PSMA-targeted zwitterionic functionalized gold nanostar complexes and their applications

Abstract

This invention discloses a PSMA-targeted zwitterionic functionalized gold nanostar composite and its applications. The invention uses hyperbranched polyethyleneimine (PEI.NH2) as a carrier, and sequentially modifies its surface with diethylenetriaminepentaacetic acid dianhydride (DTPA), succinimide ester-polyethylene glycol-PSMA targeting ligand NHS-PEG-ACUPA, succinimide ester-polyethylene glycol-thiol NHS-PEG-SH, and fluorescein isothiocyanate (FI). The composite is further modified by chelating gadolinium ions (Gd) with diethylenetriaminepentaacetic acid dianhydride. 3+ Finally, functionalized polyethyleneimine was linked to alkoxyphenylsulfonamide-modified gold nanostars APAS-Au NSs via gold-sulfur bonds. The resulting gold nanostar complex can achieve efficient enrichment in prostate cancer cells and has CT / MR dual-modal imaging and photothermal therapy functions.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a PSMA-targeted zwitterionic functionalized gold nanostar complex and its application in the preparation of diagnostic and therapeutic reagents. Background Technology

[0002] Prostate cancer (PCa) is the second most common malignant cancer among men worldwide and is considered one of the leading causes of death in men. The 5-year survival rate for prostate cancer varies; early detection leads to a 5-year survival rate exceeding 99%, but late-stage prostate cancer is often incurable (Kim, Y. et al. J. Prev. 2023, 44(2), 253-266). Currently, common diagnostic methods for prostate cancer include initial serum PSA testing, digital rectal examination, and more invasive biopsy. However, the error rate of serum prostate-specific antigen (PSA) testing is 20-25%, making it inaccurate in distinguishing between prostate cancer, benign prostatic hyperplasia (BPH), and prostatitis (Cimadamore, A. et al. Front. Oncol. 2018, 8, 653). Furthermore, because the tumor is small and cancer cells are unevenly distributed in the early stages of PCa, false negative results can occur in prostate biopsies (Cimadamore, A. et al. Front. Oncol. 2018, 8, 653). Common treatments for prostate cancer include total prostatectomy, radiotherapy, hormone therapy, immunotherapy, and chemotherapy. However, surgical removal causes significant damage to the body, and the toxicity of anti-tumor drugs can affect normal cells. Therefore, new diagnostic and treatment methods are still needed to achieve accurate diagnosis and treatment of early-stage prostate cancer.

[0003] To avoid overdiagnosis and overtreatment, one of the main goals of nanomedicine is to perform multiple functions using a single nanocarrier, that is, to achieve targeting, imaging, diagnosis and monitoring of cells using only a single nanoparticle. Molecular imaging plays an excellent role in early diagnosis. Currently, common molecular imaging techniques mainly include X-ray computed tomography (CT), magnetic resonance imaging (MR), ultrasound imaging (US), photoacoustic imaging (PA), and single-photon emission computed tomography (SPECT), which have been widely used in cancer diagnosis. Among them, the principle of CT imaging is based on the difference in density and thickness between different organs or tissues, which causes X-rays to attenuate to different degrees as they pass through the organs and tissues, thereby forming regional imaging of different gray levels. These gray levels represent the corresponding damage sites or the size of shape changes (Bohrmann, L. et al. Theranostics. 2022, 12(9), 4010-4050). Heavy metals, as common CT contrast agents, enhance image contrast by increasing X-ray attenuation, and their absorption of high-energy X-rays gradually increases with increasing atomic number (Kase, AM et al. Cancers. 2022, 14(6), 1361). Based on this, various metal-based nano-CT contrast agents have been developed for early tumor diagnosis, mainly including gold nanoparticles (Au NPs), bismuth, iodine, and hafnium-based nanoparticles. Among them, as a type of Au NPs, highly branched gold nanostars (Au NSs) exhibit higher photothermal conversion efficiency due to their easier penetration of electric fields, allowing them to reach photothermal lethal doses for cancer cells even at lower Au concentrations compared to spherical Au NPs (Mousavi, SM et al. Drug Metab. Rev. 2022, 52, 299-318). Therefore, based on their excellent morphology and outstanding photothermal conversion properties, Au NSs are more suitable for photothermal therapy of cancer. For example, Wang S. et al. modified Au NSs with long-chain amino-modified polyethylene glycol (PEG) and carboxylated PEG to obtain AuNSs-N / C with mixed charges. When the molar ratio of amino-PEG to carboxy-PEG was 4:1, the resulting Au NSs-N / C4 exhibited high affinity for cancer cells and photothermal therapeutic effects under tumor pH conditions (Wang, S. et al. Small, 2015, 11, 1801-1810). However, after intravenous administration, Au NSs showed low enrichment at tumor sites, resulting in poor diagnostic and therapeutic effects; furthermore, single CT imaging has limitations such as poor soft tissue contrast, making accurate diagnosis difficult and restricting the biomedical applications of Au NSs.

[0004] The low enrichment of Au NSs in prostate cancer limits its diagnosis and treatment. As researchers delve deeper into prostate cancer, they have discovered a prostate cancer membrane-specific antigen (PSMA) expressed on prostate cancer epithelial cells. Its expression in prostate cancer cells is 100-1000 times higher than in normal cells, making it an effective target for the diagnosis and treatment of prostate tumors (Uijen, MJMet al.Eur.J.Nucl.Med.Mol.Imaging.2021,48,4350-4368). Currently, common targeting ligands that bind to PSMA include monoclonal antibodies MLN591 and J591, as well as (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureido)glutaric acid (ACUPA) and its derivatives. ACUPA-modified nanoparticles show significantly higher uptake in PSMA-positive cells (LNCaP cells) compared to non-ACUPA-modified nanoparticles, increasing the accumulation of modified nanomaterials in LNCaP cells. Therefore, modifying Au NSs with ACUPA is expected to improve the enrichment of gold nanostars in prostate cancer, thereby enhancing the therapeutic effect.

[0005] Furthermore, the pH of the tumor environment is lower than that of normal tissues compared to normal cells (Liu, P. et al. ACS Biomother. Sci. Eng. 2018, 4, 40-46). pH-sensitive zwitterionic alkoxyphenyl sulfonamides (APAS) can be protonated at pH < 6.5, changing from electrically neutral to positively charged. Within the normal tissue pH range of 7.2-7.4, APAS maintains its electrically neutral zwitterionic state and is almost non-toxic to normal tissues. However, in the prostate tumor microenvironment of pH 6.0-6.8, because APAS becomes positively charged at pH < 6.5, it attracts negatively charged cell membranes via electrostatic attraction, causing modified nanomaterials to accumulate on the surface of cancer cells, thus enhancing the internalization of APAS-modified nanomaterials by cancer cells (Mizuhara, T. et al. Angew. Chem. Int. Ed. 2015, 54, 6567-6570). Therefore, modifying Au NSs with APAS can be an effective strategy to enable Au NSs to be efficiently taken up in prostate cancer sites.

[0006] Addressing the limitations of low soft tissue resolution in Au NSs-mediated single CT imaging, which makes it difficult to accurately obtain comprehensive and reliable information about prostate cancer sites, multimodal imaging has been developed by combining various imaging modalities to overcome their individual limitations. Currently, common multimodal imaging methods combining CT and MR imaging include CT / MR imaging, SPECT / CT imaging, and PET / CT imaging. Among these, MR imaging, with its superior soft tissue contrast, complements CT imaging, providing more detailed anatomical information for prostate cancer diagnosis. Gd-based contrast agents are the most widely used T1 contrast agents. Therefore, based on the excellent CT imaging performance and photothermal conversion efficiency of Au NSs, this invention designs a method to combine them with the targeting molecule ACUPA, pH-sensitive zwitterionic APAS, and a Gd-based contrast agent for MR imaging, achieving enhanced CT / MR dual-modal imaging and photothermal therapy for prostate cancer cells. Summary of the Invention

[0007] The purpose of this invention is to provide a PSMA-targeted zwitterionic functionalized gold nanostar complex and its application in the preparation of diagnostic and therapeutic reagents.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] A PSMA-targeted zwitterionic functionalized gold nanostar complex was prepared using the following steps:

[0010] Step 1: Dissolve the PSMA targeting ligand ACUPA in water, add sodium carbonate aqueous solution and stir to react, then add bisuccinimide ester polyethylene glycol NHS-PEG-NHS aqueous solution and stir to react to obtain NHS-PEG-ACUPA.

[0011] The molar ratio of PSMA-targeting ligand ACUPA to sodium carbonate is 3-4:2, and the molar ratio of PSMA-targeting ligand ACUPA to bisuccinimide ester polyethylene glycol NHS-PEG-NHS is 1-1.5:1.

[0012] Step 2: Dissolve hyperbranched polyethyleneimine (PEI.NH2) in water, add an aqueous solution of the chelating agent diethylenetriaminepentaacetic acid dianhydride (DTPA), stir the reaction, and obtain PEI.NH2-DTPA.

