Nanoparticles that enhance indocyanine green near-infrared II fluorescence, their preparation method and applications

Abstract

This invention discloses nanoparticles that enhance the near-infrared II fluorescence of indocyanine green, their preparation method, and applications. The nanoparticles are prepared by mixing PLGA-PEG-PLGA and ICG-NHS. The specific method is as follows: S1, dissolve PLGA-PEG-PLGA in a solvent to form a first solution; S2, dissolve ICG-NHS in a solvent to form a second solution; S3, thoroughly mix the first and second solutions, react them, and remove unreacted substances by ultrafiltration to obtain the nanoparticles. The order of steps S1 and S2 is not limited. This invention enhances the emission of ICG through PLGA-PEG-PLGA, extending the application of ICG in the near-infrared II region to the NIR-IIa region (1300nm-1400nm), and effectively prolongs the circulation time of ICG in the blood, thus removing limitations in its application in fields such as tumor and vascular disease diagnosis.

Description

Technical Field

[0001] This invention relates to the field of near-infrared II fluorescence imaging technology, and in particular to a nanoparticle that enhances indocyanine green near-infrared II fluorescence, its preparation method, and its uses. Background Technology

[0002] Near-infrared II (NIR-II, 1000-1700nm) fluorescence imaging has become a research hotspot in the field of precision disease diagnosis in recent years. It facilitates non-invasive visualization of biological systems and boasts advantages such as high sensitivity, high temporal and spatial resolution, and non-invasiveness and safety, making it a technology with great clinical application potential. However, the lack of NIR-II fluorophores available for clinical research is a significant limiting factor in the clinical application of NIR-II fluorescence imaging. Currently, NIR-II imaging using the tail emission of clinically approved or commercially available NIR-I fluorophores can effectively accelerate the application of NIR-II fluorescence imaging in clinical diagnosis, representing a highly feasible and novel approach.

[0003] Indocyanine green (ICG) is a cyanine dye that has been used clinically as an imaging medium for over fifty years, widely applied in angiography, sentinel lymph node biopsy, liver clearance experiments, and laparoscopic surgery. After being shown to exhibit tail fluorescence in the NIR-II region, it has been widely used in near-infrared II imaging. However, some inherent drawbacks of ICG limit its clinical application to some extent, such as its moderate fluorescence quantum yield and short plasma half-life. The rapid clearance of ICG by the liver after it enters the bloodstream and binds to plasma proteins is a major obstacle to its development in fluorescence imaging. The short plasma half-life limits the time available for ICG to be used in vascular imaging to diagnose vascular diseases and is also highly detrimental to the EPR (enhanced permeability and retention) effect in passive tumor imaging of solid tumors. Incorporating ICG into nanoparticles can improve this deficiency. Currently, most FDA-approved nanoparticles are organic nanoparticles. As of the end of 2018, among 255 studies involving nanoparticles, two nanoparticles using ICG had been included in clinical trials. ICG nanoparticles have enormous potential for clinical translation, and designing ICG nanoparticles with stronger luminescence properties and longer blood circulation time is of great significance. Summary of the Invention

[0004] This invention reveals that nanoparticles formed from PLGA-PEG-PLGA and ICG not only possess ultra-strong fluorescence signals, but also exhibit significantly increased circulation time in the blood, which is highly beneficial for applications such as vascular imaging and tumor imaging.

[0005] One of the objectives of this invention is to provide nanoparticles that enhance the near-infrared II fluorescence of indocyanine green.

[0006] The second objective of this invention is to provide a method for preparing nanoparticles that enhance the near-infrared fluorescence of indocyanine green.

[0007] A third objective of this invention is to provide the use of the nanoparticles that enhance the near-infrared II fluorescence of indocyanine green.

[0008] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0009] In a first aspect, the present invention proposes a nanoparticle that enhances the near-infrared fluorescence of indocyanine green, wherein the nanoparticle is prepared by a mixed reaction of PLGA-PEG-PLGA and ICG-NHS.

[0010] Specifically, this involves preparing solutions of PLGA-PEG-PLGA and ICG-NHS separately, then mixing and reacting them thoroughly.

[0011] Dimeric lactic acid-glycolic acid copolymer polyethylene glycol (PLGA-PEG-PLGA) is an amphiphilic BAB triblock copolymer composed of hydrophilic block polyethylene glycol (PEG) and hydrophobic block polylactide-glycolic acid (PLGA), with the following structure:

[0012]

[0013] x, y, m, and n represent the degree of aggregation.

