CN110791281A - Preparation method and application of macrophage tracing fluorescent probe - Google Patents
Preparation method and application of macrophage tracing fluorescent probe Download PDFInfo
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- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
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Abstract
The invention relates to a preparation method and application of a macrophage tracing fluorescent probe. The probe prepared by the invention has better macrophage targeting capability and higher biological safety.
Description
Technical Field
The invention relates to the field of material synthesis and application, in particular to a preparation method and application of a macrophage-targeting glucan nanoprobe.
Background
The malignant tumor is a disease seriously threatening the health of residents, and according to the latest statistical data, the death of the malignant tumor accounts for 23.91 percent of the total death of residents in China, the morbidity and the mortality of the malignant tumor are in a continuous rising state in recent ten years, the medical cost caused by the malignant tumor exceeds 2200 hundred million every year, the prevention and control situation is severe, and the development of a real-time, quantitative and sensitive tumor monitoring probe is urgent. Direct targeting and monitoring of tumor cells are a common idea, and a tumor area not only has tumor cells, but also infiltrates a plurality of immune cells for regulating and controlling tumor processes to form a tumor matrix environment and regulate and control tissue remodeling and angiogenesis, so that monitoring of division, apoptosis, rest, molecular expression and the like of the tumor cells is important, and monitoring of the tumor matrix environment is also important.
The regulatory cells occupying the most amount in the tumor stroma environment are macrophages, which are important components of the innate immune process, mononuclear cells in bone marrow are distributed to everywhere in the whole body to be differentiated into resident macrophages in various tissues, so that the regulatory cells can generally play positive roles of clearing foreign and harmful substances and the like, but are educated into harmful cells by cytokines secreted by tumor cells at tumor positions to form tumor-related macrophages (TAM), promote the growth, proliferation, invasion, metastasis, immunosuppression and angiogenesis of tumors, establish a stroma environment suitable for survival for the tumor cells, and further cause the poor prognosis of tumor patients, so that the macrophage targeting at the tumor position, the development of a tumor treatment strategy aiming at the macrophages and the development of a corresponding evaluation means are very important.
At present, more and more means for targeting TAM to intervene tumors are available, and the method is rapidly promoted to clinical application, mainly comprises methods of consuming macrophages in tumor regions, inhibiting recruitment of macrophages in tumor regions, promoting phenotypic transformation of TAM and the like, and some means show good tumor prognosis in preclinical and clinical researches, for example, the resistance of tumors to gemcitabine can be reduced by removing TAM in pancreatic cancer models, so that the means for screening, monitoring and evaluating are very important.
This urgently requires imaging means for tracking tumor-associated macrophages and corresponding imaging contrast agents. Current means of tracking tumor-associated macrophages are Computed Tomography (CT), Magnetic Resonance Imaging (MRI), Positron Emission Tomography (PET), and fluorescence imaging, among others. CT is the most common diagnostic means in clinic at present, but a large amount of nanoparticles are needed to trace macrophages even at a molar level, and the high atomic number elements used are generally high in toxicity, so that the clinical application of the means for tracing macrophages is limited. PET can realize quick quantitative whole-body imaging, but has higher requirements on instruments, has radiation, can only do 1-2 times a year, and can not provide accurate anatomical positioning. MRI is a common imaging means in clinic, has no ionizing radiation, has sub-millimeter resolution, can clearly display the relation between a tumor area and a blood vessel, and has an important function for judging the resectability of tumor surgery, but the spatial resolution of MRI is poor, the diagnosis effect on early tumors is limited, gadolinium (Gd) is a common MRI contrast agent in medical treatment, is a main object for researching MRI contrast agent, and has the problems of short half-life period and the like due to small molecular weight. In summary, the conventional imaging detection techniques play an important role in macrophage tracing, but all have some limitations. Compared with the imaging methods of ultrasound, MRI, CT, PET and the like which are commonly used in clinic, fluorescence imaging has the advantages of high imaging speed, high sensitivity, no ionizing radiation, simple instruments and equipment and the like, and plays an increasingly important role in early detection of cancer, guiding surgical treatment in operation and the like. Even if MRI, PET and other researches are carried out, a fluorescent tracing means is required to carry out optimization design and cell-level verification of a targeting mechanism and effect, and in fact, most results of how macrophages and other cells treat nanoparticles are obtained by fluorescence imaging nowadays, so that the fluorescence imaging has incomparable unique advantages in tracing the macrophages and screening macrophage therapy.
