An enzyme-responsive multifunctional nanodot, its preparation method, and its application in enhancing prostate cancer imaging and treatment.

Abstract

This invention provides an enzyme-responsive multifunctional nanodot, its preparation method, and its application in enhancing prostate cancer imaging and treatment, belonging to the field of nanomaterials technology. The enzyme-responsive multifunctional nanodots AMNDs-FTp significantly aggregate at the tumor site after dephosphorylation of ALP overexpressed in the prostate cancer microenvironment, exhibiting significantly enhanced imaging effects across multiple modes, including fluorescence, CT, and MRI. Enhanced CT / MRI imaging enables accurate preoperative tumor diagnosis, while enhanced fluorescence imaging facilitates intraoperative visual navigation. Furthermore, the invention significantly enhances the absorption and conversion of near-infrared light into heat, improving the efficacy of photothermal therapy for prostate cancer tumors. This invention's enzyme-responsive multifunctional nanodot material provides an effective strategy to improve the diagnostic accuracy and therapeutic efficacy of prostate cancer, demonstrating broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of polymer materials technology, specifically relating to a method for preparing alkaline phosphatase-responsive multifunctional nanodots and their application in enhancing prostate cancer imaging and treatment. Background Technology

[0002] Prostate cancer is one of the malignant tumors of the male urinary system, and its incidence is increasing year by year. Currently, the diagnosis and treatment of prostate cancer still face many clinical challenges. Designing a nanomedicine platform capable of accurately diagnosing and treating prostate cancer is of significant clinical importance.

[0003] In recent years, gold nanoparticles (AuNPs) have attracted widespread attention from researchers due to their excellent fluorescence properties, biocompatibility, and ease of surface functionalization. Manganese (Mn) is an essential trace element for maintaining human physiological functions, and Mn... 2+ It is a paramagnetic metal ion with excellent NMR imaging performance and has wide applications in biomedicine. Based on AuNPs and Mn... 2+ The advantages of this method are to develop an effective and simple way to combine AuNPs and Mn. 2+ Assembling them into single, multifunctional spherical nanoparticles can better leverage their advantages in tumor diagnosis and treatment. Notably, the antitumor effect of gold-based nanomaterials is closely related to their size. Smaller sizes (less than 20 nm) exhibit better fluorescence properties, penetrate deeper into tissues, and diffuse more uniformly within tumor tissue. Unfortunately, they are easily expelled from the body, resulting in insufficient accumulation at the tumor site and limiting their diagnostic and therapeutic effects. Larger sizes (greater than 100 nm) can exert a high permeability and retention (EPR) effect on solid tumors and have strong absorption in the near-infrared region, making them suitable for photothermal therapy. However, they are easily captured by the reticuloendothelial system, leading to less than ideal therapeutic effects. To address these contradictions, inducing the aggregation of nanoparticles through external stimuli may offer a solution, thereby fully utilizing the advantages of nanoparticles.

[0004] Researchers have discovered that nanoparticle self-assembly can be achieved using enzymes overexpressed in the tumor microenvironment. Alkaline phosphatase (ALP) has been reported to be overexpressed on the membrane structures of certain tumors, such as liver cancer, cervical cancer, osteosarcoma, and especially prostate cancer. It has become an important biomarker for targeted imaging, diagnosis, and treatment of related tumors. ALP is characterized by its dephosphorylation of substrates, generating more hydrophobic products, accompanied by the self-assembly (or aggregation) of these products. Therefore, developing an ALP-triggered self-assembly of gold-based nanomaterials at tumor sites holds promise for improving the diagnostic accuracy and therapeutic efficacy of prostate cancer. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing alkaline phosphatase-responsive multifunctional nanodots and their application in enhancing prostate cancer imaging and treatment.