[0014] In some embodiments, the mass fraction of PEG in PLGA-PEG-PLGA is 20-40%, preferably 30%; and / or

[0015] In PLGA-PEG-PLGA, the molar ratio of lactide to glycolide in PLGA is 60:40-80:20, preferably 75:25.

[0016] Indocyanine green reactive ester (ICG-NHS) is a derivative of ICG dye. ICG itself does not contain a carboxylic acid group. Indocyanine green is labeled by replacing one sulfonate group in indocyanine green with a carboxylic acid group, as shown in the following structure:

[0017]

[0018] By combining the FDA-approved NIR-I region dye ICG with PLGA-PEG-PLGA, a stable nanoparticle with effective fluorescence signal enhancement and long blood circulation time is formed. PLGA-PEG-PLGA is an amphiphilic biblock copolymer that can be used as a drug carrier. Its molecular chains are hydrophobic at both ends of PLGA. In water, the PLGA at both ends of the molecular chains spontaneously aggregate inward to form a nucleus. In this invention, hydrophobic ICG-NHS is selected to fully react with PLGA-PEG-PLGA. After the hydrophobic compound combines with the PLGA block, a stable structure can be formed.

[0019] In some embodiments, the nanoparticles are prepared by a method comprising the following steps:

[0020] S1, dissolve PLGA-PEG-PLGA in a solvent to form the first solution;

[0021] S2, dissolve ICG-NHS in a solvent to form a second solution;

[0022] S3, the first and second solutions are thoroughly mixed and reacted, and unreacted substances are removed by ultrafiltration to obtain nanoparticles.

[0023] In some embodiments, in S1, the solvent is used to dissolve PLGA-PEG-PLGA to form a first solution. In a preferred embodiment, the solvent may be water.

[0024] Preferably, in S1, the mass concentration of PLGA-PEG-PLGA in the first solution is 0.1-25%, such as 1, 2, 5, 10, 15, 20%, but not limited thereto, and preferably 5%.

[0025] In some embodiments, in S2, the solvent is used to dissolve ICG-NHS to form a second solution. In a preferred embodiment, the solvent may be dimethyl sulfoxide (DMSO).

[0026] Preferably, the concentration of ICG-NHS in the second solution is 0.1-100 μg / μL, such as 1, 2, 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, or 90 μg / μL, but is not limited thereto, and is preferably 10 μg / μL.

[0027] Preferably, the volume ratio of the first solution to the second solution is 1:1 to 999:1, such as 2:1, 5:1, 10:1, 20:1, 50:1, 100:1, 200:1, 500:1, 600:1, 800:1, 900:1, but not limited thereto, and preferably 99:1.

[0028] Preferably, after the first and second solutions are thoroughly mixed, they are placed at 4°C for overnight reaction.

[0029] Preferably, the ultrafiltration process includes: placing the reaction system in a 3.5 kDa ultrafiltration tube and centrifuging at 6500 g for 20 min; removing the waste liquid at the bottom of the ultrafiltration tube, adding deionized water, centrifuging at 6500 g for 20 min, and repeating the operation 3 times.

[0030] In some embodiments, the nanoparticles have a concentrated particle size distribution between 30-80 nm, with an average particle size of 45-55 nm, preferably around 50 nm; and / or

[0031] The average hydrated particle size of the nanoparticles is 20-30 nm, preferably around 25 nm.

[0032] In some embodiments, the nanoparticles, in aqueous solution, have an emission wavelength tail peak extending to 1300–1400 nm.

[0033] Secondly, this invention proposes a method for preparing nanoparticles that enhance indocyanine green near-infrared II fluorescence, comprising the following steps:

[0034] S1, dissolve PLGA-PEG-PLGA in a solvent to form the first solution;

[0035] S2, dissolve ICG-NHS in a solvent to form a second solution;

[0036] S3, the first and second solutions are thoroughly mixed and reacted, and unreacted substances are removed by ultrafiltration to obtain nanoparticles.

[0037] Each step is explained in detail:

[0038] In some embodiments, in S1, the solvent is used to dissolve PLGA-PEG-PLGA to form a first solution. In a preferred embodiment, the solvent may be water.

[0039] In some embodiments, in step S1, the mass concentration of PLGA-PEG-PLGA in the first solution is 0.1-20%, preferably 5%.