Indocyanine green (ICG) is a diagnostic reagent which is currently used in clinic, has high safety and good imaging effect, and is found to have good fluorescence imaging effect in a near infrared fluorescence secondary region (NIR-II,1000-1700nm) besides the imaging of the traditional near infrared fluorescence primary region in recent years. The near-infrared fluorescence two-zone has less tissue absorption and scattering and lower tissue autofluorescence in living tissues, can greatly improve the tissue penetration depth and the spatial resolution of fluorescence imaging, greatly weakens the limitations of low tissue penetration depth and low spatial resolution of the traditional fluorescence one-zone imaging, has wide application prospect in biomedical images, is deep and clear to see, has clear organ outlines during imaging, and is beneficial to clearly observing the particle distribution condition of each organ in real time. Particularly, in the aspect of pancreatic cancer in-vivo imaging, the liver and the pancreas are positioned very close to each other, traditional fluorescence imaging is difficult to distinguish, and two-region fluorescence imaging has absolute advantages. However, indocyanine green has the defects of poor optical stability and the like, and the difficulty and key problem of macrophage fluorescence imaging by using indocyanine green through means of chemical modification and the like to enhance the stability of indocyanine green.
With suitable imaging means such as fluorescence imaging and MRI imaging and contrast agents, the key problem of tracing tumor-associated macrophages is how to specifically target the contrast agents to the tumor-associated macrophages. The current strategy for targeting tumor-associated macrophages comprises modifying specific ligands, antibodies and the like of the macrophages on the surface of a contrast medium delivery system, such as modifying CD206 antibodies or small-molecule mannose to target CD206 proteins (also called mannose receptors) on the surfaces of the macrophages, wherein the antibody targeting has good specificity, but the purification is difficult and the cost is high, and the small-molecule modification has the problem of easy metabolic clearance of the kidney.
In view of the above, a new method is needed to solve the above problems.
Disclosure of Invention
The invention mainly solves the technical problem of providing a preparation method and application of a macrophage tracing probe, which has the advantages of simple method, mild reaction condition, good reproducibility, low toxicity and good biocompatibility. In order to solve the technical problems, the invention adopts a technical scheme that:
a preparation method of a macrophage tracing fluorescent probe comprises the following steps:
s110, placing the carboxymethylated glucan, one of NHS and sulfo-NHS and the activated carboxyl reagent in a buffer solution, and stirring at room temperature to react to obtain an activated carboxymethylated glucan solution;
s120, dissolving a cross-linking agent in a buffer solution, adding the activated carboxymethylated glucan solution, and stirring at room temperature to react to obtain a clear solution;
s130, dropwise adding the clear solution into precooled absolute ethyl alcohol, centrifuging to obtain a precipitate, redissolving the obtained precipitate in water, and filtering with a microfiltration membrane to obtain a filtrate;
s140, performing room temperature dialysis on the filtrate, filtering the filtrate by using a membrane after the dialysis is finished, pre-freezing the filtrate at a first temperature, transferring the filtrate to a second temperature for freezing, and performing freeze drying to obtain freeze-dried powder;
s150, dissolving the activated carboxyl reagent and NHS in dimethyl sulfoxide, adding the tracer micromolecules into the water solution of the freeze-dried powder after the activation, and reacting at room temperature in a dark place to obtain a reaction solution;
s160, removing free contrast micromolecules in the reaction liquid and then concentrating to obtain a concentrated solution;
s170, dissolving succinic anhydride in dimethyl sulfoxide, adding a catalytic amount of triethylamine to react with the concentrated solution at room temperature in a dark place, dialyzing at room temperature, and then passing through a microfiltration membrane to obtain the macrophage tracing fluorescent probe.
