Bionic fluorescent probe loaded with indocyanine green as well as preparation method and application of bionic fluorescent probe

Abstract

The invention relates to an indocyanine green-loaded bionic fluorescent probe as well as a preparation method and application thereof. The bionic fluorescent probe disclosed by the invention comprises IZ NPs loaded with indocyanine green and a tumor cell membrane, the tumor cell membrane is coated with IZ NPs (indocyanine green) loaded with the indocyanine green; the indocyanine green is modified in pores of the IZ NPs; and the IZ NPs has a crystal structure of ZIF8. The bionic fluorescent probe provided by the invention has the characteristics of high ICG loading rate and active targeting of tumor lesions, the synthesis method is simple, the tumor targeting efficiency is high, better application of ICG is realized, enrichment of tumor parts is increased while the stability of ICG is improved, and the bionic fluorescent probe can be applied to surgical navigation.

Description

Technical Field

[0001] The present invention relates to the technical field of nanoparticle preparation and application, in particular to a biomimetic fluorescent probe loaded with indocyanine green, a preparation method thereof and an application thereof. Background Art

[0002] As a fluorescent dye approved by the FDA, indocyanine green (ICG) has been widely used in clinical research since 1958 and is considered to be one of the least toxic drugs ever used by humans. The ultraviolet absorption peak of ICG is at 780 nm, and the emission peak is at 800 nm. The absorption and emission at long wavelengths make it very suitable for medical imaging. In addition, ICG can also absorb light energy and convert it into heat energy for tumor photothermal therapy; or as a photosensitizer, absorb light energy and transfer it to surrounding oxygen molecules to generate reactive oxygen species to produce cytotoxicity to kill tumor cells, and this process is called photodynamic therapy. Recent studies have shown that ICG also has sonodynamic therapy effects. In summary, ICG is a good clinical diagnosis and treatment drug.

[0003] However, the actual clinical application of ICG is limited by its disadvantages. First, ICG is easily photodegraded, and light irradiation will accelerate its degradation rate; second, ICG is easily thermally degraded, and the increase in temperature will accelerate its degradation; third, ICG has water instability, and the degradation in water will cause changes in the structure of ICG and change its ultraviolet absorption and fluorescence, and even lead to loss; fourth, the in vivo half-life of ICG is short, which is 2 min - 4 min, severely limiting its application scope; fifth, ICG is easily non-specifically bound to human serum albumin and is thus rapidly metabolized and cleared by the liver. In order to overcome these deficiencies, different preparation strategies are needed to encapsulate, dope or conjugate ICG molecules to better exert the clinical diagnosis and treatment advantages of ICG. Summary of the Invention

[0004] In order to overcome the limitations of ICG in clinical application and further expand the application of ICG in the field of tumor diagnosis and treatment, the present invention provides a biomimetic fluorescent probe loaded with indocyanine green, a preparation method thereof and an application thereof. The biomimetic fluorescent probe provided by the present invention has the characteristics of high ICG loading rate and active targeting of tumor lesions, and the synthesis method is simple, the tumor targeting efficiency is high, realizing better application of ICG, improving its stability while increasing the enrichment at the tumor site, and can be applied to surgical navigation.

[0005] The present invention is realized through the following technical solutions:

[0006] The first object of the present invention is to provide a biomimetic fluorescent probe loaded with indocyanine green, and the biomimetic fluorescent probe includes IZ NPs loaded with indocyanine green and tumor cell membranes; the tumor cell membranes coat the IZ NPs loaded with indocyanine green; the indocyanine green is modified in the pores of the IZ NPs; the IZ NPs have a crystal structure of ZIF8.

[0007] In one embodiment of the present invention, the IZ NPs loaded with indocyanine green are prepared by mixing 2-methylimidazole, indocyanine green and Zn(NO3)2·6H2O in an organic solvent.

[0008] In one embodiment of the present invention, the tumor cell membranes are selected from one or more of HepG2 cell membranes, 4T1 cell membranes and U87 cell membranes.

[0009] In one embodiment of the present invention, the hydrated particle size of the biomimetic nanoparticles is 70nm - 120nm.

[0010] The second object of the present invention is to provide a preparation method of the biomimetic fluorescent probe loaded with indocyanine green, comprising the following steps:

[0011] (1) Mix 2-methylimidazole, indocyanine green and Zn(NO3)2·6H2O in an organic solvent, perform solid-liquid separation and take the solid phase to obtain IZ NPs loaded with indocyanine green;

[0012] (2) Prepare tumor cell membranes;

[0013] (3) Mix the IZ NPs loaded with indocyanine green obtained in step (1) with the tumor cell membranes obtained in step (2), perform ultrasonic treatment under ice bath conditions, and centrifuge to obtain the biomimetic fluorescent probe loaded with indocyanine green.

