Nanoprobe responding to tumor microenvironment and preparation method of nanoprobe

Abstract

The invention discloses a nano probe for tumor microenvironment response and a preparation method of the nano probe, and particularly provides a nano probe for a near-infrared two-region (NIR) fluorescence imaging system for tumor microenvironment response and a preparation method of the nano probe. According to the present invention, the gold nanoparticles (AuNPs) capable of being removed from the kidney and the targeting-size control part (EL) are simply connected through the responder (TR) capable of being cut by the tumor microenvironment (TME) related enzyme so as to obtain the nanometer probe (ELTR (at) AuNPs) capable of being used for accurately diagnosing cancers; according to the nano probe designed by the invention, the size of the nano probe is increased by utilizing EL modification, the existence of AuNPs enables the nano probe to realize high-resolution imaging under an NIR-II window, and the nano probe has the potential of visual cancer diagnosis.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and particularly relates to a tumor microenvironment-responsive nanoprobe and a preparation method thereof. Background Art

[0002] Cancer is one of the main causes of death globally, seriously affecting human health and imposing a huge financial burden on the public healthcare system. Therefore, it is necessary to develop an accurate and simple diagnostic method to provide reliable information about the presence, progression, treatment outcome, and recurrence of cancer. Current research mainly focuses on developing probes for multiple imaging modalities. For example, Positron emission tomography (PET)-Computed tomography (CT) and Positron emission tomography-Magnetic resonance imaging (MRI). However, these methods cannot reflect the dynamic changes of tumor biomarkers and face disadvantages such as technical complexity, high radiation, and high cost. Therefore, it is necessary to construct a simple, accurate, and low-cost multimodal cancer diagnosis platform.

[0003] Fluorescence imaging in the second near-infrared window (NIR-II, 1000 - 1700 nm) has promoted the development of in vivo bioimaging towards high spatiotemporal resolution and excellent biocompatibility due to its low photon scattering, negligible autofluorescence interference, and non-radiative properties. However, most probes gradually accumulate in the reticuloendothelial system (RES) tissues (such as the liver and spleen) and non-cancerous inflammatory tissues during blood circulation, which can lead to significant background noise, interfering with tumor identification. At the same time, potential toxicity may be caused by slow excretion in the body, seriously hindering their clinical translation.

[0004] In the tumor microenvironment (TME), protease-activated probes can amplify diagnostic signals, which is beneficial to improving detection sensitivity. If TME-responsive probes can redistribute NIR-II fluorescent probes to the bladder (a non-RES organ) and then excrete them through urine, this can reduce the potential long-term toxicity of the probes. Recently, artificial enzymes based on metal nanoparticles (NPs, < 3nm) in the field of nanotechnology have opened up a new path. In particular, gold nanoparticles (AuNPs) have received extensive attention due to their easy functionalization, good stability and biocompatibility, and strong artificial enzyme activity due to the presence of a large number of catalytic sites. In addition, some types of AuNPs have also been proven to have NIR-II fluorescence properties, indicating that AuNP-based probes can achieve NIR-II fluorescence imaging responsive to the TME for accurate tumor detection. Summary of the Invention

[0005] The object of the present invention is to provide a tumor microenvironment-responsive nanoprobe and its preparation method in view of the problems existing in the prior art. The nanoprobe is a nano-detection probe designed based on the differences between the tumor microenvironment and the normal tissue microenvironment. The nanoprobe is constructed by molecular self-assembly of a targeting-size control component, a responsive component, and a fluorescent component, and can achieve specific detection of tumors.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A tumor microenvironment-responsive nanoprobe, which is composed of gold nanoparticles, a targeting-size control part, and an enzyme-responsive linker.

[0007] Preferably, the gold nanoparticles (AuNPs) are gold nanoparticles with cyclodextrin as a ligand.

[0008] Preferably, the enzyme-responsive linker (TR) is synthesized from an enzyme-responsive peptide segment and an alicyclic amine.

[0009] Preferably, the targeting-size control part is a multi-arm polyethylene glycol ester (EL).

[0010] The present invention also provides a preparation method of the nanoprobe, which includes the following steps: (1) Preparation of gold nanoparticles: Using cyclodextrin and chloroauric acid as raw materials, gold nanoparticles AuNPs are synthesized by one-step reduction. (2) Preparation of the enzyme-responsive linker: Coupling an alicyclic amine with an enzyme-responsive peptide segment to obtain the enzyme-responsive linker TR. (3) Synthesis of the nanoprobe: Effectively connecting the gold nanoparticles AuNPs and the multi-arm polyethylene glycol ester EL together through the prepared enzyme-responsive linker TR, thereby obtaining the nanoprobe ELTR@AuNPs.

