy. The Au PHCNPs nanoparticles prepared in Example 1 were prepared into a 0.2 mg / mL solution, and their ultraviolet-visible absorption spectra were determined under the conditions of pH 5, 6, 7, and 10, temperature 6°C, 25°C, 37°C, and 50°C, and at different time points (1st, 3rd, 5th, and 7th days). From the spectra, it can be seen that the peak shape is consistent and there is no shift phenomenon, indicating that the Au PHCNPs nanoparticles can maintain good colloidal stability under different pH, different temperature conditions, and for a long time. See the accompanying drawings Figure 7The Au PHCNPs nanoparticles prepared in Example 1 were dispersed in water and PBS respectively and diluted to obtain 0.1 mg / mL solutions in water and PBS respectively. The hydrodynamic size distribution of the Au PHCNPs dispersed in PBS and water at different time points was then determined by DLS method. It can be found that the hydration particle size and PDI of the Au PHCNPs nanoparticles do not change significantly, indicating that the Au PHCNPs nanoparticles dispersed in water and PBS can maintain good colloidal stability and uniformity within a certain period of time, see the accompanying drawings Figure 8 .

[0075] 3. The prepared Au PHCNPs were dissolved in aqueous solution to prepare 200 μL solutions with gold concentrations of 0.1 M, 0.08 M, 0.04 M, 0.02 M, and 0.01 M. At the same time, a small molecule iodine contrast agent commonly used in clinics, Omnipaque, was used as a control. The medical Omnipaque was diluted to solutions with iodine concentrations of 0.1 M, 0.08 M, 0.04 M, 0.02 M, and 0.01 M. Then, the CT imaging images and CT signal values of Au PHCNPs and Omnipaque were determined using a clinical medical CT imaging system, and the X-ray attenuation coefficients of Au PHCNPs and Omnipaque were compared. The results show that the X-ray attenuation intensity of Au PHCNPs is higher than that of Omnipaque at the same gold or iodine concentration. The results show that the prepared Au PHCNPs nanoparticles have higher X-ray attenuation coefficients and more excellent CT imaging performance than Omnipaque, see the accompanying drawings Figure 9 .

[0076] 4. After marking 131 I, the radiochemical stability of the obtained products 131 I-Au PCNPs and 131 I-Au PHCNPs was evaluated using ITLC. 100 μL of the radiolabeled 131 I-Au PCNPs and 131 I-Au PHCNPs were mixed with 1 mL of PBS, FBS respectively, and then ITLC was used to test the radiochemical purity of the PBS solutions of 131 I-Au PCNPs and 131 I-Au PHCNPs at different time points (0.5, 4, 8, 16 h) at room temperature, and the FBS solutions of 131 I-Au PCNPs and 131 I-Au PHCNPs at 37°C. The results show that in both PBS and FBS systems, at different time points, 131 I-Au PCNPs and 131The radiochemical purity of the I-Au PHCNPs is all above 94%, which indicates that there is no large amount of 131 I detached from the support, i.e. the prepared 131 I-Au PCNPs and 131 I-Au PHCNPs have good in vitro radiochemical stability, see the attached Figure 10 .

