Bionic nanoparticles loaded with fluorescent dye and preparation method thereof

Abstract

The present invention relates to the technical field of pharmaceutical preparations, and specifically provides a biomimetic nanoparticle loaded with a fluorescent dye and a preparation method thereof. The particles include F127, a fluorescent dye, and a macrophage membrane vesicle; the particles are negatively charged and have a particle size of less than 100 nm; the fluorescent dye is indocyanine green or DiR-BOA. The preparation method includes: preparing nanomicelles: dissolving F127 in chloroform, adding indocyanine green or DiR-BOA, and blowing with nitrogen until a layer of drug film is formed; vacuum drying the drug film, adding deionized water, stirring at 45-55°C for 25-35 minutes, filtering with a 0.22 μm filter membrane to obtain nanomicelles loaded with a fluorescent dye; preparing macrophage membrane vesicles, resuspending; adding nanomicelles and macrophage membrane vesicles, mixing, and repeatedly mechanically extruding. The advantages are: solving the shortcomings of fluorescent dyes that are prepared and used immediately; being conducive to lymphatic drainage, homologous targeting of lymph node-resident macrophages, and enhancing lymph node visualization.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical preparations, and in particular to biomimetic nanoparticles loaded with fluorescent dyes and a preparation method thereof. Background Art

[0002] At present, the main methods for tracing sentinel lymph nodes for cancer include dye method, radionuclide method, combined method and superparamagnetic particles of iron oxide (SPIO) tracing method. All of the above tracing methods have certain limitations. For example, when the dye tracer methylene blue is used alone, its visibility in high-density adipose tissue is low and its metastasis is fast, with a detection rate of only 70% to 86%. Improper operation may lead to skin necrosis, allergic reaction and hypotension. The commonly used radionuclide tracer is technetinum-99m (99mTc) labeled colloid. Its clinical application requires corresponding instruments and equipment and the cooperation of nuclear medicine departments. Therefore, it is difficult to carry out in primary hospitals and there is the problem of radioactive contamination. Combination tracing of sentinel lymph nodes (SLNs) with a blue dye and radionuclide is a standard, evidence-based tracing method, achieving an SLN detection rate exceeding 96% and a false-negative rate as low as 5%. However, this method is technically and cost-intensive, increasing the workload of the surgeon and the financial burden on the patient. The greatest advantage of SPIO tracing is its noninvasive nature, but the technology is not perfect. Studies have shown that the sensitivity of SPIO tracing for lymph nodes 5 to 10 mm in diameter is 96%, but for nodes <5 mm in diameter, the sensitivity drops to 41%. Therefore, it is necessary to develop an efficient and simple method for SLN detection.

[0003] In contrast, near-infrared fluorescence (NIRF) imaging technology has unique advantages in sentinel lymph node localization. ICG can be excited by external light with a wavelength of around 780nm and emits near-infrared light with a wavelength of around 840nm. It has better tissue penetration and can better identify lymph nodes in hypertrophic adipose tissue under visible light than other dyes, and is relatively non-toxic. Currently, sentinel lymph node navigation technology based on real-time fluorescence imaging of indocyanine green (ICG) has been widely used in a variety of tumors, including breast cancer, melanoma, colorectal cancer, and endometrial cancer. These technologies make tracer injection simple and accurate. However, ICG has inherent limitations, such as poor stability and low quantum yield. In addition, ICG is prone to nonspecific aggregation, resulting in a decrease in fluorescence intensity. These problems often lead to diffuse imaging of cancer SLNs, making it difficult to accurately distinguish SLNs from secondary lymph nodes. Although ICG can be stabilized by loading nanoparticles, there are some drawbacks: (1) the synthetic material is not FDA-approved, has weak biocompatibility, and cannot maintain stability while increasing the fluorescence intensity of ICG, which cannot improve the detection rate of SLN; (2) the nanoparticles cannot be retained in the SLN, and the indiscriminate visualization of secondary lymph nodes reduces the specificity of SLN detection, increases the area of ​​surgical trauma, and is not conducive to postoperative recovery; (3) the requirement for lymph node visualization is that the biomimetic nanoparticle size is less than 100nm and is negatively charged, but the biomimetic nanoparticles prepared by existing preparation methods usually do not meet this standard and are not suitable for lymph node visualization. Therefore, it is very necessary to develop a cancer SLN tracer with good stability and high fluorescence intensity, so as to provide a new method for the rapid and accurate detection of cancer SLN.