[0013] The molar ratio of diethylenetriaminepentaacetic acid dianhydride to hyperbranched polyethyleneimine PEI.NH2 is 20-22:1;

[0014] Step 3: Add the aqueous solution of NHS-PEG-ACUPA obtained in Step 1 to the aqueous solution of PEI.NH2-DTPA, stir and react to obtain PEI.NH2-DTPA-(PEG-ACUPA). Then, add the aqueous solution of succinimide ester-polyethylene glycol-thiol NHS-PEG-SH to the aqueous solution of PEI.NH2-DTPA-(PEG-ACUPA)-(PEG-SH), stir and react to obtain PEI.NH2-DTPA-(PEG-ACUPA)-(PEG-SH).

[0015] The molar ratio of NHS-PEG-ACUPA, NHS-PEG-SH and hyperbranched polyethyleneimine PEI.NH2 is 10-12:1;

[0016] Step 4: Add DMSO solution of fluorescein isothiocyanate to the DMSO solution of PEI.NH2-DTPA-(PEG-ACUPA)-(PEG-SH) obtained in step 3, stir and react to obtain PEI.NH2-DTPA-FI-(PEG-ACUPA)-(PEG-SH).

[0017] The molar ratio of fluorescein isothiocyanate to hyperbranched polyethyleneimine (PEI.NH2) is 5-7:1;

[0018] Step 5: Add an aqueous solution of gadolinium nitrate to the aqueous solution of PEI.NH2-DTPA-FI-(PEG-ACUPA)-(PEG-SH) obtained in Step 4, stir the reaction, and obtain PEI.NH2-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH);

[0019] The molar ratio of gadolinium nitrate to diethyltriaminepentaacetic acid dianhydride is 3-5:1;

[0020] Step 6: Add triethylamine to the aqueous solution of PEI.NH2-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) obtained in Step 5, then add acetic anhydride, stir the reaction, and obtain functionalized hyperbranched polyethyleneimine PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH);

[0021] The molar ratio of triethylamine, acetic anhydride, and PEI.NH2-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) is 120-660:100-550:1;

[0022] Step 7: Heat the aqueous solution of chloroauric acid to boiling, then add sodium citrate solution, continue heating and stirring until the solution turns wine red, then let it cool naturally to room temperature to obtain an aqueous solution of Au seeds.

[0023] Step 8: Add the Au seeds aqueous solution obtained in Step 7 to the chloroauric acid aqueous solution, then simultaneously add the silver nitrate aqueous solution and the ascorbic acid aqueous solution, and add the hexadecyltrimethylammonium bromide aqueous solution. Stir the reaction to obtain the gold nanostar Au NSs aqueous solution.

[0024] Step 9: Dissolve alkoxyphenyl sulfonamide APAS in DMSO, add it to the DMSO solution of Au NSs obtained in Step 8, stir and react to obtain APAS-Au NSs;

[0025] The mass ratio of alkoxyphenyl sulfonamide APAS to Au NSs is 3-5:1;

[0026] Step 10: Mix and stir the aqueous solution of PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) obtained in step 6 with the aqueous solution of APAS-Au NSs obtained in step 9 to obtain the PSMA-targeted zwitterionic functionalized gold nanostar complex ACUPA-APAS-Gd-Au-PENSs.

[0027] The mass ratio of Au NSs to PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) is 1:5-6.

[0028] Furthermore, in step 1, the reaction time of PSMA targeting ligand ACUPA and sodium carbonate is 6 h, and the reaction time after adding bisuccinimide ester polyethylene glycol NHS-PEG-NHS is 72 h.

[0029] Furthermore, the reaction time for hyperbranched polyethyleneimine (PEI.NH2) and diethylenetriaminepentaacetic acid dianhydride in step 2 is 24 h.

[0030] Furthermore, the reaction time for NHS-PEG-ACUPA and PEI.NH2-DTPA in step 3 is 72 h, and the reaction time after adding succinimide ester-polyethylene glycol-thiol NHS-PEG-SH is 72 h.

[0031] Furthermore, in step 4, the stirring reaction is carried out under the condition of stirring in the dark for 24 hours.

[0032] Furthermore, in step 5, the stirring reaction is carried out under light-protected stirring conditions for 24 hours.

[0033] Furthermore, in step 6, the stirring reaction is carried out under the condition of stirring in the dark for 24 hours.

[0034] Furthermore, in step 10, the stirring reaction time is 24 hours.

[0035] In one embodiment of the present invention, the concentration of chloroauric acid in step 7 is 30 mg / mL, and the mass fraction of sodium citrate in the sodium citrate solution is 1%; the concentration of HAuCl4 in step 8 is 30 mg / mL, and the molar ratio of HAuCl4, AgNO3 and AA is 50:4-6:1; the mass ratio of APAS to Au NSs in step 9 is 3-5:1.

[0036] The application of the above-mentioned PSMA-targeted zwitterionic functionalized gold nanostar complex in the preparation of tumor diagnostic and therapeutic reagents.

[0037] Furthermore, the tumor is prostate cancer.

[0038] The use of polyethylene glycol (PEG) in this invention can increase the biocompatibility of the material, thereby prolonging the time that the nanomaterial remains at the tumor site, and thus extending the imaging and treatment time of the material in vivo.

[0039] In this invention, the PSMA-targeting ligand ACUPA is used to modify the surface of PEI.NH2, which can specifically bind to PSMA on the surface of prostate cancer cells. Through the targeting effect of ACUPA, the modified nanomaterials are efficiently enriched at the tumor site.

[0040] In this invention, NHS-PEG-SH is used to modify the surface of PEI.NH2 to impart -SH to the surface of functionalized PEI.NH2, enabling functionalized PEI.NH2 to bind with Au NSs through gold-sulfur bonds.

[0041] In this invention, the chelating agent DTPA is used to chelate Gd. 3+ This is to achieve MR imaging effects of nanomaterials in vivo and in vitro.

[0042] In this invention, Au NSs with a surface plasmon resonance (SPR) peak at 808 nm were synthesized by seed growth method to realize CT imaging and photothermal therapy of nanomaterials at tumor sites.

[0043] This invention uses pH-sensitive zwitterionic APAS, which is linked to AuNSs by gold-sulfur bonds through the thiol functional group contained in its molecular structure. Under the slightly acidic conditions of tumors, APAS changes from electroneutrality to positronicity, thereby achieving efficient enrichment of nanomaterials at the tumor site.

[0044] This invention uses hyperbranched polyethyleneimine (PEI.NH2) as a carrier, and sequentially modifies its surface with diethylenetriaminepentaacetic dianhydride (DTPA), succinimide ester-polyethylene glycol-PSMA targeting ligand NHS-PEG-ACUPA, succinimide ester-polyethylene glycol-thiol NHS-PEG-SH, and fluorescein isothiocyanate (FI). Furthermore, it utilizes DTPA to chelate gadolinium ions (Gd). 3+ Finally, functionalized polyethyleneimine was linked to alkoxyphenyl sulfonamide-modified gold nanostars (APAS-Au NSs) via gold-sulfur bonds to obtain a gold nanostar complex capable of highly efficient enrichment in prostate cancer cells and possessing CT / MR dual-modal imaging and photothermal therapy functions. The PSMA-targeted zwitterionic functionalized gold nanostar complex prepared by this method has advantages such as simple preparation process and mild reaction conditions. The synthetic route can also be used to prepare photothermal reagents loaded with other functionalized polymers, showing promising application prospects in the early diagnosis and treatment of prostate cancer.

[0045] Beneficial effects

[0046] (1) The synthesis method of ACUPA-APAS-Gd-Au-PENSs prepared by this invention is simple, the reaction conditions are mild, and it is easy to operate, and it has great biomedical application value.

[0047] (2) In this invention, the modification of ACUPA and APAS can enhance the uptake of functionalized gold nanostar complex by prostate cancer cells. The modification of the MR imaging reagent Gd endows the functionalized gold nanostar complex with MR imaging capabilities. The high atomic number, good X-ray attenuation performance, and good photothermal conversion efficiency of Au NSs themselves give the functionalized gold nanostar complex good CT imaging performance and photothermal therapy capabilities. These advantages enable ACUPA-APAS-Gd-Au-PENSs to achieve high uptake by prostate cancer cells while realizing CT / MR dual-modal imaging and photothermal therapy for prostate cancer cells. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the synthesis route of ACUPA-APAS-Gd-Au-PENSs in this invention.

[0049] Figure 2The ACUPA-PEG-NHS (a), PEI.NH2 (b), PEI.NH2-DTPA (c), PEI.NH2-DTPA-(PEG-ACUPA) (d), PEI.NH2-DTPA-(PEG-ACUPA)-(PEG-SH) (e), and PEI.NH2-DTPA-FI-(PEG-ACUPA)-(PEG-SH) (f) prepared for this invention are dispersed in D2O. 1 H NMR spectrum;

[0050] Figure 3 The UV-Vis absorption spectra of Au seeds, Au NSs, APAS-Au NSs and ACUPA-APAS-Gd-Au-PENSs prepared for this invention.

[0051] Figure 4 TEM image (a), particle size distribution histogram (b), high-resolution TEM image (c, d), and selected area electron diffraction pattern (e) of ACUPA-APAS-Gd-Au-PENSs prepared for this invention.

[0052] Figure 5 Infrared spectra of Au NSs, APAS-Au NSs, PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) and ACUPA-APAS-Gd-Au-PENSs prepared in this invention.

[0053] Figure 6 Thermogravimetric curves of Au NSs, APAS-Au NSs and ACUPA-APAS-Gd-Au-PENSs prepared for this invention.