[0077] 5. The targeting specificity of the prepared Au PHCNPs nanoparticles to CD44 receptor overexpressing 4T1 cells was detected by flow cytometry. First, 4T1 cells were seeded in a 12-well plate (2 × 10 5 cells / well) and incubated for 24 h. Then the culture medium was discarded and the obtained cells were divided into two groups, one group was added with HA (10 mg / mL) containing medium and the other group was added with the same amount of pure medium. After 1 h of incubation, 4T1-LCD44 cells and 4T1-HCD44 cells were obtained, respectively. The Au PHCNPs nanoparticles prepared in Example 1 and the Au PCNPs nanoparticles prepared in Comparative Example 1 were dispersed in the culture medium (the final concentration of the material was 5 μM). Then the 4T1-LCD44 cells and 4T1-HCD44 cells were incubated with PBS, Au PCNPs and Au PHCNPs containing medium (200 μL) for 2 h, respectively. After the incubation, the cells were washed with PBS for three times. Then the cells were trypsinized and centrifuged to collect PBS treated 4T1-HCD44 cells, Au PCNPs treated 4T1-LCD44 and 4T1-HCD44 cells, Au PHCNPs treated 4T1-LCD44 and 4T1-HCD44 cells, totally 5 groups of cells, each group had three parallel samples. Then the collected cells in each group were suspended in 1 mL PBS and the flow cytometer was used to detect the samples of each group of cells. The results showed that, compared with the fluorescence intensity of the PBS group, the fluorescence intensity of 4T1-LCD44 and 4T1-HCD44 cells did not increase after 2 h of incubation with Au PCNPs, while the fluorescence intensity of 4T1-HCD44 cells incubated with Au PHCNPs was significantly enhanced and was significantly higher than that of 4T1-LCD44 cells incubated with Au PHCNPs (p < 0.001). Based on the HA mediated targeting, Au PHCNPs can achieve the targeted specificity of uptake of cancer cells with high expression of CD44 receptor, see the attached Figure 11 .

[0078] 6. The targeting specificity of the prepared Au PHCNPs nanoparticles to CD44 receptor overexpressing 4T1 cells was further proved by laser scanning confocal microscopy. First, 4T1 cells were seeded in a 12-well plate (8 × 104 After 24 h incubation, the culture medium was discarded, and the obtained cells were divided into two groups, one group was added with culture medium containing HA (10 mg / mL), and the other group was added with the same amount of pure culture medium, and 4T1-LCD44 cells and 4T1-HCD44 cells were obtained respectively after 1 h incubation. Then 4T1-LCD44 cells and 4T1-HCD44 cells were incubated with culture medium containing PBS, Au PCNPs, Au PHCNPs (material concentration 5 μM) respectively for 2 h. After incubation, the culture medium was discarded and washed with PBS for three times, then 500 μL of 2.5% glutaraldehyde was added to each well for fixation for 15 min, and then washed with PBS for 2-3 times, 500 μL of DAPI (1 μg / mL) reagent was added for staining for 20 min, and then washed with PBS for 2-3 times, and finally the cells on the coverslips were mounted on the glass slides, and photographed under the laser scanning confocal microscope. It can be clearly seen from the laser scanning confocal microscope pictures that 4T1-LCD44 cells and 4T1-HCD44 cells incubated with Au PCNPs and 4T1-LCD44 cells incubated with Au PHCNPs did not show obvious fluorescence signals, while 4T1-HCD44 cells incubated with Au PHCNPs showed strong red fluorescence signals, and the fluorescence was mainly distributed in the cytoplasm. The results show that Au PHCNPs can be specifically taken up into the cytoplasm by 4T1-HCD44 cells. At the same time, based on the specific binding property of HA and CD44 receptor, the uptake amount of Au PHCNPs by 4T1-HCD44 cells is increased. This result is consistent with the flow cytometry test result, see the description accompanying drawings Figure 12 .

[0079] 7、To further verify that the modification of HA can improve the targeting of Au PHCNPs nanoparticles to CD44 receptor overexpressing cancer cells and increase the uptake of Au PHCNPs nanoparticles by cancer cells, the gold uptake amount of 4T1-LCD44 cells and 4T1-HCD44 cells after incubation with Au PHCNPs nanoparticles of different gold concentrations was detected by ICP-OES. First, 4T1 cells were inoculated in 12-well plates (2 × 10 5Cells were incubated at 1000 cells / well for 24 h. The culture medium was then discarded, and the resulting cells were divided into two groups. One group was incubated with culture medium containing HA (10 mg / mL), and the other group was incubated with an equal volume of pure culture medium. After incubation for 1 h, 4T1-LCD44 cells and 4T1-HCD44 cells were obtained, respectively. Au PHCNPs nanoparticles prepared in Example 1 were dissolved in culture medium and gradually diluted to obtain Au PHCNPs culture medium solutions with gold concentrations of 25, 50, and 100 μM. Then, 4T1-LCD44 cells and 4T1-HCD44 cells were incubated for 2 h with PBS and culture medium containing different gold concentrations of Au PHCNPs, respectively. After incubation, the cells were washed three times with PBS. Cells were then digested with trypsin and resuspended in 1 mL of cell culture medium for cell counting. Finally, the cells were collected by centrifugation, digested with aqua regia, and after complete lysis, diluted to 3 mL with distilled water. The gold content in each cell sample was tested using ICP-OES. The results showed that the uptake of Au PHCNPs nanoparticles by both cell types gradually increased with increasing gold concentration. Specifically, at gold concentrations of 50 μM and 100 μM, the gold uptake by 4T1-HCD44 cells was significantly higher than that by 4T1-LCD44 cells (p < 0.001). This result is consistent with the results of flow cytometry and laser scanning confocal microscopy, demonstrating that the high uptake of Au PHCNPs nanoparticles by 4T1-HCD44 cells can be achieved due to the HA-mediated targeting properties to CD44 receptor-overexpressing cancer cells. (See the instruction manual appendix.) Figure 13 .

