CN114478581A - Light-treated NIR-II small molecules, compounds and complexes, and preparation method and application thereof - Google Patents

Light-treated NIR-II small molecules, compounds and complexes, and preparation method and application thereof Download PDF

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CN114478581A
CN114478581A CN202210005209.1A CN202210005209A CN114478581A CN 114478581 A CN114478581 A CN 114478581A CN 202210005209 A CN202210005209 A CN 202210005209A CN 114478581 A CN114478581 A CN 114478581A
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CN114478581B (en
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洪学传
肖玉玲
刘奕伸
李芊芊
梁科
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Tibet University
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Abstract

The invention provides a phototherapeutic NIR-II small molecule represented by formula 3:
Figure DDA0003456362280000011
the complex is a brand new compound with the maximum emission wavelength of more than 1000nm, the quantum yield in aqueous solution is 0.42%, the photothermal conversion efficiency is 41.77%, and the complex has good light stability and biocompatibility, is easy to be absorbed and metabolized by organisms, and has obvious cytotoxic effect on tumor cells.

Description

Light-treated NIR-II small molecules, compounds and complexes, and preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedical fluorescence imaging application, in particular to NIR-II small molecules, compounds and complexes for light therapy, and a preparation method and application thereof.
Background
Chemotherapy, one of the most convenient and promising treatments, has successfully increased the survival of cancer patients over the past few years. Since the first application of cisplatin in tumor therapy, there has been a growing effort to explore potential therapeutic applications of transition metal complexes. Among them, organic ruthenium (II/III) complexes, a new generation of anticancer candidate compounds, are attracting attention due to their interesting optical properties and strong interactions with cellular DNA and proteins. However, the course of therapy is greatly hampered by severe systemic toxicity due to natural or acquired resistance and nonspecific distribution. NAMI-A, the first clinically approved ruthenium complex, was found to bind to DNA, RNA and histidine residues of serum albumin, but eventually failed due to its low therapeutic efficacy.
New treatment modalities such as photothermal therapy (PTT) or photodynamic therapy (PDT) have synergistic effects with chemotherapy, improving overall therapeutic efficacy while minimizing side effects. Therefore, ru (ii) polypyridine complexes with a variety of photophysical properties and biological activities are the best candidates to combine these therapeutic modalities. In addition, due to the presence of fluorescence, ru (ii) polypyridine complexes have a variety of photophysical properties and biological activities. Ru (II) polypyridine complex [ Ru (Phen)2dppz]2+And [ (bpy)2Ru(tpphz)Ru(bpy)2]4+Has great potential in visualizing drug delivery and image-guided therapy. However, there areThe emission wavelength of the Ru (II) complex is mainly located in the strongly reflective visible region (390-780 nm). Therefore, it cannot be used for in vivo bioimaging, and the conversion of ru (ii) complexes to therapeutic platforms remains a challenge. In addition, in order to optimize the solubility, pharmacokinetics, toxicity and biodistribution of ru (ii) complexes, nanocarriers including selenium, gold nanomaterials, silica, carbon nanotubes have been used, but the particle size, complexity and poor metabolism and biodegradability of nanosystems have been a major concern.
Therefore, it is important to develop a small molecule with good light stability and photo-thermal conversion efficiency, no toxicity, good biocompatibility, easy absorption and metabolism by organisms, obvious cytotoxic effect on tumor cells, and near-infrared two-region fluorescence emission.
Disclosure of Invention
The present invention is directed to solving, at least to some extent, one of the technical problems of the prior art, and accordingly, in a first aspect of the invention, the present invention provides a phototherapeutic NIR-II small molecule according to formula 3:
Figure BDA0003456362260000021
in a second aspect of the present invention, there is provided a phototherapeutic NIR-II compound having the structure shown in formula 4,
Figure BDA0003456362260000022
wherein R1 and R2 are respectively and independently selected from-NH-PEG-OCH3One of folic acid, iRGD, CREKA and oligopeptide PPSHTPT;
preferably, the structure of the light-treated NIR-II compound is as in formula H7-PEG2KAs shown in the drawings, the above-described,
Figure BDA0003456362260000031
in a third aspect of the present invention, there is provided a phototherapeutic NIR-II complex having the structure shown in formula 5,
Figure BDA0003456362260000032
r1 and R2 are respectively and independently selected from-NH-PEG-OCH3One of folic acid, iRGD, CREKA and oligopeptide PPSHTPT; r3 is selected from one of Ru and Ir; n and a are respectively and independently taken from 1, 2, 3 and 4; m is selected from Cl, SO4(ii) a R3 is selected from one of Ru and Ir; n is selected from 1, 2, 3 and 4; m is selected from Cl, SO4；
Preferably, the structure of the light-treated NIR-II complex is as shown in formula HL-PEG2KAs shown in the drawings, the above-described,
Figure BDA0003456362260000041
in one or more embodiments of the present invention, the phototherapeutic NIR-II complex self-assembles in water to form nanoparticles having an average particle size of 125 to 130nm, preferably 127.15 nm.
In one or more embodiments of the invention, the fluorescence emission wavelength of the phototherapeutic NIR-II complex is 1028 nm.