[0011] Preferably, in step (1), the preparation method of the gold nanoparticles is as follows: Mix the HAuCl4 solution and the cyclodextrin solution and stir evenly to obtain a mixture. Subsequently, add the NaOH solution and the NaBH4 solution to the above mixture in sequence, continue to react for 2 - 4 h, and then purify to obtain the gold nanoparticles AuNPs.

[0012] Preferably, in step (1), the molar ratio of HAuCl4, cyclodextrin, NaOH, and NaBH4 is 1:(0.6 - 1):(15 - 25):(2.4 - 3). Preferably, it is 1:0.8:20:2.65.

[0013] Preferably, in step (2), the preparation method of the enzyme-responsive linker is as follows: Weigh the enzyme-responsive peptide segment, dissolve it in a solvent, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to activate for 8-15 min, then add the cycloaliphatic amine solution, adjust the pH value of the reaction system to 8, and react under magnetic stirring for 18-30 h to obtain the enzyme-responsive linker TR.

[0014] Preferably, in step (2), the mass ratio of the enzyme-responsive peptide segment, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is (4-8):(5-8):(2-4). Preferably, it is 6:7.14:3.07.

[0015] Preferably, in step (3), the preparation method of the nanoprobe is as follows: Mix the enzyme-responsive linker TR solution with the multi-arm polyethylene glycol ester EL, continue the amidation reaction for 2-4 h, remove the excess reactants, redisperse the obtained product in PBS, and then add gold nanoparticles AuNPs to prepare ELTR@AuNPs through complexation.

[0016] The beneficial effects of the present invention are as follows: (1) The nanoprobe prepared by the present invention has a relatively large size characteristic given by the multi-arm polyethylene glycol ester.

[0017] (2) The nanoprobe ELTR@AuNPs prepared by the present invention can achieve fluorescence imaging in the NIR-II window.

[0018] (3) The nanoprobe prepared by the present invention can be used for visual detection of tumors. Description of the Drawings

[0019] Figure 1 It is the transmission electron micrograph of AuNPs and ELTR@AuNPs prepared by the present invention; Figure 2 It is the particle size diagram of AuNPs, EL, and ELTR@AuNPs prepared by the present invention; Figure 3 It is the emission spectrum diagram of ELTR@AuNPs prepared by the present invention. Detailed Embodiments

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0021] Unless otherwise specified, the reagents involved in the embodiments of the present invention are all commercially available products and can be obtained through commercial channels. The methods involved in the embodiments of the present invention are all conventional methods and can be obtained through the corresponding implementation specifications, implementation standards or existing literature in this field.

[0022] Example 1 Preparation of Nano-probe ELTR@AuNPs This example provides a preparation method of nano-probe ELTR@AuNPs, including the following steps: (1) Preparation of Gold Nanoparticles AuNPs Take 1 mL of HAuCl4 solution (10 mmol / L) and 2 mL of cyclodextrin solution (4 mmol / L) and mix them. Stir magnetically for 15 min to obtain a mixture. Subsequently, add 0.2 mL of NaOH solution (1 mol / L) and 0.2 mL of NaBH4 solution (5 mg / mL) to the above mixture in sequence and continue to react for 3 h. In order to obtain pure AuNPs, transfer the product to a dialysis bag and dialyze it in water multiple times. Finally, use an ultrafiltration centrifugal tube to remove large-sized nanoparticles and collect the solution in the outer tube, that is, pure gold nanoparticles AuNPs are obtained.

[0023] (2) Preparation of Enzyme-responsive Linker TR Weigh 6 mg of enzyme-responsive peptide segment, dissolve it in a solvent, add 7.14 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 3.07 mg of N-hydroxysuccinimide (NHS) to activate for 10 min, then add the pre-prepared cycloaliphatic amine solution, adjust the pH value of the reaction system to 8 with sodium hydroxide solution, and react under magnetic stirring for 24 h to obtain the enzyme-responsive linker TR.

[0024] (3) Preparation of Nano-probe ELTR@AuNPs Mix the enzyme-responsive linker TR solution with multi-arm polyethylene glycol ester EL, continue the amidation reaction for 3 h, use an ultrafiltration centrifugal tube to centrifuge three times at 3000 rpm to remove the excessive reactants, redisperse the obtained product in PBS, then add gold nanoparticles AuNPs, and prepare ELTR@AuNPs through complexation. Use an ultrafiltration centrifugal tube to remove the unbound reactants, and store the obtained ELTR@AuNPs in a 4°C refrigerator for further characterization.

