CN111777616A - Porphyrin derivative capable of detecting hyaluronidase based on self-assembly, preparation method and application thereof - Google Patents
Porphyrin derivative capable of detecting hyaluronidase based on self-assembly, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a porphyrin derivative capable of detecting hyaluronidase based on self-assembly, a preparation method and application thereof, wherein the preparation method of the porphyrin derivative is completed by the following steps: the porphyrin parent is firstly reacted with dibromoalkane, and the obtained product is then reacted with triethylamine to obtain the porphyrin derivative. The porphyrin derivative and the hyaluronic acid chain with negative charges form a novel fluorescent nano sensor by a static self-assembly method. Compared with the existing detection technology, the fluorescence nano-sensor obtained by the invention has good biocompatibility, simple preparation method and high selectivity, can effectively subtract biological background fluorescence when the emission wavelength is in the near infrared region, has good industrial development prospect, and has huge application prospect in the technical fields of analytical chemistry, life science and the like.
Description
Technical Field
The invention belongs to the field of chemical materials and analysis and detection, and particularly relates to a preparation method and application of a porphyrin derivative for detecting hyaluronidase.
Background
Hyaluronic Acid (HA) contains multiple repeating glucuronic acid and N-acetylglucosamine disaccharide units, is negatively charged, HAs good water solubility, and is a main component constituting extracellular matrix (ECM) and intercellular matrix (ICM). Hyaluronidase (HAase) is a specific hydrolase of HA, is an endoglycosidase, can directly hydrolyze beta-1, 4 glycosidic bonds of hyaluronic acid, and the final product is mainly a metabolite of tetrasaccharide, and is used as an auxiliary drug for chemotherapy for many years to enhance the permeability of drugs. HAase has been reported to be involved in many physiological and pathological processes, and is highly expressed in malignant tumors such as bladder cancer, prostate cancer, brain cancer, and rectal cancer. Therefore, HAase has attracted extensive attention as a novel tumor marker, and the design and synthesis of a sensor capable of detecting HAase is of great significance for clinical diagnosis and treatment of early cancers.
The detection method of HAase in the prior art mainly comprises a turbidity method, a viscosity method, an zymogram method, an immunoassay method, a colorimetric method, a chemiluminescence auxiliary method and a fluorescence sensing method. Among them, the fluorescence sensing method is widely used because of its advantages such as simple operation, low loss, and high sensitivity. The fluorescence sensing method mainly depends on the mark of a fluorescence sensor to detect HAase, and the traditional fluorescence sensor is mainly divided into an organic small-molecule fluorescence sensor and a fluorescence nano sensor, wherein most of the organic small-molecule fluorescence sensors are poor in water solubility, and a large amount of organic solvent is required to be used as a cosolvent in the using process, so that the organic small-molecule fluorescence sensor is high in toxicity and poor in light stability; the fluorescence nanosensor has the defects of poor interference resistance although the fluorescence nanosensor has better water dispersibility, biocompatibility and light stability. Most fluorescence sensors in the prior art tend to cause high biological background fluorescence interference due to absorption and emission in the uv-vis range, resulting in a low signal-to-noise ratio. Therefore, the development of organic molecules and corresponding fluorescence sensors which can effectively overcome the defects of the traditional fluorescence sensing method has important practical significance and application prospect.
Disclosure of Invention
In order to solve the defects in the prior art, the invention aims to provide a porphyrin derivative capable of detecting hyaluronidase based on self-assembly and a preparation method thereof, wherein the porphyrin derivative is used as a fluorescent chromophore of a fluorescent sensor to solve the problem of biological background fluorescence interference; the invention also relates to an application of the porphyrin derivative based on self-assembly and capable of detecting hyaluronidase, and the porphyrin derivative and the negatively charged hyaluronidase chain are prepared into a fluorescent nano sensor so as to achieve the purpose of quickly and effectively identifying hyaluronidase.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
the invention relates to a porphyrin derivative capable of detecting hyaluronidase based on self-assembly, which has a general formula as follows:
wherein R1, R2 and R3 are respectively H or O (CH)2)nN(CH2CH3)3 +(ii) a The O (CH)2)nN(CH2CH3)3 +N =2~ 24.