In a fourth aspect of the invention there is provided the use of a phototherapeutic NIR-II small molecule according to the first aspect of the invention and/or a phototherapeutic NIR-II compound according to the second aspect of the invention and/or a phototherapeutic NIR-II complex according to the third aspect of the invention for near infrared two-zone tumour imaging.
In a fifth aspect, the present invention provides the use of a small phototherapeutic NIR-II molecule according to the first aspect of the present invention and/or a phototherapeutic NIR-II compound according to the second aspect of the present invention and/or a phototherapeutic NIR-II complex according to the third aspect of the present invention in the preparation of an anti-tumour medicament.
In a sixth aspect of the present invention, the present invention provides a method for preparing a phototherapeutic NIR-II small molecule according to the first aspect of the present invention, wherein the phototherapeutic NIR-II small molecule is prepared from compound 1, and the reaction formula for preparing the phototherapeutic NIR-II small molecule from compound 1 is as follows:
Figure BDA0003456362260000051
compound 1 the preparation of said phototherapeutic NIR-II small molecule comprises the following steps:
step 1): taking compound 1, 4, 7-dibromo-5, 6-dinitrodiazosulfide and K2CO3、Pd(PPh3)4Adding the mixture into a reaction container, stirring the mixture in an oil bath at 100-130 ℃ for reaction under the protection of nitrogen, after the reaction is finished, cooling the mixture, diluting the mixture with water, extracting the diluted mixture with EA, and purifying the extracted mixture to obtain a mauve compound 2;
step 2): adding the compound 2 obtained in the step 1) into a mixed solvent of DCM, methanol and water, adding 95 (w/w)% of ammonium chloride and zinc powder, placing the mixture into a reaction vessel, reacting for 1-3 h at 20-30 ℃, filtering, washing a filter cake with DCM, and carrying out rotary drying on the filtrate to obtain a yellow crude product;
step 3): dissolving 1, 10-phenanthroline-5, 6-dione and the yellow crude product obtained in the step 2) in acetic acid, adding the solution into a reaction container, refluxing in an oil bath at the temperature of 110-130 ℃ for 3-6H, cooling, concentrating to obtain a residue, and purifying to obtain a green solid compound, namely H7;
step 4): dissolving H7 in DCM at 0 ℃, slowly adding 1mL TFA, adding the mixture into a reaction container, preserving at room temperature for 30min, and concentrating to obtain a compound 3, namely the light-treated NIR-II micromolecule;
preferably, in the step 1), the compound 1, 4, 7-dibromo-5, 6-dinitrobenzothiadiazole, K2CO3、Pd(PPh3)4In a molar ratio of1:3.2:0.1；
Preferably, in the step 2), the molar ratio of the compound 2 to the ammonium chloride to the zinc powder is 0.028:1: 3.32; the volume ratio of DCM to methanol to water is 1:1: 1.
In a seventh aspect of the present invention, the present invention provides a method for preparing a phototherapeutic NIR-II compound according to the second aspect of the present invention, wherein the phototherapeutic NIR-II compound is prepared from the phototherapeutic NIR-II small molecule of claim 1, the phototherapeutic NIR-II small molecule is prepared by the following reaction formula:
Figure BDA0003456362260000061
preferably, the phototherapeutic NIR-II compound is obtained from a therapeutic NIR-II small molecule according to the first aspect of the invention modifying a polypeptide, protein, polyethylene glycol, aptamer or folate, and derivatives thereof, at a regulatable site;
preferably, the preparation of the phototherapeutic NIR-II compound by the phototherapeutic NIR-II small molecule comprises the steps of: taking-NH-PEG-OCH3Or folic acid, iRGD, CREKA, oligopeptide PPSHTPT, DIPEA and HATU are added into the DMSO solution of the light-treated NIR-II micromolecules, stirred for 5-8 h at the temperature of 20-30 ℃, and purified to obtain the light-treated NIR-II compound;
preferably, -NH-PEG-OCH3Or folic acid or iRGD or CREKA or oligopeptides PPSHTPT, DIPEA and HATU in a molar ratio of 0.028:1: 3.32.
In an eighth aspect of the present invention, there is provided a process for the preparation of a phototherapeutic NIR-II complex according to the third aspect of the present invention, wherein the phototherapeutic NIR-II complex is prepared from a phototherapeutic NIR-II compound according to the second aspect of the present invention, the phototherapeutic NIR-II compound being prepared as follows:
Figure BDA0003456362260000062
the preparation of the phototherapeutic NIR-II complex from the phototherapeutic NIR-II compound comprises the following steps: applying said phototherapeutic NIR-II compound, Ru (bpy)2Cl2Or Ru (II) -bis (4,4'-dimethyl-2,2' -dipyridine) or Ru (phen)2Cl2、Ru(dppz)2Cl2Or cis- [ Ir (2, 2' -dipyridine)2D2]PF6Placing the mixture into a reaction container, adding 40-60 (v/v)% of methanol, refluxing for 8-10 hours at 80-100 ℃, concentrating a reaction solution after the reaction is finished to obtain a residue, and purifying the residue to obtain the light-treated NIR-II complex;
preferably, the phototherapeutic NIR-II compounds are reacted with Ru (bpy)2Cl2Or Ru (II) -bis (4,4'-dimethyl-2,2' -dipyridine) or Ru (phen)2Cl2Or Ru (dppz)2Cl2Or cis- [ Ir (2, 2' -dipyridine)2D2]PF6In a molar ratio of 1: 8.
