CN107857788B

CN107857788B - Glycosylated BODIPY derivative, surface sugar modified nano micelle and preparation method and application thereof

Info

Publication number: CN107857788B
Application number: CN201710546240.5A
Authority: CN
Inventors: 张权; 尹健; 蔡颖
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2017-07-06
Filing date: 2017-07-06
Publication date: 2021-04-13
Anticipated expiration: 2037-07-06
Also published as: CN107857788A

Abstract

The invention relates to a glycosylated BODIPY derivative, a surface sugar modified nano micelle, a preparation method and an application thereof, wherein the preparation method of the surface sugar modified nano micelle comprises the following steps: dissolving glycosylated BODIPY derivative in organic solvent to prepare mother liquor, preparing aqueous solution of emulsifier, preparing the above mother liquor and aqueous solution of emulsifier into mixed solution, stirring, fully mixing, standing, dialyzing, preparing said surface sugar modified nano micelle by dialysis freeze-drying method.

Description

Glycosylated BODIPY derivative, surface sugar modified nano micelle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of surface sugar modified nano-micelles, and particularly relates to a glycosylated BODIPY derivative, a surface sugar modified nano-micelle, and a preparation method and application thereof.

Background

Photodynamic therapy (PDT) is a non-invasive technique for treating neoplastic diseases. It needs to have three basic elements of light source, molecular oxygen and photosensitizer. The process is that the light irradiation with specific wavelength makes the photosensitizer absorbed by tissue excited, and the excited photosensitizer transfers energy to ambient oxygen to produce singlet oxygen with strong activity, and the singlet oxygen and adjacent biological macromolecules undergo oxidation reaction to produce cytotoxicity action, so that the cells are damaged and even die. Thus, the phototoxicity of the photosensitizer determines the efficacy of PDT.

The first generation of photosensitizer is mainly hematoporphyrin derivative, and because the absorption of the first generation of photosensitizer in an infrared light region with the wavelength of more than 600nm is weak, the photodynamic reaction depth cannot meet the tumor treatment requirement of deeper infiltration. In addition, it has the disadvantages of insufficient conversion of light into cytotoxic substances, long exposure time and long-lasting skin photosensitization reaction. Some second-generation photosensitizers which enter clinical test stages at present mainly comprise porphyrin derivatives, intermediate substituted aryl porphins, phthalocyanines, chlorophyll a degradation product derivatives, hematoporphyrin monomethyl ether and the like, and have the advantages of single structure, high singlet oxygen yield, strong absorption in an infrared region (the wavelength is 650-800nm) and the like. However, due to the weak tumor targeting property, normal tissues except tumor tissues, such as skin, absorb the photosensitizer, so that the patient still needs to be protected from light for a long time after receiving photodynamic therapy to relieve the toxic and side effects of skin redness and swelling, pigmentation and the like.

Current clinical applications of PDT therapies are mainly limited by poor water solubility of photosensitizers and lack of targeting to tumor cells. For example, in order to improve the tumor targeting of photosensitizer molecules, some effector molecules with biological recognition function, such as monoclonal antibodies, polypeptides, folic acid and the like, are introduced into porphyrin photosensitizer molecules to obtain some coupled photosensitizers. Although the targeting property of the photosensitizer is improved to a certain extent by the effector molecules, the application of the photosensitizer is limited due to weak tissue penetration capability, complex preparation method and possible immunogenicity of the monoclonal antibody; most polypeptide compounds have short half-life in vivo and low bioavailability, and only a few polypeptide receptors have high expression in tumor cells, so the application of the polypeptide targeting photosensitizer is also greatly limited.

The BODIPY derivatives have excellent photophysical properties such as: good chemical and light stability, low photobleaching, high molar extinction coefficient at long wavelengths, and the like. In addition, it contains several reaction sites favorable to chemical modification, and its action wavelength can reach the optimal therapeutic window of PDT through reasonable modification, so that it is one kind of second generation photosensitizer with wide application foreground. Since the BODIPY derivative is an organic molecule and insoluble in water, researchers have modified polyethylene glycol and folic acid to improve its water solubility and targeting to tumor cells (refer to H.He, et. al. journal of medical Chemistry 2011,54, 3097-. However, these derivatives of BODIPY modified by PEG and folic acid still have no high stability in water, and are easy to aggregate and settle, and 1% DMSO needs to be added to increase the stability in water when in use. Because of the strong cytotoxicity of DMSO, the use of these targeted photosensitizers for intravenous and subcutaneous administration is limited.