[0004] Pluronic F127 is a triblock polymer used in the production of cosmetics, pharmaceuticals, pesticides, and more. F127 is currently an FDA-approved surfactant, and there are reports indicating its use in humans. However, there are no reports of using F127 to prepare biomimetic nanoparticles that can target lymph nodes. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a biomimetic nanoparticle loaded with fluorescent dye and a preparation method thereof.

[0006] The first object of the present invention is to provide a biomimetic nanoparticle loaded with a fluorescent dye, comprising: Pluronic F127, a fluorescent dye and macrophage membrane vesicles; the particle has a negative charge and a particle size of less than 100 nm.

[0007] Preferably, the particles are prepared by preparing nanomicelles from Pluronic F127 and a fluorescent dye, which are then co-extruded with macrophage membrane vesicles; the fluorescent dye is indocyanine green or DiR-BOA.

[0008] Preferably, the fluorescent dye is indocyanine green, and the encapsulation efficiency of the indocyanine green is 60-70%.

[0009] Preferably, the nanoparticles are used to target lymph nodes and / or macrophages.

[0010] Preferably, the mass ratio of Pluronic F127, indocyanine green and macrophage membrane vesicles is 5000-10000:1:25-100.

[0011] The second object of the present invention is to provide a method for preparing biomimetic nanoparticles loaded with fluorescent dyes, comprising the following steps:

[0012] S1. Preparation of nanomicelles: Pluronic F127 was dissolved in chloroform, indocyanine green or DiR-BOA was added, and nitrogen was blown until a film was formed; the film was vacuum dried, deionized water was added, and the mixture was stirred at 45-55°C for 25-35 minutes. The mixture was filtered through a 0.22 μm filter membrane to obtain fluorescent dye-loaded nanomicelles;

[0013] S2, prepare macrophage membrane vesicles and resuspend;

[0014] S3. Add the nanomicelles prepared in step S1 and the macrophage membrane vesicles prepared in step S2, mix well, and repeatedly mechanically extrude to obtain biomimetic nanoparticles loaded with fluorescent dye; the mass ratio of the nanomicelles to the macrophage membrane vesicles is 100:0.5-1.

[0015] Preferably, the repeated mechanical extrusion is performed by continuously passing through 400 nm, 200 nm and 100 nm polycarbonate porous membranes and repeatedly extruding 20 to 30 times.

[0016] Preferably, the preparation of the macrophage membrane vesicles in step S2 is by culturing, collecting and washing macrophages, adding cell lysis buffer to resuspend the cells, homogenizing, centrifuging at 15000-22000g at 0-4°C for 20-30 minutes, taking the supernatant, and then ultracentrifuging at 100000-150000g at 0-4°C for 55-65 minutes, discarding the supernatant, and obtaining a precipitate, which is the macrophage membrane vesicles.

[0017] Preferably, the cell lysis buffer comprises 225 mM d-mannitol, 30 mM Tris-HCl, 75 mM sucrose, 0.2 mM EGTA, pH=7.5.

[0018] Preferably, the ultracentrifugation speed is 120,000 g and the time is 60 min.

[0019] The third object of the present invention is to provide the use of biomimetic nanoparticles loaded with fluorescent dyes in the preparation of drugs for detecting sentinel lymph nodes.

[0020] Beneficial effects of the present invention:

[0021] (1) It can improve the instability of indocyanine green (ICG) in aqueous solution, solving the problem of immediate preparation for clinical use; the micelle core formed by F127 protects ICG from specific aggregation in aqueous solution, avoids fluorescence quenching, maintains its aqueous solution stability, and maintains a certain fluorescence after 3-6 months;

[0022] (2) Easy to drain through lymphatic vessels: The core particle size of micelles is between 10 and 100 nm. Substances with this size are most likely to drain to lymph nodes. In addition, the wrapping of macrophage membranes changes the charge of micelles from nearly neutral to negative, and the extracellular matrix is ​​negatively charged. Therefore, negatively charged particles are easily drained through lymphatic vessels by electrostatic repulsion.