The invention has the beneficial effects that:
1. the invention provides NIR-II micromolecules, compounds and complexes capable of realizing fluorescence emission above 1000nm and used for cooperative phototherapy, which are brand new compounds with maximum emission wavelength more than 1000nm, have better quantum yield (QY is 0.42%) above 1000nm, are nontoxic, have good biocompatibility and are easy to be absorbed and metabolized by organisms;
2. the invention provides application of the NIR-II small molecules, compounds and complexes for the phototherapy in tumor imaging of a near infrared region II.
3. The invention provides application of the light-treated NIR-II small molecules, compounds and complexes in preparation of antitumor drugs.
4. The invention provides a preparation method of the light-treated NIR-II micromolecules, compounds and complexes, which has the advantages of simple synthetic route, high reaction efficiency, high yield and higher industrial application prospect
Drawings
FIG. 1 shows HL-PEG2KMALDI-TOF-MS characterization of (matrix-assisted laser desorption ionization-time of flight-mass spectrometry);
FIG. 2 shows HL-PEG2KPerforming high performance liquid chromatography characterization;
FIG. 3 shows H7-PEG2KAnd absorbance and fluorescence emission of HL-PEG2K in aqueous solution (10 μ M);
FIG. 4 shows HL-PEG2KAnd ICG photostability in H2O, FBS and PBS;
FIG. 5 shows HL-PEG2KLight stability of (a);
FIG. 6 shows HL-PEG2KCytotoxicity against U87 cells;
FIG. 7 shows HL-PEG2KThe photothermal conversion efficiency of;
FIG. 8 shows HL-PEG2K(iii) apoptosis and cell cycle assays of; wherein (a) AM and PI (scale bar: 200 μ M) staining of U87 MG cells treated with different treatment groups (control, laser irradiation, HL-PEG2K (60 μ M), HL-PEG2K (60 μ M) + laser irradiation and cisplatin (60 μ M)); (b) flow cytometry analysis of control, laser irradiation, HL-PEG2K (60 μ M), HL-PEG2K (60 μ M) treated U87 MG apoptosis; (c) quantitative analysis results of flow cytometry; (d) flow cytometry analysis of the cell cycle of control, laser irradiated, HL-PEG2K (60 μ M), HL-PEG2K (60 μ M) plus laser irradiated and cisplatin (60 μ M) treated U87 MG cells;
FIG. 9 shows HL-PEG2KLive and dead cell staining of U87 cells was treated;
FIG. 10 shows HL-PEG2KDetermination of J-aggregates after treatment of U87 cells;
FIG. 11 shows HL-PEG2KMeasurement of NADH after treatment of U87 cells;
FIG. 12 shows HL-PEG2KImmunoblot analysis after treatment of U87 cells;
FIG. 13 shows HL-PEG2KFluorescence imaging and CPTT in glioma model mice. (a) Lying position (808nm, 90mW cm)-21000LP, 30ms) near infrared images of HL-PEG2K in subcutaneous/orthotopic glioma models and their quantitative signal intensities; (b) HL-PEG2K in supine position (808nm, 90mW cm)-21000LP, 30ms) and its quantitative signal intensity; (c) and (d) representative IR photographs and HL-PEG2K solution at 808nm laser (1W cm)2) In vivo heating profile of U87 MG tumor-bearing mice under irradiation;(e) u87 MG mouse model tumor weight was excised; (f) PBS and laser (1W cm) were used respectively25min), cisplatin (200. mu.L, 2mg/kg Pt) and HL-PEG2K (200. mu.L, 2mg/kg Ru) irradiated with 808nm laser (1W cm2Average weight of mice after 5 min); (g) separately using PBS and laser (1W cm)25min), cisplatin (200. mu.L, 2mg/kg Pt) and HL-PEG2K (200. mu.L, 2mg/kg Ru) irradiated with 808nm laser (1W cm25min) relative tumor volume of mice;
FIG. 14 shows HL-PEG2KThe biocompatibility analysis of (a);
FIG. 15 shows HL-PEG2KFluorescence signals in different pH buffers;
FIG. 16 shows HL-PEG2KFluorescence imaging of major organs in glioma model mice;
FIG. 17 is a photograph of mice during treatment;
FIG. 18 shows HL-PEG collection of mouse blood over a predetermined time2KThe intensity of fluorescence;
FIG. 19 shows HL-PEG2KTEM image and DLS of (a);
FIG. 20 shows HL-PEG2KThe photothermal curve of (a);
FIG. 21 shows IR-26(IR series dyes), H7-PEG2K，HL-PEG2KQuantum yield in dichloromethane.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications. The following examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer, by using conventional methods known in the art without specific descriptions, and by using consumables and reagents which were commercially available without specific descriptions. Unless otherwise defined, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art. In addition, any methods or materials similar or equivalent to those described herein can also be used in the present invention.
