A kind of trastuzumab modified AIE fluorescent nanoparticles and preparation method and application thereof

Abstract

The invention discloses a trastuzumab-modified AIE fluorescent nanoparticle and a preparation method and application thereof; the trastuzumab-modified AIE fluorescent nanoparticle of the invention is obtained by coupling AIE fluorescent nanoparticles and trastuzumab, the surface of the AIE fluorescent nanoparticle contains at least one of a carboxyl group and an amino group, and the AIE fluorescent nanoparticle is loaded with AIE molecules; the average particle size of the trastuzumab-modified AIE fluorescent nanoparticle is 50-300nm, and the dispersion coefficient is 0.05-0.25. The trastuzumab-modified AIE fluorescent nanoparticle of the invention is used for fluorescent targeting of breast cancer tumors, and can effectively target breast cancer tumors in the process of breast cancer surgery navigation, real-time imaging, obtain images with high signal-to-noise ratio and resolution, and accurately indicate the lesions and edges of breast cancer.

Description

Technical Field

[0001] The present invention belongs to the field of medical imaging technology, and specifically relates to trastuzumab-modified AIE fluorescent nanoparticles and a preparation method and application thereof. Background Art

[0002] There are many treatments for breast cancer, but surgical resection is still the most effective and radical treatment. The fundamental goal of tumor surgery is to completely remove tumor tissue to avoid recurrence, while maximizing the preservation of healthy tissue adjacent to the tumor to avoid unnecessary iatrogenic damage. Therefore, it is crucial to accurately identify tumor lesions and their edges in real time during breast cancer tumor resection surgery. Since the tiny lesions of early and advanced tumors do not have typical morphological structures, the subtle differences between tumors and healthy tissues are difficult to accurately identify based on the doctor's naked eye observation and hand palpation feedback.

[0003] The surgical navigation system based on fluorescence imaging technology can perform real-time dynamic imaging of tumors and other lesion tissues. It has the functional advantages of multi-angle, high-throughput and dynamic continuous acquisition of image data and structure, providing a more accurate and safe solution for surgical operations. However, the development of fluorescent contrast agents that can be used in breast cancer surgical navigation systems is not yet sufficient. This is mainly due to the biological characteristics of breast cancer itself and the limitations of fluorescence imaging technology: 1. Breast cancer tumors are highly heterogeneous, and it is difficult for fluorescent probes to accurately identify and locate all tumor cells, resulting in certain errors and uncertainties in the imaging results; 2. Breast cancer tumors and living tissues have autofluorescence characteristics. During the fluorescence imaging process, the overlap of the fluorescence signal with the fluorescent probe will cause artifacts and blurring in the imaging results, interfering with the accuracy of fluorescence imaging; 3. Breast cancer is generally located deep in the breast. Due to the absorption and scattering of light by living tissues, the intensity of the fluorescence signal will gradually weaken with the increase of imaging depth, resulting in a decrease in imaging quality, which requires deeper penetration depth for breast cancer fluorescence imaging; 4. The microenvironment of breast cancer tumors is complex, and the in situ targeted fluorescence imaging of living tumors is easily affected, which may lead to uneven distribution of fluorescence signals in tumor tissues and affect targeting accuracy.

[0004] At present, the main fluorescent contrast agent used in clinical practice is indocyanine green (ICG), but it has a certain false positive / false negative ratio in breast cancer angiography, which has great limitations in its use in breast cancer surgical navigation. Therefore, it is urgent to develop a fluorescent contrast agent that can be used for breast cancer tumor in vivo fluorescence targeting technology to achieve accurate identification of breast cancer tumors and meet the needs of breast cancer surgical navigation, which is of great significance for the early diagnosis and effective treatment of breast cancer. Summary of the invention

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a trastuzumab-modified AIE fluorescent nanoparticle and a preparation method and application thereof, which can accurately indicate the lesions and edges of breast cancer in real time during surgical navigation, reduce the difficulty of surgery, and effectively prevent postoperative recurrence.

[0006] The purpose of the present invention is achieved by at least one of the following technical solutions.

[0007] A trastuzumab-modified AIE fluorescent nanoparticle, obtained by coupling an AIE fluorescent nanoparticle with trastuzumab, wherein the surface of the AIE fluorescent nanoparticle contains at least one of a carboxyl group and an amino group, and the interior of the AIE fluorescent nanoparticle is loaded with an AIE molecule;

[0008] The average particle size of the trastuzumab-modified AIE fluorescent nanoparticles is 50-300 nm, and the dispersion coefficient is 0.05-0.25.

[0009] Preferably, the average particle size of the trastuzumab-modified AIE fluorescent nanoparticles is 90-150 nm;

[0010] Preferably, the dispersion coefficient of the trastuzumab-modified AIE fluorescent nanoparticles is 0.05-0.1;

[0011] Preferably, the AIE fluorescent nanoparticles are spherical AIE fluorescent nanoparticles in which AIE molecules are encapsulated by an amphiphilic polymer as a carrier, and the amphiphilic polymer contains at least one of a carboxyl group and an amino group.

[0012] Further preferably, the amphiphilic polymer comprises at least one of Formula I and Formula II:

[0013]

[0014] Formula I

[0015]

[0016] Formula II

[0017] In Formula I and Formula II, n is independently an integer ≥45.

[0018] Preferably, the average particle size of the AIE fluorescent nanoparticles is 90-150 nm, and the coefficient of variation is 0.1-0.25;

[0019] Preferably, the total density of amino and carboxyl groups on the surface of the AIE fluorescent nanoparticles is 4-20 mmol / mL, based on a 2 mg / mL AIE fluorescent nanoparticle solution;

[0020] Preferably, the maximum emission wavelength of the AIE molecule is greater than 700 nm.

[0021] Further preferably, the maximum emission wavelength of the AIE molecule is 700-1200 nm.