[0040] In some embodiments, in S2, the solvent is used to dissolve ICG-NHS to form a second solution. In a preferred embodiment, the solvent may be DMSO.

[0041] In some embodiments, in step S2, the concentration of ICG-NHS in the second solution is 0.1-100 μg / μL, preferably 10 μg / μL.

[0042] In this invention, there is no restriction on the order of steps S1 and S2. One of them can be performed first, followed by the remaining step; or both steps can be performed simultaneously.

[0043] In some embodiments, in step S3, the volume ratio of the first solution to the second solution is 1:1 to 999:1, preferably 99:1.

[0044] In some embodiments, after the first and second solutions are thoroughly mixed in step S3, the mixture is placed at 4°C and reacted overnight.

[0045] In some embodiments, ultrafiltration in step S3 includes: placing the reaction system in a 3.5 kDa ultrafiltration tube and centrifuging at 6500 g for 20 min; removing the waste liquid at the bottom of the ultrafiltration tube, then adding deionized water, centrifuging at 6500 g for 20 min, and repeating the operation 3 times.

[0046] The nanoparticles of the first aspect can be obtained according to the method of the second aspect.

[0047] Thirdly, this invention proposes the application of the nanoparticles that enhance the near-infrared II fluorescence of indocyanine green in the preparation of NIR-IIa region fluorescent imaging agents.

[0048] Fourthly, this invention proposes the application of the nanoparticles that enhance the near-infrared II fluorescence of indocyanine green in the preparation of NIR-IIa region angiography agents or tumor imaging agents.

[0049] Contrast agents include, but are not limited to, lymph node localization and extrahepatic bile duct contrast agents.

[0050] Beneficial effects

[0051] This invention enhances the emission of ICG through PLGA-PEG-PLGA, extending the application of ICG in the near-infrared II region to the NIR-IIa region (1300nm-1400nm), and effectively prolongs the circulation time of ICG in the blood, thus removing its limitations in the diagnosis of tumors and vascular diseases.

[0052] The present invention has been described in detail above; however, the above embodiments are merely illustrative in nature and are not intended to limit the invention. Furthermore, this document is not limited to the foregoing prior art or the invention itself, or to any theory described in the following embodiments.

[0053] Unless otherwise expressly stated, numerical ranges throughout the application include any subranges therein and any numerical values ​​incremented by the smallest subunit of a given value. Unless otherwise expressly stated, numerical values ​​throughout the application represent approximate measures or limitations on the range of embodiments including minor deviations from a given value and having approximately the mentioned value as well as having the mentioned precise value. Except in the detailed description of the working embodiments provided at the end, all numerical values ​​of parameters (e.g., quantities or conditions) in this application (including the appended claims) should in all cases be understood to be modified by the term “approximately,” regardless of whether “approximately” actually precedes the numerical value. “Approximately” indicates that the stated numerical value allows for slight inaccuracies (some close to precision at that value; approximately or reasonably close to the value; approximate). If the inaccuracy provided by “approximately” is not understood in this common sense in the art, then “approximately” as used herein at least indicates a variation that can be produced by common methods of measuring and using these parameters. For example, “approximately” can include variations less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5%. Attached Figure Description

[0054] Figure 1 The near-infrared emission spectra of ICG-NHS in PBS, 10% FBS, and ICG@PLGA NPs are shown. (a) shows the near-infrared emission spectra of PBS solution containing 2.5 μg / mL ICG-NHS, 10% FBS solution containing 2.5 μg / mL ICG-NHS, and ICG@PLGA NPs. (b) shows the near-infrared emission spectra in the 1300-1500 nm band selected from (a). (c) shows the near-infrared fluorescence spectra of ICG@PLGA NPs after gradient dilution.

[0055] Figure 2 Transmission electron microscopy (TEM) images of ICG@PLGA NPs and graphs showing the changes in hydrated particle size of ICG@PLGA NPs in water and 1×PBS are shown, where (a) is a TEM image; (b) shows the hydrated particle size results of ICG@PLGA NPs in water and 1×PBS; and (c) shows the changes in hydrated particle size of ICG@PLGA NPs in PBS over a continuous week.

[0056] Figure 3 The in vivo vascular imaging of ICG@PLGA NPs is shown, where (a) is the near-infrared 2-zone vascular imaging under different filters after tail vein injection of ICG@PLGA NPs, and (b) is the quantitative analysis of (a).