In one embodiment, the cross-linking agent is lysine.
In one embodiment, the glucan is at least one of carboxymethyl glucan with the molecular weight of 2-40 kD and the carboxyl substitution degree of 2% -10%.
In one embodiment, the activated carboxylic reagent is at least one of EDC, DCC, CDI and DIC.
In one embodiment, the tracer small molecule is at least one of a fluorescent imaging small molecule or an MRI imaging small molecule.
In one embodiment, the tracer small molecule is at least one of COOH-ICG, CN-ICG, and Gd-DOT.
In one embodiment, the microfiltration membrane is a 0.22 μm filter.
In one embodiment, the room temperature dialysis is room temperature dialysis for 0.5-5 days with a 10kD dialysis bag using ultrapure water as a dialysis medium.
In one embodiment, the pre-freezing at the first temperature is followed by transferring to the second temperature for freezing, and then freeze-drying to obtain the lyophilized powder as follows: pre-freezing for 2h in a-20 deg.C refrigerator, transferring to a-80 deg.C refrigerator, freezing for 24h, and freeze-drying for 48h in a freeze-drying machine to obtain lyophilized powder.
In order to solve the technical problems, the invention adopts a technical scheme that: the macrophage tracing fluorescent probe obtained by the preparation method of the macrophage tracing fluorescent probe is applied to targeting of macrophages.
The invention has the beneficial effects that: compared with the prior art, the macrophage-targeting glucan nanoprobe synthesized by the invention has better macrophage targeting capability and higher biological safety, the probe chemically cross-links carboxymethyl glucan by lysine which is one of essential amino acids of a human body to form uniform and stable glucan cross-linked nanoparticles, and the nanoparticles are connected with imaging small molecules such as indocyanine green and the like by covalent bonds to construct the nanoparticles with both macrophage targeting capability and imaging capability. The invention is helpful to promote the research and development of macrophage tracing, and simultaneously provides a new theory and a new method for early diagnosis and prognosis of tumors. In addition, the preparation method is simple and convenient, the reaction condition is mild, the reproducibility is good, the toxicity is low, the biocompatibility is good, and the application prospect is wide.
Drawings
FIG. 1 is a schematic diagram of a synthetic route for a macrophage tracer probe according to one embodiment;
FIG. 2 is a particle size and TEM characterization of the macrophage tracing fluorescent probe of example 1, with a scale bar of 50 μm;
FIG. 3 is a graph showing the effect of the same amount of the probe prepared in example 1 on the cell activity of three cells, respectively;
FIG. 4 is a graph showing the near infrared two-zone imaging of the same amount of probes prepared in example 1 when added to different cells.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer.
A preparation method of a macrophage tracing fluorescent probe comprises the following steps:
s110, placing the carboxymethylated glucan, one of NHS and sulfo-NHS and the activated carboxyl reagent in a buffer solution, and stirring at room temperature to react to obtain the activated carboxymethylated glucan solution.
Specifically, in one embodiment, the glucan is at least one of carboxymethyl glucan with the molecular weight of 2-40 kD and the carboxyl substitution degree of 2% -10%. The activated carboxyl reagent is at least one of EDC, DCC, CDI and DIC. The buffer may be MES buffer or PBS buffer.
And S120, dissolving the cross-linking agent in the buffer solution, adding the buffer solution into the activated carboxymethylated glucan solution, and stirring at room temperature for reaction to obtain a clear solution.
Specifically, in one embodiment, the cross-linking agent is lysine.
S130, dropwise adding the clear solution into precooled absolute ethyl alcohol, centrifuging to obtain a precipitate, redissolving the obtained precipitate in water, and filtering with a microfiltration membrane to obtain a filtrate;
specifically, in one embodiment, the microfiltration membrane is a 0.22 μm filter.