[0022] Further preferably, the AIE molecule is selected from at least one of AIE-1 molecules to AIE-15 molecules;

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030] AIE-7 molecules

[0031]

[0032]

[0033]

[0034]

[0035]

[0036]

[0037]

[0038]

[0039] The AIE fluorescent nanoparticles can be purchased commercially or prepared by oneself.

[0040] Preferably, the AIE fluorescent nanoparticles are prepared by a nanoprecipitation method.

[0041] Preferably, the method for preparing the AIE fluorescent nanoparticles comprises the following steps:

[0042] (1) dissolving the AIE molecules and the amphiphilic polymer in a volatile organic solvent to obtain an organic phase solution;

[0043] (2) adding the organic phase solution to deionized water under ultrasonic and mixed stirring conditions; after the ultrasonic treatment, removing the volatile organic solvent under stirring, filtering, and obtaining AIE fluorescent nanoparticles.

[0044] Further preferably, in step (1), the mass ratio of the AIE molecule to the amphiphilic polymer is 1:(0.3-10); more preferably 1:1.

[0045] Further preferably, in step (1), the volatile organic solvent is tetrahydrofuran (THF) or dichloromethane (DCM);

[0046] Further preferably, in step (1), the mass ratio of the AIE molecule to the volume of the volatile organic solvent is 1 mg: (0.5-5) mL;

[0047] More preferably, in step (2), the volume amount of the volatile organic solvent is 2%-10% of the deionized water; more preferably, it is 2%-5%.

[0048] Further preferably, in step (2), the ultrasonic power is 60W-90W, the ultrasonic time is 2min-5min, and the mixing speed is 600rpm-800rpm;

[0049] Further preferably, in step (2), the stirring speed is 600 rpm-800 rpm, and the stirring time is 12 h-24 h; the filtration is performed using an aqueous phase filter, and the pore size of the filter membrane of the aqueous phase filter is 0.4-0.45 μm;

[0050] Further preferably, step (1) and step (2) are carried out at room temperature (20±5°C).

[0051] The preparation method of the above-mentioned trastuzumab-modified AIE fluorescent nanoparticles comprises the following steps:

[0052] The AIE fluorescent nanoparticles are added to a buffer, and EDC (1- (3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) are added for activation. After centrifugation and resuspending, a trastuzumab aqueous solution is added for reaction. After the reaction is completed, a crude product is obtained, and after purification, trastuzumab-modified AIE fluorescent nanoparticles are obtained; the buffer (the buffer pair therein) does not contain NH2.

[0053] Preferably, the buffer is MES (2-(N-morpholine)ethanesulfonic acid) buffer, the pH of the buffer is 6.0-6.5, and the concentration of the buffer is 0.020-0.050 mM;

[0054] Preferably, the usage ratio of the mass of the AIE fluorescent nanoparticles to the volume of the buffer solution is (0.007-0.010) g: 10 mL;

[0055] Preferably, the mass ratio of EDC to NHS is 1:(1-15); more preferably 1:(3-5);

[0056] Preferably, the mass ratio of the AIE fluorescent nanoparticles to EDC is 0.007: (0.00015-0.0225);

[0057] Preferably, the concentration of EDC in the activation system is 1-150 mg / mL; the concentration of NHS in the activation system is 1-650 mg / mL;

[0058] Preferably, the AIE fluorescent nanoparticles are AIE fluorescent nanoparticle solution with a concentration of 2-10 mg / mL;

[0059] Preferably, the mass ratio of the AIE fluorescent nanoparticles to trastuzumab is 1:(0.01-0.08); more preferably, it is 1:(0.02-0.04).

[0060] Preferably, the activation time is 0.5 to 1 h;

[0061] Preferably, the reaction time is 2 to 4 hours;

[0062] Preferably, the preparation method is carried out at room temperature (20±5°C).

[0063] Preferably, the purification is performed by differential centrifugation;

[0064] Further preferably, the differential centrifugation method is: the crude product is first centrifuged at 4000-5000 rpm for 5-10 min under vacuum conditions, the precipitate is removed and the supernatant is retained; then centrifuged at 10000-12000 rpm for 10-20 min under vacuum conditions, the supernatant is removed and the precipitate is retained, and then re-dissolved in ultrapure water; then centrifuged at 5000-6000 rpm for 5-10 min under vacuum conditions, the precipitate is removed and the supernatant is retained to obtain trastuzumab-modified AIE fluorescent nanoparticles.

[0065] An AIE fluorescent contrast agent comprises the above-mentioned trastuzumab-modified AIE fluorescent nanoparticles.

[0066] Application of the above-mentioned trastuzumab-modified AIE fluorescent nanoparticles or the above-mentioned AIE fluorescent contrast agent in fluorescent targeting of breast cancer tumors for non-disease diagnosis and / or treatment purposes.

[0067] The AIE fluorescent contrast agent of the present invention is suitable for in vivo fluorescence targeting technology of breast cancer tumors. It can accurately indicate the lesions and edges of breast cancer in real time during breast cancer surgical navigation, reduce the difficulty of surgery, and effectively prevent postoperative recurrence.

[0068] Compared with the prior art, the present invention has the following advantages:

[0069] (1) The trastuzumab-modified AIE fluorescent nanoparticles of the present invention are suitable for in vivo fluorescence targeting technology of breast cancer tumors. They can effectively target breast cancer tumors during breast cancer surgical navigation, perform real-time imaging, obtain images with high signal-to-noise ratio and resolution, accurately indicate breast cancer lesions and edges, help doctors make more accurate judgments and treatments on lesions, reduce the difficulty of surgery, and effectively prevent postoperative recurrence.

[0070] (2) The trastuzumab-modified AIE fluorescent nanoparticles of the present invention have the characteristics of long circulation and good biocompatibility. After being injected into the body, they have a long circulation time in the blood, which effectively increases the contact time and opportunity between the contrast agent and breast cancer cells, thereby enabling accurate targeting of breast cancer cells and achieving accurate identification of breast cancer tumor lesions and their edges.