[0080] 8. To test the in vitro biocompatibility of the prepared HA-targeted Au PHCNPs material and 131 The cytotoxicity of I-AuPHCNPs materials was determined using the CCK-8 assay with different gold concentrations of Au PCNPs, Au PHCNPs, and different radioactive concentrations. 131 I-Au PCNPs 131 Cell viability of 4T1 cells after 24 hours of incubation with I-Au PHCNPs. 4T1 cells were seeded in 96-well plates (1×10⁶ cells / wells). 4 Cells / well), and incubated overnight with 200 μL of medium supplemented with 10% fetal bovine serum. Then, they were incubated with different gold concentrations (0, 5, 10, 20, 50, and 100 μM) of Au PHCNPs and Au PCNPs, and different radioactive concentrations (0, 10, 25, 50, 100, and 200 μCi / mL). 131 I-Au PHCNPs, 131I-Au PCNPs treated cells for 24 h. Subsequently, the absorbance of cells at 450 nm wavelength was measured by CCK-8 method to evaluate the toxicity of materials to cells. The results showed that the cell viability of cells incubated with Au PCNPs, Au PHCNPs maintained above 90% in a certain gold concentration range, indicating that the material Au PHCNPs as well as Au PCNPs had good biocompatibility, see the attached drawings Figure 14 a. Materials 131 I-Au PHCNPs and 131 I-Au PCNPs showed obvious cytotoxicity, and when the radioactivity concentration was 100 μCi / mL, 200 μCi / mL, the cell viability of 4T1 cells treated with 131 I-Au PHCNPs was significantly lower than 131 I-Au PCNPs treated 4T1 cells (p < 0.001). The results showed that the cytotoxicity of 131 I-Au PHCNPs was mainly derived from the labeled radionuclide 131 I, while based on the targeting of HA-mediated, 131 I-Au PHCNPs were specifically taken up by 4T1 cells overexpressing CD44 receptors, thereby achieving the in vitro targeted cancer cell anticancer effect, see the attached drawings Figure 14 b.

[0081] Similarly, 4T1 cells were incubated with Au PHCNPs, Au PCNPs of different gold concentrations (20 and 100 μM) and 131 I-Au PHCNPs, 131 I-Au PCNPs of different radioactivity concentrations (50 and 200 μCi / mL) for 24 h. The cell morphology was observed by inverted biological microscope to further verify the biocompatibility and 131 I-AuPCNPs, 131 I-Au PHCNPs of in vitro targeted cancer cell anticancer effect. The results showed that the 4T1 cells incubated with PBS and Au PHCNPs, Au PCNPs of Au concentration of 20, 100 μM had good morphology. The 4T1 cells incubated with 131 I-Au PHCNPs, 131 I-Au PCNPs of radioactivity concentration of 50, 200 μCi / mL, the cells were rounded and had obvious signs of apoptosis. And when the radioactivity concentration was 200 μCi / mL, 131 I-Au PHCNPs treated 4T1 cells were significantly more than 131The number of 4T1 cell apoptosis after I-Au PCNPs treatment. The results show that, 131 The labeling of I endows the material with good radionuclide treatment effect on cancer cells, while based on HA-mediated targeting, 131 I-Au PHCNPs can be specifically high uptaken by 4T1 cells overexpressing CD44 receptors, achieving targeted anticancer effect on cancer cells in vitro, see the attached drawings of the specification Figure 15 .