[0023] (3) Homologous targeting of macrophages: The biomimetic nanoparticle carriers synthesized from cell membrane-derived vesicles have surface proteins of the source cells, such as cell adhesion molecules, and a variety of inherent functional molecules residing on the cell membrane surface functionalize the nanoparticles. The biomimetic nanoparticles wrapped in the macrophage membrane can selectively target macrophages after being drained to the lymph nodes through the lymphatic duct system, thereby enhancing the accumulation of tracers in the lymph nodes and improving the lymph node imaging effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 (a) Particle size and (b) charge results of F127-ICG and MF127-ICG provided in the examples of the present invention.

[0025] Figure 2 Absorption spectra of ICG, F127-ICG and MF127-ICG provided in the embodiments of the present invention.

[0026] Figure 3 Fluorescence spectra of ICG, F127-ICG and MF127-ICG provided in the embodiments of the present invention.

[0027] Figure 4 The absorption peaks (a, b, c), fluorescence intensity change trends over time (d, e, f) and fluorescence imaging results (g) of ICG, F127-ICG and MF127-ICG provided in the embodiments of the present invention are shown.

[0028] Figure 5 These are fluorescence imaging images of SLN at different time points after subcutaneous injection of MF127-ICG into the footpad provided by an embodiment of the present invention.

[0029] Figure 6 The cytotoxicity results of MF127-ICG provided in the examples of the present invention are shown.

[0030] Figure 7 Targeting effect of MF127 on macrophages in vitro provided by an embodiment of the present invention; (a) fluorescence imaging; (b) fluorescence intensity comparison.

[0031] Figure 8 This is the in vivo targeting effect of MF127 on lymph node macrophages provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0032] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and do not constitute a limitation of the present invention.

[0033] The present invention provides a biomimetic nanoparticle loaded with a fluorescent dye, comprising Pluronic F127, a fluorescent dye, and macrophage membrane vesicles; the particle is negatively charged and has a particle size of less than 100 nm; the particle is prepared by preparing nanomicelles from Pluronic F127 and the fluorescent dye, and then co-extruding the nanomicelles with the macrophage membrane vesicles;

[0034] The fluorescent dye is indocyanine green or DiR-BOA;

[0035] In a specific embodiment, the fluorescent dye is indocyanine green, and the encapsulation efficiency reaches 60-70%;

[0036] In specific embodiments, the particles are used to target lymph nodes or to target macrophages;

[0037] The mass ratio of Pluronic F127, indocyanine green, and macrophage membrane vesicles was 5000-10000:1:25-100;

[0038] In a specific embodiment, the mass ratio of Pluronic F127, indocyanine green, and macrophage membrane vesicles is 5000:1:25;

[0039] In a specific embodiment, the mass ratio of Pluronic F127, indocyanine green, and macrophage membrane vesicles is 10000:1:100;

[0040] Macrophages were either mononuclear macrophage cell line RAW264.7 or human macrophages.

[0041] The present invention provides a method for preparing biomimetic nanoparticles loaded with fluorescent dyes, comprising the following steps:

[0042] S1. Preparation of nanomicelles: Dissolve 200-400 mg of Pluronic F127 in chloroform, add 0.04 mg of indocyanine green or DiR-BOA, and blow nitrogen through the solution until a film is formed; vacuum dry the film, add deionized water, stir at 45-55°C for 25-35 minutes, and filter with a 0.22 μm filter membrane to obtain fluorescent dye-loaded nanomicelles;

[0043] S2, prepare macrophage membrane vesicles and resuspend;

[0044] S3. Add the nanomicelles prepared in step S1 and the macrophage membrane vesicles prepared in step S2, mix well, and repeatedly mechanically extrude to obtain biomimetic nanoparticles loaded with fluorescent dye; the mass ratio of the nanomicelles to the macrophage membrane vesicles is 100:0.5-1.