[0071] (3) The trastuzumab-modified AIE fluorescent nanoparticles of the present invention have high fluorescence intensity, excellent photostability and deeper tissue penetration depth, and can accurately target breast cancer tumor cells in vivo, which is conducive to longer-term deep in vivo real-time imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 is the particle size distribution and scanning electron microscopy image of the trastuzumab-modified AIE fluorescent nanoparticles prepared in Example 1;

[0073] Figure 2 is the particle size distribution and scanning electron microscopy image of the trastuzumab-modified AIE fluorescent nanoparticles prepared in Example 2;

[0074] Figure 3 is the particle size distribution and scanning electron microscopy image of the trastuzumab-modified AIE fluorescent nanoparticles prepared in Example 3;

[0075] Figure 4 is the uptake image of AIE NPs 1 in HCC1954 breast cancer 3D cell spheroids;

[0076] Figure 5 is the uptake image of the HCC1954 breast cancer 3D cell sphere of the trastuzumab-modified AIE fluorescent nanoparticles in Example 1;

[0077] Figure 6is the uptake image of the HCC1954 breast cancer 3D cell sphere of the trastuzumab-modified AIE fluorescent nanoparticles in Example 2;

[0078] Figure 7 is the uptake image of the HCC1954 breast cancer 3D cell sphere of the trastuzumab-modified AIE fluorescent nanoparticles in Example 3;

[0079] Figure 8 is the flow cytometric fluorescence distribution diagram of HCC1954 breast cancer cells of AIE NPs 1 (control group) and trastuzumab-modified AIE fluorescent nanoparticles in Example 1 (experimental group);

[0080] Fig. 9 This is the targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles in Example 1 on nude mice with breast cancer HCC1954 subcutaneous tumors;

[0081] Fig.10 This is the targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles in Example 1 on nude mice with breast cancer HCC1954 orthotopic tumors;

[0082] Fig.11 This is the targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles on nude mice with breast cancer HCC1954 orthotopic tumors in Example 2;

[0083] Fig.12 This is the targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles on nude mice with breast cancer HCC1954 orthotopic tumors in Example 3;

[0084] Fig.13 This is a partial picture of the targeted imaging of nude mice with breast cancer HCC1954 orthotopic tumors by trastuzumab-modified AIE fluorescent nanoparticles in Example 1;

[0085] Fig.14 The particle size distribution and scanning electron microscopy image of the trastuzumab-modified AIE fluorescent nanoparticles prepared in Comparative Example 1;

[0086] Fig.15 This is the uptake image of the HCC1954 breast cancer 3D cell spheres of the trastuzumab-modified AIE fluorescent nanoparticles in Comparative Example 1;

[0087] Fig.16 This is a targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles on nude mice with breast cancer HCC1954 orthotopic tumors in comparative example 1;

[0088] Fig.17 This is the targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles on nude mice with breast cancer HCC1954 orthotopic tumor in comparative example 2;

[0089] Fig.18 This is the targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles on nude mice with breast cancer HCC1954 orthotopic tumor in comparative example 3;

[0090] Fig.19 This is the targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles on nude mice with breast cancer HCC1954 orthotopic tumors in comparative example 4;

[0091] Fig. 20 This is the targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles on nude mice with breast cancer HCC1954 orthotopic tumor in comparative example 5;

[0092] Fig.21 This is the targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles on nude mice with breast cancer HCC1954 orthotopic tumors in comparative example 6;

[0093] Fig. 22 This is the targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles on nude mice with breast cancer HCC1954 orthotopic tumor in comparative example 7;

[0094] Fig.23 This is a targeted imaging image of the trastuzumab-modified AIE fluorescent nanoparticles in comparative example 8 on nude mice with breast cancer HCC1954 orthotopic tumors;

[0095] Fig.24 This is a local image of the targeted imaging of nude mice with breast cancer HCC1954 orthotopic tumors by trastuzumab-modified AIE fluorescent nanoparticles in comparative example 1;

[0096] Fig.25 This is a local image of the targeted imaging of nude mice with breast cancer HCC1954 orthotopic tumors by trastuzumab-modified AIE fluorescent nanoparticles in comparative example 2;

[0097] Fig.26 This is a local image of the targeted imaging of nude mice with breast cancer HCC1954 orthotopic tumors by trastuzumab-modified AIE fluorescent nanoparticles in comparative example 3;

[0098] Fig. 27 This is a local image of the targeted imaging of nude mice with breast cancer HCC1954 orthotopic tumors by trastuzumab-modified AIE fluorescent nanoparticles in comparative example 4;

[0099] Fig.28 This is a local image of the targeted imaging of nude mice with breast cancer HCC1954 orthotopic tumors by trastuzumab-modified AIE fluorescent nanoparticles in comparative example 5;

[0100] Fig.29This is a local image of the targeted imaging of nude mice with breast cancer HCC1954 orthotopic tumors by trastuzumab-modified AIE fluorescent nanoparticles in comparative example 6;

[0101] Fig.30 This is a local image of the targeted imaging of nude mice with breast cancer HCC1954 orthotopic tumors by trastuzumab-modified AIE fluorescent nanoparticles in comparative example 7;

[0102] Fig.31 This is a local image of the targeted imaging of nude mice with breast cancer HCC1954 orthotopic tumors using trastuzumab-modified AIE fluorescent nanoparticles in comparative example 8. DETAILED DESCRIPTION

[0103] The present invention is described in detail below with reference to examples, but the implementation manner and protection scope of the present invention are not limited to the following examples.

[0104] The AIE fluorescent nanoparticles used in the following examples were prepared as follows.

[0105] Preparation method of AIE NPs1:

[0106] Weigh 0.001g AIE-1 molecule (maximum emission wavelength is 1100nm) and 0.001g DSPE-PEG2000-COOH molecule and dissolve them in 1mL tetrahydrofuran solvent to obtain solution A. At room temperature, solution A was quickly injected into 50mL deionized water under ultrasonic and mixed stirring (600rpm), the ultrasonic power was 90W, ultrasonic for 2min, and placed in a fume hood and stirred (600rpm) overnight to remove the organic solvent, and filtered with a 0.45µm syringe filter to obtain an AIE nanoparticle solution. The average particle size is 90nm, and the coefficient of variation is 0.12.