A glycosylated BODIPY derivative and a preparation method thereof are disclosed in the Chinese patent with the publication number of CN103755753A, glucose is covalently linked to the BODIPY derivative, and a biological activity experiment shows that the tumor targeting property of the photosensitizer is obviously improved after the photosensitizer is modified by the glucose. However, because the glycosylated BODIPY derivative has poor water solubility, 1% DMSO is required to increase the solubility in water when the glycosylated BODIPY derivative is co-cultured with cells, and the DMSO has strong cytotoxicity, so that the targeted photosensitizer is limited to be used for intravenous injection and subcutaneous administration. Therefore, the development of the photodynamic tumor targeting has satisfied effect, better stability in water and very important theoretical and clinical significance for improving the curative effect of the photodynamic tumor therapy.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a glycosylated BODIPY derivative, a surface sugar modified nano micelle, a preparation method and an application thereof.

The invention adopts the following technical scheme:

the glycosylated BODIPY derivative has a chemical structural formula shown as a formula I:

wherein, Z in formula I₁Is a functional fragment of a monosaccharide, disaccharide, oligosaccharide, or oligosaccharide linked to a fluorescent label, Z₂Is a functional fragment of monosaccharide, disaccharide, oligosaccharide, or oligosaccharide connected with a fluorescent marker, and Z is a functional fragment of monosaccharide, disaccharide, oligosaccharide, or oligosaccharide connected with a fluorescent marker;

b is an ester bond (-COO-), an amide bond (-CONR)₁-₂) -), 1, 3-triazole ring

One of (1);

the repeating unit structure containing ethylene glycol in the formula 1, wherein n₁And n₂Equal or unequal numbers of repeating units, with values of 1-4.

Further, said R₁H or CH₃Said R is₂＝H，CH₃Or CH₃CH₂。

Furthermore, the monosaccharide is one of mannose, glucose, galactose or fructose; the disaccharide is one of maltose, sucrose or lactose; the oligosaccharide is one of oligosaccharide fragments or chitosan oligosaccharide containing mannose, glucose, galactose and fructose.

Further, the chemical structural formula is shown as formula II:

the invention provides a surface sugar modified nano micelle, which is prepared from the glycosylated BODIPY derivative and an emulsifier.

Further, the emulsifier is a tween emulsifier or a span emulsifier.

Further, the Tween emulsifier is one of Tween80, Tween20 or Tween60, and the Span emulsifier is one of Span80, Span60 or San 20.

The invention provides a preparation method of a surface sugar modified nano micelle, which comprises the following steps: dissolving the glycosylated BODIPY derivative in an organic solvent to prepare a mother solution for later use, preparing an aqueous solution of an emulsifier in addition, preparing the mother solution and the aqueous solution of the emulsifier into a mixed solution, stirring, fully mixing, standing, dialyzing, and preparing the surface sugar modified nano micelle by a dialysis freeze-drying method.

Further, the specific steps are as follows: dissolving the glycosylated BODIPY derivative in DMF to prepare 1mg/mL mother solution for later use, preparing 0.01-1% emulsifier aqueous solution, preparing the mother solution and the emulsifier aqueous solution into a mixed solution according to the volume ratio of 3/7, stirring, fully mixing, standing, dialyzing, and preparing the surface sugar modified nano micelle by a dialysis freeze-drying method.

The invention provides application of the surface sugar modified nano-micelle in preparation of a photodynamic cancer treatment medicament.