[0045] The repeated mechanical extrusion is to continuously pass through 400nm, 200nm, and 100nm polycarbonate porous membranes and repeatedly squeeze 20 to 30 times;

[0046] The preparation of the macrophage membrane vesicles in step S2 is carried out by culturing, collecting and washing macrophages, adding cell lysis buffer to resuspend the cells, homogenizing, centrifuging at 15,000 to 22,000 g at 0 to 4°C for 20 to 30 minutes, taking the supernatant, and then ultracentrifuging at 100,000 to 150,000 g at 0 to 4°C for 55 to 65 minutes, discarding the supernatant, and obtaining a precipitate, which is the macrophage membrane vesicles;

[0047] In a specific embodiment, the cell lysis buffer comprises 225 mM d-mannitol, 30 mM Tris-HCl, 75 mM sucrose, 0.2 mM EGTA, pH = 7.5;

[0048] In a specific embodiment, the homogenization in step S2 is followed by centrifugation at 20,000 g for 25 min at 4°C;

[0049] In a specific embodiment, the ultracentrifugation speed is 120,000 g and the time is 60 min;

[0050] In a specific embodiment, indocyanine green is added in step S1, and the mass ratio of Pluronic F127 to indocyanine green is 100:0.02.

[0051] Example 1 Preparation of biomimetic nanoparticles loaded with indocyanine green

[0052] This embodiment provides a method for preparing biomimetic nanoparticles loaded with indocyanine green, comprising the following steps:

[0053] 1. Preparation of F127-ICG Nanomicelles

[0054] Take 200mg of Pluronic F127 (English name: Pluronic F127, chemical formula: (C3H 60 .C2H4O) x The polymer was dissolved in 4 ml of chloroform, 0.04 mg of indocyanine green (ICG) was added, and nitrogen was blown for 2 h until a film was formed. The film was evenly dried under vacuum at room temperature for 2 h, and then 2 ml of deionized water was added. The mixture was stirred at 50°C for 30 min and filtered through a 0.22 μm filter membrane to obtain ICG-loaded F127-ICG nanomicelles.

[0055] 2. Extraction of Macrophage Membrane Vesicles

[0056] The mononuclear macrophage cell line RAW264.7 was cultured in large quantities in a carbon dioxide incubator, and then the cells were collected and washed three times with PBS buffer solution (800g, 4°C, centrifugation for 5 minutes), resuspended in cell lysis buffer (buffer containing 225mM d-mannitol, 30mM Tris-HCl, 75mM sucrose, 0.2mM EGTA, pH 7.5), and transferred to a Dounce homogenizer; the cells were repeatedly ground 40 times in the homogenizer, and the mixture was centrifuged at 20,000g for 25 minutes at 4°C. The supernatant was removed and subjected to ultrahigh-speed centrifugation (120,000g, 4°C for 60 minutes). The supernatant was discarded to obtain a precipitate, which is the macrophage membrane vesicles; the precipitate was resuspended in distilled water, and the mass of the cell membrane vesicles was determined using a Lowry protein concentration assay kit;

[0057] 3. Preparation of MF127-ICG biomimetic nanoparticles

[0058] Take 100 mg of F127-ICG nanomicelles synthesized in step S1 and 0.5 mg of macrophage membrane vesicles prepared in step S2 and mix them thoroughly; use an Avanti micro-extruder to continuously pass through 400 nm, 200 nm, and 100 nm polycarbonate porous membranes, and squeeze back and forth more than 20 times to obtain MF127-ICG biomimetic nanoparticles.

[0059] Example 2 Performance Determination of MF127-ICG Biomimetic Nanoparticles

[0060] 1. Particle size charge

[0061] The biomimetic nanoparticles MF127-ICG prepared in Example 1 were diluted to 0.01 mM, and 2 ml was taken to measure the particle size and Zeta potential of the nanoparticles using a Nano-ZS90 nanoparticle size analyzer.

[0062] The results are as follows Figure 1As shown: the average particle size of F127-ICG is 19.23nm, the charge is -2.3mV, and the average particle size of MF127-ICG biomimetic nanoparticles is 52.79nm, the charge is -7.8mV; relatively speaking, the larger particle size and more negative charge of MF127-ICG are more conducive to targeting lymph nodes from the extracellular matrix.