[0006] This method first prepares gold-manganese nanodots (AMNDs) via electrochemical reduction, then links an ALP-responsive polypeptide (Phe-Phe-Tyr(H2PO3)-OH, abbreviated as FTp) to AMNDs through chemical bonds to synthesize enzyme-responsive multifunctional nanodots (AMNDs-FTp). These multifunctional nanodots can undergo dephosphorylation with ALP overexpressed in the prostate cancer microenvironment. The resulting product exhibits strong hydrophobicity and intermolecular interactions such as π-π stacking, enabling self-assembly. It shows significant aggregation at the tumor site, increased size and density, enhancing the imaging and therapeutic effects of prostate cancer. We validated the application of this multifunctional nanodot material for prostate cancer imaging and treatment through in vivo / in vitro experiments. The multifunctional nanodots AMNDs-FTp possess certain multimodal imaging capabilities (fluorescence / CT / MRI). When they react with and aggregate with ALP, the imaging effects for each mode (fluorescence, CT, MRI, etc.) are significantly improved. Enhanced CT / MRI imaging enables accurate preoperative tumor diagnosis, while enhanced fluorescence imaging facilitates intraoperative visual navigation, demonstrating potential applications in clinical tumor detection and surgical guidance. Furthermore, AMNDs-FTp exhibit significantly enhanced near-infrared light absorption and heat conversion due to aggregation, improving the efficacy of photothermal therapy for tumors. In conclusion, this enzyme-responsive, multifunctional nanodot AMNDs-FTp provides an effective strategy to improve the diagnostic accuracy and therapeutic efficacy of prostate cancer.

[0007] The preparation method of the enzyme-responsive multifunctional nanodots described in this invention comprises the following specific steps:

[0008] (1) Preparation of gold-manganese nanodots (AMNDs): 10 mL of H2O, 1 mL of template / reducing agent silver nanodots, and different molar ratios of HAuCl4 and MnO2 were added sequentially to a round-bottom flask. The mixture was stirred continuously for 30 minutes at room temperature. Next, NaOH solution was slowly added to the system to adjust the pH to 7–9. After the mixture was stirred evenly, it was reacted in an oil bath at 80 °C for 4 hours. After cooling to room temperature, the product solution was obtained. The product solution was centrifuged at 8800 rpm for 15 minutes, and excess isopropanol was added to the supernatant. After mixing evenly, a suspension containing solid particles was obtained. The suspension was then centrifuged at 6600 rpm for 8 minutes, and the precipitate was dried to obtain the product gold-manganese nanodots (AMNDs). 10 mg of AMNDs was weighed and dissolved in 5 mL of deionized water to obtain an AMNDs solution with a concentration of 2 mg / mL. The solution was stored at room temperature in the dark.

[0009] (2) Preparation of enzyme-responsive multifunctional nanodots AMNDs-FTp: 3 mL of AMNDs solution with a concentration of 2 mg / mL, 7.2 mg of 1-ethyl-(3-dimethylaminoethyl)carbodiimide hydrochloride, and 3.6 mg of N-hydroxysuccinimide were added sequentially to a round-bottom flask and stirred at room temperature for 15 min. 75 μL of Phe-Phe-Tyr(H2PO3)-OH (abbreviated as FTp) solution was added to the mixture, and the reaction was carried out at room temperature for 8 h. After the reaction was completed, the obtained product was dialyzed in deionized water for 12 h, with the water changed every 3 h to remove unreacted substances and impurities. The final solution was lyophilized to obtain a solid sample. 10 mg of the solid was weighed and dissolved in 5 mL of deionized water to obtain an AMNDs-FTp solution with a concentration of 2 mg / mL, which was stored at room temperature away from light for later use.