Compared with the prior art, the invention has the beneficial effects that:

1) the particles of the surface sugar modified nano micelle prepared by the invention in a good biological solvent are spherical and have uniform particle size distribution (see figure 1). Under the condition of no illumination, the nano-micelle is respectively incubated with human breast cancer MDA-MB-231 cells and human normal mammary epithelial MCF-10A cells for 24 hours, and the cell survival rate is higher than 90 percent (see figure 2). And the nano micelle can specifically recognize mannose receptors on the cell surface of the breast cancer MDA-MB-231, selectively enters cell lysosomes (shown in figure 3) after being absorbed by the breast cancer MDA-MB-231 cells through the receptor recognition effect, and then under the illumination of specific wavelength, the photosensitive drug is excited to induce the generation of singlet oxygen (shown in figure 4), and kills cells to enable the cells to shrink and damage cell nuclei (shown in figure 5). In addition, the method for preparing the nano-micelle provided by the invention is used for trying to prepare the micelle from the glycosylated BODIPY derivative disclosed in the Chinese patent with the publication number of CN103755753A, and the result of a transmission electron microscope photo shows that the nano-micelle with uniform particle size is not obtained and the aggregation phenomenon is serious (shown in figure 6);

2) the surface sugar-modified nano micelle prepared by the method is spherical in aqueous solution, has uniform particle size distribution and high stability;

3) the traditional photosensitizer medicine has poor water solubility and lacks the targeting property to tumor cells. Therefore, in the light treatment, the active oxygen generated by the photosensitizer is excited to cause great toxic and side effects on normal tissues and cells. The surface sugar-modified nano micelle is formed by co-assembling the glycosylated BODIPY derivative and the emulsifier, can be stably dispersed in an aqueous solution, a cell culture solution or physiological saline, and has good biocompatibility. Under experimental conditions, the micelle and human breast cancer MDA-MB-231 cells are incubated for 24 hours together, and the cell survival rate is higher than 90% in a dark environment;

4) the surface-modified mannose nano-micelle can specifically identify mannose receptors on the surfaces of breast cancer MDA-MB-231 cells, is selectively taken in by the breast cancer MDA-MB-231 cells through the identification effect of the mannose receptors, and further is LED (20 mW/cm) at the specific 665nm wavelength²) Under the illumination, the photosensitizer BTM is excited to induce the generation of active oxygen and inhibit the growth of cancer cells; the nano micelle prepared from the glycosylated BODIPY derivative in the Chinese invention patent CN103755753A is co-cultured with breast cancer MDA-MB-231 cells, so that the amount of the breast cancer cells endocytosed is small, and the LED illumination (665nm, 20 mW/cm)²) No obvious nuclear damage was observed later (see figure 7).

Drawings

FIG. 1 is a transmission electron micrograph of a surface sugar-modified nanomicelle;

FIG. 2 is a graph showing the relative survival rates of breast cancer MDA-MB-231 cells and breast epithelial MCF-10A cells after 24 hours of incubation in the presence of nanomicelles of different concentrations;

fig. 3 is a fluorescence confocal microscope photograph demonstrating that nanomicelle is endocytosed into the lysosome of breast cancer MDA-MB-231 cells by cell surface sugar receptor mediated mode, a: LysoTracker stained lysosomes of cells; b: BTM fluorescence imaging; c: merged images of the first two images;

FIG. 4 is a confocal fluorescence microscope photograph showing LED lamp illumination (20 mW/cm) at a specific 665nm wavelength²) Then, the breast cancer MDA-MB-231 intracellular photosensitizer BTM is excited and induced to generate active oxygen, and the active oxygen in the cells is stained by a green fluorescent probe 2',7' -dichlorofluorescein diacetate; wherein, A: bright field photographs of the cells at the illuminated border; b, fluorescent photograph of the illuminated boundary; c: merged images of the first two images;

FIG. 5 is a fluorescence confocal microscope photograph showing that under the illumination of LED light with a specific 665nm wavelength, the photosensitizer BTM in the breast cancer MDA-MB-231 cell is excited and induced to generate singlet oxygen, and kills the cell to shrink the cell and damage the cell nucleus; wherein, A: before illumination; b: after illumination; c: DAPI stained nuclei prior to illumination; d: DAPI stained nuclei after illumination, with a scale of 20 μm;

FIG. 6 is a transmission electron microscope photograph showing that the glycosylated BODIPY derivative disclosed in the Chinese patent with the publication number of CN103755753A cannot be assembled with the emulsifier Tween80 to form a nano micelle with uniform particle size;

FIG. 7 is a fluorescence confocal microscope photograph showing that after coculture of nano-micelles prepared from the glycosylated BODIPY derivative of CN103755753A of the present invention with MDA-MB-231 cells of breast cancer, the amount of endocytosis of the nano-micelles entering the breast cancer cells is small, and LED illumination (665nm, 20 mW/cm)²) No obvious cell damage was found; wherein, A: DAPI stained nuclei after illumination; b: a photosensitizer fluorescence imaging photo after illumination;

FIG. 8 is a chemical structural formula of BTM prepared by the present invention;

FIG. 9 is a chemical structural formula of BTM1 prepared in accordance with the present invention;

FIG. 10 shows the chemical structure of BTM2 prepared according to the present invention.