[0082] 9, Based on the good X-ray attenuation characteristics of the material Au PHCNPs and the targeting of CD44 receptor overexpressing cancer cells in vitro, the in vivo tumor targeting CT imaging performance of Au PHCNPs will be tested. Select 4-6 week old adult female BALB / c nude mice (20-25 g) and divide them into two groups (n = 3), subcutaneously inoculate 4T1 cells (3 × 10 6 cells / mouse), and after 3 weeks, when the tumor grows to 0.5-1.2 cm 3 , it can be used for in vivo targeted tumor CT imaging research. First, pentobarbital sodium (40 mg / kg) is injected intraperitoneally into the two groups of tumor-bearing mice for anesthesia, and then Au PHCNPs ([Au] = 68.4 mM, 100 μL) PBS solution is injected into one group of tumor-bearing mice, and Au PCNPs ([Au] = 68.4 mM, 100 μL) PBS solution is injected into the other group of tumor-bearing mice. At different time points (0, 0.5, 1, 2, 4, 6, 8, 12, 16, 24 h), the tumor-bearing mice are scanned by the MicroCT imaging system for in vivo CT imaging. As can be seen from the figure, the CT image brightness and the corresponding CT signal value of the tumor site of the two groups of tumor-bearing mice treated with Au PHCNPs and Au PCNPs respectively show a trend of first enhancement and then weakening, reaching a peak at 6 h. And 6 h after injection, the CT signal value of the tumor site of the tumor-bearing mice treated with Au PHCNPs is significantly higher than that of the tumor site of the tumor-bearing mice treated with Au PCNPs (p < 0.001). The results show that based on HA-mediated targeting, HA-modified Au PHCNPs can be specifically delivered to the tumor site and highly enriched in the tumor site, achieving in vivo targeted tumor CT imaging, see the attached drawings of the specification Figure 16 .

[0083] 10、The foregoing results have demonstrated that the modification of Cy7 endows Au PHCNPs and Au PCNPs with excellent in vitro fluorescence imaging performance. Meanwhile, based on the demonstrated targeting specificity of Au PHCNPs to CD44 receptor overexpressing cancer cells, the in vivo targeted tumor fluorescence imaging performance of Au PHCNPs will be tested. The tumor model is established by the foregoing method, and the tumor-bearing mice are divided into two groups (n = 3). All the tumor-bearing mice are anesthetized, and then the tail vein injection method is adopted to inject the PBS solution of Au PHCNPs (5 nmol, 100 μL) into one group of tumor-bearing mice and the PBS solution of Au PCNPs (5 nmol, 100 μL) into the other group of tumor-bearing mice. At different time points (0.5, 1, 2, 4, 6, 8, 12, 16, 24 h), the fluorescence imaging of the tumor-bearing mice is performed using the in vivo imaging system (IVIS) (EX = 730-750 nm, EM = 750-780 nm), and the fluorescence values of the target sites are recorded. After 24 h of injection of the materials, the two groups of tumor-bearing mice are euthanized, and the main organs (heart, liver, spleen, lung, kidney) and tumor tissues are obtained, and the fluorescence imaging is performed and the corresponding fluorescence intensity values are recorded. The in vivo FL imaging results show that, at 6 h after administration, the fluorescence brightness and the corresponding fluorescence signal values of the tumor sites of the 4T1 tumor-bearing mice treated with Au PHCNPs and Au PCNPs respectively reach the peak values, and the fluorescence signal intensity of the tumor site of the 4T1 tumor-bearing mice treated with Au PHCNPs is significantly higher than that of the 4T1 tumor-bearing mice treated with Au PCNPs (p < 0.001). The T / M value quantitative analysis results show that, at 4 and 6 h after injection, the T / M values of the 4T1 tumor-bearing mice treated with Au PHCNPs are significantly higher than those of the 4T1 tumor-bearing mice treated with Au PCNPs (p < 0.001). At the same time, the fluorescence imaging of the ex vivo organs and tumors and the corresponding fluorescence signal intensity value quantitative analysis results show that, in addition to the tumor, the materials also have a higher uptake in the liver, lung and kidney organs. In addition, it can be clearly seen that the fluorescence brightness and the fluorescence signal intensity values of the tumor site of the 4T1 tumor-bearing mice treated with Au PHCNPs are significantly higher than those of the 4T1 tumor-bearing mice treated with Au PCNPs. The above results show that, based on the HA-mediated targeting effect, Au PHCNPs can be specifically delivered to the tumor site and highly enriched at the tumor site, thereby realizing higher sensitivity in vivo targeted tumor FL imaging, see the accompanying drawings Figure 17 .