[0054] Figure 7 Zeta potential diagrams of ACUPA-APAS-Gd-Au-PENSs and ACUPA-Gd-Au-PENSs prepared in this invention dissolved in phosphate buffers of different pH values.

[0055] Figure 8 The pseudo-color images of T1-weighted MR imaging of ACUPA-APAS-Gd-Au-PENSs prepared for this invention at different Gd concentrations (a) and the linear fitting of 1 / T1 as a function of Gd concentration (b).

[0056] Figure 9 For NIR laser (808nm, 1.2W / cm) 2Photothermal heating curves (a) and temperature changes (b) of ACUPA-APAS-Gd-Au-PENSs aqueous solutions with different Au concentrations after irradiation; photothermal heating curves (c) of ACUPA-APAS-Gd-Au-PENSs aqueous solutions (0.5 mM, 0.1 mL) under NIR laser irradiation with different powers; photothermal heating and natural cooling temperature change curves of ACUPA-APAS-Gd-Au-PENSs aqueous solutions for five cycles (d) and one cycle (e); linear fitting plot of natural cooling time and -lnθ of ACUPA-APAS-Gd-Au-PENSs aqueous solutions (f).

[0057] Figure 10 ICP-OES assay results of Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and ACUPA-APAS-Gd-Au-PENSs prepared for this invention after incubating LNCaP cells for 3 hours under different pH conditions.

[0058] Figure 11 Flow cytometry results are shown for LNCaP cells incubated with PBS (a), Gd-Au-PENSs (b), APAS-Gd-Au-PENSs (c), ACUPA-Gd-Au-PENSs (d), and ACUPA-APAS-Gd-Au-PENSs (e) at pH 7.4, and with Gd-Au-PENSs (f), APAS-Gd-Au-PENSs (g), ACUPA-Gd-Au-PENSs (h), and ACUPA-APAS-Gd-Au-PENSs (i) at pH 6.0 for 3 h.

[0059] Figure 12 The mean fluorescence intensity (a) and cell uptake rate (b) of LNCaP cells incubated for 3 h with PBS, Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs and ACUPA-APAS-Gd-Au-PENSs, respectively, at pH 7.4 and pH 6.0.

[0060] Figure 13 The results are obtained by laser scanning confocal microscopy after incubating LNCaP cells for 3 hours with PBS and different materials at different pH values ​​(7.4, 6.0). In the numbered figures (a), (b), (c), (d), (e), (f), (g), (h), and (i), the left figure shows DAPI fluorescence, the right figure shows FI fluorescence, and the bottom figure shows combined fluorescence. The scale bar is 20 μm.

[0061] Figure 14 CT images (a) and CT signal values ​​(b) of LNCaP cells after incubation for 3 h with Gd-Au-PENSs (1), APAS-Gd-Au-PENSs (2), ACUPA-Gd-Au-PENSs (3) and ACUPA-APAS-Gd-Au-PENSs (4) at pH 6.0; and MR images (c) and MR signal values ​​(d) of LNCaP cells after incubation for 3 h with Gd-Au-PENSs (1), APAS-Gd-Au-PENSs (2), ACUPA-Gd-Au-PENSs (3) and ACUPA-APAS-Gd-Au-PENSs (4) at pH 6.0.

[0062] Figure 15 CCK-8 assay results after 6 hours of incubation of LNCaP cells with different gold nanostar complexes. Detailed Implementation

[0063] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0065] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0066] PSMA-targeting ligand ACUPA was purchased from Qiangyao Biotechnology Co., Ltd., with a purity of 95%.

[0067] The bipolar succinimide polyethylene glycol NHS-PEG-NHS was purchased from Shanghai Yayi Biotechnology Co., Ltd., with a molecular weight of 2000 and a purity of 95%.

[0068] Succinimide ester-polyethylene glycol-thiol NHS-PEG-SH was purchased from Shanghai Yayi Biotechnology Co., Ltd., with a molecular weight of 2000 and a purity of 95%.

[0069] Alkoxyphenyl sulfonamides (APAS) were synthesized using existing techniques, the synthetic methods of which can be found in Langmuir, 2019, 35, 13405-13412; J. Am. Chem. Soc. 2010, 132, 5285-5289; Angew. Chem., Int. Ed. 2015, 54, 6567-6570.

[0070] Example 1

[0071] (1) Weigh 20 mg of the PSMA-targeting ligand ACUPA and dissolve it in 4 mL of pure water. While stirring, add a solution of Na2CO3 (3.319 mg) dissolved in 2 mL of pure water dropwise to the above solution. Stir the reaction at room temperature for 6 h, with the molar ratio of ACUPA to Na2CO3 being 3:2. Then, dissolve 125.27 mg of NHS-PEG-NHS (Mw = 2000) in 3 mL of pure water and add it dropwise to the treated ACUPA aqueous solution. Stir the reaction for 72 h, with the molar ratio of ACUPA to NHS-PEG-NHS being 1:1. After the reaction is complete, place the solution in a cellulose dialysis membrane with a molecular weight cutoff of 1000 Da and dialyze it in ultrapure water for 3 days. After freeze-drying, obtain the solid product NHS-PEG-ACUPA and store it in a refrigerator at -20°C.

[0072] (2) Weigh 50 mg of PEI.NH2 (Mw = 25000) and dissolve it in 5 mL of pure water. Add a solution of diethylenetriaminepentaacetic acid dianhydride (DTPA) (57.1712 mg) dissolved in 5 mL of pure water and stir for 24 h. The DTPA is 20 times the molar equivalent of PEI.NH2. The reaction solution is collected in a dialysis bag with MWCO = 8000-14000 Da and dialyzed in ultrapure water for 3 days. After freeze-drying, the product PEI.NH2-DTPA is obtained and stored in a refrigerator at -20°C.

[0073] (3) Weigh 50 mg of NHS-PEG-ACUPA obtained in step (1) and 70.67 mg of PEI.NH2-DTPA obtained in step (2), and dissolve them in 5 mL of ultrapure water until completely dissolved. Mix and stir for 72 h. The molar ratio of PEI.NH2 to NHS-PEG-ACUPA is 1:10. Transfer the mixed solution after the above reaction to a dialysis bag with MWCO = 8000-14000 Da and dialyze in ultrapure water for 3 days. Finally, freeze dry to obtain the solid product PEI.NH2-DTPA-(PEG-ACUPA). Weigh 44.28 mg of NHS-PEG-SH (Mw = 2000) solid powder and dissolve it in 3.5 mL of ultrapure water. Add the solution dropwise to a 9 mL pure water solution containing 100 mg of completely dissolved PEI.NH2-DTPA-(PEG-ACUPA). Stir the reaction for 72 h, with a molar ratio of PEI.NH2 to NHS-PEG-SH of 1:10. The solution after reaction is purified by dialysis for 3 days using a cellulose dialysis membrane with a molecular weight cutoff of 8000-14000 Da. After freeze-drying, the product PEI.NH2-DTPA-(PEG-ACUPA)-(PEG-SH) is obtained and stored in a refrigerator at -20°C.

[0074] (4) Take 100 mg of PEI.NH2-DTPA-(PEG-ACUPA)-(PEG-SH) obtained in step (3) and 3.23 mg of fluorescein isothiocyanate (FI), and dissolve them in 5 mL of DMSO. After they are completely dissolved, mix and stir them for 24 h under light-protected conditions, with the molar ratio of PEI.NH2 to FI being 1:5. After the reaction is completed, dialyze the mixed solution in pure water for 3 days under light-protected conditions using a cellulose dialysis membrane with a molecular weight cutoff of 8000-14000 Da to remove the organic solvent DMSO and unreacted FI. After freeze-drying, PEI.NH2-DTPA-FI-(PEG-ACUPA)-(PEG-SH) is obtained and stored in a refrigerator at -20°C under light-protected conditions.

[0075] (5) Weigh 38.4 mg of gadolinium nitrate (Gd(NO3)3), dissolve it completely in pure water (5 mL), and add it dropwise to the pure water (8 mL) solution of PEI.NH2-DTPA-FI-(PEG-ACUPA)-(PEG-SH) (100 mg) obtained in step (4). Stir the reaction in the dark for 24 h, where the molar ratio of Gd(NO3)3 to DTPA is 3:1. After the reaction is complete, place the solution in a cellulose dialysis membrane (MWCO = 8000-14000 Da), dialyze it with pure water for 3 days, and freeze-dry it to obtain PEI.NH2-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH). Store it in a refrigerator at -20°C in the dark.

[0076] (6) Dissolve 100 mg of PEI.NH2-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) in 3 mL of pure water. After it is completely dissolved, add triethylamine (808.4 μL) and stir for 0.5 h. Then add acetic anhydride (461.7 μL) and stir for 24 h in the dark. After that, dialyze with a cellulose dialysis membrane with a molecular weight cutoff of 8000-14000 Da for 3 days, changing the pure water used for dialyzing 3-4 times a day. After freeze drying, functionalized hyperbranched polyethyleneimine PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) is obtained.