S140, performing room temperature dialysis on the filtrate, filtering the filtrate by using a membrane after the dialysis is finished, pre-freezing the filtrate at a first temperature, transferring the filtrate to a second temperature for freezing, and performing freeze drying to obtain freeze-dried powder;
specifically, in one embodiment, the room temperature dialysis is performed for 0.5 to 5 days in a 10kD dialysis bag using ultrapure water as a dialysis medium.
Specifically, in one embodiment, the pre-frozen powder is transferred to the second temperature for freezing after being pre-frozen at the first temperature, and then the pre-frozen powder is freeze-dried to obtain the freeze-dried powder as follows: pre-freezing for 2h in a-20 deg.C refrigerator, transferring to a-80 deg.C refrigerator, freezing for 24h, and freeze-drying for 48h in a freeze-drying machine to obtain lyophilized powder.
S150, dissolving the activated carboxyl reagent and NHS in dimethyl sulfoxide, adding the tracer micromolecules into the water solution of the freeze-dried powder after the activation, and reacting at room temperature in a dark place to obtain a reaction solution;
specifically, in one embodiment, the tracer small molecule is at least one of a fluorescent imaging small molecule or an MRI imaging small molecule. Specifically, the tracer micromolecules are at least one of COOH-ICG, CN-ICG and Gd-DOT.
S160, removing ionographic small molecules in the reaction liquid and concentrating to obtain a concentrated solution;
s170, dissolving succinic anhydride in dimethyl sulfoxide, adding a catalytic amount of triethylamine to react with the concentrated solution at room temperature in a dark place, dialyzing at room temperature, and then passing through a microfiltration membrane to obtain the macrophage tracing fluorescent probe.
Specifically, in one embodiment, the microfiltration membrane is a 0.22 μm filter. The room temperature dialysis is carried out for 0.5-5 days by using ultrapure water as a dialysis medium and using a 10kD dialysis bag.
Specifically, the preparation method of the macrophage tracing fluorescent probe comprises the following steps:
1. carboxymethylated dextran (0.00055-0.55g) was weighed out accurately, EDC activated carboxyl reagent (0.0024-2.4g) and NHS (0.0004572-0.4572mg) were added, dissolved in MES buffer (50mM, pH 6.0-6.5) (0.0062-6.2mL) and the reaction was stirred gently at room temperature (0.25-48 h).
2. The crosslinker L-lysine (0.0004-0.4g) was weighed out accurately and dissolved in MES buffer (0.0007-0.7mL) (50mM, pH 6.0-6.5) and added to the carboxymethylated dextran solution activated in step 1, and the reaction was stirred gently at room temperature (5-48 h).
3. Dropwise adding the clear solution obtained in the step 2 into (0.03-30mL) precooled absolute ethyl alcohol, centrifuging (0.25-2.5 kXg, 3-30min), collecting white precipitate, re-dissolving the obtained white precipitate in water, and filtering with a 0.22 mu m filter membrane to obtain filtrate.
4. And 3, performing room-temperature dialysis (for 0.5-5 days) on the filtrate obtained in the step 3 by using a 10kD dialysis bag by using ultrapure water as a dialysis medium, passing through a 0.22-micrometer filter membrane after the dialysis is finished, pre-freezing for 2h in a-20-DEG refrigerator, transferring to a-80-DEG refrigerator for freezing for 24h, and performing freeze drying for 48h in a freeze dryer to obtain the freeze-dried powder.
5. Accurately weighing (0.00002-0.02g) the freeze-dried powder obtained in the step 4, adding (0.000003-0.003g) EDC and (0.000001-0.001g) NHS, dissolving in (0.0002-0.2mL) dimethyl sulfoxide, adding (0.000001-0.001g) carboxylated ICG or gadoteric acid (Gd-DOTA) tracer micromolecules, and carrying out light-shielding and room-temperature mild reaction (1-48 h).
6. Dialyzing the solution obtained in step 5 with ultrapure water as dialysis medium at room temperature (0.5-5 days) with 10kD dialysis bag to remove free small contrast molecules, and concentrating to (50-200 μ L) with 10kD ultrafiltration tube to obtain concentrated solution.