Examples 1-3 below are methods for synthesizing light-treated NIR-II small molecules (compound 3), with the following reaction formulas:
Figure BDA0003456362260000091
example 1: synthesis of Compound 2
The preparation method of the compound 2 comprises the following steps:
in the presence of 1, 4, 7-dibromo-5, 6-dinitrobenzothiadiazole (296.7mg, 0.77mmol) and K2CO3(342mg,2.5mmol) in solution, Pd (PPh) was added3)4(89.3mg, 0.077mmol), stirred in an oil bath at 115 ℃ under nitrogen. After the reaction was completed, the mixture was cooled and diluted with water (100mL), extracted with EA (50mL × 3), and further purified by a conventional procedure to obtain the magenta compound 2(160mg, yield 20%).1H NMR(400MHz,CDCl3)δ7.45–7.39(m,4H),7.33(dd,J＝20.2,12.8Hz,5H),7.25–7.09(m,17H),4.24–4.18(m,4H),2.97(t,J＝7.8Hz,4H),2.65(t,J＝7.8Hz,4H),1.06–0.96(m,4H),0.07(s,18H).13C NMR(101MHz,CDCl3)δ173.1,153.2,149.8,146.7,144.8,142.3,137.0,130.2,129.6,129.5,127.8,126.2,125.8,124.3,122.1,120.4,62.8,36.0,30.4,17.3,1.4.
Example 2: synthesis of Compound H7
The preparation method of the compound H7 comprises the following steps:
compound 2(80mg, 0.076mmol) obtained in example 1 was added to 5mL of EDCM and 5mL of methanol, and 95 (w/w)% ammonium chloride (146mg,2.73mmol.) and zinc powder (594mg,9.08mmol.) were added. The mixture was reacted at 25 ℃ for 2h, then filtered, the filter cake was washed with 20ml of lcm and the filtrate was spin dried to give a yellow crude product (80mg) which was used directly in the subsequent synthesis. 1, 10-phenanthroline-5, 6-dione (20mg, 0.09mmol) and the above crude product were dissolved in 5mL of acetic acid, refluxed in an oil bath at 120 ℃ for 4h, then cooled and concentrated under a rotary evaporator to give a residue. The above residue was purified to obtain desired compound H7(35mg, yield 39.3%) as a green solid.1H NMR(400MHz,CDCl3)δ9.23-9.21(m,4H),8.00(d,J＝8.4Hz,4H),7.70(dd,J＝7.7,4.6Hz,2H),7.39-7.29(m,12H),7.28-7.25(m,4H),7.21-7.19(m,4H),7.12(t,J＝6.8Hz,2H),4.20(t,J＝7.8Hz,4H),2.97(t,J＝7.8Hz,4H),2.65(t,J＝7.8Hz,4H),0.99(t,J＝7.8Hz,4H),0.04(s,18H).13C NMR(101MHz,CDCl3)δ173.1,153.0,152.9,149.4,148.3,147.5,145.6,142.1,137.3,136.2,134.4,134.1,129.5,129.4,129.2,128.1,127.9,125.6,125.2,124.6,123.6,121.1,62.8,36.1,30.5,17.3,1.4.
Example 3: synthesis of light-treated NIR-II small molecule compound 3
Compound H7-PEG2KThe preparation method comprises the following steps:
1mL of TFA was added slowly to 2mL of a solution of H7(10mg, 8.54mmol) obtained in example 2 in DCM, and the mixture was stored at room temperature for 30min at 0 ℃. And concentrating the crude product to obtain a compound 3, namely the NIR-II micromolecule for phototherapy.
Example 4 below illustrates the synthesis of a light-treatable NIR-II compound exemplified by H7-PEG2k
The reaction formula is shown as follows:
Figure BDA0003456362260000101
example 4: compound H7-PEG2KSynthesis of (2)
NH was added to a 250. mu.L solution of the phototherapeutic NIR-II small molecules (10mg, 10.3. mu. mol) obtained in example 3 in dimethyl sulfoxide2PEG-OME (61.8mg, 30.9. mu. mol, M.W.2000, Ponsure Biotechnology), DIPEA (13.3mg, 103.2. mu. mol), HATU (39.2mg, 103.2. mu. mol). The above solution was stirred at 25 ℃ for 6h and then purified using Amicon Ultra-0.5ml 3.5K (6 washes). The green compound H7-PEG2k was obtained in 83% yield (42.2 mg). The purity of H7-PEG2k was analyzed using a high performance liquid chromatography column (Thermo Science Hypersil Gold C8).1H NMR(400MHz,CDCl3)δ9.32(d,J＝8.2Hz,2H),9.25(s,2H),8.07(d,J＝8.7Hz,4H),7.79(s,3H),7.42–7.30(m,12H),7.24(m,J＝8.1Hz,6H),7.14(t,J＝6.8Hz,2H),6.59(s,2H),3.66(m,354H),3.40(s,6H),3.04–2.96(t,4H),2.61–2.52(t,4H),2.27–2.20(t,4H).