[0077] (7) Add 339.9 mg of chloroauric acid (HAuCl4) to 11.33 mL of pure water to prepare an aqueous solution with an Au concentration of 30 mg / mL. Add 100 mg of sodium citrate to 9.9 mL of pure water to prepare a 1% sodium citrate aqueous solution. Place the prepared HAuCl4 aqueous solution in a round-bottom flask and heat it to boiling (130-140℃) in an oil bath. Then, while stirring at 700 r / min, add 1.5 mL of the prepared 1% sodium citrate aqueous solution. Continue stirring at 700 r / min at 130-140℃ until the solution turns wine red, then stop the reaction and allow it to cool naturally to room temperature to obtain an aqueous solution of gold seeds (Au seeds).

[0078] (8) Dissolve 1.698 g of HAuCl4 in 56.6 mL of pure water to obtain an aqueous solution with an Au concentration of 30 mg / mL. Place the solution in a round-bottom flask and add 0.5 mL of the Au seeds aqueous solution obtained in step (7) while stirring at 700 r / min. Immediately add 2 mL of 3 mM silver nitrate (AgNO3) aqueous solution and 1 mL of 0.1 M ascorbic acid (AA) aqueous solution. Stir for 1 min and then add 2 mL of 0.1 M hexadecyltrimethylammonium bromide (CTAB). Continue stirring for 5 min and terminate the reaction when the solution turns dark blue. Then, place the solution in a 50 mL centrifuge tube and centrifuge at 5000 r / min for 15 min. Take the precipitate, rehydrate it with pure water, and wash it by centrifugation 3-4 times. Disperse the obtained precipitate in 5 mL of pure water and freeze-dry to obtain pure gold nanostars (Au NSs).

[0079] (9) Weigh 30 mg of alkoxyphenyl sulfonamide (APAS) and 10 mg of Au NSs, and dissolve them separately in 3 mL of DMSO. Mix the two solutions and react them under magnetic stirring for 2 days. Then, centrifuge at 6000 r / min for 20 min to remove DMSO and unreacted APAS. Then, reconstitute the precipitate with DMSO and pure water in sequence, centrifuge (6000 r / min, 10 min), remove the supernatant, disperse the purified precipitate in 4 mL of pure water, and freeze-dry to obtain APAS-AuNSs.

[0080] (10) Weigh 50 mg of PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) and 10 mg of APAS-Au NSs, dissolve them separately in 3 mL of pure water, and mix and stir at room temperature for 48 h. After the reaction is complete, centrifuge to remove unreacted PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH), reconstitute the precipitate with pure water, centrifuge, and repeat 3 times. Finally, disperse the collected precipitate in 5 mL of pure water, freeze-dry to obtain the final product ACUPA-APAS-Gd-Au-PENSs. The synthetic route is as follows: Figure 1 As shown.

[0081] Comparative Example 1

[0082] (1) Weigh 50 mg of mPEG-NHS with a molecular weight of 2000 and 77.29 mg of PEI.NH2-DTPA from Example 1, and dissolve them in 5 mL of ultrapure water until completely dissolved. Mix and stir for 72-80 h, wherein the molar ratio of PEI.NH2 to mPEG-NHS is 1:10. Transfer the mixed solution after the above reaction to a dialysis bag with MWCO = 8000-14000 Da, dialyze in ultrapure water for 3 days, and finally freeze-dry to obtain the solid product PEI.NH2-DTPA-mPEG. Weigh 40.88 mg of NHS-PEG-SH solid powder with a molecular weight of 2000, dissolve it in 3.5 mL of ultrapure water, and add it dropwise to a pure water solution (9 mL) containing 100 mg of completely dissolved PEI.NH2-DTPA-mPEG. Stir the reaction for 72-80 h, wherein the molar ratio of PEI.NH2 to NHS-PEG-SH is 1:10. The solution after reaction is purified by dialysis for 3 days using a cellulose dialysis membrane with a molecular weight cutoff of 8000-14000 Da. After freeze-drying, the product PEI.NH2-DTPA-mPEG-(PEG-SH) is obtained and stored in a refrigerator at -20°C.

[0083] (2) Weigh 100 mg of PEI.NH2-DTPA-mPEG-(PEG-SH) and 3.04 mg of FI obtained in step (1) respectively, and dissolve them in 5 mL of DMSO. After they are completely dissolved, mix and stir them for 24-26 h under light-protected conditions, with the molar ratio of PEI.NH2 to FI being 1:5. After the reaction is completed, dialyze the mixed solution in pure water for 3 days under light-protected conditions using a cellulose dialysis membrane with a molecular weight cutoff of 8000-14000 Da to remove the organic solvent DMSO and unreacted FI. After freeze-drying, PEI.NH2-DTPA-FI-mPEG-(PEG-SH) is obtained and stored in a refrigerator at -20°C under light-protected conditions.

[0084] (3) Weigh 36.2 mg of gadolinium nitrate (Gd(NO3)3), dissolve it completely in pure water (5 mL), and add it dropwise to the pure water (8 mL) solution of PEI.NH2-DTPA-FI-mPEG-(PEG-SH) (100 mg) obtained in step (2). Stir the reaction in the dark for 24 h, where the molar ratio of Gd(NO3)3 to DTPA is 3:1. Place the solution after the reaction in a cellulose dialysis membrane (MWCO = 8000-14000 Da), dialyze it with pure water for 3 days, and freeze-dry it to obtain PEI.NH2-DTPA(Gd)-FI-mPEG-(PEG-SH). Store it in a refrigerator at -20℃ in the dark.

[0085] (4) Dissolve 100 mg of PEI.NH2-DTPA(Gd)-FI-mPEG-(PEG-SH) prepared in step (3) in 3 mL of pure water. After it is completely dissolved, add triethylamine (795.1 μL) and stir for 0.5 h. Then add acetic anhydride (456.8 μL) and stir for 24 h in the dark. After that, dialyze with a cellulose dialysis membrane with a molecular weight cutoff of 8000-14000 Da for 3 days, changing the pure water used for dialyzing 3-4 times a day. After freeze drying, functionalized hyperbranched polyethyleneimine PEI.NHAc-DTPA(Gd)-FI-mPEG-(PEG-SH) without ACUPA is obtained.

[0086] (5) Weigh 50 mg of PEI.NHAc-DTPA(Gd)-FI-mPEG-(PEG-SH) prepared in step (4) and 10 mg of APAS-Au NSs prepared in Example 1, and dissolve them separately in 3 mL of pure water. Mix and stir at room temperature for 48 h. After the reaction is complete, centrifuge to remove unreacted PEI.NHAc-DTPA(Gd)-FI-mPEG-(PEG-SH), and re-dissolve the precipitate in pure water and centrifuge. Repeat this process 3 times. Finally, disperse the collected precipitate in 5 mL of pure water and freeze-dry to obtain the comparative material APAS-Gd-Au-PENSs.

[0087] Comparative Example 2

[0088] Weigh 50 mg of PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) prepared in Example 1 and 10 mg of Au NSs prepared in Example 1, and dissolve them separately in 3 mL of pure water. Mix and stir at room temperature for 48 h. After the reaction is complete, centrifuge to remove unreacted PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH). Redissolve the precipitate with pure water and centrifuge, repeating the process three times. Finally, disperse the collected precipitate in 5 mL of pure water and freeze-dry to obtain the comparative material ACUPA-Gd-Au-PENSs.

[0089] Comparative Example 3

[0090] Weigh 50 mg of PEI.NHAc-DTPA(Gd)-FI-mPEG-(PEG-SH) prepared in Comparative Example 1 and 10 mg of Au NSs prepared in Example 1, and dissolve them separately in 3 mL of pure water. Mix and stir at room temperature for 48 h. After the reaction is complete, centrifuge to remove unreacted PEI.NHAc-DTPA(Gd)-FI-mPEG-(PEG-SH). Redissolve the precipitate with pure water and centrifuge, repeating this process three times. Finally, disperse the collected precipitate in 5 mL of pure water and freeze-dry to obtain the comparative material Gd-Au-PENSs.

[0091] The following uses nuclear magnetic resonance hydrogen spectrum (NMR spectroscopy) 1 The materials prepared in the above embodiments were characterized by methods including ¹H NMR, UV-Vis absorption spectroscopy, transmission electron microscopy (TEM), FTIR, thermogravimetric analysis (TGA), zeta potential measurement, T1 relaxation rate measurement, inductively coupled plasma atomic emission spectroscopy (ICP-OES), flow cytometry, laser scanning confocal microscopy, cell CT / MR imaging, and cell proliferation-toxicity assay (CCK-8).

[0092] (1) 1 H NMR test

[0093] like Figure 2 As shown, 1 1H NMR test results showed that the PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) prepared in Example 1 had 17.44 DTPA, 7.61 NHS-PEG-ACUPA, 7.75 NHS-PEG-SH and 3.01 FI on its surface.

[0094] (2) UV-Vis test

[0095] like Figure 3As shown, UV-Vis test results indicate that the UV absorption peak of Au seeds prepared in Example 1 is located at 520 nm, and the UV absorption peak of Au NSs is located at 808 nm, consistent with the literature. This indicates that Au seeds and Au NSs were successfully synthesized in this invention. After modification with APAS, the UV absorption peak of APAS-Au NSs was still observed to be located at 808 nm, indicating that the modification with APAS did not change the optical properties of Au NSs. By observing the UV-Vis spectrum of ACUPA-APAS-Gd-Au-PENSs, a UV absorption peak at 500 nm attributed to FI and a SPR characteristic peak at 808 nm attributed to Au NSs were simultaneously observed. This indicates that PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) was successfully modified on the surface of Au NSs, and its modification did not change the optical properties of Au NSs.