A preparation method of porphyrin derivative based on self-assembly and capable of detecting hyaluronidase comprises the following steps in sequence:
adding SM1 into dry N, N-dimethylformamide, starting stirring, adding anhydrous potassium carbonate, stirring at room temperature, adding SM2 after stirring and mixing uniformly, refluxing and stirring for 24-48 h under the protection of inactive gas, detecting by thin layer chromatography, cooling to room temperature after reaction, washing with water to remove the N, N-dimethylformamide, purifying the obtained solid material by column chromatography to obtain SM3, wherein the chemical reaction equation is as follows:
wherein, X1，X2，X3Are respectively H or OH; at X1=X2=X3When = H, A1=A2=A3= H; at X1=X2=H，X3When OH is not zero, A1=A2=H，A3=O（CH2）nBr; at X1=H，X2=X3When OH is not zero, A1=H，A2=A3=O（CH2）nBr at X1=X2=X3When OH is not zero, A1=A2=A3=O（CH2）nBr; the O (CH)2）nN = 2-24 in Br;
adding SM3 into dry N, N-dimethylformamide, adding excessive triethylamine, refluxing and stirring for 10-20 hours under the protection of inactive gas, monitoring the reaction by thin-layer chromatography, cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4, namely the porphyrin derivative, wherein the reaction equation is as follows:
wherein A is1，A2，A3Are each H or O (CH)2）nBr; in A1=A2=A3When H, R1=R2=R3= H; in A1=A2=H，A3= O（CH2）nBr is, R1=R2=H，R3= O(CH2)nN(CH2CH3)3 +(ii) a In A1=H，A2=A3=O（CH2）nBr is, R1=H，R2=R3= O(CH2)nN(CH2CH3)3 +(ii) a In A1=A2=A3= O（CH2）nBr is, R1=R2=R3= O(CH2)nN(CH2CH3)3 +(ii) a The O (CH)2）nBr and O (CH)2)nN(CH2CH3)3 +N =2~ 24.
As a limitation to the preparation method of the invention, the reflux temperature is 110-140 ℃.
As a further limitation to the above preparation method of the present invention, the SM 1: SM 2: the molar ratio of the anhydrous potassium carbonate is 1: 15-25: 15-30.
The thin-layer chromatography liquid is a mixed liquid of petroleum ether and dichloromethane, wherein the volume ratio of the petroleum ether to the dichloromethane is 1-10: 1; the column chromatography eluent is a mixed solution of petroleum ether and dichloromethane, wherein the volume ratio of the petroleum ether to the dichloromethane is 1-5: 1.
As another limitation to the preparation method of the invention, the molar ratio of the SM3 to the triethylamine is 1: 20-40.
The invention also provides an application of the porphyrin derivative based on self-assembly and capable of detecting hyaluronidase, and the porphyrin derivative and the hyaluronan chain are mixed to prepare the fluorescent nano-device capable of detecting hyaluronidase.
As a limitation of the invention relating to the use of porphyrin derivatives, the hyaluronic acid chain is
Wherein m =100~ 1000.
As another limitation of the application of the porphyrin derivative, the mass ratio of the porphyrin derivative to the hyaluronic acid chain is 2-6: 1.
Due to the adoption of the technical scheme, compared with the prior art, the porphyrin derivative and the corresponding fluorescence sensor have the following beneficial effects:
(1) according to the invention, a functional hydroxyl porphyrin matrix is synthesized by triethyl quaternary ammonium salt to obtain a porphyrin derivative (Mito TPP) with positive charge as a near infrared fluorescence chromophore, so that a sensor is targeted by mitochondria;
(2) the invention is a fluorescence nano-sensor which is formed by self-assembling Mito TPP with positive charges and hyaluronic acid chain with negative charges, the hyaluronic acid further increases the biocompatibility of the nano-sensor and reduces the adverse reaction generated after the fluorescence nano-sensor is injected into the body;
(3) compared with the traditional organic small molecule fluorescence sensor, the fluorescence nano sensor prepared by the porphyrin derivative provided by the invention has the advantages that the water solubility of the sensor is improved, so that the use of organic solvents is reduced, and the toxicity of the sensor is reduced;
(4) the emission spectrum of the porphyrin derivative is near 650nm, and compared with the traditional fluorescent nano sensor, the emission spectrum of the porphyrin derivative can effectively solve the problem of interference generated in the detection process of biological background fluorescence on hyaluronidase dissolved in water.