[0107] Preparation method of AIE NPs2:

[0108] 0.001g AIE-2 molecule (maximum emission wavelength is 780nm) and 0.0005g DSPE-PEG2000-COOH molecule were weighed and dissolved in 1mL tetrahydrofuran solvent to obtain solution A. Solution A was quickly injected into 50mL deionized water under ultrasonic and mixed stirring (700rpm) at room temperature. The ultrasonic power was 90W and the ultrasonic was performed for 2min. The solution was placed in a fume hood and stirred (700rpm) overnight to remove the organic solvent. The solution was filtered using a 0.45µm syringe filter to obtain an AIE nanoparticle solution with an average particle size of 110nm and a coefficient of variation of 0.18.

[0109] Preparation method of AIE NPs3:

[0110] 0.001g AIE-6 molecule (maximum emission wavelength is 900nm), 0.0003g DSPE-PEG2000-NH2 molecule, 0.0005g DSPE-PEG2000 were weighed and dissolved in 1mL tetrahydrofuran solvent to obtain solution A; at room temperature, solution A was quickly injected into 50mL deionized water under ultrasonic and mixed stirring (800rpm), the ultrasonic power was 90W, ultrasonic for 2min, placed in a fume hood and stirred (800rpm) overnight to remove the organic solvent, and filtered with a 0.45µm syringe filter to obtain an AIE nanoparticle solution. The average particle size is 95nm, and the coefficient of variation is 0.20.

[0111] Preparation method of AIE NPs4:

[0112] 0.001g AIE-1 molecule, 0.0002g DSPE-PEG2000-COOH molecule, and 0.0006g DSPE-PEG2000 were weighed and dissolved in 1mL tetrahydrofuran solvent to obtain solution A. Solution A was quickly injected into 20mL deionized water under ultrasonic and mixed stirring (650rpm) at room temperature, with an ultrasonic power of 90W for 2min, and then placed in a fume hood and stirred (650rpm) overnight to remove the organic solvent, and filtered using a 0.45µm syringe filter to obtain an AIE nanoparticle solution with an average particle size of 180nm and a coefficient of variation of 0.18.

[0113] Preparation method of AIE NPs5: (AIE molecular structure: , the maximum emission wavelength is 610nm)

[0114] 0.001g AIE molecules and 0.001g DSPE-PEG2000-COOH molecules were weighed and dissolved in 1mL tetrahydrofuran solvent to obtain solution A. Solution A was quickly injected into 50mL deionized water under ultrasonic and mixed stirring (600rpm) at room temperature. The ultrasonic power was 90W and the ultrasonic was performed for 2min. The solution was placed in a fume hood and stirred (600rpm) overnight to remove the organic solvent. The solution was filtered using a 0.45µm syringe filter to obtain an AIE nanoparticle solution with an average particle size of 90nm and a coefficient of variation of 0.17.

[0115] The surface functional groups and their contents of the AIE NPs1-5 prepared above are shown in Table 1.

[0116] Table 1

[0117]

[0118] Among them, the content of surface functional groups is measured by the following method:

[0119] Take 1mL of 2mg / mL AIE nanoparticle solution and gradually add 0.1 M / L alkaline standard solution (sodium hydroxide) into the AIE nanoparticle solution. During the titration process, measure the conductivity of the solution in real time. According to the standard curve formed by the potentiometric titrator, determine the difference between the two jump points, and calculate the carboxyl content (the same for the amino group) according to the formula.

[0120] Example 1: Preparation and characterization of trastuzumab modified AIE fluorescent nanoparticles

[0121] Take 0.007g solid content of AIE NPs 1 solution (1.75mL, 4mg / mL), add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 150µL EDC (concentration of 100mg / mL, pH6.5 0.05mM MES buffer) and 900µL NHS (concentration of 100mg / mL, pH6.5 0.05mM MES buffer), activate under rotating mixing conditions for 0.5h, collect the precipitate after centrifugation to obtain carboxyl-activated AIE nanoparticles, then add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 0.12mg solid content of trastuzumab solution (0.0057mL, 21 mg / mL, purchased from Aladdin, product number T305227) was rotated and mixed for 2 hours, and the obtained solution was first subjected to ultracentrifugation at 5000 rpm under vacuum conditions for 10 min to remove the precipitate, and the supernatant was centrifuged at 12000 rpm for 20 min to remove the supernatant, and the precipitate was re-dissolved in ultrapure water and then centrifuged at 5000 rpm for 5 min to remove the precipitate, and the supernatant was collected to obtain the trastuzumab-modified AIE fluorescent nanoparticle solution.

[0122] The surface morphology of trastuzumab-modified AIE fluorescent nanoparticles was tested by dynamic light scattering and scanning electron microscopy. The average particle size was about 100 nm and the dispersion coefficient PDI was 0.07 (e.g. Figure 1 as shown).

[0123] Example 2: Preparation and characterization of trastuzumab modified AIE fluorescent nanoparticles

[0124] Take 0.007g solid content of AIE NPs 2 solution (1.75mL, 4mg / mL), add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 150µL EDC (concentration of 100mg / mL, pH6.5 0.05mM MES buffer) and 900µL NHS (concentration of 100mg / mL, pH6.5 0.05mM MES buffer), activate under rotating mixing conditions for 0.5h, collect the precipitate after centrifugation to obtain carboxyl-activated AIE nanoparticles, then add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 0.08mg solid content of trastuzumab solution (0.0038mL, 21 mg / mL, purchased from Aladdin, product number T305227) was rotated and mixed for 2 hours, and the obtained solution was subjected to ultracentrifugation at 5000 rpm under vacuum conditions for 10 min to remove the precipitate, and the supernatant was centrifuged at 12000 rpm for 20 min to remove the supernatant. The precipitate was re-dissolved in ultrapure water and then centrifuged at 6000 rpm for 10 min to remove the precipitate, and the supernatant was collected to obtain trastuzumab-modified AIE fluorescent nanoparticle solution.