[0057] Figure 4The circulation time plots (1300 LP) of ICG and ICG@PLGA NPs in blood are shown, where (a) shows the changes in near-infrared II fluorescence signal of the femoral artery at different time points after tail vein injection of ICG; (b) shows the quantitative analysis of the area marked by the dashed line in (a); (c) shows the changes in near-infrared II fluorescence signal of the femoral artery at different time points after tail vein injection of ICG@PLGA NPs; and (d) shows the quantitative analysis of the area marked by the dashed line in (c). Detailed Implementation

[0058] The present invention will be further described below with reference to the embodiments. It should be noted that the following embodiments are provided for illustrative purposes only and do not constitute a limitation on the scope of protection of the present invention.

[0059] Unless otherwise specified, the raw materials, reagents, and methods used in the embodiments are all conventional raw materials, reagents, and methods in the art.

[0060] reagents and medicines

[0061] PLGA-PEG-PLGA: Jinan Daigang Biotechnology Co., Ltd. 1740 (the ratio of lactide to glycolide is 75:25, and the mass fraction of PEG is 30%).

[0062] ICG-NHS: Xi'an Ruixi Biotechnology Co., Ltd. RH-3110;

[0063] DMSO: Aladdin Chemical Reagent, analytical grade;

[0064] 0.2M PBS (pH=7.2-7.4) by Meilun Biotechnology.

[0065] Equipment and Instruments

[0066] Near-infrared emission spectrometer in region II: a self-built platform by Northeastern University;

[0067] Transmission electron microscope: JEOL Ltd., JEM-2100;

[0068] Malvern Potential Particle Size Analyzer: Malvern Instruments Malvern Mastersizer 2000;

[0069] Near-infrared zone II camera: Hengxin Imaging Technology Hengxin NIR-II;

[0070] Centrifuge: Beckman Coulter 64R.

[0071] Unless otherwise specified, all experimental methods used are conventional methods.

[0072] Example 1

[0073] Preparation: Dissolve PLGA-PEG-PLGA in water to obtain a 5% (50 mg / mL) solution at room temperature until it becomes colorless and transparent;

[0074] Dissolve ICG-NHS in DMSO to a concentration of 10 μg / μL, and sonicate until completely dissolved;

[0075] Take 990 μL of 5% PLGA-PEG-PLGA solution, add 10 μL of 10 μg / μL ICG-NHS solution to the system, and let it react overnight at 4°C.

[0076] Purification: The reaction system was placed in a 3.5 kDa ultrafiltration tube and centrifuged at 6500 g for 10 min. After removing the waste liquid at the bottom of the ultrafiltration tube, 2.5 mL of deionized water was added and centrifuged at 6500 g for 20 min. This operation was repeated three times. Finally, 1 mL of deionized water was added to make up the volume to obtain nanoparticles (denoted as ICG@PLGA NPs, with a concentration of 100 μg / mL calculated by ICG). The nanoparticles were stored at 4 °C for later use.

[0077] Example 2

[0078] 1×PBS solution of ICG@PLGA NPs: Take 5 μL of ICG@PLGA NPs obtained in Example 1, add 185 μL of water, mix thoroughly, and then add 10 μL of 0.2M PBS (pH = 7.2-7.4). The concentration is 2.5 μg / mL based on ICG.

[0079] Example 3

[0080] Aqueous solution of ICG@PLGA NPs: Take 5 μL of ICG@PLGA NPs obtained in Example 1, add 195 μL of water, and mix thoroughly. The concentration, calculated as ICG, is 2.5 μg / mL.

[0081] Example 4

[0082] 1×PBS solution of ICG@PLGA NPs: Take 200 μL of ICG@PLGA NPs obtained in Example 1, add 22 μL of 0.2M PBS (pH = 7.2-7.4), and mix well.

[0083] The structure of nanoparticles was characterized by absorption spectroscopy, emission spectroscopy, transmission electron microscopy, and hydrated particles.

[0084] Experimental Example 1: Emission Spectroscopy Measurement

[0085] 200 μL of 2.5 μg / mL ICG@PLGA NPs in 1×PBS solution (Example 2), 200 μL of 2.5 μg / mL ICG-NHS in 10% FBS solution (control group), and 200 μL of 2.5 μg / mL ICG-NHS in 1×PBS solution (control group) were used to measure the emission spectra using a near-infrared II emission spectrometer. The results are as follows: Figure 1 As shown in (a) and (b).