[0096] (3) TEM test

[0097] like Figure 4 As shown in Figures a and b, the synthesized ACUPA-APAS-Gd-Au-PENSs exhibit a star-shaped structure with multiple tips, a size ranging from 74.1 ± 3.9 nm, and a uniform normal distribution. Figure 4 The high-resolution TEM images of c and d clearly show the well-organized crystal structure, proving that ACUPA-APAS-Gd-Au-PENSs possesses crystal-specific properties. Figure 4 Selected area electron diffraction patterns of the middle e show characteristic diffraction rings corresponding to the (111), (200), (220) and (311) planes of Au NSs, which proves that ACUPA-APAS-Gd-Au-PENSs still has the face-centered cubic crystal structure of Au NSs, indicating that the modification of APAS and PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) did not change the optical properties of Au NSs.

[0098] (4) FTIR test

[0099] like Figure 5 As shown, the FTIR test results indicate that, compared with the infrared spectrum of Au NSs, APAS-Au NSs exhibits a significantly larger infrared spectrum at 1392 cm⁻¹. -1 and 1077cm -1A vibrational peak is observed at 1670 cm⁻¹, which coincides with the stretching vibration peak of the O=S=O bond of the sulfonyl group in APAS as described in the literature. This indicates that APAS was successfully modified onto the surface of Au NSs. Compared with the infrared spectrum of APAS-Au NSs, the infrared spectrum of ACUPA-APAS-Gd-Au-PENSs shows a peak at 1670 cm⁻¹. -1 and 1109cm -1 Two vibration peaks appeared, corresponding to the elution positions of the stretching vibration peaks of the C=O and CN bonds in PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH), respectively. This indicates that PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) was successfully modified on the surface of APAS-Au NSs in this invention.

[0100] (5) TGA test

[0101] Weigh out 8-10 mg each of Au NSs, APAS-Au NSs and ACUPA-APAS-Gd-Au-PENSs, and place them under nitrogen protection, heating them from 25℃ to 700℃ at a rate of 20℃ / min.

[0102] like Figure 6 It can be seen that the mass loss of unmodified Au NSs is 11.26%. After surface modification with APAS and PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH), the mass losses of APAS-Au NSs and ACUPA-APAS-Gd-Au-PENSs are 13.52% and 70.58%, respectively. Compared with Au NSs, the mass loss of APAS-Au NSs is due to the ignition of the non-metallic organic component APAS; compared with APAS-Au NSs, the mass loss of ACUPA-APAS-Gd-Au-PENSs is due to the ignition of the non-metallic organic component PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH). The calculated mass fractions of APAS and PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) loaded on Au NSs were 2.26% and 57.06%, respectively.

[0103] (6) Zeta potential test results

[0104] The zeta potential of ACUPA-APAS-Gd-Au-PENSs prepared in Example 1 and ACUPA-Gd-Au-PENSs prepared in Comparative Example 2 was measured to evaluate the charge-flipping performance of APAS in ACUPA-APAS-Gd-Au-PENSs. ACUPA-APAS-Gd-Au-PENSs and ACUPA-Gd-Au-PENSs were dissolved in 1 mL of phosphate buffer at different pH values ​​(5.5, 6.0, 6.5, 7.0, 7.5, 8.0) to prepare 1.5 μM solutions, and the zeta potential was measured using a particle size and zeta potential analyzer.

[0105] like Figure 7 As shown, ACUPA-APAS-Gd-Au-PENSs are electroneutrally neutral in phosphate buffer at pH 8.0-7.0, and positively charged at pH 6.5-5.5, with the potential increasing with increasing acidity. In contrast, unmodified ACUPA-Gd-Au-PENSs are electroneutrally neutral at pH 8.0-5.5. This demonstrates that the APAS functionalization in this invention enables ACUPA-APAS-Gd-Au-PENSs to exhibit charge-switching properties, changing from electroneutrally neutral to positively charged under acidic conditions.

[0106] (7) Results of T1 relaxation rate test

[0107] First, the Gd content in the prepared ACUPA-APAS-Gd-Au-PENSs was determined using ICP-OES. Then, the above material was weighed and thoroughly dissolved in ultrapure water to prepare aqueous solutions with Gd concentrations of 0.025 mM, 0.05 mM, 0.1 mM, 0.2 mM, 0.4 mM, and 0.8 mM, respectively. The T1 relaxation time of ACUPA-APAS-Gd-Au-PENSs with different Gd concentrations was measured sequentially using magnetic resonance imaging. The slope of the curve obtained by linearly fitting the reciprocal of the T1 relaxation time (1 / T1) to the corresponding Gd ion concentration represents the T1 relaxation rate of the nanomaterial.

[0108] like Figure 8 As shown, the brightness in the MR imaging pseudocolor image increases with concentration dependence as Gd concentration increases. By linearly fitting the Gd concentration to the reciprocal of the relaxation time (1 / T1), the T1 relaxation rate of ACUPA-APAS-Gd-Au-PENSs is found to be 8.49 mM. -1 s -1It exhibits a relatively high T1 relaxation rate. This indicates that the ACUPA-APAS-Gd-Au-PENSs prepared in this invention possess excellent MR imaging capabilities.

[0109] (8) Photothermal performance test results

[0110] The photothermal heating test was conducted on the ACUPA-APAS-Gd-Au-PENSs nanomaterials prepared in Example 1 through the following three experiments to evaluate the photothermal conversion efficiency and photothermal stability. First, aqueous solutions of ACUPA-APAS-Gd-Au-PENSs with different Au concentrations (0.1 mM, 0.5 mM, 1 mM, and 5 mM) were prepared, with an equal volume of distilled water as a control. Near-infrared laser (808 nm, 1.2 W / cm²) was used. 2 The laser was irradiated for 300 seconds, and the temperature change was recorded every 5 seconds using thermocouples. The temperature difference (ΔT) before and after near-infrared laser irradiation was also recorded. Secondly, different powers (0.5, 1, 1.2, 1.5, 2 W / cm²) were used. 2 A100 μL of ACUPA-APAS-Gd-Au-PENSs aqueous solution ([Au] = 0.5 mM) was irradiated with near-infrared laser (808 nm) for 300 s, and the temperature change was recorded every 5 s using a thermocouple. Third, the temperature of the ACUPA-APAS-Gd-Au-PENSs aqueous solution (100 μL, [Au] = 0.5 mM) under near-infrared laser (808 nm, 1.2 W / cm²) was observed. 2 The temperature was increased for 250 seconds under irradiation and then cooled naturally for 250 seconds after the laser was turned off. This process was repeated 5 times, and the temperature change was recorded every 5 seconds using a thermocouple.

[0111] like Figure 9 As shown in Figure a, after laser treatment (1.2W / cm²), 2 After irradiation, the water temperature remained almost unchanged, while the temperature of aqueous solutions of ACUPA-APAS-Gd-Au-PENSs with different Au concentrations increased significantly. The higher the concentration, the greater the temperature increase; when the Au concentration reached 0.5 mM, the temperature could rise to 53.5℃. Furthermore, aqueous solutions of ACUPA-APAS-Gd-Au-PENSs with different Au concentrations were irradiated with a near-infrared laser (808 nm, 1.2 W / cm²). 2 The change in temperature difference (ΔT) before and after irradiation indicates that the higher the Au concentration, the greater the temperature difference. Figure 9 (As shown in b). Figure 9Figure c shows the temperature change curves of an aqueous solution of ACUPA-APAS-Gd-Au-PENSs with an Au concentration of 0.5 mM irradiated by lasers of different power. Under laser irradiation, the temperature shows a clear upward trend, with higher power resulting in a higher temperature rise. At a power of 1.2 W / cm², the temperature increase is observed. 2 Under these conditions, the temperature can rise to 55.8℃, sufficient to kill cancer cells. After undergoing five cycles of heating and cooling, from... Figure 9 It can be observed that the highest temperature of the ACUPA-APAS-Gd-Au-PENSs aqueous solution after each heating is almost the same as the highest temperature of the first heating. By linearly fitting the negative of the natural logarithm of the cooling time and temperature, the photothermal conversion efficiency of ACUPA-APAS-Gd-Au-PENSs can be calculated to be 72.9%. Figure 9 (As shown in e and f). The above results demonstrate that the ACUPA-APAS-Gd-Au-PENSs prepared in this invention have good photothermal conversion efficiency and photothermal stability, and have the potential for photothermal therapy of tumors.

[0112] (9) ICP-OES test results

[0113] LNCaP cells were seeded in 12-well plates (2.5 × 10⁻⁶). 5 Cells / well were incubated overnight at 37°C and 5% CO2. After cell attachment, the culture medium was removed. RPMI-1640 media with different pH values ​​(5.5, 6.0, 6.5, 7.0, 7.5) were prepared using phosphate buffer. ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs were dissolved in RPMI-1640 media with different pH values ​​to prepare RPMI-1640 media containing ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs with an Au concentration of 100 μM. The prepared solution was added to adherent LNCaP cells and incubated for 3 hours, with 1 mL of solution added to each well, and 3 replicates per group. After incubation, the cells were washed three times with PBS, digested with trypsin, and the digestion was terminated by adding RPMI-1640 medium. The cells were then dispersed by agitation, centrifuged, and collected. Subsequently, 400 μL of aqua regia was added to the collected cells for lysis for 24 hours, followed by dilution with PBS to 6 mL. The Au content was measured by ICP-OES.