[0014] In one embodiment of the present invention, in step (1), the organic solvent is selected from dimethyl sulfoxide and / or N,N-dimethylformamide.

[0015] In one embodiment of the present invention, in step (2), the tumor cell membranes are prepared by the following method:

[0016] Digest the tumor cells with trypsin, collect the cells and centrifuge, discard the supernatant, wash the obtained cell precipitate, and then add solution A of the cell membrane protein and cell plasma protein extraction kit containing PMSF protease inhibitor to lyse and break the cells under ice bath conditions. After the breaking is completed, perform low-temperature gradient centrifugation to obtain tumor cell membranes.

[0017] The third object of the present invention is to provide the application of the biomimetic fluorescent probe loaded with indocyanine green in the preparation of anti-tumor drugs.

[0018] In one embodiment of the present invention, the drug is a drug for combined photothermal and photodynamic therapy.

[0019] In one embodiment of the present invention, the laser wavelength for photothermal therapy is 800 nm - 820 nm, preferably 808 nm.

[0020] In one embodiment of the present invention, the dose of the biomimetic fluorescent probe loaded with indocyanine green is less than or equal to 800 μg / mL.

[0021] In one embodiment of the present invention, the tumor is selected from one or more of liver cancer, breast cancer, and glioma.

[0022] The biomimetic fluorescent probe IZM NPs of the present invention are subjected to in vitro and in vivo experiments after preparation for tumor imaging and safety evaluation, and can be used for surgical navigation.

[0023] The above technical solution of the present invention has the following advantages compared with the prior art:

[0024] The present invention provides a biomimetic fluorescent probe (IZM NPs) loaded with indocyanine green, its preparation method and application. The IZM NPs provided by the present invention have the ability to image tumors. Since the surface of the nanoparticles is modified with tumor cell membranes, tumors have specific uptake of them, can target and enrich in tumor sites, and ICG can be excited by near-infrared laser to generate heat energy and ROS, realizing the combined photothermal and photodynamic therapy effect on tumors.

[0025] The IZM NPs prepared by the present invention show excellent targeting at both the cellular and in vivo levels, and are expected to be applied to surgical navigation imaging; they have low toxicity at therapeutic doses and can be tolerated by mice, showing excellent therapeutic effects and are expected to be used for further preclinical studies. Description of the Drawings

[0026] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to specific embodiments of the present invention in combination with the drawings, wherein,

[0027] Figure 1 are the characterization diagrams of IZ NPs and IZM NPs in the present invention; wherein A is the ultraviolet-visible absorption spectrum of IZ NPs; B is the X-ray powder diffraction pattern of IZ NPs; C and D are the transmission electron microscope characterizations of IZ NPs before and after encapsulating cell membranes;

[0028] Figure 2 are the potential and hydrodynamic diameter diagrams of IZM NPs and IZ NPs in the present invention; wherein A is the potential diagram of IZM NPs and IZNPs; B is the hydrodynamic diameter diagram of IZM NPs and IZ NPs;

[0029] Figure 3 It is the specific uptake experimental result of IZM NPs in the present invention;

[0030] Figure 4 It is the experimental result of the IZM NPs producing active oxygen under laser irradiation in the present invention;

[0031] Figure 5 is the temperature rise curve of IZM NPs in the present invention;

[0032] Figure 6 It is a real-time image of the temperature rise of IZM NPs in the present invention;

[0033] Figure 7 is a photothermal stability cycle diagram of IZM NPs in the present invention;

[0034] Figure 8 This is the fluorescence imaging of IZM NPs in the present invention in mice;

[0035] Figure 9 The results of different groups of nanoparticles in the present invention on tumor treatment; A is the tumor weight result; B is the tumor volume result; C is the actual picture of the tumor;

[0036] Figure 10 It is the safety evaluation result of IZM NPs of different concentrations in the present invention. DETAILED DESCRIPTION

[0037] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0038] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0039] Indocyanine green was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0040] Zinc nitrate hexahydrate (Zn(NO3)2·6H2O) was purchased from Sinopharm Chemical Reagent Co., Ltd.;

[0041] Dimethylimidazole (2-MI) was purchased from Beijing Bailingwei Technology Co., Ltd.;