[0125] The surface morphology of trastuzumab-modified AIE fluorescent nanoparticles was tested by dynamic light scattering and scanning electron microscopy. The average particle size was about 50 nm and the dispersion coefficient PDI was 0.09 (e.g. Figure 2 as shown).

[0126] Example 3: Preparation and characterization of trastuzumab modified AIE fluorescent nanoparticles

[0127] Take 0.007g solid content of AIE NPs 3 solution (1.75mL, 4mg / mL), add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 150µL EDC (concentration of 100mg / mL, pH6.5 0.05mM MES buffer) and 900µL NHS (concentration of 100mg / mL, pH6.5 0.05mM MES buffer), activate under rotating mixing conditions for 0.5h, collect the precipitate after centrifugation to obtain carboxyl-activated AIE nanoparticles, then add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 0.56mg solid content of trastuzumab solution (0.0267mL, 21 mg / mL, purchased from Aladdin, product number T305227) was rotated and mixed for 2 hours, and the obtained solution was subjected to ultracentrifugation at 5000 rpm under vacuum conditions for 10 min to remove the precipitate, and the supernatant was centrifuged at 12000 rpm for 20 min to remove the supernatant. The precipitate was re-dissolved in ultrapure water and then centrifuged at 6000 rpm for 10 min to remove the precipitate, and the supernatant was collected to obtain trastuzumab-modified AIE fluorescent nanoparticle solution.

[0128] The surface morphology of trastuzumab-modified AIE fluorescent nanoparticles was tested by dynamic light scattering and scanning electron microscopy. The average particle size was about 300 nm and the dispersion coefficient PDI was 0.08 (e.g. Figure 3 as shown).

[0129] Performance Testing:

[0130] Specific uptake imaging of trastuzumab modified AIE fluorescent nanoparticles in HCC1954 human breast cancer cells

[0131] (1) 3D spheroid uptake test

[0132] AIE NPs 1 (control group) and trastuzumab-modified AIE fluorescent nanoparticles prepared in Example 1-3 (experimental group) were prepared into 50 μg / mL solutions (in terms of AIE molecule content) with 10 mM phosphate buffer (PBS), and added to the 3D cell spheres of HCC1954 human breast cancer cells (cells were purchased from Wuhan Pronocell Life Science Co., Ltd., and the following examples are the same) for staining. After incubation for 4 h, the results showed that the AIE NPs 1 and trastuzumab-modified AIE fluorescent nanoparticles prepared in Example 1-3 were 50 μg / mL and 50 μg / mL, respectively. Figure 4 ), experimental group (as shown Figure 5-7 The uptake of trastuzumab in 3D cell spheres (as shown) is more obvious, indicating that trastuzumab can specifically bind to breast cancer and exhibit excellent breast cancer cell targeting.

[0133] (2) Flow cytometer test

[0134] AIE NPs 1 (control group) and trastuzumab-modified AIE fluorescent nanoparticles prepared in Example 1 (experimental group) were prepared into 50 μg / mL solutions (in terms of AIE molecule content) with 10 mM phosphate buffer (PBS), and added to a culture dish of HCC1954 human breast cancer cells with a density of about 60% for staining. After incubation for 4 h, the cell uptake in the experimental group was more obvious than that in the control group (e.g. Figure 8 As shown), it shows that trastuzumab can specifically bind to breast cancer and exhibit excellent breast cancer cell targeting.

[0135] In vivo fluorescence imaging of trastuzumab-modified AIE fluorescent nanoparticles in nude mice with breast cancer HCC1954

[0136] (1) In vivo fluorescence imaging of HCC1954 breast cancer subcutaneous tumors in nude mice

[0137] The trastuzumab-modified AIE fluorescent nanoparticles prepared in Example 1 were prepared into a solution with a concentration of 1 mg / mL (in terms of AIE molecule content) using physiological saline. 150 μL of the solution was injected into nude mice with breast cancer HCC1954 subcutaneous tumors (nude mice were purchased from Zhuhai Baishitong Biotechnology Co., Ltd., and the following examples are the same) through the tail vein, and optical signals were collected 5 min, 3 h, 5 h, 24 h, and 48 h after administration (using the near-infrared zone II small animal in vivo imaging system of Guangzhou Boluteng Biotechnology Co., Ltd., and the excitation light source was selected to be 808 nm (2 W / cm 2 ), the emission filter selected was 1300nm LP, the exposure time was set to 5s, and the following embodiments were the same). After 3h of administration, obvious signal enrichment was detected at the site of the breast cancer subcutaneous tumor, which lasted until 48h, and the signal at the site of the breast cancer subcutaneous tumor became stronger and stronger (such as Fig. 9 As shown), it shows that trastuzumab can specifically bind to breast cancer and exhibit excellent breast cancer targeting.

[0138] (2) In vivo fluorescence imaging of breast cancer HCC1954 orthotopic tumors in nude mice

[0139] The trastuzumab-modified AIE fluorescent nanoparticles prepared in Example 1-3 were prepared into a solution with a concentration of 1 mg / mL (in terms of AIE molecule content) using physiological saline. 150 μL of the solution was injected into nude mice bearing breast cancer HCC1954 tumors in situ via the tail vein using a syringe, and optical signals were collected 2 hours after administration. Obvious signal enrichment (e.g. Figure 10-12As shown), it shows that the trastuzumab-modified AIE fluorescent nanoparticles of the present invention have high fluorescence intensity and deeper tissue penetration depth, can accurately target breast cancer in situ tumor cells in vivo, and are conducive to longer-term deep in vivo real-time imaging.