[0010] The advantages of this invention are as follows: 1. After dephosphorylation of ALP overexpressed in the prostate cancer microenvironment, enzyme-responsive multifunctional nanodots AMNDs-FTp generate nanoaggregates at the tumor site, significantly improving multimodal imaging (fluorescence / CT / MRI) and enabling precise diagnosis; 2. Enhanced CT / MRI imaging enables accurate preoperative tumor diagnosis, and enhanced fluorescence imaging enables "intraoperative visual navigation," showing potential application value in clinical tumor detection and surgical guidance; 3. AMNDs-FTp exhibits strong absorption in the near-infrared region after aggregation, further improving photothermal therapy effects; 4. Aggregation increases the concentration and residence time of nanodots at the tumor site; 5. Multifunctional nanodots AMNDs-FTp possesses good biocompatibility and can be used as a safe formulation for in vivo tumor imaging and treatment; 6. The preparation process is simple, green, and pollution-free; 7. Multifunctional nanodots AMNDs-FTp achieves multiple functions such as in-situ aggregation at the prostate cancer tumor site, imaging diagnosis, and treatment, with broad application prospects. Attached Figure Description

[0011] Figure 1 : Transmission electron microscopy (TEM) images of the enzyme-responsive multifunctional nanodots prepared in Example 1 before (a) and after (b) in vitro simulated aggregation. The size of the nanodots before aggregation was approximately 4.6 nm. After reaction with ALP, the nanodots exhibited significant aggregation, with the size increasing to approximately 165 nm.

[0012] Figure 2Figure a shows the fluorescence spectra of the enzyme-responsive multifunctional nanodots prepared in Example 1 before and after simulated aggregation in vitro. Figure b shows the in vitro fluorescence imaging. As shown, the fluorescence imaging effect is significantly enhanced after the nanodots aggregate. Figures c and d are the in vitro CT and MRI images of the multifunctional nanodots, respectively. Nanodots at different concentrations (50, 100, 150, 200 μg / mL) showed obvious aggregation after reacting with ALP, and the CT and MRI signals gradually increased, both exceeding the imaging effect of the same concentration of unaggregated nanodots.

[0013] Figure 3 The image shows in vivo multimodal imaging (fluorescence / CT / MRI) of the enzyme-responsive multifunctional nanodots prepared in Example 1. As shown in the figure, in a mouse model of prostate cancer, the aggregated group exhibited enhanced fluorescence, CT, and MRI imaging effects, indicating that after ALP overexpression catalyzes dephosphorylation in the prostate cancer microenvironment, the nanodots showed significant aggregation at the tumor site, enhancing the imaging effect of prostate cancer.

[0014] Figure 4 Figure 1 shows the in vitro photothermal heating curve (a) and cell photothermal killing curve (b) of the enzyme-responsive multifunctional nanodots prepared in Example 1. As shown in the figure, compared with the non-aggregated group, the nanodots exhibited a better heating effect after aggregation. Under the same light irradiation conditions, the aggregated nanodots showed a stronger tumor cell killing effect, enhancing the therapeutic effect of prostate cancer. Detailed Implementation

[0015] Example 1:

[0016] (1) Preparation of gold-manganese nanodots (AMNDs): 10 mL of H2O, 1 mL of template / reducing agent silver nanodots, HAuCl4 and MnO2 in a molar ratio of 1:0.3 were added sequentially to a round-bottom flask, and the mixture was stirred continuously for 30 minutes at room temperature. Next, NaOH solution was slowly added to the system to adjust the pH to 7. After the mixture was stirred evenly, it was reacted in an oil bath at 80℃ for 4 hours. After cooling to room temperature, the product solution was obtained. The product solution was centrifuged at 8800 rpm for 15 minutes, and excess isopropanol was added to the supernatant. After mixing evenly, a suspension containing solid particles was obtained, which was then centrifuged at 6600 rpm for 8 minutes. The precipitate was dried to obtain the product gold-manganese nanodots (AMNDs). 10 mg of AMNDs was weighed and dissolved in 5 mL of deionized water to obtain an AMNDs solution with a concentration of 2 mg / mL, which was stored at room temperature in the dark.