Detailed Description

The present invention will be described in further detail with reference to examples.

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. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Abbreviations: DMSO, dimethylformamide; DCM, dichloromethane; DMF, N-dimethylformamide; BTN, azido-substituted BODIPY photosensitizer; BTMn, glycosylated fluoroboron dipyrrole derivative, n represents number; a D-mannose-modified BODIPY photosensitizer; tween X, emulsifier tween, X representing tween serial No.; span X, emulsifier span, X represents span serial number.

Example 1

Will be provided with

(86mg, 394.12. mu. mol) and

(100mg, 78.67. mu. mol) was dissolved in DMSO, bubbled under argon atmosphere for 10min, and ascorbic acid (8.7mg, 43.92. mu. mol) and CuSO were weighed₄·5H₂O (6.5mg, 26.09. mu. mol) was dissolved in deionized water (DMSO: H)₂O15: 1, v/v, 8mL) was dissolved and bubbled for 5 minutes. Then the aqueous solution was added to the reaction system and reacted at 25 ℃ for 48 hours. Terminating the reaction, filtering to remove insoluble substances, adding appropriate amount of saturated water solution of disodium edetate into the filtrate, mixing well, transferring into dialysis bag (MW 1000) for dialysis for 3 days, and changing water every 6 hr. Finally, freeze-drying the clear green solution, purifying by high performance liquid chromatography to obtain pure BTM, and carrying out chemical treatmentThe structural formula is shown in figure 8.¹H NMR(400MHz,DMSO-d₆)δ＝1.47(s,6H),3.36–3.63(m,30H),3.66–3.79(m,10H),3.84(m,5H),4.15(m,6H),4.46–4.59(m,14H),4.67(dd,J＝12.2,2.9,3H),4.73(m,7H),7.07(d,J＝7.3,4H),7.16(d,J＝8.3,2H),7.34(d,J＝8.3,2H),7.44(d,J＝5.3,2H),7.58(d,J＝8.4,4H),8.10(m,5H).ESI-MS:m/z calcd for C₇₈H₁₀₀BF₂I₂N₁₁O₂₇:1925.49；found:1949.31[M+Na]⁺。

Example 2

Will be provided with

(86mg, 394.12. mu. mol) and

(100mg, 78.67. mu. mol) was dissolved in DMSO, bubbled under argon atmosphere for 10min, and ascorbic acid (8.7mg, 43.92. mu. mol) and CuSO were weighed₄·5H₂O (6.5mg, 26.09. mu. mol) was dissolved in deionized water (DMSO: H)₂O15: 1, v/v, 8mL) was dissolved and bubbled for 5 minutes. Then the aqueous solution was added to the reaction system and reacted at 25 ℃ for 48 hours. Terminating the reaction, filtering to remove insoluble substances, adding appropriate amount of saturated water solution of disodium edetate into the filtrate, mixing well, transferring into dialysis bag (MW 1000) for dialysis for 3 days, and changing water every 6 hr. And finally, freeze-drying the clear green solution, and purifying by high performance liquid chromatography to obtain a pure product BTM1, wherein the chemical structural formula is shown in figure 9.¹H NMR(400MHz,DMSO-d₆)δ＝1.47(s,6H),3.36–3.63(m,30H),3.65–3.79(m,10H),3.84(m,5H),4.15(m,6H),4.45–4.59(m,14H),4.67(dd,J＝12.2,2.9,3H),4.73(m,7H),7.07(d,J＝7.3,4H),7.16(d,J＝8.3,2H),7.34(d,J＝8.3,2H),7.44(d,J＝5.3,2H),7.58(d,J＝8.4,4H),8.10(m,5H).ESI-MS:m/z calcd for C₇₈H₁₀₀BF₂I₂N₁₁O₂₇:1925.49；found:1949.20[M+Na]⁺。