[0114] like Figure 10As shown, within the pH range of 7.0-7.5, at the same pH, the Au phagocytosis (i.e., material uptake) in LNCaP cells followed the following order: ACUPA-APAS-Gd-Au-PENSs group ≈ ACUPA-Gd-Au-PENSs group > APAS-Gd-Au-PENSs group ≈ Gd-Au-PENSs group. Furthermore, the Au phagocytosis of LNCaP cells treated with ACUPA-APAS-Gd-Au-PENSs was significantly higher than that of LNCaP cells treated with APAS-Gd-Au-PENSs and Gd-Au-PENSs under the same conditions (p<0.001). This result indicates that the targeting effect of ACUPA can enhance the uptake of nanomaterials by LNCaP cells. Under pH conditions of 5.5–6.5, the Au phagocytosis of LNCaP cells incubated with ACUPA-APAS-Gd-Au-PENSs and APAS-Gd-Au-PENSs gradually increased with decreasing pH. Specifically, under pH conditions of 5.5–6.0, the Au phagocytosis of LNCaP cells followed the order: ACUPA-APAS-Gd-Au-PENSs group > APAS-Gd-Au-PENSs group > ACUPA-Gd-Au-PENSs group > Gd-Au-PENSs group. Notably, within the pH range of 5.5–6.0, at the same pH, the Au phagocytosis of LNCaP cells treated with ACUPA-APAS-Gd-Au-PENSs was significantly higher than that treated with APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs (p<0.001). In conclusion, the pH-responsive charge-flipping properties of APAS under acidic conditions can enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells. Furthermore, under acidic conditions, the targeting effect of ACUPA and the pH-responsive charge-flipping properties of APAS jointly enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells.

[0115] (10) Flow cytometry test results

[0116] LNCaP cells were seeded in 12-well plates (2.5 × 10⁻⁶). 5Cells / well were incubated overnight at 37°C and 5% CO2. After cell adhesion, the culture medium was removed. RPMI-1640 media with different pH values ​​(6.0, 7.4) were prepared using phosphate-buffered saline. ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs were dissolved in RPMI-1640 media with different pH values ​​to prepare RPMI-1640 media containing ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs at an Au concentration of 100 μM. Pure RPMI-1640 media diluted with PBS was used as a blank control. The prepared solution was added to each well at a rate of 1 mL, and the cells were incubated for 3 hours, with 3 replicates per group. After incubation, the cells were washed three times with PBS, digested with trypsin, and the digestion was terminated by adding RPMI-1640 medium. The cells were then dispersed by agitation, centrifuged, and collected. Finally, the cells were resuspended in 3 mL of PBS. Flow cytometry was used to analyze each LNCaP cell sample at FL1 fluorescence channel (488 nm). 10,000 cells were collected from each sample group.

[0117] like Figure 11 and 12As shown, under pH 7.4 conditions, the mean fluorescence intensity in LNCaP cells followed the order: ACUPA-APAS-Gd-Au-PENSs group ≈ ACUPA-Gd-Au-PENSs group > APAS-Gd-Au-PENSs group ≈ Gd-Au-PENSs group ≈ PBS group. Importantly, the mean fluorescence intensity of LNCaP cells treated with ACUPA-APAS-Gd-Au-PENSs was significantly higher than that of LNCaP cells treated with APAS-Gd-Au-PENSs and Gd-Au-PENSs (p<0.001), indicating that the targeting effect of ACUPA can enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells. Under pH 6.0 conditions, the mean fluorescence intensity in LNCaP cells followed the order: ACUPA-APAS-Gd-Au-PENSs group > APAS-Gd-Au-PENSs group ≈ ACUPA-Gd-Au-PENSs group > Gd-Au-PENSs group. Furthermore, the mean fluorescence intensity of LNCaP cells treated with ACUPA-APAS-Gd-Au-PENSs was significantly higher than that of LNCaP cells treated with APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs (p<0.001). A higher average fluorescence intensity indicates that LNCaP cells phagocytose more gold nanostar complexes. These results further confirm that the pH-responsive charge-flipping property of APAS can enhance the ability of LNCaP cells to take up ACUPA-APAS-Gd-Au-PENSs under acidic conditions. Furthermore, the targeting effect of ACUPA and the pH-responsive charge-flipping properties of APAS can jointly enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells under simulated tumor microacidic conditions (pH 6.0). Quantitative analysis of the cellular uptake rate of LNCaP cells revealed that the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells under pH 7.4 and 6.0 conditions was similar to the conclusions drawn from the above statistical average fluorescence intensity analysis. ACUPA-APAS-Gd-Au-PENSs exhibited higher LNCaP cell uptake than ACUPA-Gd-Au-PENSs, APAS-Gd-Au-PENSs, and Gd-Au-PENSs under both pH 7.4 and 6.0 conditions.In summary, both the targeting effect of ACUPA and the pH-responsive charge-flipping effect of APAS under simulated tumor microacidity conditions can enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells. Furthermore, under simulated tumor microacidity conditions (pH 6.0), the two can jointly enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells.

[0118] (11) Results of laser scanning confocal microscopy test

[0119] To further observe the uptake of ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs by LNCaP cells at different pH levels (7.4, 6.0), the intensity of intracellular green fluorescence can be directly observed using laser scanning confocal microscopy, based on the unique fluorescence properties of surface-modified FI, to assess this. LNCaP cells were seeded in 12-well plates (1.0 × 10⁻⁶). 5 Cells were cultured in wells at 37°C overnight in an incubator containing 5% CO2. After cell adhesion, the RPMI-1640 medium was removed. ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs were added to RPMI-1640 media at different pH values ​​(7.4, 6.0) to prepare eight different Au concentrations of 100 μM. The prepared media containing different materials were added to the adherent LNCaP cells and incubated for 3 hours, with three replicates per group. Cells incubated in RPMI-1640 medium prepared with PBS served as a control. After incubation, the cells were washed three times with PBS and fixed with glutaraldehyde (0.5 mL, 2.5%) (4°C, 15 min). Glutaraldehyde was then aspirated, and the cells were washed 3-4 times with PBS. Cell nuclei were stained with DAPI (1 mL, 1 μg / mL) (37℃, 30 min), and washed 3-4 times with PBS. Finally, cell samples were imaged using a laser scanning confocal microscope, and the green fluorescence emitted by FI was observed through the FL1 channel to assess the uptake of ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs by LNCaP cells under different pH conditions (6.0, 7.4).

[0120] like Figure 13As shown, LNCaP cells treated with PBS showed almost no green fluorescence. LNCaP cells incubated with ACUPA-APAS-Gd-Au-PENSs at pH 6.0 exhibited the brightest green fluorescence, indicating that LNCaP cells underwent the highest uptake of ACUPA-APAS-Gd-Au-PENSs under this condition. At pH 7.4, similar to LNCaP cells incubated with PBS, LNCaP cells incubated with APAS-Gd-Au-PENSs and Gd-Au-PENSs showed almost no green fluorescence, while LNCaP cells incubated with ACUPA-Gd-Au-PENSs and ACUPA-APAS-Gd-Au-PENSs showed significant green fluorescence. This suggests that the targeting effect of ACUPA can enhance the uptake of nanomaterials by LNCaP cells. Under pH 6.0 conditions, the fluorescence intensity of LNCaP cells followed the order: ACUPA-APAS-Gd-Au-PENSs group > ACUPA-Gd-Au-PENSs group ≈ APAS-Gd-Au-PENSs group > Gd-Au-PENSs group ≈ PBS group. This indicates that under simulated tumor microacidity (pH 6.0), the pH-responsive charge-flipping property of APAS can enhance the uptake of nanomaterials by LNCaP cells, and the targeting effect of ACUPA and the pH-responsive charge-flipping property of APAS can jointly enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells under simulated tumor microacidity (pH 6.0). (See appendix) Figure 13 ).

[0121] (12) Results of in vitro cellular CT imaging

[0122] LNCaP cells were seeded in 6-well plates (2.0 × 10⁶ cells / well). 6Incubate the cells overnight at 37°C and 5% CO2 in cells / wells. Once the cells have adhered, remove the culture medium. Prepare RPMI-1640 medium at pH 6.0. Dissolve ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs in pH 6.0 medium to prepare RPMI-1640 medium containing different Au concentrations (0, 50, 100, 150, 200 μM) at pH 6.0. RPMI-1640 medium containing the four materials mentioned above was added to adherent LNCaP cells and incubated for 3 hours (2 mL per well, 3 replicates per group). After incubation, the LNCaP cells were washed with PBS, digested, centrifuged, and finally resuspended in 100 μL of PBS. The collected LNCaP cell suspension was then subjected to CT imaging using a CT imaging system.