[0063] 2. Drug-loading assay of MF127-ICG biomimetic nanoparticles

[0064] A certain volume of MF127-ICG biomimetic nanoparticles was weighed and dissolved in a certain volume of deionized water, and its absorption spectrum was measured using an ultraviolet spectrophotometer;

[0065] The results are as follows Figure 2 As shown: the absorption peak of ICG is around 780nm, and no obvious change was found in the absorption peak position of MF127-ICG, indicating that the nanoparticles successfully loaded ICG. The peak value increased, which may be due to the absorption of the nanoparticles in the near-infrared.

[0066] 3. Fluorescence Enhancement Assay of MF127-ICG Biomimetic Nanoparticles

[0067] A certain volume of MF127-ICG nanodrug was weighed and dissolved in a certain volume of deionized water, and its fluorescence spectrum was measured using a fluorescence spectrophotometer.

[0068] like Figure 3 As shown: Under 780nm excitation light, the fluorescence peak of MF127-ICG at 840nm is 39.43. Compared with the free ICG solution with the same concentration, the fluorescence intensity of F127-ICG increased to 213.8%, which is significantly higher than that of free ICG.

[0069] IV. Aqueous Stability of MF127-ICG Biomimetic Nanoparticles

[0070] A certain volume of MF127-ICG nanodrug was weighed and dissolved in a certain volume of deionized water, and stored at room temperature away from light. Its absorption spectrum and fluorescence spectrum at 0d and 7d were measured, and fluorescence imaging was performed using an in vivo imaging system to observe changes in fluorescence intensity.

[0071] like Figure 4 As shown in the figure: within 7 days, the absorption peak of free ICG dropped to 25.6% of the initial intensity, but MF127-ICG remained at 62.5%. At the same time, the fluorescence intensity of free ICG was close to 0, but the fluorescence intensity of MF127-ICG did not decrease. The fluorescence intensity of MF127-ICG was further detected by fluorescence imaging system, which was consistent with the above conclusions ( Figure 4 g).

[0072] Currently, ICG is prepared and used immediately in clinical practice. However, during the experiment, it was found that the MF127-ICG biomimetic nanoparticles prepared by the method of the present invention are still stable after being placed for 3-6 months. Therefore, the present invention can solve the shortcomings of the existing technology.

[0073] 5. MF127-ICG in vivo lymph node tracing

[0074] MF127-ICG was injected subcutaneously into the footpad of normal Balb / c mice. Free ICG and F127-ICG nanomicelles were used as controls. The fluorescence signal of ICG at 2 min, 5 min, 10 min, and 30 min was observed using a small animal in vivo imaging system.

[0075] The results are as follows Figure 5 As shown: 2 minutes after F127-ICG and MF127-ICG were injected into the mouse footpad, obvious fluorescence signals were observed in the popliteal lymph nodes. A relatively weak fluorescence signal was observed at 30 minutes after free ICG. Compared with the gradually decreasing fluorescence signal of the popliteal lymph nodes in the F127-ICG group, the fluorescence signal of the primary lymph nodes in the MF127-ICG group was more stable and had a greater difference in fluorescence intensity from the secondary lymph nodes. This can be attributed to the fact that the macrophage membrane-coated nanoparticles can target lymph node-resident macrophages, allowing MF127-ICG to be retained in the lymph nodes. These advantages are more conducive to improving the sensitivity and specificity of sentinel lymph node (SLN) detection.

[0076] 6. Cytotoxicity of MF127-ICG Biomimetic Nanoparticles

[0077] RAW264.7 cells and human umbilical vein cell fusion cells EA.hy926 were plated on 96-well plates in advance, and the ICG concentration was set to 0.01 mM. Three replicate wells were set for each group. Free ICG, F127-ICG, and MF127-ICG were added to the cells, respectively, and incubated in a carbon dioxide incubator for 24 hours. Then, 20 μl of MTS reagent (cell proliferation and toxicity detection) was added, and the cells were incubated in the incubator for 1 to 4 hours. The absorbance value was measured at 490 nm using a microplate reader.

[0078] The results are as follows Figure 6 As shown: After 24 hours of treatment of macrophages and endothelial cells with free ICG and nanoparticles, the cell viability was basically above 90%, indicating that the macrophage membrane-wrapped nanoparticles had good biocompatibility.