[0017] (2) Preparation of enzyme-responsive multifunctional nanodots AMNDs-FTp: 3 mL of 2 mg / mL AMNDs solution, 7.2 mg of 1-ethyl-(3-dimethylaminoethyl)carbodiimide hydrochloride, and 3.6 mg of N-hydroxysuccinimide were added sequentially to a round-bottom flask and stirred at room temperature for 15 min. 75 μL of Phe-Phe-Tyr(H2PO3)-OH (FTp) solution was added to the mixture, and the reaction was carried out at room temperature for 8 h. After the reaction, the product was dialyzed in deionized water for 12 h, with the water changed every 3 h to remove unreacted substances and impurities. The final solution was lyophilized to obtain a solid sample. 10 mg of the solid was weighed and dissolved in 5 mL of deionized water to obtain a 2 mg / mL AMNDs-FTp solution, which was stored at room temperature away from light for later use.

[0018] Example 2:

[0019] (1) Preparation of gold-manganese nanodots (AMNDs): 10 mL of H2O, 1 mL of template / reducing agent silver nanodots, and HAuCl4 and MnO2 in a molar ratio of 1:0.4 were added sequentially to a round-bottom flask. The mixture was stirred continuously for 30 minutes at room temperature. Next, NaOH solution was slowly added to the system to adjust the pH to 8. After the mixture was stirred evenly, it was reacted in an oil bath at 80℃ for 4 hours. After cooling to room temperature, the product solution was obtained. The product solution was centrifuged at 8800 rpm for 15 minutes, and excess isopropanol was added to the supernatant. After mixing evenly, a suspension containing solid particles was obtained. The suspension was then centrifuged at 6600 rpm for 8 minutes, and the precipitate was dried to obtain the product gold-manganese nanodots (AMNDs). 10 mg of AMNDs was weighed and dissolved in 5 mL of deionized water to obtain an AMNDs solution with a concentration of 2 mg / mL. The solution was stored at room temperature in the dark.

[0020] (2) Preparation of enzyme-responsive multifunctional nanodots AMNDs-FTp: 3 mL of 2 mg / mL AMNDs solution, 7.2 mg of 1-ethyl-(3-dimethylaminoethyl)carbodiimide hydrochloride, and 3.6 mg of N-hydroxysuccinimide were added sequentially to a round-bottom flask and stirred at room temperature for 15 min. 75 μL of Phe-Phe-Tyr(H2PO3)-OH (FTp) solution was added to the mixture, and the reaction was carried out at room temperature for 8 h. After the reaction, the product was dialyzed in deionized water for 12 h, with the water changed every 3 h to remove unreacted substances and impurities. The final solution was lyophilized to obtain a solid sample. 10 mg of the solid was weighed and dissolved in 5 mL of deionized water to obtain a 2 mg / mL AMNDs-FTp solution, which was stored at room temperature away from light for later use.

[0021] Example 3:

[0022] (1) Preparation of gold-manganese nanodots (AMNDs): 10 mL of H2O, 1 mL of template / reducing agent silver nanodots, and HAuCl4 and MnO2 in a molar ratio of 1:0.5 were added sequentially to a round-bottom flask. The mixture was stirred continuously for 30 minutes at room temperature. Next, NaOH solution was slowly added to the system to adjust the pH to 9. After the mixture was stirred evenly, it was reacted in an oil bath at 80℃ for 4 hours. After cooling to room temperature, the product solution was obtained. The product solution was centrifuged at 8800 rpm for 15 minutes, and excess isopropanol was added to the supernatant. After mixing evenly, a suspension containing solid particles was obtained. The suspension was then centrifuged at 6600 rpm for 8 minutes, and the precipitate was dried to obtain the product gold-manganese nanodots (AMNDs). 10 mg of AMNDs was weighed and dissolved in 5 mL of deionized water to obtain an AMNDs solution with a concentration of 2 mg / mL. The solution was stored at room temperature in the dark.

[0023] (2) Preparation of enzyme-responsive multifunctional nanodots AMNDs-FTp: 3 mL of 2 mg / mL AMNDs solution, 7.2 mg of 1-ethyl-(3-dimethylaminoethyl)carbodiimide hydrochloride, and 3.6 mg of N-hydroxysuccinimide were added sequentially to a round-bottom flask and stirred at room temperature for 15 min. 75 μL of Phe-Phe-Tyr(H2PO3)-OH (FTp) solution was added to the mixture, and the reaction was carried out at room temperature for 8 h. After the reaction, the product was dialyzed in deionized water for 12 h, with the water changed every 3 h to remove unreacted substances and impurities. The final solution was lyophilized to obtain a solid sample. 10 mg of the solid was weighed and dissolved in 5 mL of deionized water to obtain a 2 mg / mL AMNDs-FTp solution, which was stored at room temperature away from light for later use.