[0025] I. Performance Verification 1. Transmission Electron Microscopy Scanning Analysis: Characterize and analyze the morphology and structure of AuNPs and ELTR@AuNPs by transmission electron microscopy (TEM). The results are as Figure 1As shown. It can be observed from the TEM image that the particle size of AuNPs is about 2 nm. It is worth noting that the particle size of the surface-modified nanoprobe ELTR@AuNPs has increased significantly to about 10 nm, and the significant difference in the particle size distribution further confirms the effective modification of the EL and TR components on the surface of AuNPs.

[0026] 2. Hydrodynamic particle size analysis: The hydrodynamic particle sizes of AuNPs, EL, and ELTR@AuNPs were systematically measured using dynamic light scattering (DLS) technology, and the results are as Figure 2 shown. The test data show that the hydrodynamic particle size of AuNPs is about 2 nm, which is basically consistent with the TEM characterization result, indicating that AuNPs have good dispersibility and stability in solution. After introducing the EL component, the hydrodynamic particle size of the prepared nanoprobe ELTR@AuNPs has increased significantly to about 10 nm. This significant change in particle size indicates that the EL molecules have been successfully grafted onto the surface of AuNPs.

[0027] 3. Near-infrared second-window fluorescence analysis: To deeply study the optical properties of ELTR@AuNPs, its emission spectrum was systematically characterized using a transient / steady-state fluorescence spectrometer, and the results are as Figure 3 shown. ELTR@AuNPs exhibit significant optical response characteristics in the near-infrared second window (NIR-II, 1000 - 1700 nm), and its maximum emission peak is located at about 1060 nm.

[0028] The above research results show that the ELTR@AuNPs nanoprobe has been successfully synthesized, and its NIR-II fluorescence property can be used for visual monitoring of tumors.

[0029] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A tumor microenvironment-responsive nanoprobe, characterized in that: The described nanoprobe is composed of gold nanoparticles, a targeting-size control part, and an enzyme-responsive linker.

2. The nanosensor according to claim 1, wherein: The gold nanoparticles are gold nanoparticles with cyclodextrin as a ligand.

3. The nanoprobe according to claim 1, characterized in that: The enzyme-responsive linker is synthesized from an enzyme-responsive peptide segment and an alicyclic amine.

4. The nanoprobe according to claim 1, characterized in that: The targeting-size control part is a multi-arm polyethylene glycol ester.

5. A method for preparing the nanoprobe according to any one of claims 1-4, characterized in that: It includes the following steps: (1) Preparation of gold nanoparticles: Using cyclodextrin and chloroauric acid as raw materials, gold nanoparticles AuNPs are synthesized by one-step reduction. (2) Preparation of the enzyme-responsive linker: Coupling the alicyclic amine with the enzyme-responsive peptide segment to obtain the enzyme-responsive linker TR. (3) Synthesis of the nanoprobe: Effectively connecting the gold nanoparticles AuNPs and the multi-arm polyethylene glycol ester EL together through the prepared enzyme-responsive linker TR, thereby obtaining the nanoprobe ELTR@AuNPs.

6. The preparation method according to claim 5, characterized in that: In step (1), the preparation method of the gold nanoparticles is as follows: Mix the HAuCl4 solution and the cyclodextrin solution and stir evenly to obtain a mixture. Subsequently, add the NaOH solution and the NaBH4 solution to the above mixture in sequence, continue the reaction for 2 - 4 h, and then purify to obtain the gold nanoparticles AuNPs.

7. The preparation method according to claim 5, characterized in that: In step (2), the preparation method of the enzyme-responsive linker is as follows: Weigh the enzyme-responsive peptide segment, dissolve it in a solvent, add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide to activate for 8 - 15 min, then add the alicyclic amine solution, adjust the pH value of the reaction system to 8, and react under magnetic stirring for 18 - 30 h to obtain the enzyme-responsive linker TR.

8. The preparation method according to claim 5, characterized in that: In step (3), the preparation method of the nanoprobe is as follows: Mix the enzyme-responsive linker TR solution with the multi-arm polyethylene glycol ester EL, continue the amidation reaction for 2 - 4 h, remove the excess reactants, disperse the obtained product in PBS, and then add the gold nanoparticles AuNPs to prepare ELTR@AuNPs through complexation.

9. The preparation method according to claim 6, characterized in that: In step (1), the molar ratio of HAuCl4, cyclodextrin, NaOH, and NaBH4 is 1:(0.6 - 1):(15 - 25):(2.4 - 3).

10. The preparation method according to claim 7, characterized in that: In step (2), the mass ratio of the enzyme-responsive peptide segment, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide is (4 - 8):(5 - 8):(2 - 4).

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