[0140] (3) In vivo fluorescence imaging of breast cancer HCC1954 orthotopic tumors in nude mice

[0141] The trastuzumab-modified AIE fluorescent nanoparticles prepared in Example 1 were prepared into a solution with a concentration of 1 mg / mL (in terms of AIE molecule content) using physiological saline. 150 μL of the solution was injected into nude mice with breast cancer HCC1954 tumors in situ through the tail vein using a syringe, and optical signals were collected 1 hour after administration. Obvious signal enrichment was detected at the site of the breast cancer tumor in situ, and the tumor boundary was clearly visible (e.g. Fig.13 As shown), it shows that the trastuzumab-modified AIE fluorescent nanoparticles of the present invention can accurately target breast cancer in situ tumor cells in vivo, improve the signal-to-noise ratio and resolution of breast cancer imaging, and accurately indicate the lesions and edges of breast cancer.

[0142] Comparative Example 1: Preparation and characterization of trastuzumab modified AIE fluorescent nanoparticles

[0143] 0.007 g of AIE NPs 1 solution (1.75 mL, 4 mg / mL) with a solid content was taken and added to 10 mL of deionized water, and then 0.12 mg of trastuzumab solution (0.0057 mL, 21 mg / mL, purchased from Aladdin, product number T305227) with a solid content was added, followed by 150 µL of EDC (concentration of 100 mg / mL, prepared with deionized water) and 900 µL of NHS (concentration of 100 mg / mL, prepared with deionized water), and the mixture was reacted for 4 h under rotating mixing conditions. The obtained solution was first ultracentrifuged at 5000 rpm under vacuum conditions for 10 min to remove the precipitate, and the supernatant was centrifuged at 12000 rpm for 20 min to remove the supernatant. The precipitate was re-dissolved with ultrapure water and centrifuged at 6000 rpm for 10 min to remove the precipitate, and the supernatant was collected to obtain a trastuzumab-modified AIE fluorescent nanoparticle solution.

[0144] The surface morphology of trastuzumab-modified AIE fluorescent nanoparticles was tested by dynamic light scattering and scanning electron microscopy. The average particle size was about 500 nm and the dispersion coefficient PDI was 0.25 (e.g. Fig.14 as shown).

[0145] Comparative Example 2: Preparation and Characterization of Trastuzumab-Modified AIE Fluorescent Nanoparticles

[0146] Take 0.007g solid content of AIE NPs 1 solution (1.75mL, 4mg / mL), add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 150µL EDC (concentration of 100mg / mL, pH6.5 0.05mM MES buffer) and 900µL NHS (concentration of 100mg / mL, pH6.5 0.05mM MES buffer), activate under rotating mixing conditions for 0.5h, collect the precipitate after centrifugation to obtain carboxyl-activated AIE nanoparticles, then add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 0.06mg solid content of trastuzumab solution (0.00286mL, 21mg / mL, purchased from Aladdin, item number T305227) was mixed by rotation for 2h, and the obtained solution was first centrifuged at 5000rpm under vacuum conditions for 10min to remove the precipitate, and the supernatant was centrifuged at 12000rpm for 20min to remove the supernatant, and the precipitate was re-dissolved in ultrapure water and centrifuged at 5000rpm for 5min to remove the precipitate, and the supernatant was collected to obtain the trastuzumab-modified AIE fluorescent nanoparticle solution. The average particle size is about 40nm, and the dispersion coefficient PDI is 0.23.

[0147] Comparative Example 3: Preparation and characterization of trastuzumab modified AIE fluorescent nanoparticles

[0148] Take 0.007g solid content of AIE NPs 1 solution (1.75mL, 4mg / mL), add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 150µL EDC (concentration of 100mg / mL, pH6.5 0.05mM MES buffer) and 900µL NHS (concentration of 100mg / mL, pH6.5 0.05mM MES buffer), activate under rotating mixing conditions for 0.5h, collect the precipitate after centrifugation to obtain carboxyl-activated AIE nanoparticles, then add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 0.6mg solid content of trastuzumab solution (0.0286mL, 21mg / mL, purchased from Aladdin, item number T305227) was rotated and mixed for 2h, and the obtained solution was first centrifuged at 5000rpm under vacuum conditions for 10min to remove the precipitate, and the supernatant was centrifuged at 12000rpm for 20min to remove the supernatant, and the precipitate was re-dissolved in ultrapure water and centrifuged at 5000rpm for 5min to remove the precipitate, and the supernatant was collected to obtain the trastuzumab-modified AIE fluorescent nanoparticle solution. The average particle size is about 450nm, and the dispersion coefficient PDI is 0.29.

[0149] Comparative Example 4: Preparation and Characterization of Trastuzumab-Modified AIE Fluorescent Nanoparticles

[0150] Take 0.007g solid content of AIE NPs 4 (AIE fluorescent nanoparticles with a surface carboxyl content of 3mmol / mL) solution (1.75mL, 4mg / mL), add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 150µL EDC (concentration of 100mg / mL, 0.05mM MES buffer, pH6.5) and 900µL NHS (concentration of 100mg / mL, 0.05mM MES buffer, pH6.5), activate under rotating mixing conditions for 0.5h, collect the precipitate after centrifugation to obtain carboxyl-activated AIE nanoparticles, then add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 0.12mg solid content of trastuzumab solution (0.0057mL, 21mg / mL, purchased from Aladdin, item number T305227) was mixed by rotation for 2h, and the obtained solution was first centrifuged at 5000rpm under vacuum conditions for 10min to remove the precipitate, and the supernatant was centrifuged at 12000rpm for 20min to remove the supernatant, and the precipitate was re-dissolved in ultrapure water and centrifuged at 5000rpm for 5min to remove the precipitate, and the supernatant was collected to obtain the trastuzumab-modified AIE fluorescent nanoparticle solution. The average particle size is about 70nm, and the dispersion coefficient PDI is 0.1.