7. Weighing (0.00015-0.015g) succinic anhydride, dissolving in (0.0001-0.1mL) dimethyl sulfoxide, adding a catalytic amount of triethylamine (0.00001-0.01 μ L) to react with the concentrated solution at room temperature in a dark place for 0.5-48h, dialyzing at room temperature (0.5-5 days) by using a 10kD dialysis bag by using ultrapure water as a dialysis medium, and filtering through a 0.22 μm filter membrane to obtain the probe.
Example 1:
1. 0.55g of carboxymethylated dextran was accurately weighed, 2.4g of EDC and 0.4572g of NHS were added, and the mixture was dissolved in 6.2mL of MES buffer (50mM, pH 6.0-6.5) and reacted at room temperature with gentle stirring for 10 minutes.
2. 0.4g of L-lysine was accurately weighed, dissolved in 0.7mL of MES buffer (50mM, pH 6.0-6.5), added to the carboxymethylated dextran solution activated in step 1, and reacted at room temperature with gentle stirring for 5 hours.
3. Dropwise adding the clear solution obtained in the step 2 into 30mL of precooled absolute ethyl alcohol, centrifuging (2.5 kXg, 3min), collecting white precipitate, re-dissolving the obtained white precipitate in water, and filtering with a 0.22 mu m filter membrane.
4. And (3) performing room-temperature dialysis (3 days) on the solution obtained in the step (3) by using ultrapure water as a dialysis medium and using a 10kD dialysis bag, passing through a 0.22-micrometer filter membrane after the dialysis is finished, pre-freezing for 2h in a refrigerator with the temperature of-20 ℃, transferring to a refrigerator with the temperature of-80 ℃ for freezing for 24h, and performing freeze drying for 48h in a freeze dryer to obtain freeze-dried powder.
5. Accurately weighing 0.05g of the freeze-dried powder obtained in the step 4, adding 0.003g of EDC and 0.001g of NHS, dissolving in 0.2mL of dimethyl sulfoxide, adding 0.001g of carboxylated ICG, and carrying out mild reaction for 7h at room temperature in a dark place.
6. The solution obtained in step 5 was dialyzed at room temperature (0.5 day) using a 10kD dialysis bag using ultrapure water as a dialysis medium, to remove free ICG, and concentrated to (150. mu.L) concentrate using a 10kD ultrafiltration tube.
7. Weighing 0.015g succinic anhydride, dissolving in 0.1mL dimethyl sulfoxide, adding a catalytic amount of triethylamine, reacting with the concentrated solution at room temperature in a dark place for 18h, dialyzing with a 10kD dialysis bag at room temperature (for 0.5 day) by using ultrapure water as a dialysis medium, and filtering with a 0.22 μm filter membrane to obtain the final probe solution.
Example 2:
1. 0.11g of carboxymethylated dextran was accurately weighed, 0.48g of EDC and 0.09144g of NHS were added, and the mixture was dissolved in 1.24 mM MES buffer (50mM, pH 6.0-6.5) and reacted at room temperature with gentle stirring for 10 minutes.
2. 0.08g of L-lysine was accurately weighed, dissolved in 0.14mL of MES buffer (50mM, pH 6.0-6.5), added to the carboxymethylated dextran solution activated in step 1, and reacted at room temperature with gentle stirring for 5 hours.
3. Dropwise adding the clear solution obtained in the step 2 into 6mL of precooled absolute ethyl alcohol, centrifuging (2.5 kXg, 3min), collecting white precipitate, re-dissolving the obtained white precipitate in water, and filtering with a 0.22 mu m filter membrane.
4. And (3) performing room-temperature dialysis (3 days) on the solution obtained in the step (3) by using ultrapure water as a dialysis medium and using a 10kD dialysis bag, passing through a 0.22-micrometer filter membrane after the dialysis is finished, pre-freezing for 2h in a refrigerator with the temperature of-20 ℃, transferring to a refrigerator with the temperature of-80 ℃ for freezing for 24h, and performing freeze drying for 48h in a freeze dryer to obtain freeze-dried powder.