[0086] Relationship between ICG@PLGA NPs fluorescence signal and concentration: After Example 2 was diluted eight times in a 2-fold gradient, the emission spectrum was measured using a near-infrared II fluorescence spectrometer. The results are as follows: Figure 1 As shown in (c).

[0087] Figure 1 In (a), peak area analysis using Graghpad Prism 9 showed that the peak area of ​​the ICG@PLGA NPs solution was 263 times that of ICG-NHS and 4.2 times that of ICG-NHS in 10% FBS solution. Furthermore, the emission spectrum of ICG@PLGA NPs extended into the NIR-IIa region beyond 1300 nm. In (b), peak area analysis using Graghpad Prism 9 showed that the peak area of ​​the ICG@PLGA NPs solution was 10.3 times that of ICG-NHS in 10% FBS solution. This phenomenon indicates that in the NIR-IIa region, ICG@PLGA NPs exhibits a stronger fluorescence signal than ICG-NHS in 10% FBS solution. In (c), the results showed that the NIR-IIa spectrum gradually decreased with decreasing concentration, indicating that the enhancement effect of PLGA-PEG-PLGA on the fluorescence signal of ICG-NHS does not change with concentration, forming a relatively stable structure. Example 2: Transmission electron microscopy observation and hydration particle size determination of nanoparticles

[0088] Transmission electron microscopy: 10 μL of an aqueous solution of 2.5 μg / mL ICG@PLGANPs (Example 3) was dropped onto a copper grid and dried under vacuum at 35 °C for 2 h. The synthesized nanoparticles were characterized using transmission electron microscopy at a working voltage of 200 kV. The results are as follows: Figure 2 As shown in (a), this indicates that the diameter of the nanoparticle is approximately 50 nm.

[0089] Hydrated particle size: The hydrated particle size of ICG@PLGANPs in water (Example 3) and 1×PBS (pH = 7.2) (Example 2) solution was measured using a Malvern potentiometric particle size analyzer. Results are as follows: Figure 2As shown in (b), the hydrated particle size of ICG@PLGANPs remains unchanged in water and 1×PBS, indicating that the nanoparticles have a certain degree of stability and will not aggregate due to the influence of salt in PBS.

[0090] Hydrated particle size stability: 1 mL of ICG@PLGANPs (Example 2) containing 2.5 μg / mL ICG-NHS dissolved in 1×PBS was used to monitor changes in hydrated particle size at 1, 2, 3, 5, and 7 days using a Malvern particle size analyzer. Results are as follows: Figure 2 As shown in (c). The results indicate that there was no significant change in the hydrated particle size of ICG@PLGANPs after a week of continuous monitoring. The nanoparticles are relatively stable in PBS solution and do not aggregate or disintegrate over time within a week.

[0091] Experimental Case 3: Vascular Imaging Study

[0092] Fluorescence signals in mouse blood vessels: Three nude mice (Huafukang, 6-8 weeks old, 16-20g) were injected intravenously with 200μL of ICG@PLGANPs in PBS (0.01M, pH=7.2-7.4) solution (Example 4). Fluorescence signals in the femoral artery of the nude mice were collected at wavelengths of 1000LP, 1100LP, 1200LP, and 1300LP using a near-infrared dual-zone camera. Quantitative analysis of the regions of interest was performed using ImageJ. The results are as follows: Figure 3 As shown in the figure, the results indicate that with increasing wavelength, the background signal in blood vessel imaging is lower, the resolution is higher, and the signal-to-noise ratio is also higher.

[0093] Experimental Example 4: Study on Blood (In Vivo) Circulation Time

[0094] Blood circulation time: Six 6-8 week old nude mice were randomly divided into two groups. Each group received a tail vein injection of 200 μL LICG@PLGANPs in PBS (0.01 M, pH 7.2-7.4) solution (Example 4). Images were collected at different time points using a near-infrared dual-zone camera (1300 LP). Quantitative analysis of the regions of interest was performed using ImageJ. Results are as follows: Figure 4 As shown in the figure. The results indicate that after tail vein injection of ICG, under the same parameters, only a very weak fluorescence signal could be collected at approximately 5 minutes, similar to the reported plasma half-life of ICG of 2-5 minutes. After tail vein injection of ICG@PLGANPs, a faint fluorescence signal was still visible in the femoral artery at 180 minutes. This phenomenon suggests that ICG@PLGANPs have a longer blood circulation time compared to ICG.