Example 5 below is with HL-PEG2KSynthesis of NIR-II complexes for illustration of phototherapy
The reaction formula is shown as follows:
Figure BDA0003456362260000111
example 5: HL-PEG2KSynthesis of (2)
HL-PEG2KThe preparation method comprises the following steps:
the compound H7-PEG prepared in example 3 was weighed2K(10mg,2.03μmol)、Ru(bpy)2Cl2(7.8mg, 16.21. mu. mol) in a single-neck flask, adding 2mL of 50 (v/v)% methanol, refluxing at 90 ℃ for 8-10 hours, concentrating to remove the solvent, and purifying the residue by HPLC to obtain HL-PEG2K(5.8mg, yield 53%). FIG. 1 shows HL-PEG2KAnd (3) MALDI-TOF-MS characterization. FIG. 2 shows HL-PEG2KAnd (3) performing high performance liquid chromatography characterization. FIG. 3 shows H7-PEG2KAnd absorbance and fluorescence emission of HL-PEG2K in aqueous solution (10. mu.M). Measurement of HL-PEG2KHas a maximum emission wavelength of 1028nm and a tail of 1400 nm. Therefore, the nano particles have the near-infrared two-region emission characteristic and can be used for imaging research of the near-infrared two-region. FIG. 15 shows HL-PEG2KFluorescence signals in different pH buffers. FIG. 19 shows HL-PEG2KTEM image and DLS of (a); FIG. 20 shows HL-PEG2KThe photothermal curve of (a); FIG. 21 shows IR-26, H7-PEG2K,HL-PEG2KQuantum yield in dichloromethane. HL-PEG2KThe quantum yield in aqueous solution was 0.42%
Example 6
The following experiment is the HL-PEG obtained in example 52KThe light stability test of (2).
HL-PEG2KThe light stability test of (2).
The method comprises the following specific steps:
mixing HL-PEG2kICG (indocyanine green) was dissolved in FBS, H2O, and PBS. Using 808nm laser (180mW cm)2) The irradiation was continued for 60min, the fluorescence intensity was calculated with ImageJ software, and the time-dependent fluorescence intensity of each group was compared.
FIG. 4 shows HL-PEG2KAnd ICG at H2O, FBS graph comparing light stability in PBS. As can be seen, 808nm laser continuously irradiates HL-PEG in different media2KOne hour for the nanoparticles and ICG, and the laser power density is 0.09W/cm2ICG fluorescence signals in PBS, water and fetal calf serum are obviously attenuated, and HL-PEG2KThe fluorescent signal of the nanoparticles remained good. The above results show that the prepared HL-PEG2KHas better light stability than ICG. FIG. 5 shows HL-PEG2KGraph of photostability results.
Example 7
The following experiment is the HL-PEG obtained in example 52KCytotoxicity assay on U87 cells.
HL-PEG2KCytotoxicity assay on U87 cells.
The method comprises the following specific steps:
u87 MG cells were seeded on 96-well plate, cultured in cell incubator for 12h, and then separately cultured with HL-PEG2kAnd replacing the cell culture solution with the cisplatin cell culture solution. After 24 hours, respectively measuring HL-PEG by using a CCK-8 colorimetric method2KHL-PEG under laser irradiation2KViability of cell cultures under cisplatin treatment.
FIG. 6 shows HL-PEG2KHL-PEG under laser irradiation2KComparison of the results of the cytotoxicity test of cisplatin on U87 cells. As can be seen, HL-PEG2KIs significantly less cytotoxic than cisplatin.
Example 8
The following experiment is the HL-PEG obtained in example 52KThe photothermal properties of (a).
HL-PEG2KThe photothermal properties of (a).
The method comprises the following specific steps:
taking HL-PEG2KPlacing 300 μ L sample in EP tube, continuously irradiating with 808nm laser, removing laser source when the temperature reaches constant temperature, and the same process as aboveThe temperature of the sample was recorded with a photo-thermal camera every 30s and a temperature rise and cooling curve was plotted. And simultaneously testing the ultraviolet absorption emission curve of the sample, and recording the absorbance A808 at 808 nm. The photothermal conversion efficiency (η) is calculated by the following equation:
Figure BDA0003456362260000121
hs can be calculated by a formula of hs-mc/tau, m refers to the mass of a test sample, c refers to the specific heat capacity of water, tau is a cooling coefficient and can be obtained by fitting the relation between a cooling curve and temperature, delta Tmax refers to the difference between the highest temperature and the ambient temperature, and Qs is the heat dissipation capacity of pure water, so that the temperature rise of the pure water sample is not obvious, and the temperature rise can be ignored here. A808 is the absorbance of the sample at 808 nm. Substituting the above result into formula to obtain HL-PEG2KThe light-heat conversion efficiency. FIG. 7 shows HL-PEG2KThe light-heat conversion efficiency. As can be seen, HL-PEG2KThe photothermal conversion efficiency was 41.77%.
Example 9
The following experiment is the HL-PEG obtained in example 52KApoptosis and cell cycle assays of (1).
HL-PEG2KApoptosis and cell cycle assays of (1).
The method comprises the following specific steps:
with 60 μ M HL-PEG2kAnd 60 μ M cisplatin in 1W cm under the condition of 808nm laser irradiation or not2Irradiating U87 MG cells for 5min with laser power density, incubating in cell culture box for 12 hr, and adding 60 μ MHL-PEG2kAnd 60 mu M cisplatin in a novel DMEM medium for 24 h. Cells were then harvested and stored in PBS. The cells were stained with Annexin V-FITC/PI apoptosis kit (China Multi science) and PI cell cycle staining kit (China Multi science) respectively, and apoptosis and cell cycle were detected with a Beckman CytExpert flow cytometer. FIG. 8 shows HL-PEG2KApoptosis and cell cycle assays of (1). Wherein (a) different treatment groups (control group, laser irradiation,HL-PEG2KAM and PI (scale bar: 200 μm) staining; (b) flow cytometry analysis of control group, laser irradiation, HL-PEG2K(60μM)、HL-PEG2KApoptosis of (60 μ M) treated U87 MG cells; (c) quantitative analysis results of flow cytometry; (d) flow cytometer analysis control group, laser irradiation and HL-PEG2K(60μM)、HL-PEG2K(60 μ M) cell cycle of U87 MG cells irradiated with laser and treated with cisplatin (60 μ M).