Example 3

Will be provided with

(154mg, 405.46. mu. mol) and

(100mg, 78.67. mu. mol) was dissolved in DMSO, bubbled under an argon atmosphere for 10 minutes, and ascorbic acid (8.7mg, 43.92. mu. mol) and CuSO were weighed₄·5H₂O (6.5mg, 26.09. mu. mol) was dissolved in deionized water (DMSO: H)₂O15: 1, v/v, 8mL) was dissolved and bubbled for 5 minutes. Then the aqueous solution was added to the reaction system and reacted at 25 ℃ for 48 hours. The reaction was terminated, insoluble matter was removed by filtration, and DMSO was removed from the filtrate by distillation under the reduced pressure. The clear green solution was finally lyophilized to obtain pure BTM 2. The chemical structural formula is shown in figure 10.¹H NMR(400MHz,DMSO-d₆)1.50(s,6H),3.35–3.80(m,62H),3.92(m,5H),4.10–4.59(m,38H),6.97(d,J＝8.8,4H),7.06(d,J＝8.6,2H),7.16(d,J＝8.6,2H),7.59(m,6H),8.12(m,5H).MALDI-TOF MS:m/z calcd for C₉₆H₁₃₀BF₂I₂N₁₁O₄₂:2412.72；found:2435.36[M+Na]⁺。

Example 4

Mannose was replaced by galactose similarly to example 1.

Example 5

Mannose was replaced by fructose similarly to example 1.

Example 6

Similar to example 3, maltose was replaced by lactose.

Example 7

Maltose was replaced with chitosan oligosaccharide similarly to example 3.

Example 8: preparation of BTM @ Tween80 micelle

Weighing 1mg BTM solid powder, dissolving with 1mL DMF to prepare 1mg/mL mother liquor for later use; and preparing 0.1% Tween80 aqueous solution (volume ratio). Preparing the two solutions into a mixed solution according to a volume ratio of 3/7, stirring for 30min, fully mixing, standing for a moment, transferring the mixed solution into a dialysis bag (MW 15000) for dialysis for 3 days, and changing water every 6 hours. FIG. 1 is a transmission electron micrograph of BTM @ Tween80 micelles.

Example 9: preparation of BTM1@ Tween80 micelle

Weighing 1mg of BTM1 solid powder, dissolving with 1mL of DMF to prepare 1mg/mL of mother solution for later use; a0.01% aqueous solution of Tween80 was prepared (by volume). Preparing the two solutions into a mixed solution according to a volume ratio of 3/7, stirring for 30min, fully mixing, standing for a moment, transferring the mixed solution into a dialysis bag (MW 15000) for dialysis for 3 days, and changing water every 6 hours.

Example 10: preparation of BTM2@ Tween80 micelle

Weighing 1mg of BTM2 solid powder, dissolving with 1mL of DMF to prepare 1mg/mL of mother solution for later use; and preparing 1% Tween80 aqueous solution (volume ratio). Preparing the two solutions into a mixed solution according to a volume ratio of 3/7, stirring for 30min, fully mixing, standing for a moment, transferring the mixed solution into a dialysis bag (MW 15000) for dialysis for 3 days, and changing water every 6 hours.

Example 11: preparation of BTM @ Tween20 micelles

Similar to the preparation method. Weighing 1mg BTM solid powder, dissolving with 1mL DMF to prepare 1mg/mL mother liquor for later use; a0.1% aqueous solution of Tween20 was prepared (by volume). Preparing the two solutions into a mixed solution according to a volume ratio of 3/7, stirring for 30min, fully mixing, standing for a moment, transferring the mixed solution into a dialysis bag (MW 15000) for dialysis for 3d, and changing water every 6 hours. Preparing the BTM micelle powder by a dialysis freeze-drying method.

Example 12: preparation of BTM @ Tween60 micelle

The same procedure was used for the preparation of BTM @ Tween80 as described above. Weighing 1mg BTM solid powder, dissolving with 1mL DMF to prepare 1mg/mL mother liquor for later use; a0.1% aqueous solution of Tween60 was prepared (by volume). Preparing the two solutions into a mixed solution according to a volume ratio of 3/7, stirring for 30min, fully mixing, standing for a moment, transferring the mixed solution into a dialysis bag (MW 15000) for dialysis for 3d, and changing water every 6 hours. Preparing the BTM micelle powder by a dialysis freeze-drying method.