5. Accurately weighing 0.05g of the freeze-dried powder obtained in the step 4, adding 0.003g of EDC and 0.001g of NHS, dissolving in 0.2mL of dimethyl sulfoxide, adding 0.0005g of carboxylated ICG, and carrying out mild reaction for 7h at room temperature in a dark place.
6. The solution obtained in step 5 was dialyzed at room temperature (0.5 day) using a 10kD dialysis bag using ultrapure water as a dialysis medium, to remove free ICG, and concentrated to (150. mu.L) concentrate using a 10kD ultrafiltration tube.
7. Weighing 0.015g succinic anhydride, dissolving in 0.1mL dimethyl sulfoxide, adding a catalytic amount of triethylamine, reacting with the concentrated solution at room temperature in a dark place for 18h, dialyzing with a 10kD dialysis bag at room temperature (for 0.5 day) by using ultrapure water as a dialysis medium, and filtering with a 0.22 μm filter membrane to obtain the final probe solution.
Example 3:
1. 0.22g of carboxymethylated dextran was accurately weighed, 0.96g of EDC and 0.18288g of NHS were added, and the mixture was dissolved in 2.48 mM MES buffer (50mM, pH 6.0-6.5) and reacted at room temperature with gentle stirring for 10 minutes.
2. 0.16g of L-lysine was accurately weighed, dissolved in 0.28mL of MES buffer (50mM, pH 6.0-6.5), added to the carboxymethylated dextran solution activated in step 1, and reacted at room temperature with gentle stirring for 5 hours.
3. Dropwise adding the clear solution obtained in the step 2 into 12mL of precooled absolute ethyl alcohol, centrifuging (2.5 kXg, 3min), collecting white precipitate, re-dissolving the obtained white precipitate in water, and filtering with a 0.22 mu m filter membrane.
4. And (3) performing room-temperature dialysis (3 days) on the solution obtained in the step (3) by using ultrapure water as a dialysis medium and using a 10kD dialysis bag, passing through a 0.22-micrometer filter membrane after the dialysis is finished, pre-freezing for 2h in a refrigerator with the temperature of-20 ℃, transferring to a refrigerator with the temperature of-80 ℃ for freezing for 24h, and performing freeze drying for 48h in a freeze dryer to obtain freeze-dried powder.
5. Accurately weighing 0.05g of the freeze-dried powder obtained in the step 4, adding 0.003g of EDC and 0.001g of NHS, dissolving in 0.2mL of dimethyl sulfoxide, adding 0.002g of carboxylated ICG, and carrying out mild reaction for 7h at room temperature in a dark place.
6. The solution obtained in step 5 was dialyzed at room temperature (0.5 day) using a 10kD dialysis bag using ultrapure water as a dialysis medium, to remove free ICG, and concentrated to (150. mu.L) concentrate using a 10kD ultrafiltration tube.
7. Weighing 0.015g succinic anhydride, dissolving in 0.1mL dimethyl sulfoxide, adding a catalytic amount of triethylamine, reacting with the concentrated solution at room temperature in a dark place for 18h, dialyzing with a 10kD dialysis bag at room temperature (for 0.5 day) by using ultrapure water as a dialysis medium, and filtering with a 0.22 μm filter membrane to obtain the final probe solution.
Example 4:
1. 0.22g of carboxymethylated dextran was accurately weighed, 0.96g of EDC and 0.18288g of NHS were added, and the mixture was dissolved in 2.48 mM MES buffer (50mM, pH 6.0-6.5) and reacted at room temperature with gentle stirring for 10 minutes.
2. 0.16g of L-lysine was accurately weighed, dissolved in 0.28mL of MES buffer (50mM, pH 6.0-6.5), added to the carboxymethylated dextran solution activated in step 1, and reacted at room temperature with gentle stirring for 5 hours.
3. Dropwise adding the clear solution obtained in the step 2 into 12mL of precooled absolute ethyl alcohol, centrifuging (2.5 kXg, 3min), collecting white precipitate, re-dissolving the obtained white precipitate in water, and filtering with a 0.22 mu m filter membrane.