[0079] Example 3 Preparation of biomimetic nanoparticles loaded with indocyanine green

[0080] This embodiment provides a method for preparing biomimetic nanoparticles loaded with indocyanine green, comprising the following steps:

[0081] 1. Preparation of F127-ICG Nanomicelles

[0082] 400 mg of F127 polymer was dissolved in 4 ml of chloroform, 0.04 mg of indocyanine green (ICG) was added, and nitrogen was blown for 2 h until a film was formed. The film was evenly dried by vacuum drying for 2 h, and then 2 ml of deionized water was added. The mixture was stirred at 50°C for 30 min and filtered through a 0.22 μm filter membrane to obtain ICG-loaded F127-ICG nanomicelles.

[0083] 2. Extraction of Macrophage Membrane Vesicles

[0084] Mouse mononuclear macrophage cell line RAW264.7 was cultured in large quantities in a carbon dioxide incubator, then collected and washed three times with PBS buffer (800g, 4°C, centrifugation for 5 minutes), resuspended in cell lysis buffer (buffer containing 225mM d-mannitol, 30mM Tris-HCl, 75mM sucrose, 0.2mM EGTA, pH 7.5), and transferred to a Dounce homogenizer; the cells were repeatedly ground 40 times in the homogenizer, and the mixture was centrifuged at 20,000g for 25 minutes at 4°C. The supernatant was removed and subjected to ultrahigh-speed centrifugation (120,000g, 4°C for 60 minutes). The supernatant was discarded to obtain a precipitate, which is the macrophage membrane vesicles; the precipitate was resuspended in distilled water, and the mass of the cell membrane vesicles was measured using a Lowry protein concentration assay kit;

[0085] 3. Preparation of MF127-ICG biomimetic nanoparticles

[0086] 100 mg of F127-ICG nanomicelles synthesized in step S1 and 1 mg of macrophage membrane vesicles prepared in step S2 were mixed thoroughly. MF127-ICG biomimetic nanoparticles were obtained by continuously extruding through 400 nm, 200 nm, and 100 nm polycarbonate porous membranes using an Avanti microextruder more than 20 times. The prepared nanoparticles had an average particle size of 56.5 nm and a charge of -10 mV.

[0087] Example 4 Preparation and Performance Measurement of DiR-BOA-Loaded Biomimetic Nanoparticles I. Preparation Method

[0088] A method for preparing biomimetic nanoparticles loaded with DiR-BOA comprises replacing ICG with the more stable fluorescent dye DiR-BOA in the method described in Example 1. The mass ratio of Pluronic F127 to DiR-BOA is 200:1, resulting in the synthesis of F127 (DiR-BOA) nanomicelles, which are then used to synthesize MF127 (DiR-BOA) biomimetic nanoparticles. The prepared MF127 (DiR-BOA) has an average particle size of 53.2 nm and a charge of -8.01 mV.

[0089] 2. In vitro macrophage targeting experiments of MF127 (DiR-BOA)

[0090] Macrophages RAW264.7 were plated in 96-well plates and confocal dishes in advance, and the drug concentrations of DiR-BOA were set at 2.5 μg / mL, 5 μg / mL, 10 μg / mL, and 20 μg / mL, with three replicate wells in each group. Free F127 (DiR-BOA) and MF127 (DiR-BOA) were added to macrophages RAW264.7, respectively, and the cells were incubated in a carbon dioxide incubator for 1 hour, and then rinsed twice with PBS. The flow cytometry section was digested with trypsin and then loaded, and the confocal section was fixed with 4% paraformaldehyde and stained with DAPI for 10 minutes, and then the cell uptake was analyzed using a confocal laser scanning microscope.

[0091] like Figure 7 As shown in the figure, with the increase of DiR-BOA concentration, the fluorescence signals were enhanced; compared with the RAW264.7 cells incubated with F127 (DiR-BOA), the blue and red fluorescence signals in the RAW264.7 cells incubated with MF127 (DiR-BOA) were significantly enhanced, which was consistent with the results of the mean fluorescence intensity of flow cytometry, indicating that the introduction of macrophage membrane-coated nanoparticles can enhance the uptake of macrophages.