Example 13: preparation of BTM @ Tween X micelle

The same procedure was used for the preparation of BTM @ Tween80 as described above. Weighing 1mg BTM solid powder, dissolving with 1mL DMF to prepare 1mg/mL mother liquor for later use; and preparing 0.1% Tween X (Tween other series) aqueous solution (volume ratio). Preparing the two solutions into a mixed solution according to a volume ratio of 3/7, stirring for 30min, fully mixing, standing for a moment, transferring the mixed solution into a dialysis bag (MW 15000) for dialysis for 3d, and changing water every 6 hours. Preparing the BTM micelle powder by a dialysis freeze-drying method.

Example 14: preparation of BTM1@ Tween X micelle

The same procedure was used for the preparation of BTM1@ Tween80 as described above. Weighing 1mg BTM solid powder, dissolving with 1mL DMF to prepare 1mg/mL mother liquor for later use; 0.01% Tween X (Tween other series) aqueous solution (volume ratio) is also prepared. Preparing the two solutions into a mixed solution according to a volume ratio of 3/7, stirring for 30min, fully mixing, standing for a moment, transferring the mixed solution into a dialysis bag (MW 15000) for dialysis for 3d, and changing water every 6 hours. Preparing the BTM micelle powder by a dialysis freeze-drying method.

Example 15: preparation of BTM2@ Tween X micelle

The same procedure was used for the preparation of BTM2@ Tween80 as described above. Weighing 1mg BTM solid powder, dissolving with 1mL DMF to prepare 1mg/mL mother liquor for later use; and preparing 1% Tween X (Tween other series) aqueous solution (volume ratio). Preparing the two solutions into a mixed solution according to a volume ratio of 3/7, stirring for 30min, fully mixing, standing for a moment, transferring the mixed solution into a dialysis bag (MW 15000) for dialysis for 3d, and changing water every 6 hours. Preparing the BTM micelle powder by a dialysis freeze-drying method.

Example 16: preparation of BTM @ SpanX micelle

The same procedure was used for the preparation of BTM @ Tween80 as described above. The only difference is that the emulsifier is Span80, Span60 or Span 20.

Example 17: TEM characterization of BTM @ Tween80 micelles

The particle size, the hydrodynamic particle size distribution and the long-term stability of the prepared BTM @ Tween80 micelle are characterized. The actual particle size of the BTM micelle was directly observed by a dialysis electron microscope (TEM), and as can be seen from the TEM photograph of FIG. 1, the nanomicelle was rounded, the average particle size was 29nm, and the dispersibility was good.

Example 18: experiment of cytotoxicity of photosensitizer-loaded micelle

The photosensitizer-loaded micelles obtained in example 8 are added into culture solution respectively for cell culture experiments, and then the relative survival rate of cells is determined by using an MTT method, and the specific experimental method can refer to: zhang, y.cai, x. -j.wang, et.al.acs Applied Materials & Interfaces 2016,8, 33405. FIG. 2 shows the toxicity of BTM @ Tween80 micelle on two cells, the micelle is respectively incubated with human breast cancer MDA-MB-231 cells and normal breast cancer MCF-10A cells for 24 hours, and the cell survival rate is higher than 90%.

Similarly, the nano-micelles of the present invention prepared in other examples were verified, and the results showed that the survival rate of cells was high, showing that these nano-micelles were very little or no toxic.

Example 19: experiment of phototoxicity of photosensitizer-loaded cells

The photosensitizer-loaded micelles obtained in example 8 were added to culture solutions, respectively, to perform cell culture experiments. After 24 hours of incubation, 665nm LED lamps (20 mW/cm) were used²) The cells were illuminated for 30 minutes. The relative viability of the cells was then determined by the MTT method. As can be seen, the phototoxicity of the nano-micelle on breast cancer MDA-MB-231 cells is obviously higher than that of normal breast MCF-10A cells.

Similarly, the nanomicelles of the present invention prepared in other examples were confirmed, and the results showed that they all had the above-described properties.