4. And (3) performing room-temperature dialysis (3 days) on the solution obtained in the step (3) by using ultrapure water as a dialysis medium and using a 10kD dialysis bag, passing through a 0.22-micrometer filter membrane after the dialysis is finished, pre-freezing for 2h in a refrigerator with the temperature of-20 ℃, transferring to a refrigerator with the temperature of-80 ℃ for freezing for 24h, and performing freeze drying for 48h in a freeze dryer to obtain freeze-dried powder.
5. Accurately weighing 0.05g of the freeze-dried powder obtained in the step 4, adding 0.003g of EDC and 0.001g of NHS, dissolving in 0.2mL of dimethyl sulfoxide, adding 0.002g of gadoteric acid (DOTA), and reacting at room temperature for 7 h.
6. The solution obtained in step 5 was dialyzed at room temperature (0.5 day) against a 10kD dialysis bag using ultrapure water as a dialysis medium to remove free gadoteric acid (DOTA) and concentrated to (150. mu.L) concentrate using a 10kD ultrafiltration tube.
7. Weighing 0.015g succinic anhydride, dissolving in 0.1mL dimethyl sulfoxide, adding a catalytic amount of triethylamine, reacting with the concentrated solution at room temperature in a dark place for 18h, dialyzing with a 10kD dialysis bag at room temperature (for 0.5 day) by using ultrapure water as a dialysis medium, and filtering with a 0.22 μm filter membrane to obtain the final probe solution.
Example 5: test for probe to no toxicity of cell
The cytotoxicity of the glucan-ICG nano probe on two pancreatic cancer tumor cells SW1990, SW1990-mcherry-Luc and macrophage RAW264.7 is compared, and the biocompatibility of the probe is proved.
1. Pancreatic cancer cell strains SW1990, SW1990-mcherry-Luc and a macrophage cell strain RAW264.7 are preserved in the laboratory, and all the three cell strains belong to cell strains commonly used in tumor research and can be purchased and obtained from the market. The dextran-ICG nanoparticles obtained in example 1 were diluted in DMEM medium at equal ratio to appropriate concentrations, and the cytotoxicity of the probes on SW1990, SW1990-mcherry-Luc, and RAW264.7 was determined by CCK-8 rapid colorimetry.
2. SW1990, SW1990-mcherry-Luc and RAW264.7 cells in logarithmic growth phase are added into ninety-six-well plates at the rate of 1-5 × 104 cells/well, are cultured overnight until the cells are attached to the wall, and then are respectively cultured for 24 hours by DMEM culture medium containing glucan-ICG obtained in the corresponding concentration example 1, 10ul CCK-8 is added into each well, the culture medium is incubated for 1 hour at 37 ℃, 5% CO2 and a saturated humidity incubator, and the cells are detected by a 450nm microplate reader.
As shown in FIG. 3, it can be seen from FIG. 3 that the probe obtained in example 1 has no significant killing effect on cells, demonstrating that the probe is non-toxic to cells.
Example 6: the probe targets macrophages.
1. The pancreatic cancer cell strain SW1990 is preserved in the laboratory, the cell is a common cell strain which can be purchased in the market, the macrophage is extracted from the bone marrow of the mouse, and the extraction method is a relatively mature and simple cell extraction method. The dextran probe obtained in example 1 was diluted to an appropriate concentration with a DMEM medium or the like, added to each of the two cells, incubated for 4 hours, and the cells were observed for near-infrared two-region imaging (wavelength band after 1000 nm) with a confocal microscope.
2. SW1990 cells in the logarithmic growth phase and extracted macrophages obtained by extraction were added to an eight-well chamber at 1-5 × 104 cells per well, respectively, and cultured overnight until they adhere to the wall, then cultured with a dextran probe DMEM medium obtained in example 1 at an appropriate concentration for 4 hours at 37 ℃, unbound probes were aspirated, and the cells were washed 3 times with PBS to remove unbound dextran probes, and the near-infrared two-zone imaging of the cells was observed with a confocal microscope (band after 1000 nm).