[0042] HepG2 human liver cancer cell line was purchased from Wuhan Pronocell Life Science Technology Co., Ltd.;

[0043] 100× protease inhibitor (PMSF) was purchased from Hangzhou Fude Biotechnology Co., Ltd.;

[0044] The cell membrane protein and cytoplasmic protein extraction kit was purchased from Fuzhou Feijing Biotechnology Co., Ltd.;

[0045] The high-glucose DMEM complete medium was purchased from Shanghai Yuanpei Biotechnology Co., Ltd.;

[0046] The Hoechst33342 staining solution was purchased from Shanghai Beyotime Biotechnology Co., Ltd.;

[0047] The mice were purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

[0048] Example 1:

[0049] This example provides a preparation method of a biomimetic fluorescent probe loaded with indocyanine green, and the specific steps are as follows:

[0050] (1) Weigh 550 mg of 2-MI and 5 mg of ICG in the dark, dissolve them in 4 mL of an aqueous solution containing 30% DMSO, and then weigh 100 mg of Zn(NO3)2·6H2O and dissolve it in 1 mL of an aqueous solution containing 30% DMSO. Then mix the above two solutions at room temperature, stir them in the dark with a magnetic stirrer for 30 min to obtain a green solution. Then centrifuge at a speed of 15000 rpm for 10 min, discard the supernatant, and obtain a dark green precipitate. Wash this precipitate three times with ultrapure water, and then redisperse it in ultrapure water to obtain IZ NPs.

[0051] (2) Cell culture: HepG2 cells were cultured using the high-glucose DMEM complete medium containing 10% fetal bovine serum and 1% penicillin / streptomycin, and the culture conditions were a constant temperature of 37 °C, 5% CO2, and 21% O2. When the cells were cultured to the logarithmic phase, digest the cells with trypsin, collect them and centrifuge at a speed of 1000 rpm for three minutes, then discard the supernatant, resuspend and pipette evenly with the complete medium, and then re-inoculate them into a new culture dish. All the above operations were carried out under sterile conditions.

[0052] Take ten dishes of confluent HepG2 cells, digest them with trypsin, collect the cells and centrifuge them at 1000 rpm for three minutes. Discard the supernatant, wash the obtained cell pellet twice with PBS, and finally obtain the cell pellet. Add 8 mL of Solution A of the cell membrane protein and cytoplasmic protein extraction kit containing 1% PMSF protease inhibitor to the cells in 10 dishes, and lyse the cells on ice bath for 2 h. Subsequently, use an ultrasonic crusher to further break the lysed cells. Set the power of the ultrasonic crusher to 65 W, work for 2 s, pause for 4 s, and the working duration is 6 min to break the cells under ice bath conditions. After the breaking is completed, perform low-temperature gradient centrifugation. First, centrifuge at 7500×g for 10 min to remove cell organelles, and then continue to centrifuge the supernatant at 30000×g for 1 h. Resuspend the obtained pellet with a small amount of PBS to obtain the HepG2 tumor cell membrane. Take a small amount of the cell membrane suspension, lyophilize and quantify it, and store the rest in a -80°C refrigerator for later use.

[0053] (3) Take 0.5 mL of IZ NPs (4 mg / mL) and 1 mL of the cell membrane suspension (2 mg / mL), and ultrasonicate for 5 min under ice bath conditions. Specifically, set the power of the ultrasonic crusher to 60 W, work for 2 s, pause for 4 s, for a total of 5 min. After completion, aspirate the suspension into a centrifuge tube, and under the condition of 4°C, first centrifuge at 7500 rpm for 10 min, take the supernatant and then centrifuge at 15000 rpm for 60 min. Finally, resuspend the nanoparticles with 1 mL of PBS to obtain IZM NPs, and the loading rate of ICG is 8.5%. The results of its successful synthesis are as Figure 1 shown.

[0054] The results show that IZ NPs have the same crystal structure as ZIF8, and after successfully loading ICG, the characteristic ultraviolet absorption peak of ICG appears in the ultraviolet spectrum of IZ NPs. The results of transmission electron microscopy show the successful modification of tumor cell membranes on the nanoparticles, and the changes in the hydrodynamic diameter and surface potential of the nanoparticles before and after modification ( Figure 2 ) can also prove the successful coating of the tumor cell membrane.