[0084] 11、Based on the prepared materials 131 I-Au PHCNPs have good radioactivity stability and in vitro targeting specificity to CD44 receptor overexpressing cancer cells, which will be further tested 131I-Au PHCNPs in vivo targeted tumor SPECT imaging performance. A tumor model was established using the aforementioned method, and tumor-bearing mice were divided into two groups (n = 3). The tumor-bearing mice were anesthetized, and then the I-Au PHCNPs were injected via tail vein. 131 I-Au PHCNPs ([ 131 A PBS solution (I = 2.5 mCi / mL, 100 μL) was injected into a group of tumor-bearing mice. 131 I-Au PCNPs ([ 131 A PBS solution containing 2.5 mCi / mL (100 μL) was injected into another group of tumor-bearing mice. The mice were scanned using a SPECT imaging system at different time points (0.5, 1, 2, 4, 6, 8, 12, 16, 24 h) for in vivo SPECT imaging, and the SPECT signal values ​​of the tumor and background at the corresponding time points were recorded to obtain the TBR value. Twenty-four h after injection, both groups of tumor-bearing mice were euthanized, and the radioactivity of major organs (heart, liver, spleen, lung, kidney, stomach, intestine, and soft tissue) and tumors was tested using a gamma counter. Finally, the ex vivo tumors from both groups of tumor-bearing mice were individually SPECT-imagined, and the corresponding relative SPECT signal intensities were recorded. The in vivo SPECT imaging showed that 6 h after administration, the tumor signal intensity was significantly higher than that of the tumors in the PBS solution containing 2.5 mCi / mL (100 μL). 131 The signal values ​​of tumor sites in tumor-bearing mice treated with I-Au PHCNPs were significantly higher than those treated with... 131 Signal values ​​of tumor sites in tumor-bearing mice treated with I-Au PCNPs. Quantitative analysis of TBR values ​​showed that, compared to those treated with... 131 Tumor-bearing mice treated with I-Au PCNPs, within the study time range (0.5-24h), after... 131 Tumor-bearing mice treated with I-Au PHCNPs exhibited higher TBR values. Quantitative analysis of the radioactivity intensity of isolated tumors and various organs revealed a large amount of... 131 I-Au PHCNPs and 131 I-Au PCNPs are distributed in the liver and intestines, with smaller amounts in the lungs, stomach, spleen, kidneys, and tumors, and even less in the heart and soft tissues. 131 The accumulation of I-Au PHCNPs at the tumor site is significantly higher than that of... 131 The accumulation of I-Au PCNPs at the tumor site, therefore 131 Tumor sites in tumor-bearing mice treated with I-Au PHCNPs showed higher radioactivity (p < 0.001). Finally, ex vivo tumor SPECT imaging and quantitative analysis of relative signal intensity showed that... 131 I-Au PHCNPs-treated tumor-bearing mice showed stronger SPECT signals at the tumor sites, with significantly higher relative SPECT signal intensity at the tumor sites compared to mice treated with other methods. 131The relative SPECT signal intensity at the tumor site in tumor-bearing mice treated with I-Au PCNPs (p < 0.001). These results indicate that, based on HA-mediated targeting, 131 I-Au PHCNPs can be specifically delivered to tumor sites, enabling highly sensitive in vivo targeted tumor SPECT imaging. See the instruction manual for details. Figure 18 .