The results are shown in FIG. 4, and it can be seen from FIG. 4 that after the addition of an equal amount of dextran probe, significant macrophage imaging is bright in pancreatic cancer cells SW1990, indicating that macrophages engulf more probes than pancreatic cancer cells, which can demonstrate targeting of the probes to macrophages.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (10)
1. A preparation method of a macrophage tracing fluorescent probe is characterized by comprising the following steps:
s110, placing the carboxymethylated glucan, one of NHS or sulfo-NHS and the activated carboxyl reagent in a buffer solution, and stirring at room temperature to react to obtain an activated carboxymethylated glucan solution;
s120, dissolving a cross-linking agent in a buffer solution, adding the activated carboxymethylated glucan solution, and stirring at room temperature to react to obtain a clear solution;
s130, dropwise adding the clear solution into precooled absolute ethyl alcohol, centrifuging to obtain a precipitate, redissolving the obtained precipitate in water, and filtering with a microfiltration membrane to obtain a filtrate;
s140, performing room temperature dialysis on the filtrate, filtering the filtrate by using a membrane after the dialysis is finished, pre-freezing the filtrate at a first temperature, transferring the filtrate to a second temperature for freezing, and performing freeze drying to obtain freeze-dried powder;
s150, dissolving the activated carboxyl reagent and NHS in dimethyl sulfoxide, adding the tracer micromolecules, adding the activated tracer micromolecules into the aqueous solution of the freeze-dried powder, and reacting at room temperature in a dark place to obtain a reaction solution;
s160, removing free contrast micromolecules in the reaction liquid and then concentrating to obtain a concentrated solution;
s170, dissolving succinic anhydride in dimethyl sulfoxide, adding a catalytic amount of triethylamine to react with the concentrated solution at room temperature in a dark place, dialyzing at room temperature, and then passing through a microfiltration membrane to obtain the macrophage tracing fluorescent probe.
2. The method for preparing a macrophage tracing fluorescent probe according to claim 1, wherein said cross-linking agent is lysine.
3. The method for preparing a macrophage tracing fluorescent probe according to claim 1, wherein the dextran is at least one of carboxymethyl dextran having a molecular weight of 2-40 kD and a carboxyl substitution degree of 2% -10%.
4. The method for preparing a macrophage tracing fluorescent probe according to claim 1, wherein the activated carboxyl reagent is at least one of EDC, DCC, CDI and DIC.
5. The method of claim 1, wherein the labeled small molecule is at least one of a fluorescent imaging small molecule or an MRI imaging small molecule.
6. The method for preparing a macrophage tracing fluorescent probe according to claim 5, wherein the tracing small molecule is at least one of COOH-ICG, CN-ICG and Gd-DOT.
7. The method for preparing a macrophage tracing fluorescent probe as claimed in claim 1, wherein said microfiltration membrane is a 0.22 μm filtration membrane.
8. The method for preparing a macrophage tracing fluorescent probe according to claim 1, wherein the dialysis at room temperature is performed for 0.5-5 days by using ultrapure water as a dialysis medium and using a 10kD dialysis bag at room temperature.
9. The method for preparing a macrophage tracing fluorescent probe according to claim 1, wherein the pre-freezing at the first temperature is followed by transferring to the second temperature for freezing, and then freeze-drying to obtain a freeze-dried powder: pre-freezing for 2h in a-20 deg.C refrigerator, transferring to a-80 deg.C refrigerator, freezing for 24h, and freeze-drying for 48h in a freeze-drying machine to obtain lyophilized powder.
10. The use of the macrophage tracing fluorescent probe obtained by the method for preparing the macrophage tracing fluorescent probe according to any one of claims 1-9 in targeting of macrophages.
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| WO2021115072A1 (en) * | 2019-12-13 | 2021-06-17 | 深圳先进技术研究院 | Preparation method and application of macrophage tracer fluorescent probe |
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