In conclusion, the invention provides a porphyrin derivative capable of detecting hyaluronidase based on self-assembly, and a preparation method and application thereof. The synthesis route of the porphyrin derivative is simple, the whole process is easy to operate, the steps are short, and the cost investment is low; the prepared fluorescent nano sensor has excellent water solubility and biocompatibility, weak biological background fluorescence and low cytotoxicity, and can directly realize specific recognition on hyaluronidase.
The method is suitable for detecting hyaluronidase and is used for specifically targeting the hyaluronic acid receptor overexpressed on the surface of the cancer cell.
Drawings
The invention is described in further detail below with reference to the figures and the embodiments.
FIG. 1 is a schematic diagram of hyaluronidase recognition by the prepared fluorescent nanosensor;
FIG. 2 is a nuclear magnetic data plot of 5- (4- (triethylamine) -butyloxyphenyl) -10,15, 20-triphenylporphyrin;
FIG. 3 is a particle size distribution diagram of a fluorescent nanosensor;
FIG. 4 is a graph showing the change of fluorescence emission spectrum of the fluorescent nanosensor with the addition time of hyaluronic acid (excitation wavelength: 425 nm);
FIG. 5 is a graph showing the change of fluorescence emission spectra of fluorescence nanosensors when HAase was added at different concentrations;
FIG. 6 is a graph of a fitted curve corresponding to the change in the fluorescence intensity value with the change in the hyaluronidase concentration and a function corresponding to the curve;
FIG. 7 is a graph of interference contrast data for fluorescence recovery of various ions on a fluorescent nanosensor;
FIG. 8 is a graph of interference contrast data for various ions on the fluorescence intensity of a fluorescent nanosensor;
FIG. 9 is a graph of HeLa cell viability data for fluorescent nanoprobes of different concentrations.
Detailed Description
Preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the description of the preferred embodiment is only for purposes of illustration and understanding, and is not intended to limit the invention.
EXAMPLE 1 porphyrin derivative and preparation method thereof
A porphyrin derivative has a molecular formula of
The preparation method comprises
Firstly, adding 0.317 mmol of SM1-1 into 8ml of dry N, N-Dimethylformamide (DMF), starting stirring, adding 4.76 mmol of anhydrous potassium carbonate, stirring at room temperature, adding 4.76 mmol of SM2-1 after stirring and mixing uniformly, refluxing and stirring at 140 ℃ for 24h under the protection of nitrogen, detecting by thin layer chromatography (petroleum ether PE: dichloromethane DCM =10: 1), cooling to room temperature after the reaction is finished, washing with water to remove DMF, purifying the obtained solid material by column chromatography (petroleum ether PE: dichloromethane DCM =5: 1) to obtain SM3-1, wherein the chemical reaction equation is as follows:
secondly, adding 0.131 mmol of SM3-1 into 4ml of dry DMF, adding 5.24mmol of triethylamine, refluxing and stirring at 110 ℃ for 20h under the protection of argon, monitoring the reaction by thin layer chromatography (PE: DCM =1: 1), cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4-1, wherein the reaction equation is as follows:
examples 2-7 porphyrin derivatives and preparation methods thereof
The experimental procedures and experimental conditions in the preparation methods of examples 2 to 7 were the same as those in example 1, except that the raw materials used were the same
Wherein n =2,5,9,11,17,24, the final porphyrin derivative has the formula
Example 8A porphyrin derivative and a method for preparing the same
A porphyrin derivative has a molecular formula of
The preparation method comprises