Example 10
The following experiment is the HL-PEG obtained in example 52KLive and dead cell staining experiments after treatment of U87 cells.
HL-PEG2KLive and dead cell staining experiments after treatment of U87 cells.
The method comprises the following specific steps:
cells were washed with assay buffer, treated with 60 μ M HL-PEG2k and 60 μ M cisplatin for 24h, and laser irradiated at 808 nm. The cell cultures of all samples were then partitioned and the cells were further fixed with paraformaldehyde for 10 min. Subsequently, cells were gently washed and stained with 100 μ L of working solution containing calcein-AM/PI kit. After culturing U87 MG cells in a cell culture chamber (37 ℃) for 30min, live/dead cells were detected by an inverted fluorescence microscope (Olympus, USA). FIG. 9 shows the results of different treatment groups (control group, laser irradiation, cisplatin (60. mu.M), HL-PEG2K(60. mu.M) and HL-PEG2KGraph comparing the staining results of viable and dead cells after (60. mu.M) + laser irradiation) treatment of U87 cells.
Example 11
The following experiment is the HL-PEG obtained in example 52KMitochondrial membrane potential measurements after treatment of U87 cells.
HL-PEG2KMitochondrial membrane potential measurements after treatment of U87 cells.
The method comprises the following specific steps:
u87 MG cells were treated with 60. mu.M HL-PEG, respectively2kAnd 60 μ M cisplatin for 24h, followed by irradiating with 808nm laser at power density of 1W/cm2 for 5min, respectively, collecting cell samples, washing with PBS, and dark-treating with JC-1 for 0.5 h. Subsequently, the process of the present invention,the intensity of green fluorescence representing JC-1 monomer and red fluorescence representing J-aggregate in the above cell samples was measured using a spark fluorescence microplate reader (TECAN). Matrix Metalloproteases (MMPs) were further obtained by calculating the rate of decrease in red/green fluorescence intensity of each group. FIG. 10 shows the results of different treatment groups (control group, laser irradiation, cisplatin (60. mu.M), HL-PEG2K(60. mu.M) and HL-PEG2KGraph comparing the results of the measurement of J-aggregates after treatment of U87 cells (60. mu.M) + laser irradiation).
Example 12
The following experiment is the HL-PEG obtained in example 52KIntracellular NADH determination after treatment of U87 cells.
HL-PEG2KIntracellular NADH determination after treatment of U87 cells.
The method comprises the following specific steps:
u87 MG cells were treated with 60. mu.M HL-PEG2k and 60. mu.M cisplatin for 24h, irradiated with laser at 808nm or less and at a power density of 1W cm-2 for 5min, and then injected with an equal amount of NAD +/NADH detection reagent (Beyotime, China) in the cell lysate, respectively. Storing at 25 deg.C for 10min, and measuring fluorescence intensity with spark fluorescence microplate reader. FIG. 11 shows the results of different treatment groups (control group, laser irradiation, cisplatin (60. mu.M), HL-PEG2K(60. mu.M) and HL-PEG2KGraph showing the results of NADH measurement comparison after (60. mu.M) + laser irradiation) treatment of U87 cells.
Example 13
The following experiment was conducted for HL-PEG obtained in example 52KImmunoblot analysis after treatment of U87 cells.
HL-PEG2KImmunoblot analysis after treatment of U87 cells.
The method comprises the following specific steps:
each set of cell cultures was washed 3 times with cold PBS and then 100. mu.l of lysis buffer was added. The lysates were incubated in ice for half an hour, centrifuged at 12000 Xg for 25min to extract the protein, and their content was determined by BCA method. The protein sample is then loaded onto a gel for separation. The gel was then transferred to a filter, which was finally blocked with 5% skim milk and incubated with the following antibodies: beta-actin (1: 10000, protein technologies, China), BAX (1: 10000, protein technologies, China), Bcl-2 (1: 2500, protein technologies, China), c-caspase-3 (1: 1000, USA) and corresponding secondary antibody (1: 10000, protein technologies, China). The film was then treated with enhanced ECL reagent and further developed after rinsing. FIG. 12 is an immunoblot analysis of U87 cells treated with different treatment groups (control, laser irradiation, HL-PEG2K (60 μ M), HL-PEG2K (60 μ M) + laser irradiation and cisplatin (60 μ M)).
Example 14
The following experiment is the HL-PEG obtained in example 52KFluorescence imaging in mice.
HL-PEG2KFluorescence imaging in mice.