[0085] 12. Establish a tumor model using the aforementioned method, and wait for the tumor volume to grow to 0.5-1.2 cm. 3 Thirty tumor-bearing mice were randomly divided into 6 groups (n = 5). Subsequently, the tumor was injected via tail vein into each group. 131 I-Au PHCNPs ([ 131 I] = 1.0 mCi / mL, 200 μL), 131 I-Au PCNPs ([ 131 I] = 1.0 mCi / mL, 200 μL), Au PHCNPs (200 μL and 131 I-Au PHCNPs concentration was the same), Au PCNPs (200 μL and 131 I-Au PCNPs concentration is the same), Na 131 I ([ 131 Six groups of tumor-bearing mice were injected with physiological saline solution (I = 1.0 mCi / mL, 200 μL) and physiological saline (200 μL) every 3 days. Before injection, the tumor size and body weight of all tumor-bearing mice were measured using digital calipers. Tumor size and body weight were then measured every 3 days, and tumor volume (V = L × W) was calculated. 2 / 2, L is the long diameter of the tumor, W is the short diameter of the tumor) and relative tumor volume (RTV = V t / V0, where V0 is the tumor volume measured before administration of the drug in a separate container, V t (Tumor volume at each measurement) to assess changes in tumor volume and body weight in tumor-bearing mice.

[0086] Tumor-bearing mice were subjected to 131 I-Au PHCNPs, 131 I-Au PCNPs, Au PHCNPs, Au PCNPs, Na 131After 21 days of treatment with saline, saline and I, the tumor-bearing mice were euthanized and the tumor tissues were harvested. The tumor tissues were then fixed with 10% neutral formalin and embedded in paraffin, and then the corresponding tissue sections with a thickness of 4 μm were prepared. Finally, the tissue sections were treated by H&E and TUNEL staining, and all the staining images were taken and collected under an optical microscope, and then the TUNEL positive cells in randomly selected areas were statistically analyzed by using ImageJ software to obtain the tumor cell apoptosis rate.

[0087] The results of the relative tumor volume and body weight quantitative analysis showed that, 131 The tumor-bearing mice in the I-Au PHCNPs treatment group had the slowest tumor growth rate, and after the end of the treatment period, 131 The relative tumor volume of the tumor-bearing mice in the I-Au PHCNPs treatment group was much smaller than that of the tumor-bearing mice in the other treatment groups. The body weight and body weight change trend of the tumor-bearing mice in each treatment group were almost the same. The above results showed that, based on the HA-mediated targeting effect, 131 I-Au PHCNPs showed excellent in vivo targeted radionuclide treatment effect on tumors, and the prepared material had little effect on the body weight of the tumor-bearing mice, see the attached drawings in the specification Figure 19 To further evaluate the in vivo targeted anti-tumor effect of the prepared material, the obtained tumor sections were subjected to H&E and TUNEL staining, and the number of tumor cell apoptosis (TUNEL positive cells) was counted. It can be seen that, compared with other treatment groups, 131 The I-Au PHCNPs treatment group showed the most number of tumor cell apoptosis. The results of quantitative analysis showed that, 131 The tumor cell apoptosis rate of the tumor-bearing mice in the I-Au PHCNPs treatment group was significantly higher than that of the tumor-bearing mice in the other treatment groups 131 The tumor cell apoptosis rate of the tumor-bearing mice in the I-Au PHCNPs treatment group, see the attached drawings in the specification Figure 20 、 21 The above results showed that the effect of inducing tumor cell apoptosis mainly came from 131 the radionuclide treatment effect of I, and based on the HA-mediated targeting effect, I-Au PHCNPs could be specifically delivered to the tumor site, thereby exhibiting in vivo targeted anti-tumor effect. 131