Claims

1. A method for preparing enzyme-responsive multifunctional nanodots, comprising the following steps: (1) Preparation of gold manganese nanodots AMNDs: 10 mL H2O, 1 mL template agent / reducing agent silver nanodots, HAuCl4 and MnO2 in different molar ratios were added to a round bottom flask in sequence, and stirred continuously for 30 minutes at room temperature; next, NaOH solution was slowly added to the system to adjust the pH of the system to 7 ~ 9; after the mixture was stirred evenly, it was reacted in an oil bath at 80℃ for 4 h, and the product solution was obtained after cooling to room temperature; the product solution was centrifuged at 8800 rpm for 15 minutes, and excess isopropanol was added to the supernatant; after mixing evenly, a suspension with solid particles was obtained, and then centrifuged at 6600 rpm for 8 minutes. The precipitate was dried to obtain the product gold manganese nanodots AMNDs; 10 mg AMNDs was weighed and dissolved in 5 mL deionized water to obtain an AMNDs solution with a concentration of 2 mg / mL, which was stored at room temperature in the dark. (2) Preparation of enzyme-responsive multifunctional nanodots AMNDs-FTp: 3 mL of AMNDs solution with a concentration of 2 mg / mL, 7.2 mg of 1−ethyl−(3−dimethylaminoethyl)carbodiimide hydrochloride, and 3.6 mg of N−hydroxysuccinimide were added to a round-bottom flask and stirred at room temperature for 15 min; 75 μL of Phe-Phe-Tyr(H2PO3)-OH (abbreviated as FTp) solution was added to the mixture and reacted at room temperature for 8 h; after the reaction was completed, the product was dialyzed in deionized water for 12 h, with the water changed every 3 h to remove unreacted substances and impurities; the final solution was freeze-dried to obtain a solid sample, and 10 mg of the solid was weighed and dissolved in 5 mL of deionized water to obtain an enzyme-responsive multifunctional nanodots AMNDs-FTp solution with a concentration of 2 mg / mL, which was stored at room temperature in the dark for later use.

2. The method for preparing enzyme-responsive multifunctional nanodots as described in claim 1, characterized in that: The molar ratio of HAuCl4 and MnO2 in step (1) is 1:0.3~0.

5.

3. The enzyme-responsive multifunctional nanodots prepared by the method for preparing enzyme-responsive multifunctional nanodots according to any one of claims 1 to 2, characterized in that, Multifunctional nanodots combine the functions of in-situ aggregation at the prostate cancer tumor site, multimodal imaging, and photothermal therapy.

4. The enzyme-responsive multifunctional nanodot according to claim 3, characterized in that, After undergoing dephosphorylation with alkaline phosphatase ALP, which is overexpressed in the prostate cancer microenvironment, nanodots significantly aggregate at the tumor site, increasing in size and density. This significantly enhances the multimodal imaging effect of the multifunctional nanodots, enabling accurate detection of prostate cancer.

5. The enzyme-responsive multifunctional nanodot according to claim 3, characterized in that, Nanodots aggregate after reacting with ALP at the site of prostate cancer tumors, thereby achieving enhanced fluorescence imaging and enabling "intraoperative visual navigation," demonstrating significant application value in prostate cancer tumor detection and intraoperative guidance.

6. The enzyme-responsive multifunctional nanodot according to claim 3, characterized in that, After nanodots react with and aggregate in the ALP at the site of prostate cancer tumors, their ability to absorb near-infrared light and convert it into heat is enhanced, significantly improving the effect of photothermal ablation of prostate cancer tumors.

Images