[0055] Example 2: Specific uptake experiment of tumor cells HepG2 on IZM NPs:

[0056] 15,000 HepG2 cells were seeded in a confocal dish and cultured at 37 °C for 12 h. Dead and poorly viable cells were washed away with 1×PBS. IZ NPs and IZM NPs with a volume of 400 μL and a concentration of 60 μg / mL were slowly added to the confocal dish and incubated for different times. Subsequently, they were washed with PBS to remove the nanoparticles. Then, the cells were fixed with 4% paraformaldehyde. After 20 min, they were washed with PBS. Finally, the cell nuclei were stained with DAPI staining solution. After 3 min, the staining solution was aspirated, and the cells were washed three times with PBS. Finally, 1 mL of PBS was added for confocal fluorescence microscopy imaging. The results are as Figure 3 shown.

[0057] The results showed that IZM NPs modified with tumor cell membranes exhibited stronger cell uptake than IZ NPs. Moreover, with the increase in time, the cell uptake of IZM NPs could remain stable, demonstrating a more excellent tumor cell targeting ability.

[0058] Example 3: Experiment on the generation of reactive oxygen species (ROS) by IZM NPs under laser irradiation:

[0059] 15,000 HepG2 cells were seeded in a confocal dish and cultured at 37 °C for 12 h. Dead and poorly viable cells were washed away with 1×PBS. IZM NPs with a volume of 400 μL and a concentration of 60 μg / mL were slowly added to the confocal dish and incubated for 8 h to allow HepG2 cells to fully uptake IZM NPs. Subsequently, they were washed with PBS to remove the nanoparticles. Then, an 808 nm laser was used to irradiate the cells at a power density of 1 W / cm 2 for 5 min. Then, DCFH-DA was used to stain the ROS in the cells at room temperature for 20 min. Finally, the cell nuclei were stained with Hoechst33342 staining solution. After 20 min, the staining solution was aspirated, and the cells were washed three times with PBS. 1 mL of PBS was added for confocal fluorescence microscopy imaging. The results are as Figure 4 shown.

[0060] The experimental results showed that compared with the IZM NPs group and the group irradiated with a simple 808 nm laser, the IZM NPs + 808 group generated the most ROS, indicating that IZM NPs have the potential for photodynamic therapy. Pure IZM NPs generated a small amount of ROS due to their material properties, but it was difficult to cause cell killing. However, when combined with 808 nm laser irradiation, three times more ROS than the nanoparticles themselves were generated, showing the potential to kill tumor cells.

[0061] Example 4: In vitro photothermal effect and photothermal stability experiment of IZM NPs:

[0062] To investigate the in vitro photothermal effect and photothermal stability of IZM NPs, IZM NPs with different concentrations were irradiated with an 808 nm laser, and a thermal imager was used to record the heating curve and real-time image of IZM NPs. The specific experimental method is as follows:

[0063] IZM NPs solutions with concentrations of 0 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL were prepared with PBS at pH 7.4, and were irradiated with an 808 nm laser for 10 min at a power density of 1 W / cm 2 . The real-time heating image and temperature were recorded by a thermal imager, and the heating curve was plotted. The results are shown in Figure 5 and Figure 6 . As can be seen from Figure 5 and Figure 6 , IZM NPs have excellent photothermal effects. Prepare IZM NPs with different concentrations of 0 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL. Irradiate IZM NPs at a power density of 1 W / cm 2 , and record the real-time image and temperature within 10 min. After irradiation, the temperatures of IZM NPs with concentrations of 0 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL reached 42.8 °C, 47.1 °C, 53.3 °C, respectively. Compared with the ambient temperature of 28 °C, the temperatures increased by 14.8 °C, 19.1 °C, and 27.3 °C, respectively, showing good photothermal performance.

[0064] Subsequently, to verify the photothermal stability of IZM NPs, IZM NPs were irradiated at a light intensity density of 1 W / cm 2 for 3 min, and then paused for 9 min to allow the solution to cool down to room temperature sufficiently. This was taken as one cycle, and a total of 5 cycles were carried out. The results are shown in Figure 7 . The results show that IZM NPs have good photothermal stability, and the temperature increase is almost the same, about 13.5 °C. The photothermal experiment and stability experiment show that IZM NPs have excellent photothermal performance and have the potential for photothermal therapy as an anti-tumor drug.

[0065] Example 5 In vivo fluorescence imaging experiment:

[0066] First, a tumor animal model was constructed. Logarithmic growth phase HepG2 cells were digested and made into a suspension with a cell concentration of 2×10 7 / mL; after anesthetizing the nude mice and making marks, 100 μL of the cell suspension was aspirated with a 0.5 mL syringe and inoculated with tumors on the left leg, with an injection cell amount of 2×10 6 / mouse.