Firstly, adding 0.317 mmol of SM1-2 into 8ml of dry N, N-Dimethylformamide (DMF), starting stirring, adding 6.02 mmol of anhydrous potassium carbonate, stirring at room temperature, adding 5.39 mmol of SM2-2 after stirring and mixing uniformly, refluxing and stirring at 130 ℃ for 30h under the protection of nitrogen, detecting by thin layer chromatography (petroleum ether PE: dichloromethane DCM =8: 1), cooling to room temperature after the reaction is finished, washing with water to remove DMF, purifying the obtained solid material by column chromatography (petroleum ether PE: dichloromethane DCM =4: 1) to obtain SM3-2, wherein the chemical reaction equation is as follows:
secondly, adding 0.131 mmol of SM3-2 into 4ml of dry DMF, adding 4.72mmol of triethylamine, refluxing and stirring for 17h at 125 ℃ under the protection of argon, monitoring the reaction by thin layer chromatography (PE: DCM =2: 1), cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4-2, wherein the reaction equation is as follows:
examples 9 to 14 preparation methods of porphyrin derivatives
The experimental procedures and experimental conditions in the preparation methods of examples 9 to 14 were the same as those in example 1, except that the raw materials used were the same
Wherein n =5, 9,11,17, 22, 24, the final porphyrin derivative has the formula
Example 15A porphyrin derivative and a method for preparing the same
A porphyrin derivative has a molecular formula of
The preparation method comprises
Firstly, adding 0.317 mmol of SM1-3 into 8ml of dry N, N-Dimethylformamide (DMF), starting stirring, adding 7.29mmol of anhydrous potassium carbonate, stirring at room temperature, adding 6.34 mmol of SM2-3 after stirring and mixing uniformly, refluxing and stirring at 120 ℃ for 36h under the protection of nitrogen, detecting by thin layer chromatography (petroleum ether PE: dichloromethane DCM =6: 1), cooling to room temperature after the reaction is finished, washing with water to remove DMF, purifying the obtained solid material by column chromatography (petroleum ether PE: dichloromethane DCM =3: 1) to obtain SM3-3, wherein the chemical reaction equation is as follows:
secondly, adding 0.131 mmol of SM3-3 into 4ml of dry DMF, adding 4.32mmol of triethylamine, refluxing and stirring for 14h at 135 ℃ under the protection of argon, monitoring the reaction by thin layer chromatography (PE: DCM =3: 1), cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4-3, wherein the reaction equation is as follows:
examples 16 to 21 porphyrin derivatives and preparation methods thereof
The experimental procedures and experimental conditions in the preparation methods of examples 16 to 21 were the same as those in example 1, except that the raw materials used were the same
N =2,5, 11,17, 22, 24, the fraction of porphyrin derivative finally produced, leads to different productsSub-formula is
Example 22A porphyrin derivative and a process for the preparation thereof
A porphyrin derivative has a molecular formula of
The preparation method comprises
Firstly, adding 0.317 mmol of SM4-1 into 8ml of dry N, N-Dimethylformamide (DMF), starting stirring, adding 8.24mmol of anhydrous potassium carbonate, stirring at room temperature, after stirring and mixing uniformly, adding 6.97mmol of SM4-2, refluxing and stirring at 110 ℃ for 40h under the protection of nitrogen, detecting by thin layer chromatography (petroleum ether PE: dichloromethane DCM =4: 1), after the reaction is finished, cooling to room temperature, washing with water to remove DMF, purifying the obtained solid material by column chromatography (petroleum ether PE: dichloromethane DCM =2: 1) to obtain SM4-3, wherein the chemical reaction equation is as follows:
secondly, adding 0.131 mmol of SM4-3 into 4ml of dry DMF, adding 3.14mmol of triethylamine, refluxing and stirring at 115 ℃ for 12h under the protection of argon, monitoring the reaction by thin layer chromatography (PE: DCM =7: 1), cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4-4, wherein the reaction equation is as follows:
examples 23 to 28 porphyrin derivatives and preparation methods thereof
The experimental procedures and experimental conditions in the preparation methods of examples 23 to 28 were the same as those in example 1, except that the raw materials used were the same
Wherein n =2,5, 13, 17, 22, 24, the final porphyrin derivative has the formula
Example 29A porphyrin derivative and its preparation method
A porphyrin derivative has a molecular formula of
The preparation method comprises
Firstly, adding 0.317 mmol of SM1-5 into 8ml of dry N, N-Dimethylformamide (DMF), starting stirring, adding 9.51 mmol of anhydrous potassium carbonate, stirring at room temperature, adding 7.93 mmol of SM2-5 after stirring and mixing uniformly, refluxing and stirring at 140 ℃ for 48h under the protection of nitrogen, detecting by thin layer chromatography (petroleum ether PE: dichloromethane DCM =10: 1), cooling to room temperature after the reaction is finished, washing with water to remove DMF, purifying the obtained solid material by column chromatography (petroleum ether PE: dichloromethane DCM =5: 1) to obtain SM3-5, wherein the chemical reaction equation is as follows:
secondly, adding 0.131 mmol of SM3-5 into 4ml of dry DMF, adding 2.62mmol of triethylamine, refluxing and stirring at 110 ℃ for 10h under the protection of argon, monitoring the reaction by thin layer chromatography (PE: DCM =1: 1), cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4-5, wherein the reaction equation is as follows:
example 30A fluorescent nanosensor that can detect hyaluronic acid
Taking synthetic SM4-1 to prepare 0.5 mg/mL Tetrahydrofuran (THF) solution, taking hyaluronic acid
(m = 100) is prepared into 0.05 mg/mL aqueous solution, then 40 μ L of hyaluronic acid is put into a 1.5 mL centrifuge tube, then a pipette is used for taking 16 μ L of SM4-1 solution to be evenly mixed with the hyaluronic acid, 100 μ L of 1mol/L HEPES buffer solution with pH of 7.0 is added, 844 μ L of water is added for ultrasonic treatment for 1 min, and the reaction is continued for 5 h, so that the fluorescence nano-sensor can be obtained.
Example 31A fluorescent nanosensor that can detect hyaluronic acid
Taking synthetic SM4-2 to prepare into 0.5 mg/mL Tetrahydrofuran (THF) solution, taking hyaluronic acid
(m = 300) is prepared into 0.05 mg/mL aqueous solution, then 40 μ L of hyaluronic acid is put into a 1.5 mL centrifuge tube, then a liquid transfer gun is used for taking 12 μ L of SM4-2 solution to be uniformly mixed with the hyaluronic acid, 100 μ L of 1mol/L HEPES buffer solution with pH of 7.2 is added, 844 μ L of water is added for ultrasonic treatment for 1.2 min, and the reaction is continued for 6h, so that the fluorescence nanosensor can be obtained.
Example 32A fluorescent nanosensor that can detect hyaluronic acid
Taking synthetic SM4-3 to prepare into 0.5 mg/mL Tetrahydrofuran (THF) solution, taking hyaluronic acid
(m = 500) is prepared into 0.05 mg/mL aqueous solution, then 40 μ L of hyaluronic acid is put into a 1.5 mL centrifuge tube, then a pipette is used for taking 8 μ L of SM4-3 solution to be evenly mixed with the hyaluronic acid, 100 μ L of 1 mol/LpH HEPES buffer solution is added, 844 μ L of water is added for ultrasonic treatment for 1.5 min, and the reaction is continued for 7h, so that the fluorescence nanosensor can be obtained.
Example 33A fluorescent nanosensor that can detect hyaluronic acid
Taking synthetic SM4-4 to prepare 0.5 mg/mL of tetrahydroExtracting hyaluronic acid with furan (THF) solution
(m = 700) preparing 0.05 mg/mL aqueous solution, then putting 40 μ L hyaluronic acid into a 1.5 mL centrifuge tube, then using a pipette to take 20 μ L SM4-4 solution to be uniformly mixed with the hyaluronic acid, adding 100 μ L1 mol/L HEPES buffer solution with pH of 7.6, then adding 844 μ L water, performing ultrasonic treatment for 1.8 min, and continuing to react for 8h to obtain the fluorescent nano-sensor.