[0151] Comparative Example 5: Preparation and Characterization of Trastuzumab Modified AIE Fluorescent Nanoparticles

[0152] Take 0.007g solid content of AIE NPs 1 solution (1.75mL, 4mg / mL), add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 150µL EDC (concentration of 100mg / mL, pH6.5 0.05mM MES buffer) and 900µL NHS (concentration of 100mg / mL, pH6.5 0.05mM MES buffer), activate under rotating mixing conditions for 0.5h, collect the precipitate after centrifugation to obtain carboxyl-activated AIE nanoparticles, then add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 0.12mg solid content of trastuzumab solution (0.0057mL, 21mg / mL, purchased from Aladdin, item number T305227) was mixed by rotation for 2h, and the obtained solution was first subjected to ultracentrifugation at 5000rpm under vacuum conditions, centrifuged for 5min to remove the supernatant, and the precipitate was collected and re-dissolved to obtain a trastuzumab-modified AIE fluorescent nanoparticle solution. The average particle size was about 400nm, and the dispersion coefficient PDI was 0.26.

[0153] Comparative Example 6: Preparation and Characterization of Trastuzumab Modified AIE Fluorescent Nanoparticles

[0154] Take 0.007g solid content of AIE NPs 1 solution (1.75mL, 4mg / mL), add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 150µL EDC (concentration of 100mg / mL, pH6.5 0.05mM MES buffer) and 900µL NHS (concentration of 100mg / mL, pH6.5 0.05mM MES buffer), activate under rotating mixing conditions for 0.5h, collect the precipitate after centrifugation to obtain carboxyl-activated AIE nanoparticles, then add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 0.12mg solid content of trastuzumab solution (0.0057mL, 21mg / mL, purchased from Aladdin, item number T305227) was mixed by rotation for 2h, and the obtained solution was first centrifuged at 5000rpm under vacuum conditions for 10min to remove the precipitate, and the supernatant was centrifuged at 12000rpm for 20min to remove the supernatant, and the precipitate was re-dissolved in ultrapure water and centrifuged at 6000rpm for 10min to remove the precipitate, and the supernatant was collected to obtain the trastuzumab-modified AIE fluorescent nanoparticle solution. The average particle size is about 40nm, and the dispersion coefficient PDI is 0.22.

[0155] Comparative Example 7: Preparation and Characterization of Trastuzumab Modified AIE Fluorescent Nanoparticles

[0156] Take 0.007g solid content of AIE NPs 5 solution (1.75mL, 4mg / mL), add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 150µL EDC (concentration of 100mg / mL, pH6.5 0.05mM MES buffer) and 900µL NHS (concentration of 100mg / mL, pH6.5 0.05mM MES buffer), activate under rotating mixing conditions for 0.5h, collect the precipitate after centrifugation to obtain carboxyl-activated AIE nanoparticles, then add 10mL pH6.5 0.05mM MES buffer, mix thoroughly, add 0.12mg solid content of trastuzumab solution (0.0057mL, 21mg / mL, purchased from Aladdin, item number T305227) was mixed by rotation for 2h, and the obtained solution was first centrifuged at 5000rpm under vacuum conditions for 10min to remove the precipitate, and the supernatant was centrifuged at 12000rpm for 20min to remove the supernatant, and the precipitate was re-dissolved in ultrapure water and centrifuged at 5000rpm for 5min to remove the precipitate, and the supernatant was collected to obtain the trastuzumab-modified AIE fluorescent nanoparticle solution. The average particle size was about 100nm, and the dispersion coefficient PDI was 0.09.

[0157] Comparative Example 8: Preparation and characterization of pertuzumab modified AIE fluorescent nanoparticles

[0158] 0.007 g solid content of AIE NPs 1 solution (1.75 mL, 4 mg / mL) was taken, and 10 mL of 0.05 mM MES buffer at pH 6.5 was added. After thorough mixing, 150 µL of EDC (concentration of 100 mg / mL, 0.05 mM MES buffer at pH 6.5) and 900 µL of NHS (concentration of 100 mg / mL, 0.05 mM MES buffer at pH 6.5) were added. The mixture was activated for 0.5 h under rotating mixing conditions. After centrifugation, the precipitate was collected to obtain carboxyl-activated AIE nanoparticles. 10 mL of 0.05 mM MES buffer at pH 6.5 was added. After thorough mixing, 0.12 mg solid content of Pertuzumab solution (0.0239 mL, 5.02mg / mL, purchased from Selleck, item number A2008) was mixed by rotation for 2h, and the obtained solution was first centrifuged at 5000rpm under vacuum conditions for 10min to remove the precipitate, and the supernatant was centrifuged at 12000rpm for 20min to remove the supernatant, and the precipitate was re-dissolved in ultrapure water and centrifuged at 5000rpm for 5min to remove the precipitate, and the supernatant was collected to obtain the trastuzumab-modified AIE fluorescent nanoparticle solution. The average particle size is about 100nm, and the dispersion coefficient PDI is 0.12.

[0159] Performance Testing:

[0160] Specific uptake imaging of trastuzumab modified AIE fluorescent nanoparticles in HCC1954 human breast cancer cells

[0161] 3D cell sphere uptake test: The trastuzumab-modified AIE fluorescent nanoparticles prepared in Comparative Example 1 (experimental group) were prepared into a 50 µg / mL solution (in terms of AIE molecule content) with 10 mM phosphate buffer (PBS), and added to the 3D cell spheres of HCC1954 human breast cancer cells (cells purchased from Wuhan Pronocell Life Science Co., Ltd.) constructed in advance for staining. After incubation for 4 hours, the results were compared with the control group (such as Figure 4 ), experimental group (as shown Fig.15 The 3D cell spheroid uptake of trastuzumab (as shown) is quite obvious, indicating that in the 3D cell spheroid uptake test, trastuzumab-modified AIE fluorescent nanoparticles with an average particle size of >300nm can also show a certain breast cancer cell targeting ability.

[0162] In vivo fluorescence imaging of monoclonal antibody-modified AIE fluorescent nanoparticles in nude mice with breast cancer HCC1954

[0163] (1) Fluorescence imaging of nude mice with breast cancer HCC1954 orthotopic tumors: The monoclonal antibody-modified AIE fluorescent nanoparticles prepared in Comparative Examples 1-8 were prepared into a solution with a concentration of 1 mg / mL (in terms of AIE molecule content) using physiological saline. 150 μL of the solution was injected into nude mice with breast cancer HCC1954 orthotopic tumors via the tail vein using a syringe, and optical signals were collected 2 h after administration (e.g. Figure 16-23 A certain amount of signal enrichment can be detected in the breast cancer in situ tumor, but the targeting is not strong.