Example 20: experiment for proving that BTM @ Tween80 micelle is endocytosed by breast cancer cells to enter lysosome by fluorescent labeling method

The photosensitizer-loaded micelle obtained in example 8 was added to a culture solution to perform a cell culture experiment, and then lysosomes of cells were stained with a lysosome staining kit. Since the photosensitizer BTM fluoresces red and the lysosome staining reagent fluoresces green, the excitation wavelength was 488/561nm by observing the degree of coincidence between the red fluorescence and the green fluorescence with a confocal laser microscope. Fig. 3 is a confocal photograph of the entry of nanomicelle into the lysosome of the cells, and it can be seen that the nanomicelle is endocytosed by the cancer cells into the lysosome.

Similarly, the nanomicelles of the present invention prepared in other examples were examined and the results showed that they all were endocytosed by breast cancer cells into lysosomes.

Example 21: experiment for generating active oxygen by illumination of nano-micelle endocytosed by breast cancer cell through fluorescent labeling method, the photosensitizer-loaded micelle obtained in example 8 is added into a culture solution for cell culture experiment, and after 24 hours of culture, a 665nm LED lamp (20 mW/cm) is used²) The cells were illuminated for 30 minutes. Then, active oxygen in cells is stained to be green fluorescence by using an active oxygen detection kit (2',7' -dichlorofluorescence yellow diacetate, DCFH-DA), the green fluorescence in the cells is observed by a laser confocal microscope, and the excitation wavelength is 488 nm. FIG. 4 is a confocal photograph of active oxygen detection in breast cancer MDA-MB-231 cells, and it can be seen that a photosensitizer endocytosed into the cancer cells generates a large amount of active oxygen after being irradiated by light.

Similarly, the nanomicelles of the present invention prepared in other examples were examined and the results showed that they all produced intracellular reactive oxygen species.

Example 22: experiment for proving damage of breast cancer cells caused by active oxygen generated by light photosensitizer through fluorescence labeling method

The photosensitizer-loaded micelle obtained in example 8 was added to the culture solution to conduct a cell culture experiment, and after 24 hours of culture, a 665nm LED lamp (20 mW/cm) was used²) The cells were illuminated for 30 minutes. Then, the change of the cell morphology of the breast cancer MDA-MB-231 is observed by a confocal microscope. Fig. 5A is a photograph showing the morphology of cancer cells before light irradiation, and fig. 5B is a photograph showing the morphology of cancer cells after light irradiation. In addition, cells were fixed with formaldehyde and then stained for blue fluorescence with 4', 6-diamidino-2-phenylindole (DAPI). And observing the damage condition of cell nuclei before and after illumination by using a confocal microscope, wherein the excitation wavelength is 405 nm. FIGS. 5C and 5D are LED lamps (20 mW/cm) with cancer cell nuclei at a specific 665nm wavelength²) The confocal photographs before and after the illumination show that the cell nucleus of the cancer cell is obviously damaged by the illumination.

It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A glycosylated BODIPY derivative characterized in that it has a chemical formula as shown in formula II, III or IV:

2. the surface sugar-modified nanomicelle, characterized by being prepared from the glycosylated BODIPY derivative according to claim 1 and an emulsifier.

3. The surface sugar-modified nanomicelle according to claim 2, characterized in that the emulsifier is a tween-based emulsifier or a span-based emulsifier.

4. The surface sugar modified nanomicelle according to claim 3, characterized in that the Tween emulsifier is one of Tween80, Tween20 or Tween60, and the Span emulsifier is one of Span80, Span60 or Span 20.

5. The method for preparing the surface sugar-modified nanomicelle according to claim 2, comprising the steps of: dissolving the glycosylated BODIPY derivative in DMF to prepare 1mg/mL mother solution for later use, preparing 0.01-1% emulsifier aqueous solution, preparing the mother solution and the emulsifier aqueous solution into a mixed solution according to the volume ratio of 3/7, stirring, fully mixing, standing, dialyzing, and preparing the surface sugar modified nano micelle by a dialysis freeze-drying method.

6. Use of the surface sugar-modified nanomicelle according to claim 2 for the preparation of a medicament for the photodynamic treatment of cancer.