EXAMPLE 34A fluorescent nanosensor that can detect hyaluronic acid
Taking synthetic SM4-5 to prepare into 0.5 mg/mL Tetrahydrofuran (THF) solution, taking hyaluronic acid
(m = 1000) is prepared into 0.05 mg/mL aqueous solution, then 40 μ L of hyaluronic acid is put into a 1.5 mL centrifuge tube, then a liquid transfer gun is used for taking 24 μ L of SM4-5 solution to be uniformly mixed with the hyaluronic acid, 100 μ L of 1 mol/LpH HEPES buffer solution is added, 844 μ L of water is added for 2 min, and the reaction is continued for 5 h, so that the fluorescence nano-sensor can be obtained.
EXAMPLE 35 identification of fluorescent nanosensors
The fluorescence nanosensor obtained in example 30 was selected, the particle size distribution of the nanosensor was 148 nm, as shown in fig. 3, the fluorescence intensity after self-assembly rapidly decreased with the increase of response time, and when the response time reached 6h, the fluorescence intensity was almost 1/6 before hyaluronic acid was added, and the fluorescence intensity at this time tended to a stable value, as shown in fig. 4, the fluorescence nanosensor obtained based on the above steps, i.e., a fluorescence nanosensor capable of detecting hyaluronidase.
Example 36 fluorescence titration experiment of fluorescence nanosensor for detection of Hyaluronidase
10 samples of 1.5 mL are taken and added to the fluorescence sensor solution obtained in example 6, and the fluorescence emission spectra of 12 samples are obtained by incubating 12 samples in 12 samples bottles at constant temperature of 37 ℃ for 1 h, using 425nm as the excitation wavelength, and measuring the fluorescence emission spectra of each sample, in turn, at concentrations of [ HAase ] =0 (a), 10U/mL (b), 20U/mL (c), 30U/mL (d), 50U/mL (e), 60U/mL (f), 70U/mL (g), 80U/mL (h), 90U/mL (i), 100U/mL (j), 150U/mL (k), 200U/mL (l), respectively, as shown in FIG. 5. The measurement result shows that: the fluorescence intensity of the polymeric fluorescence sensor at 656 nm gradually increased with increasing hyaluronidase concentration. The corresponding fitted relatively ideal functional graph and the corresponding functional graph (y = a + b x, a =0.9427, b =0.01701, R2= 0.9967) can be made from the 656 nm fluorescence intensity variation versus HA ase concentration in fig. 4 (see fig. 6).
EXAMPLE 37 comparative assays for the detection of the Effect of other molecules, ions, on hyaluronidase
15 centrifugal tubes of 1.5 mL are respectively filled with the fluorescence nanosensor solution obtained in example 30, and then HAase, NaCl, KCl and CaCl are respectively added at the concentration of 100U/mL, 0.1 mol/L2、0.1 mol/LMgCl2Solutions of 10mmol/L alanine, 10mmol/L threonine, 10mmol/L glycine, 10mmol/L histidine, 10mmol/L phenylalanine, 10mmol/L glucose, 5 mmol/L GSH (glutathione), 1 mmol/L Cys (cysteine) and 5 mmol/L Hcy (homocysteine) are respectively added into a No. 2-15 sample bottle, and the No. 1 sample is a blank sample. The fluorescence spectrum data of 15 samples under 425nm wavelength excitation were then measured, respectively, to obtain the fluorescence change at 656 nm wavelength emission, and the results are shown in FIG. 7. The measurement result shows that: the above-mentioned various substances, except hyaluronidase, had no significant effect on the fluorescence intensity of the prepared fluorescent nanosensor.