[0067] Ten days after tumor inoculation, the major axis of the tumor in nude mice was between 1 cm and 1.2 cm, and they were randomly divided into the IZ NPs group and the IZM NPs group. The drugs were administered via the tail vein, and the dose of ICG was 1 mg / Kg. Subsequently, IVIS small animal in vivo imaging was performed at different time points after injection to observe the enrichment of the two kinds of nanoparticles at the tumor site. The results are as Figure 8 shown. It can be seen from Figure 8 that after modifying the tumor cell membrane, the nanoparticles can achieve better enrichment and retention at the tumor site.

[0068] Example 6 Evaluation of therapeutic efficacy:

[0069] Seven days after tumor inoculation, the major axis of the tumor in nude mice was between 4 mm and 8 mm, and they were randomly divided into the PBS group, the IZM NPs group, and the IZM NPs + 808 group. The nanoparticles were injected into the tumor. The injection day was recorded as the 0th day of treatment. The tumor volume and body weight were measured every two days. The treatment results on the 12th day after treatment are as Figure 9 shown. It can be seen from Figure 9 that the combination of IZ NPs and 808 nm laser irradiation has a good anti-tumor effect, and the insignificant change in the body weight of the mice indicates good biosafety.

[0070] Example 7 Safety evaluation:

[0071] Different amounts of nanoparticles were taken and mixed with mouse red blood cells and incubated for 2 h. PBS was set as the negative control and ultrapure water as the positive control. After completion, centrifugation was performed at 3000 rpm at low temperature for 15 min. 100 μL of the supernatant was taken and added to a 96-well plate, and the absorbance at 545 nm was measured. The results are as Figure 10 shown.

[0072] The results show that in the concentration range of 0 - 800 μg / mL, the nanoparticles all showed good biosafety and no hemolysis occurred.

[0073] Obviously, the above examples are only for clearly illustrating the examples and are not limitations on the implementation modes. For those of ordinary skill in the art, other different forms of changes or variations can be made based on the above description. It is not necessary and impossible to list all the implementation modes here. And the obvious changes or variations derived therefrom are still within the protection scope of the present invention.

Claims

1. A biomimetic fluorescent probe loaded with indocyanine green, characterized in that, The bionic fluorescent probe includes indocyanine green-loaded IZ NPs and tumor cell membranes; the tumor cell membranes coat the indocyanine green-loaded IZ NPs; the indocyanine green is modified in the pores of the IZ NPs; the IZ NPs have a crystal structure of ZIF8.

2. The biomimetic fluorescent probe loaded with indocyanine green according to claim 1, wherein The indocyanine green-loaded IZ NPs are prepared by mixing 2-methylimidazole, indocyanine green, and Zn(NO3)2·6H2O in an organic solvent.

3. The biomimetic fluorescent probe loaded with indocyanine green according to claim 1, characterized in that, The tumor cell membranes are selected from one or more of HepG2 cell membranes, 4T1 cell membranes, and U87 cell membranes.

4. The biomimetic fluorescent probe loaded with indocyanine green according to claim 1, characterized in that, The hydrated particle size of the bionic nanoparticles is 70 nm - 120 nm.

5. The preparation method of the indocyanine green-loaded biomimetic fluorescent probe according to any one of claims 1-4, characterized in that, It includes the following steps: (1) Mix 2-methylimidazole, indocyanine green, and Zn(NO3)2·6H2O in an organic solvent, perform solid-liquid separation to obtain the solid phase, and obtain indocyanine green-loaded IZ NPs; (2) Prepare tumor cell membranes; (3) Mix the indocyanine green-loaded IZ NPs obtained in step (1) with the tumor cell membranes obtained in step (2), ultrasonicate under ice bath conditions, and centrifuge to obtain the indocyanine green-loaded bionic fluorescent probe.

6. The preparation method according to claim 5, characterized in that, In step (1), the organic solvent is selected from dimethyl sulfoxide and / or N,N-dimethylformamide.

7. Use of the indocyanine green-loaded bionic fluorescent probe according to any one of claims 1 - 4 in the preparation of a drug for treating tumors.

8. The application according to claim 7, characterized in that, The drug is for combined photothermal and photodynamic therapy.

9. The application according to claim 7, wherein The dosage of the indocyanine green-loaded bionic fluorescent probe is less than or equal to 800 μg / mL.

10. The application according to claim 7, characterized in that The tumors are selected from one or more of liver cancer, breast cancer, and glioma.

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