[0123] like Figure 14 As shown in Figures a and b, under pH 6.0 conditions, although the brightness change was not clearly visible on the CT images, the CT signal value (HU) clearly showed that with the increase of Au concentration, the HU value of LNCaP cells incubated with RPMI-1640 medium containing ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs all showed an increasing trend. When the Au concentration reached 200 μM, the HU value of LNCaP cells incubated with ACUPA-APAS-Gd-Au-PENSs was significantly higher than that of the other three groups (p<0.05). At an Au concentration of 200 μM, the HU value of LNCaP cells incubated with ACUPA-APAS-Gd-Au-PENSs was 69.87, which was 1.20 times, 1.17 times, and 1.43 times that of LNCaP cells incubated with APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs in RPMI-1640 medium at the same Au concentration, respectively. This indicates that under pH 6.0 conditions, the targeting effect of ACUPA and the pH-responsive charge-flipping effect of APAS can jointly enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells, thereby improving the CT imaging effect of LNCaP cells in vitro.

[0124] (13) In vitro cell MR imaging effect

[0125] LNCaP cells were seeded in 6-well plates (2.0 × 10⁶ cells / well). 6 Cells / well were incubated overnight at 37°C and 5% CO2. After cell attachment, the culture medium was removed. RPMI-1640 medium at pH 6.0 was prepared by dissolving ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs in pH 6.0 medium to prepare RPMI-1640 media containing different Gd concentrations (0, 20, 50, 100, 200 μM). RPMI-1640 medium containing the above four materials was added to adherent LNCaP cells and incubated for 3 hours (2 mL per well, 3 replicates per group). After incubation, the LNCaP cells were washed with PBS, digested, centrifuged, and finally resuspended in 100 μL of PBS.

[0126] like Figure 14 As shown in Figures c and d, after cells phagocytosed the material, the MR imaging signals increased to varying degrees with the increase of Gd concentration. In particular, the MR imaging signal of LNCaP cells incubated with ACUPA-APAS-Gd-Au-PENSs showed the greatest change. At a Gd concentration of 100 μM, the MR imaging signal values ​​of LNCaP cells incubated with ACUPA-APAS-Gd-Au-PENSs began to show a significant difference from those of LNCaP cells incubated with Gd-Au-PENSs (p<0.001). Among them, at a Gd concentration of 200 μM, the MR imaging signal value of LNCaP cells incubated with ACUPA-APAS-Gd-Au-PENSs was 260.30, significantly higher than that of LNCaP cells incubated with APAS-Gd-Au-PENSs (225.50), ACUPA-Gd-Au-PENSs (232.93), and Gd-Au-PENSs (211.20) (p<0.001). This result indicates that the targeting effect of ACUPA and the pH-responsive charge-flipping performance of APAS can jointly enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells under simulated tumor microacidic conditions (pH 6.0), thereby improving the MR imaging effect of LNCaP cells under these conditions.

[0127] (14) Determination of the effect of in vitro cell photothermal therapy by CCK-8 assay

[0128] Using LNCaP cells as a model, the in vitro photothermal therapeutic effects of ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs on prostate cancer cells were evaluated using CCK-8 assay. LNCaP cells were seeded in 96-well plates (1.0 × 10⁻⁶ wells). 4 Cells / well were incubated overnight at 37°C and 5% CO2. After cell attachment, the culture medium was removed, and the 96-well plates were numbered from 1 to 5. The four materials ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs were dissolved in RPMI-1640 medium to prepare RPMI-1640 medium with different Au concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mM), containing ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs, respectively. LNCaP cells were incubated in RPMI-1640 medium containing different Au concentrations (ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs) for 6 hours in wells (3 replicates per group, 100 μL per well) after 6 hours of incubation. After 6 hours, the LNCaP cells were washed with PBS, and the medium in each well was replaced with fresh RPMI-1640 medium. Then, near-infrared laser (808 nm, 1.2 W / cm²) was used. 2 LNCaP cells in well 2 of a 96-well plate were irradiated for 300 s. Then, 20 μL of CCK-8 solution was added to each well of each cell group, and the cells were incubated at 37°C and 5% CO2 for 4 h. Finally, the absorbance of each cell at 450 nm was measured using a microplate reader to calculate the cell viability of the LNCaP cells.

[0129] To investigate the effect of the pH-responsive charge-flipping properties of APAS on the photothermal therapy efficiency of various gold nanostar complexes in LNCaP cells in vitro, RPMI-1640 media with pH 7.4 and pH 6.0 were prepared. ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs were dissolved in RPMI-1640 media with pH 7.4 and pH 6.0 to prepare RPMI-1640 media with different Au concentrations (0, 0.2, 0.4, 0.6, 0.8, 1.0 mM) and different pH (7.4, 6.0) of ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs. Different pH (7.4, 6.0) media containing different Au concentrations of APAS-Gd-Au-PENSs and Gd-Au-PENSs were added to 96-well plates (well 3), and different pH (7.4, 6.0) media containing different Au concentrations of ACUPA-APAS-Gd-Au-PENSs and ACUPA-Gd-Au-PENSs were added to 96-well plates (well 4). The adherent LNCaP cells were incubated for 6 hours (3 replicates per group, 100 μL per well). After 6 hours of incubation, the LNCaP cells were washed with PBS, and the medium in each well was replaced with fresh RPMI-1640 medium. Then, near-infrared laser (808 nm, 1.2 W / cm²) was used for further analysis. 2 LNCaP cells in 96-well plates (wells 3 and 4) were irradiated for 300 s. Then, 20 μL of CCK-8 solution was added to each well of each group of cells, and the cells were incubated at 37°C and 5% CO2 for 4 h. Finally, the absorbance of each well at 450 nm was measured using a microplate reader to calculate the cell viability of the LNCaP cells.

[0130] To comprehensively evaluate the in vitro LNCaP cell photothermal therapy efficacy of ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs under simulated tumor microacidity (pH 6.0). Four materials, ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs, were dissolved in RPMI-1640 medium at pH 6.0 to prepare RPMI-1640 media with different Au concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mM), containing ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs respectively, at pH 6.0. The prepared media containing different materials at pH 6.0 were added to 96-well plates (numbered 5) and LNCaP cells were incubated for 6 hours after cell adhesion (3 replicates per group, 100 μL per well). After 6 hours of incubation, LNCaP cells were washed with PBS, and the culture medium in each well was replaced with fresh RPMI-1640 medium. Subsequently, near-infrared laser (808 nm, 1.2 W / cm²) was used. 2 LNCaP cells in well 5 of a 96-well plate were irradiated for 300 s. Then, 20 μL of CCK-8 solution was added to each well of each group of cells, and the cells were incubated at 37°C and 5% CO2 for 4 h. Finally, the absorbance of each well at 450 nm was measured using a microplate reader to calculate the cell viability of LNCaP cells.

[0131] like Figure 15 As shown in Figure a, after co-culturing LNCaP cells with different concentrations of ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs for 6 hours, even with an Au concentration of 1 mM, the cell viability remained above 80.68%, indicating that ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs all possess good biocompatibility. Figure 15As shown in Figure b, after laser irradiation, ACUPA-APAS-Gd-Au-PENSs+Laser, APAS-Gd-Au-PENSs+Laser, ACUPA-Gd-Au-PENSs+Laser, and Gd-Au-PENSs+Laser all exhibited varying degrees of cancer cell inhibition effects on LNCaP cells. The cancer cell inhibition efficiency increased with increasing Au concentration. Specifically, when the Au concentration was 0.8 mM and 1 mM, the cancer cell inhibition efficiency was: ACUPA-APAS-Gd-Au-PENSs+Laser group ≈ ACUPA-Gd-Au-PENSs+Laser group > APAS-Gd-Au-PENSs+Laser group ≈ Gd-Au-PENSs+Laser group. Furthermore, at Au concentrations of 0.8 mM and 1 mM, the cell viability of LNCaP cells treated with ACUPA-APAS-Gd-Au-PENSs+Laser was 39.59% and 32.17%, respectively, which was significantly lower than that of LNCaP cells treated with APAS-Gd-Au-PENSs+Laser and Gd-Au-PENSs+Laser at the same Au concentration (p<0.001). This indicates that the targeting effect of ACUPA can enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells, thereby improving the efficiency of ACUPA-APAS-Gd-Au-PENSs-mediated photothermal therapy of LNCaP cells in vitro.