[0088] 13、In the verification 131 ​After in vivo and in vitro SPECT / CT / FL multimodality imaging of I-Au PHCNPs and radionuclide therapy effect, based on the presence of gold in the material, the gold content of each main organ (heart, liver, spleen, lung, kidney) and tumor tissue in the body at different time points (24, 48, 72 h) was tested to evaluate the metabolism of the material in the body. The tumor model was established by the method described above, and 12 tumor-bearing mice were divided into four groups (n = 3). Each group of tumor-bearing mice was sacrificed at different time points (0, 24, 48, 72 h) after tail vein injection of Au PHCNPs, and the main organs and tumor tissues were obtained and weighed. Finally, each organ and tumor tissue was digested with aqua regia, and the gold content in each tissue organ was measured by ICP-OES and quantitatively analyzed. It can be seen that 72 h after injection, the gold concentration of each organ has approached or even reached the level before injection. The results show that the prepared material can be well metabolized out of the body and will not be long-term retained in the body, see attached Figure 22 .

Claims

1. A functionalized polyethyleneimine-encapsulated gold nanoparticle composite material modified with near-infrared fluorescent dye Cy7, characterized in that: It is prepared by the following steps: Step 1: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS are added to NH2-PEG-COOH solution for activation. Then, polyethyleneimine PEI.NH2 solution is added and the reaction is stirred to obtain PEI.NH2-PEG-NH2. In step 1, the sum of the amounts of EDC and NHS is in the ratio of NH2-PEG-COOH to 5:1, the ratio of EDC to NHS is 1:1, the ratio of NH2-PEG-COOH to PEI.NH2 is 20:1, and the stirring reaction time is 3 days. Step 2: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS are added to the hyaluronic acid (HA) solution for activation. Then, PEI.NH2-PEG-NH2 prepared in Step 1 is added and the reaction is stirred to obtain PEI.NH2-(PEG-HA). In step 2, the sum of the amounts of EDC and NHS is in the ratio of the amount of HA to 5:1, the ratio of the amounts of EDC and NHS is 1:1, and the ratio of the amounts of HA to PEI.NH2-PEG-NH2 is 30:1; the stirring reaction time is 3 days. Step 3: Add a dimethyl sulfoxide solution of N-hydroxysuccinimide ester (HPAO) of 3-(4-hydroxyphenyl)propionic acid to the PEI.NH2-(PEG-HA) obtained in Step 2, stir and react to obtain PEI.NH2-(PEG-HA)-HPAO. In step 3, the molar ratio of HPAO to PEI.NH2-(PEG-HA) is 10:1; the stirring reaction time is 1-2 days. Step 4: Add sulfonic acid-cyanine 7-N-hydroxysuccinimide Cy7 to the PEI.NH2-(PEG-HA)-HPAO obtained in Step 3, stir the reaction in the dark, and obtain PEI.NH2-(PEG-HA)-Cy7-HPAO. In step 4, the molar ratio of Cy7 to PEI.NH2-(PEG-HA)-HPAO is 16:1; the stirring reaction time is 3 days. Step 5: Add chloroauric acid solution to PEI.NH2-(PEG-HA)-Cy7-HPAO obtained in step 4, stir and react for 20-40 minutes, then quickly add sodium borohydride solution to react, then add triethylamine, react for 20-40 minutes, then add acetic anhydride, stir and react to obtain Au PHCNP nanoparticles. In step 5, the molar ratio of chloroauric acid to sodium borohydride is 1:5, the molar ratio of chloroauric acid to PEI.NH2-(PEG-HA)-Cy7-HPAO is 100:1, the molar ratio of triethylamine, acetic anhydride, and PEI.NH2-(PEG-HA)-Cy7-HPAO is 120-660:100-550:1, and the stirring reaction time is 24 hours. Step 6: Add chloramine T and Na to the Au PHCNPs nanoparticles obtained in step 5. 131 I. After stirring for 3 minutes, Na2S2O5 and KI were added while stirring, and the reaction was continued for 5 minutes. Finally, the composite material was obtained by separation and purification using a desalting column.

2. The composite material according to claim 1, characterized in that: In step 6, Na 131 The radioactivity of I is 185-365 MBq.

3. The application of the composite material according to claim 1 in the preparation of tumor diagnostic and therapeutic reagents.

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