[0164] (2) Fluorescence imaging of nude mice with breast cancer HCC1954 orthotopic tumors: The monoclonal antibody-modified AIE fluorescent nanoparticles prepared in Comparative Examples 1-8 were prepared into a solution with a concentration of 1 mg / mL (in terms of AIE molecule content) using physiological saline. 150 μL of the solution was injected into nude mice with breast cancer HCC1954 orthotopic tumors via the tail vein using a syringe, and optical signals were collected 1 hour after administration (e.g. Figure 24-31 Signal enrichment can be detected in the breast cancer in situ tumor, but the tumor boundary is not obvious, and the resolution and signal-to-noise ratio of breast cancer imaging are also poor.

Claims

1. A method for preparing trastuzumab-modified AIE fluorescent nanoparticles, characterized in that: The following steps are involved: The AIE fluorescent nanoparticles are added to a buffer, EDC and NHS are added for activation, and after centrifugation and resuspension, a trastuzumab aqueous solution is added for reaction. After the reaction, a crude product is obtained, and after purification, trastuzumab-modified AIE fluorescent nanoparticles are obtained; the buffer does not contain -NH2; The mass ratio of the AIE fluorescent nanoparticles to trastuzumab is 1:(0.01-0.08); The surface of the AIE fluorescent nanoparticles contains at least one of a carboxyl group and an amino group, and the interior of the AIE fluorescent nanoparticles is loaded with AIE molecules; The AIE fluorescent nanoparticles are spherical AIE fluorescent nanoparticles that use an amphiphilic polymer as a carrier to encapsulate the AIE molecules, wherein the amphiphilic polymer contains at least one of a carboxyl group and an amino group; The average particle size of the AIE fluorescent nanoparticles is 90-150 nm, and the coefficient of variation is 0.1-0.25; The total density of amino and carboxyl groups on the surface of the AIE fluorescent nanoparticles is 4-20 mmol / mL, based on a 2 mg / mL AIE fluorescent nanoparticle solution; The maximum emission wavelength of the AIE molecule is greater than 700 nm; The purification adopts differential centrifugation; the differential centrifugation method is: the crude product is first centrifuged at 4000-5000rpm for 5-10min under vacuum conditions, the precipitate is removed and the supernatant is retained; then centrifuged at 10000-12000rpm for 10-20min under vacuum conditions, the supernatant is removed and the precipitate is retained, and then ultrapure water is added for re-dissolution; then centrifuged at 5000-6000rpm for 5-10min under vacuum conditions, the precipitate is removed and the supernatant is retained, to obtain trastuzumab-modified AIE fluorescent nanoparticles; The average particle size of the trastuzumab-modified AIE fluorescent nanoparticles is 50-300 nm, and the dispersion coefficient is 0.05-0.25; The amphiphilic polymer comprises at least one of Formula I and Formula II: Formula I Formula II In Formula I and Formula II, n is independently an integer ≥ 45; The AIE molecule is selected from at least one of AIE-1 molecules to AIE-15 molecules; AIE-7 molecules 。 2. The preparation method according to claim 1, characterized in that: The buffer is MES buffer, the pH of the buffer is 6.0-6.5, and the concentration of the buffer is 0.020-0.050 mM; The mass ratio of the AIE fluorescent nanoparticles to the volume of the buffer solution is (0.007-0.010) g: 10 mL; The mass ratio of EDC to NHS is 1:(1-15); The mass ratio of the AIE fluorescent nanoparticles to EDC is 0.007: (0.00015-0.0225); The activation time is 0.5 to 1 h; The reaction time is 2 to 4 hours; The preparation method is carried out at room temperature.

3. The preparation method according to claim 1, characterized in that: The preparation method of the AIE fluorescent nanoparticles comprises the following steps: (1) dissolving the AIE molecules and the amphiphilic polymer in a volatile organic solvent to obtain an organic phase solution; (2) adding the organic phase solution to deionized water under ultrasonic and mixed stirring conditions; after the ultrasonic treatment, removing the volatile organic solvent under stirring, filtering, and obtaining AIE fluorescent nanoparticles.

4. The preparation method according to claim 3, characterized in that: In step (1), the mass ratio of the AIE molecule to the amphiphilic polymer is 1:(0.3-10); the volatile organic solvent is tetrahydrofuran or dichloromethane; the mass ratio of the AIE molecule to the volatile organic solvent is 1 mg:(0.5-5) mL; In step (2), the volume amount of the volatile organic solvent is 2%-10% of the deionized water; the power of the ultrasound is 60W-90W, the ultrasound time is 2min-5min, and the mixing speed is 600rpm-800rpm; In step (2), the stirring speed is 600 rpm-800 rpm, and the stirring time is 12 h-24 h; the filtration is performed using an aqueous phase filter, and the pore size of the filter membrane of the aqueous phase filter is 0.4-0.45 μm; Step (1) and step (2) are carried out at room temperature.

5. Trastuzumab-modified AIE fluorescent nanoparticles prepared by the preparation method according to any one of claims 1 to 4.

6. The trastuzumab-modified AIE fluorescent nanoparticles according to claim 5, characterized in that: The average particle size of the trastuzumab-modified AIE fluorescent nanoparticles is 90-150 nm; the dispersion coefficient of the trastuzumab-modified AIE fluorescent nanoparticles is 0.05-0.

1.

7. An AIE fluorescent contrast agent, characterized in that The invention relates to a trastuzumab-modified AIE fluorescent nanoparticle comprising the trastuzumab-modified AIE fluorescent nanoparticle according to any one of claims 5 to 6.

8. Use of the trastuzumab-modified AIE fluorescent nanoparticles according to any one of claims 5 to 6 in the preparation of fluorescent targeted contrast agents for breast cancer tumors.

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