[0092] MF127 (DiR-BOA) targets lymph node macrophages

[0093] Two groups, MF127 (DiR-BOA) and F127 (DiR-BOA), were set up. Normal Balb / c mice were injected subcutaneously with either drug via the footpad. Lymph nodes were removed 1 hour later and fixed in paraformaldehyde (PFA) (protected from light). Lymph nodes were dehydrated with 30% sucrose for 24 hours and then frozen and sectioned at a thickness of 10 μm. Sections were blocked with a blocking solution containing 1% BSA and 0.3% triton. One hour after sectioning, macrophages were stained with an antibody against the mouse growth factor-like motif, mucin-like hormone receptor F4 / 80. Nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI), and sections were mounted. Cellular uptake was analyzed using confocal laser scanning microscopy.

[0094] like Figure 8 As shown: Compared with F127(DiR-BOA), the green fluorescence of macrophages in the MF127(DiR-BOA) group co-localized more strongly with the red fluorescence of nanoparticles, further indicating that the wrapping of macrophage membranes is conducive to the targeting of nanoparticles to macrophages.

[0095] Although the embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. A person of ordinary skill in the art may make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention. The above specific embodiments of the present invention do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made based on the technical concept of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. Biomimetic nanoparticles loaded with fluorescent dyes, characterized in that: include: Pluronic F127, a fluorescent dye, and macrophage membrane vesicles; the particles are negatively charged and have a particle size of less than 100 nm; the fluorescent dye is indocyanine green, and the encapsulation efficiency of the indocyanine green is 60-70%; the mass ratio of Pluronic F127, indocyanine green, and macrophage membrane vesicles is 5000-10000:1:25-100; The particles are prepared by preparing nanomicelles from Pluronic F127 and a fluorescent dye, and then co-extruding them with macrophage membrane vesicles. The macrophage membrane vesicles are prepared by culturing, collecting and washing macrophages, adding a cell lysis buffer to resuspend the cells, homogenizing, centrifuging at 15,000 to 22,000 g at 0 to 4°C for 20 to 30 minutes, taking the supernatant, and then ultracentrifuging at 100,000 to 150,000 g at 0 to 4°C for 55 to 65 minutes, discarding the supernatant, and obtaining a precipitate, which is the macrophage membrane vesicle.

2. The biomimetic nanoparticles loaded with fluorescent dye according to claim 1, characterized in that: The nanoparticles are used for targeting lymph nodes and / or targeting macrophages.

3. A method for preparing fluorescent dye-loaded biomimetic nanoparticles, for preparing the fluorescent dye-loaded biomimetic nanoparticles according to claim 1, characterized in that: The steps include: S1. Preparation of nanomicelles: Pluronic F127 was dissolved in chloroform, indocyanine green was added, and nitrogen was blown until a film was formed. The film was vacuum dried, deionized water was added, and the mixture was stirred at 45-55°C for 25-35 minutes. The mixture was filtered through a 0.22 μm filter membrane to obtain fluorescent dye-loaded nanomicelles. S2, prepare macrophage membrane vesicles and resuspend; S3. Add the nanomicelles prepared in step S1 and the macrophage membrane vesicles prepared in step S2, mix well, and repeatedly mechanically extrude to obtain biomimetic nanoparticles loaded with fluorescent dye; the mass ratio of the nanomicelles to the macrophage membrane vesicles is 100:0.5-1.

4. The method for preparing fluorescent dye-loaded biomimetic nanoparticles according to claim 3, wherein: The repeated mechanical extrusion is to continuously pass through 400nm, 200nm and 100nm polycarbonate porous membranes and repeatedly squeeze 20 to 30 times.

5. The method for preparing fluorescent dye-loaded biomimetic nanoparticles according to claim 4, characterized in that: The cell lysis buffer includes 225 mM d-mannitol, 30 mM Tris-HCl, 75 mM sucrose, 0.2 mM EGTA, and pH=7.

5.

6. The method for preparing fluorescent dye-loaded biomimetic nanoparticles according to claim 5, characterized in that: The ultracentrifugation was performed at a speed of 120,000 g and for 60 min.

7. Use of the fluorescent dye-loaded biomimetic nanoparticles according to any one of claims 1 to 2 in the preparation of a drug for detecting sentinel lymph nodes.

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