Example 38 comparative assay for Effect of other Small molecules, ions and Hyaluronidase Co-existence
15 1.5 mL centrifuge tubes were filled with the fluorescent nanosensor solution obtained in example 30, respectively, sample No. 1 was blank, and then HAase, NaCl, KCl, and LCaCl were added at concentrations of 100U/mL, 0.1 mol/L, and2、0.1 mol/L MgCl210mmol/L alanine, 10mmol/L threonine, 10mmol/L glycine, 10mmol/L histidine, 10mmol/LAdding mmol/L phenylalanine, 10mmol/L glucose, 5 mmol/L GSH (glutathione), 1 mmol/L Cys (cysteine) and 5 mmol/L Hcy (homocysteine) solution into a 2-14 sample bottle. The fluorescence emission spectrum data of 15 samples under 425nm wavelength excitation were then measured to obtain the fluorescence change value at 656 nm wavelength emission, and the results are shown in FIG. 8. The measurement result shows that: in addition to hyaluronidase, the various coexisting ions and molecules described above do not interfere with the response of the fluorescent nanosensor to hyaluronidase.
Example 39 Hela cell viability assay with fluorescent nanoprobes at different concentrations
First, HeLa cells were placed in DMEM medium containing 10% fetal bovine serum, and then cultured in a constant temperature (37 ℃) and humidity incubator containing 5% CO2 for 24 hours, after removing the medium, washed 3 times with PBS buffer solution, added with DMEM medium containing fluorescent nanoprobes of different concentrations (0. mu.g/mL, 5. mu.g/mL, 10. mu.g/mL, 15. mu.g/mL, 20. mu.g/mL) for culture, and cultured for further 24 hours, cytotoxicity was measured according to ISO10993-5 standard using MTT assay, and the results are shown in FIG. 9.
Although the present invention has been described in detail with reference to the above embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described above, or equivalents may be substituted for elements thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A porphyrin derivative based on self-assembly and capable of detecting hyaluronidase is characterized in that the general formula of the porphyrin derivative is as follows:
wherein R1, R2 and R3 are respectively H or O (CH)2)nN(CH2CH3)3 +(ii) a The O (CH)2)nN(CH2CH3)3 +N =2~ 24.
2. A preparation method of a porphyrin derivative capable of detecting hyaluronidase based on self-assembly is characterized by sequentially carrying out the following steps:
adding SM1 into dry N, N-dimethylformamide, starting stirring, adding anhydrous potassium carbonate, stirring at room temperature, adding SM2 after stirring and mixing uniformly, refluxing and stirring for 24-48 h under the protection of inactive gas, detecting by thin layer chromatography, cooling to room temperature after reaction, washing with water to remove the N, N-dimethylformamide, purifying the obtained solid material by column chromatography to obtain SM3, wherein the chemical reaction equation is as follows:
wherein, X1，X2，X3Are respectively H or OH; at X1=X2=X3When = H, A1=A2=A3= H; at X1=X2=H，X3When OH is not zero, A1=A2=H，A3=O（CH2）nBr; at X1=H，X2=X3When OH is not zero, A1=H，A2=A3=O（CH2）nBr at X1=X2=X3When OH is not zero, A1=A2=A3= O（CH2）nBr; the O (CH)2）nN = 2-24 in Br;
adding SM3 into dry N, N-dimethylformamide, adding excessive triethylamine, refluxing and stirring for 10-20 hours under the protection of inactive gas, monitoring the reaction by thin-layer chromatography, cooling to room temperature after the reaction is finished, adding anhydrous ether, stirring until a solid is separated out, and performing suction filtration to obtain SM4, namely the porphyrin derivative, wherein the reaction equation is as follows:
wherein A is1，A2，A3Are each H or O (CH)2）nBr; in A1=A2=A3When H, R1=R2=R3= H; in A1=A2=H，A3= O（CH2）nBr is, R1=R2=H，R3= O(CH2)nN(CH2CH3)3 +(ii) a In A1=H，A2=A3=O（CH2）nBr is, R1=H，R2=R3= O(CH2)nN(CH2CH3)3 +(ii) a In A1=A2=A3= O（CH2）nBr is, R1=R2=R3= O(CH2)nN(CH2CH3)3 +(ii) a The O (CH)2）nBr and O (CH)2)nN(CH2CH3)3 +N =2~ 24.