The method comprises the following specific steps:
u87 MG tumor model mouse is anesthetized with pentobarbital sodium solution (50MG/kg), fixed under laser irradiation after anesthesia, and injected with HL-PEG into tail vein2KProbe (200. mu.L, 2mg/kg Ru) with laser (90mW cm) at 808nm2) NIR-II imaging was performed with different wavelength filters (1000nm and 1250nm) in prone position at predetermined time points under irradiation. FIG. 13 shows HL-PEG2KFluorescence imaging and CPTT in glioma model mice. Wherein, (a) near infrared images of HL-PEG2K in the decubitus (808nm, 90mW cm-2,1000LP, 30ms) in the subcutaneous/orthotopic glioma model and their quantitative signal intensities; (b) HL-PEG2K in supine position (808nm, 90mW cm)-21000LP, 30ms) and its quantitative signal intensity; (c) and (d) representative IR photograph and HL-PEG2K solution at 808nm laser (1W cm)2) In vivo heating profile under irradiation for U87 MG tumor-bearing mice.
Example 15
The following experiment is the HL-PEG obtained in example 52KCPTT under live NIR-II image guidance.
HL-PEG2KCPTT under live NIR-II image guidance. FIG. 13(e) U87 MG mouse model tumor weight resection; (f) PBS and laser (1W cm) were used respectively25min), cisplatin (200. mu.L, 2mg/kg Pt) and HL-PEG2K (200. mu.L, 2mg/kg Ru) irradiated with 808nm laser (1W cm2Average weight of mice after 5 min); (g) are respectively provided withUsing PBS, laser (1W cm)25min), cisplatin (200. mu.L, 2mg/kg Pt) and HL-PEG2K (200. mu.L, 2mg/kg Ru) irradiated with 808nm laser (1W cm25min) relative tumor volume of mice. Figure 17 is a photograph of mice during treatment. FIG. 16 shows HL-PEG2KFluorescence imaging of major organs in glioma model mice; FIG. 18 shows HL-PEG collection of mouse blood over a predetermined time2KFluorescence intensity.
The method comprises the following specific steps:
u87 MG mice were randomly divided into 5 groups (n ═ 4): (A) control group, (B)808nm laser group, (C) cisplatin injection group (200. mu.L, 2mg/kg Pt), (D) HL-PEG2kInjection group (200. mu.L, 2mg/kg Ru), (E) HL-PEG2k injection group (200. mu.L, 2mg/kg Ru), given 808nm laser irradiation. (B) Group (E) has a power density of 1W/cm for 12h after injection2The 808nm laser continuously irradiates the tumor for 5 min. The length and width of the mouse tumor was measured with digital calipers every 4d throughout the treatment period (22 d). And the tumor volume (mm3) was calculated according to the following formula. Tumor volume is length x width2/2. Organs were isolated from the tumor model, fixed with tissue fixative and analyzed by HE staining.
FIG. 14 shows HL-PEG2KThe biocompatibility analysis of (2). Acute toxicity tests were performed by intravenous injection. PBS (200. mu.L), HL-PEG were administered separately2K(200. mu.L, 2mg/kg Ru), cisplatin (200. mu.L, 2mg/kg Pt), 3 per group. Mice were sacrificed after 7d of treatment and HE staining was observed for histopathological changes. PBS or HL-PEG2kNo obvious inflammatory lesions or injuries were observed in the group, while renal tubular epithelial cell edema, cytoplasma loosening or vacuolization was observed in the cisplatin group. In addition, biochemical markers were also analyzed to assess liver and kidney toxicity. ALP, ALT, AST and T-Bil in each group have no significant difference, but BUN (reflecting the sensitive index of renal insufficiency) in the cis-platinum group is reduced by about 6.49 times compared with that in the control group, which indicates that cisplatin causes severe renal insufficiency. And HLPEG2kThe treatment group has no abnormal index. ICR mice treated 3d after injection with 1mg/kg Ru and 2mg/kg Ru, respectively, had 3 weights per group, and recorded every 2d, with no statistical difference (P) from the control group>0.05). Studies have shown that HL-PEG2K has non-negligible toxicity and good pharmacokineticsAnd (4) the chemical property.
Although the embodiments of the present invention have been shown and described, it is understood that the above embodiments are illustrative and not restrictive, and that those skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention and that they should be included in the protection scope of the present invention.

Claims (10)

1. A phototherapeutic NIR-II small molecule according to formula 3:
Figure FDA0003456362250000011
2. a phototherapeutic NIR-II compound having the structure shown in formula 4,
Figure FDA0003456362250000012
wherein R1 and R2 are respectively and independently selected from-NH-PEG-OCH3One of folic acid, iRGD, CREKA and oligopeptide PPSHTPT;
preferably, the structure of the light-treated NIR-II compound is as in formula H7-PEG2KAs shown in the drawings, the above-described,
Figure FDA0003456362250000021
3. a phototherapeutic NIR-II complex, wherein the structure of the phototherapeutic NIR-II complex is shown as formula 5,
Figure FDA0003456362250000022
r1 and R2 are respectively and independently selected from-NH-PEG-OCH3One of folic acid, iRGD, CREKA and oligopeptide PPSHTPT; r3 is selected from one of Ru and Ir; n and a are respectively and independently taken from 1, 2, 3 and 4; m is selected from Cl, SO4；
Preferably, the structure of the light-treated NIR-II complex is as shown in formula HL-PEG2KAs shown in the drawings, the above-described,
Figure FDA0003456362250000031
4. the phototherapeutic NIR-II complex of claim 3, wherein the phototherapeutic NIR-II complex self-assembles in water to form nanoparticles having an average particle size of 125 to 130 nm.