[0095] The above description is merely the preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A nanoparticle that enhances indocyanine green near-infrared II fluorescence, characterized in that, The nanoparticles were prepared by a mixed reaction of PLGA-PEG-PLGA and ICG-NHS. The PLGA-PEG-PLGA structure is as follows: x, y, m, n represent the degree of aggregation; In PLGA-PEG-PLGA, the mass fraction of PEG is 20-40%. In PLGA-PEG-PLGA, the molar ratio of lactide to glycolide in PLGA is 60:40-80:

20. The nanoparticles are prepared by a method comprising the following steps: S1, dissolve PLGA-PEG-PLGA in a solvent to form the first solution; S2, dissolve ICG-NHS in a solvent to form a second solution; S3: The first and second solutions are thoroughly mixed and reacted. Unreacted substances are removed by ultrafiltration to obtain nanoparticles. There is no restriction on the order of steps S1 and S2. In the first solution, the solvent is water, and the mass concentration of PLGA-PEG-PLGA is 0.1-25%. In the second solution, the solvent is dimethyl sulfoxide, and the concentration of ICG-NHS is 0.1-100 µg / µL.

2. The nanoparticles according to claim 1, characterized in that, The mass fraction of PEG in PLGA-PEG-PLGA is 30%.

3. The nanoparticles according to claim 1, characterized in that, In PLGA-PEG-PLGA, the molar ratio of lactide to glycolide in PLGA is 75:

25.

4. The nanoparticles according to claim 1, characterized in that, The mass concentration of PLGA-PEG-PLGA in the first solution is 5%.

5. The nanoparticles according to claim 1, characterized in that, In the second solution, the concentration of ICG-NHS is 10 µg / µL.

6. The nanoparticles according to claim 1, characterized in that, The volume ratio of the first solution to the second solution is 1:1 to 999:

1.

7. The nanoparticles according to claim 1, characterized in that, The volume ratio of the first solution to the second solution is 99:

1.

8. The nanoparticles according to any one of claims 1-3, characterized in that, The nanoparticles have a concentrated particle size distribution between 30-80 nm, with an average particle size of 45-55 nm; and / or The average hydrated particle size of the nanoparticles is 20-30 nm.

9. The nanoparticles according to claim 8, characterized in that, The average particle size is 50 nm.

10. The nanoparticles according to claim 8, characterized in that, The average hydrated particle size of the nanoparticles is 25 nm.

11. The nanoparticles according to any one of claims 1-3, characterized in that, In aqueous solution, the emission wavelength tail peak of the nanoparticles extends to 1300–1400 nm.

12. A method for preparing nanoparticles with enhanced indocyanine green near-infrared II fluorescence as described in any one of claims 1-11, characterized in that, Includes the following steps: S1, dissolve PLGA-PEG-PLGA in a solvent to form the first solution; S2, dissolve ICG-NHS in a solvent to form a second solution; S3: The first and second solutions are thoroughly mixed and reacted. Unreacted substances are removed by ultrafiltration to obtain nanoparticles. There is no restriction on the order of steps S1 and S2. In the first solution, the solvent is water, and the mass concentration of PLGA-PEG-PLGA is 0.1-25%; in the second solution, the solvent is dimethyl sulfoxide, and the concentration of ICG-NHS is 0.1-100 µg / µL.

13. The preparation method according to claim 12, characterized in that, The mass concentration of PLGA-PEG-PLGA in the first solution is 5%.

14. The preparation method according to claim 12, characterized in that, In the second solution, the concentration of ICG-NHS is 10 µg / µL.

15. The preparation method according to claim 12, characterized in that, The volume ratio of the first solution to the second solution is 1:1 to 999:

1.

16. The preparation method according to claim 12, characterized in that, The volume ratio of the first solution to the second solution is 99:

1.

17. The use of nanoparticles that enhance indocyanine green near-infrared II fluorescence as described in any one of claims 1-11 in the preparation of NIR-IIa region fluorescent imaging agents.

18. The use of nanoparticles that enhance indocyanine green near-infrared II fluorescence as described in any one of claims 1-11 in the preparation of NIR-IIa region angiography agents or tumor imaging agents.

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