3. The preparation method of the porphyrin derivative based on self-assembly detectable hyaluronidase according to claim 2, characterized in that the reflux temperature is 110-140 ℃.
4. The method for preparing a porphyrin derivative based on self-assembly detectable hyaluronidase according to claim 3, the SM 1: SM 2: the molar ratio of the anhydrous potassium carbonate is 1: 15-25: 15-30.
5. The preparation method of the porphyrin derivative based on self-assembly detectable hyaluronidase according to any one of claims 2-4, characterized in that the thin layer chromatography liquid is a mixed liquid of petroleum ether and dichloromethane, wherein the volume ratio of petroleum ether to dichloromethane is 1-10: 1; the column chromatography eluent is a mixed solution of petroleum ether and dichloromethane, wherein the volume ratio of the petroleum ether to the dichloromethane is 1-5: 1.
6. The preparation method of the porphyrin derivative capable of detecting hyaluronidase based on self-assembly as claimed in claim 5, wherein the molar ratio of SM3 to triethylamine is 1: 20-40.
7. Use of a porphyrin derivative based on self-assembly for detecting hyaluronidase, characterized in that a fluorescent nanoorgan for detecting hyaluronidase is prepared by mixing the porphyrin derivative as described in claim 1 with a hyaluronic acid chain.
8. The application of the porphyrin derivative capable of detecting hyaluronidase based on self-assembly as claimed in claim 7 is characterized in that a tetrahydrofuran solution of the porphyrin derivative and an aqueous solution of a hyaluronic acid chain are placed in a centrifuge tube, a HEPES buffer solution with pH of 7-8 is added for dilution, and after ultrasonic treatment is performed for 1-2 min, the mixed solution is continuously reacted for 5-8 h, so as to obtain the fluorescent nano-sensor capable of detecting hyaluronidase.
9. The use of a porphyrin derivative self-assembly based detectable hyaluronidase according to claim 7, wherein the hyaluronan chain has the formula:
wherein m =100~ 1000.
10. The use of the self-assembly based porphyrin derivative detectable hyaluronidase according to claim 8, wherein the mass ratio of the porphyrin derivative to the hyaluronan chain is 2-6: 1.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1753896A (en) * | 2002-12-23 | 2006-03-29 | 命运之神药品有限公司 | Novel compounds and uses thereof |
| US20070015742A1 (en) * | 2003-02-26 | 2007-01-18 | Gokhan Yahioglu | Porphyrin derivatives |
| WO2013142886A1 (en) * | 2012-03-30 | 2013-10-03 | Joanneum Research Forschungsgesellschaft Mbh | Opto-chemical sensor |
| CN104390947A (en) * | 2014-11-20 | 2015-03-04 | 南京邮电大学 | Fluorescence method for detecting hyaluronidase |
| CN105085710A (en) * | 2015-08-12 | 2015-11-25 | 长春理工大学 | Amino-sulfonyl phenyl porphyrin-hyaluronic acid polymer and preparation method and application thereof |
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| CN1753896A (en) * | 2002-12-23 | 2006-03-29 | 命运之神药品有限公司 | Novel compounds and uses thereof |
| US20070015742A1 (en) * | 2003-02-26 | 2007-01-18 | Gokhan Yahioglu | Porphyrin derivatives |
| WO2013142886A1 (en) * | 2012-03-30 | 2013-10-03 | Joanneum Research Forschungsgesellschaft Mbh | Opto-chemical sensor |
| CN104390947A (en) * | 2014-11-20 | 2015-03-04 | 南京邮电大学 | Fluorescence method for detecting hyaluronidase |
| CN105085710A (en) * | 2015-08-12 | 2015-11-25 | 长春理工大学 | Amino-sulfonyl phenyl porphyrin-hyaluronic acid polymer and preparation method and application thereof |
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