5. A phototherapeutic NIR-II complex according to claim 3, wherein the fluorescence emission wavelength of the phototherapeutic NIR-II complex is 1028 nm.
6. Use of a phototherapeutic NIR-II small molecule according to claim 1 and/or a phototherapeutic NIR-II compound according to claim 2 and/or a phototherapeutic NIR-II complex according to claim 3 for near infrared two-zone tumor imaging.
7. Use of a phototherapeutic NIR-II small molecule according to claim 1 and/or a phototherapeutic NIR-II compound according to claim 2 and/or a phototherapeutic NIR-II complex according to claim 3 for the preparation of an anti-tumor medicament.
8. A method for preparing a small phototherapeutic NIR-II molecule of claim 1, wherein said small phototherapeutic NIR-II molecule is prepared from compound 1, and the reaction formula for preparing said small phototherapeutic NIR-II molecule from compound 1 is as follows:
Figure FDA0003456362250000041
compound 1 preparation of the phototherapeutic NIR-II small molecule comprises the following steps:
step 1): taking the compounds 1, 4, 7-dibromo-5, 6-dinitrobenzothiadiazole and K2CO3、Pd(PPh3)4Adding the mixture into a reaction container, stirring the mixture in an oil bath at 100-130 ℃ for reaction under the protection of nitrogen, after the reaction is finished, cooling the mixture, diluting the mixture with water, extracting the diluted mixture with EA, and purifying the extracted mixture to obtain a mauve compound 2;
step 2): adding the compound 2 obtained in the step 1) into a mixed solvent of DCM, methanol and water, adding 95 (w/w)% of ammonium chloride and zinc powder, placing the mixture into a reaction vessel, reacting for 1-3 h at 20-30 ℃, filtering, washing a filter cake with DCM, and carrying out rotary drying on the filtrate to obtain a yellow crude product;
step 3): dissolving 1, 10-phenanthroline-5, 6-dione and the yellow crude product obtained in the step 2) in acetic acid, adding the solution into a reaction container, refluxing in an oil bath at the temperature of 110-130 ℃ for 3-6H, cooling, concentrating to obtain a residue, and purifying to obtain a green solid compound, namely H7;
step 4): dissolving H7 in DCM at 0 ℃, slowly adding 1mL TFA, adding the mixture into a reaction container, preserving at room temperature for 30min, and concentrating to obtain a compound 3, namely the light-treated NIR-II micromolecule;
preferably, in the step 1), the compound 1, 4, 7-dibromo-5, 6-dinitrobenzothiadiazole, K2CO3、Pd(PPh3)4In a molar ratio of 1:3.2: 0.1;
preferably, in the step 2), the molar ratio of the compound 2 to the ammonium chloride to the zinc powder is 0.028:1: 3.32; the volume ratio of DCM to methanol to water is 1:1: 1.
9. A method of preparing a phototherapeutic NIR-II compound of claim 2, wherein the phototherapeutic NIR-II compound is prepared from the phototherapeutic NIR-II small molecule of claim 1, wherein the phototherapeutic NIR-II small molecule is prepared by the reaction formula:
Figure FDA0003456362250000051
preferably, the phototherapeutic NIR-II compound is obtained from a therapeutic NIR-II small molecule of claim 1 modifying a polypeptide, protein, polyethylene glycol, aptamer or folate and derivatives thereof at a regulatable site;
preferably, the preparation of the phototherapeutic NIR-II compound by the phototherapeutic NIR-II small molecule comprises the steps of: taking NH2Adding PEG-OME or folic acid or iRGD or CREKA or oligopeptide PPSHTPT, DIPEA and HATU into the DMSO solution of the light-treated NIR-II micromolecule, stirring for 5-8 h at 20-30 ℃, and purifying to obtain the light-treated NIR-II compound;
preferably, NH2The molar ratio of-PEG-OME or folic acid or iRGD or CREKA or oligopeptide PPSHTPT, DIPEA and HATU is 0.028:1: 3.32.
10. A method of preparing a phototherapeutic NIR-II complex of claim 3, wherein the phototherapeutic NIR-II complex is prepared from the phototherapeutic NIR-II compound of claim 2, wherein the phototherapeutic NIR-II compound is prepared as follows:
Figure FDA0003456362250000052
the preparation of the phototherapeutic NIR-II complex from the phototherapeutic NIR-II compound comprises the following steps: applying said phototherapeutic NIR-II compound, Ru (bpy)2Cl2Or Ru (II) -bis (4,4'-dimethyl-2,2' -dipyridine) or Ru (phen)2Cl2、Ru(dppz)2Cl2Or cis- [ Ir (2, 2' -dipyridine)2D2]PF6Placing the mixture into a reaction container, adding 40-60 (v/v)% of methanol, refluxing for 8-10 hours at 80-100 ℃, concentrating a reaction solution after the reaction is finished to obtain a residue, and purifying the residue to obtain the light-treated NIR-II complex;
preferably, the phototherapeutic NIR-II compounds are reacted with Ru (bpy)2Cl2Or Ru (II) -bis (4,4'-dimethyl-2,2' -dipyridine) or Ru (phen)2Cl2、Ru(dppz)2Cl2Or cis- [ Ir (2, 2' -dipyridine)2D2]PF6In a molar ratio of 1: 8.
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