[0132] Meanwhile, to investigate the inhibitory effect of APAS-modified nanomaterials on cancer cells in the tumor microacidic environment, the photothermal therapeutic effects of APAS-Gd-Au-PENSs and Gd-Au-PENSs on LNCaP cells in vitro were first tested under different pH conditions (7.4, 6.0). Figure 15As shown in Figure c, under the same Au concentration, the cell viability of LNCaP cells treated with Gd-Au-PENSs+Laser at different pH conditions (7.4, 6.0) and APAS-Gd-Au-PENSs+Laser at pH 7.4 was similar, while the cell viability of LNCaP cells treated with APAS-Gd-Au-PENSs+Laser at pH 6.0 was lower. When the Au concentration was 1 mM, the cell viability of LNCaP cells treated with APAS-Gd-Au-PENSs+Laser at pH 6.0 (32.51%) was significantly lower than that of LNCaP cells treated with Gd-Au-PENSs+Laser at pH 6.0 (43.08%, p<0.001). These results indicate that under simulated tumor microacidic conditions, the pH-responsive charge-flipping properties of APAS can enhance the uptake of APAS-Gd-Au-PENSs by LNCaP cells, thereby improving its in vitro photothermal therapeutic effect on LNCaP cells. Subsequently, after modifying the targeted small molecule ACUPA, ACUPA-APAS-Gd-Au-PENSs were obtained and tested in vitro using LNCaP cell photothermal therapy. The results are as follows: Figure 15 As shown in Figure d, LNCaP cells treated with ACUPA-Gd-Au-PENSs+Laser at different pH (7.4, 6.0) and at pH 7.4 showed similar cell viability under the same Au concentration. However, the cell viability of LNCaP cells treated with ACUPA-APAS-Gd-Au-PENSs+Laser at pH 6.0 was significantly lower, with the most significant difference observed at Au concentrations of 0.8 mM and 1.0 mM. At an Au concentration of 1 mM, the cell viability of LNCaP cells treated with ACUPA-APAS-Gd-Au-PENSs+Laser at pH 6.0 (16.30%) was significantly lower than that treated with ACUPA-Gd-Au-PENSs+Laser at pH 6.0 (33.23%) and ACUPA-APAS-Gd-Au-PENSs+Laser at pH 7.4 (32.85%) (p<0.001). This further demonstrates that under simulated tumor microacidity (pH 6.0), the pH-responsive charge-flipping properties of APAS can enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells, thereby improving the efficiency of ACUPA-APAS-Gd-Au-PENSs-mediated in vitro LNCaP cell photothermal therapy, and that modification of ACUPA does not affect the pH-responsive charge-flipping performance of APAS.

[0133] Finally, the photothermal therapeutic efficacy of ACUPA-APAS-Gd-Au-PENSs, APAS-Gd-Au-PENSs, ACUPA-Gd-Au-PENSs, and Gd-Au-PENSs on LNCaP cells under simulated tumor microacidic conditions (pH 6.0) was comprehensively evaluated. The results are as follows: Figure 15 As shown in Figure e, with increasing Au concentration, the cell inhibition efficiencies of ACUPA-APAS-Gd-Au-PENSs+Laser, ACUPA-Gd-Au-PENSs+Laser, APAS-Gd-Au-PENSs+Laser, and Gd-Au-PENSs+Laser against LNCaP cells gradually increased. At Au concentrations of 0.8 mM and 1.0 mM, the cell inhibition efficiencies at the same Au concentration were: ACUPA-APAS-Gd-Au-PENSs+Laser > ACUPA-Gd-Au-PENSs+Laser ≈ APAS-Gd-Au-PENSs+Laser > Gd-Au-PENSs+Laser. Among them, at an Au concentration of 1.0 mM, the cell viability of LNCaP cells treated with ACUPA-APAS-Gd-Au-PENSs+Laser (13.78%) was significantly lower than that of LNCaP cells treated with ACUPA-Gd-Au-PENSs+Laser (31.54%), APAS-Gd-Au-PENSs+Laser (33.12%), and Gd-Au-PENSs+Laser (43.96%) (p<0.001). This result indicates that the targeting effect of ACUPA and the pH-responsive charge-flipping performance of APAS can jointly enhance the uptake of ACUPA-APAS-Gd-Au-PENSs by LNCaP cells under simulated tumor microacidic conditions (pH 6.0), thereby greatly improving the in vitro photothermal therapy efficiency of ACUPA-APAS-Gd-Au-PENSs on LNCaP cells.

Claims

1. A PSMA-targeted zwitterionic functionalized gold nanostar complex, characterized in that, It is prepared by the following steps: Step 1: Dissolve the PSMA targeting ligand (S)-2-(3-((S)-5-amino-1-carboxypentyl)ureo)glutaric acid ACUPA in water, add sodium carbonate aqueous solution and stir to react, then add bi-terminated succinimide ester polyethylene glycol NHS-PEG-NHS aqueous solution and stir to react to obtain NHS-PEG-ACUPA. The molar ratio of PSMA-targeting ligand ACUPA to sodium carbonate is 3-4:2, and the molar ratio of PSMA-targeting ligand ACUPA to bisuccinimide ester polyethylene glycol NHS-PEG-NHS is 1-1.5:

1. Step 2: Dissolve hyperbranched polyethyleneimine (PEI.NH2) in water, add an aqueous solution of the chelating agent diethylenetriaminepentaacetic acid dianhydride, stir and react to obtain PEI.NH2-DTPA; The molar ratio of diethylenetriaminepentaacetic acid dianhydride to hyperbranched polyethyleneimine PEI.NH2 is 20-22:1; Step 3: Add the aqueous solution of NHS-PEG-ACUPA obtained in Step 1 to the aqueous solution of PEI.NH2-DTPA, stir and react to obtain PEI.NH2-DTPA-(PEG-ACUPA). Then, add the aqueous solution of succinimide ester-polyethylene glycol-thiol NHS-PEG-SH to the aqueous solution of PEI.NH2-DTPA-(PEG-ACUPA)-(PEG-SH), stir and react to obtain PEI.NH2-DTPA-(PEG-ACUPA)-(PEG-SH). The molar ratio of NHS-PEG-ACUPA, NHS-PEG-SH and hyperbranched polyethyleneimine PEI.NH2 is 10-12:1; Step 4: Add DMSO solution of fluorescein isothiocyanate to the DMSO solution of PEI.NH2-DTPA-(PEG-ACUPA)-(PEG-SH) obtained in step 3, stir and react to obtain PEI.NH2-DTPA-FI-(PEG-ACUPA)-(PEG-SH). The molar ratio of fluorescein isothiocyanate to hyperbranched polyethyleneimine (PEI.NH2) is 5-7:1; Step 5: Add an aqueous solution of gadolinium nitrate to the aqueous solution of PEI.NH2-DTPA-FI-(PEG-ACUPA)-(PEG-SH) obtained in Step 4, stir the reaction, and obtain PEI.NH2-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH); The molar ratio of gadolinium nitrate to diethyltriaminepentaacetic acid dianhydride is 3-5:1; Step 6: Add triethylamine to the aqueous solution of PEI.NH2-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) obtained in Step 5, then add acetic anhydride, stir the reaction, and obtain functionalized hyperbranched polyethyleneimine PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH); The molar ratio of triethylamine, acetic anhydride, and PEI.NH2-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) is 120-660:100-550:1; Step 7: Heat the aqueous solution of chloroauric acid to boiling, then add sodium citrate solution, continue heating and stirring until the solution turns wine red, then let it cool naturally to room temperature to obtain an aqueous solution of Au seeds. Step 8: Add the Au seeds aqueous solution obtained in Step 7 to the chloroauric acid aqueous solution, then simultaneously add the silver nitrate aqueous solution and the ascorbic acid aqueous solution, and add the hexadecyltrimethylammonium bromide aqueous solution. Stir the reaction to obtain the gold nanostar Au NSs aqueous solution. Step 9: Dissolve alkoxyphenyl sulfonamide APAS in DMSO, add it to the DMSO solution of Au NSs obtained in Step 8, stir and react to obtain APAS-Au NSs; Step 10: Mix and stir the aqueous solution of PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) obtained in step 6 with the aqueous solution of APAS-Au NSs obtained in step 9 to obtain the PSMA-targeted zwitterionic functionalized gold nanostar complex ACUPA-APAS-Gd-Au-PENSs. The mass ratio of Au NSs to PEI.NHAc-DTPA(Gd)-FI-(PEG-ACUPA)-(PEG-SH) is 1:5-6.

2. The PSMA-targeted zwitterionic functionalized gold nanostar complex according to claim 1, characterized in that, In step 1, the reaction time of PSMA targeting ligand ACUPA and sodium carbonate is 6 h, and the reaction time after adding bisuccinimide ester polyethylene glycol NHS-PEG-NHS is 72 h.

3. The PSMA-targeted zwitterionic functionalized gold nanostar complex according to claim 1, characterized in that, In step 2, the reaction time for hyperbranched polyethyleneimine (PEI.NH2) and diethylenetriaminepentaacetic acid dianhydride is 24 h.

4. The PSMA-targeted zwitterionic functionalized gold nanostar complex according to claim 1, characterized in that, The reaction time for NHS-PEG-ACUPA and PEI.NH2-DTPA in step 3 is 72 h, and the reaction time after adding succinimide ester-polyethylene glycol-thiol NHS-PEG-SH is 72 h.

5. The PSMA-targeted zwitterionic functionalized gold nanostar complex according to claim 1, characterized in that, In step 4, the stirring reaction is carried out under the condition of stirring in the dark for 24 hours.

6. The PSMA-targeted zwitterionic functionalized gold nanostar complex according to claim 1, characterized in that, In step 5, the stirring reaction is carried out under light-protected stirring conditions for 24 hours.

7. The PSMA-targeted zwitterionic functionalized gold nanostar complex according to claim 1, characterized in that, In step 6, the stirring reaction is carried out under the condition of stirring in the dark for 24 hours.

8. The PSMA-targeted zwitterionic functionalized gold nanostar complex according to claim 1, characterized in that, In step 10, the stirring reaction time is 24 hours.

9. The application of the PSMA-targeted zwitterionic functionalized gold nanostar complex of claim 1 in the preparation of tumor diagnostic and therapeutic reagents, wherein the tumor is prostate cancer.

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