CN114681611A - Poly 3-thiopheneacetic acid modified PCN-224 composite material and preparation method and application thereof - Google Patents
Poly 3-thiopheneacetic acid modified PCN-224 composite material and preparation method and application thereof Download PDFInfo
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- CN114681611A CN114681611A CN202210395833.7A CN202210395833A CN114681611A CN 114681611 A CN114681611 A CN 114681611A CN 202210395833 A CN202210395833 A CN 202210395833A CN 114681611 A CN114681611 A CN 114681611A
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- thiopheneacetic acid
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- RCNOGGGBSSVMAS-UHFFFAOYSA-N 2-thiophen-3-ylacetic acid Chemical compound OC(=O)CC=1C=CSC=1 RCNOGGGBSSVMAS-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 239000002131 composite material Substances 0.000 title claims abstract description 37
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
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- 239000002904 solvent Substances 0.000 claims abstract description 10
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- 229920001167 Poly(triaryl amine) Polymers 0.000 claims abstract 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
- 238000003756 stirring Methods 0.000 claims description 19
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 13
- 206010028980 Neoplasm Diseases 0.000 claims description 12
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 claims description 12
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- 238000002428 photodynamic therapy Methods 0.000 claims description 10
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- -1 4-carboxyphenyl Chemical group 0.000 claims description 4
- 229910021578 Iron(III) chloride Inorganic materials 0.000 claims description 4
- VZJJZMXEQNFTLL-UHFFFAOYSA-N chloro hypochlorite;zirconium;octahydrate Chemical compound O.O.O.O.O.O.O.O.[Zr].ClOCl VZJJZMXEQNFTLL-UHFFFAOYSA-N 0.000 claims description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene chloride Substances ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- RKCAIXNGYQCCAL-UHFFFAOYSA-N porphin Chemical compound N1C(C=C2N=C(C=C3NC(=C4)C=C3)C=C2)=CC=C1C=C1C=CC4=N1 RKCAIXNGYQCCAL-UHFFFAOYSA-N 0.000 claims description 4
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
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- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- 206010034972 Photosensitivity reaction Diseases 0.000 description 2
- 238000000862 absorption spectrum Methods 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
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- SMOZAZLNDSFWAB-UHFFFAOYSA-N 4-[10,15,20-tris(4-carboxyphenyl)-21,24-dihydroporphyrin-5-yl]benzoic acid Chemical compound C1=CC(C(=O)O)=CC=C1C(C=1C=CC(N=1)=C(C=1C=CC(=CC=1)C(O)=O)C1=CC=C(N1)C(C=1C=CC(=CC=1)C(O)=O)=C1C=CC(N1)=C1C=2C=CC(=CC=2)C(O)=O)=C2N=C1C=C2 SMOZAZLNDSFWAB-UHFFFAOYSA-N 0.000 description 1
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- RZWIIPASKMUIAC-VQTJNVASSA-N thromboxane Chemical compound CCCCCCCC[C@H]1OCCC[C@@H]1CCCCCCC RZWIIPASKMUIAC-VQTJNVASSA-N 0.000 description 1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
- A61K41/0076—PDT with expanded (metallo)porphyrins, i.e. having more than 20 ring atoms, e.g. texaphyrins, sapphyrins, hexaphyrins, pentaphyrins, porphocyanines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
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- A61K49/0013—Luminescence
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- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
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Abstract
The invention discloses a PCN-224 composite material modified by poly 3-thiopheneacetic acid and a preparation method and application thereof. The preparation method of the PCN-224 composite material modified by poly-3-thiopheneacetic acid comprises the following steps: mixing and uniformly dispersing PCN-224 and PTAA in a solvent, and then centrifuging, washing and drying the mixture to obtain the PCN-224 composite material modified by poly-3-thiopheneacetic acid. According to the invention, the PCN-224 surface is coated with the poly-3-thiopheneacetic acid, so that the dispersibility can be improved, and the obtained PCN-224 composite material modified by the poly-3-thiopheneacetic acid has excellent biocompatibility, lower biotoxicity and high killing rate on tumor cells under the illumination condition.
Description
Technical Field
The invention relates to the technical field of medical materials, in particular to a PCN-224 composite material modified by poly-3-thiopheneacetic acid and a preparation method and application thereof.
Background
Despite significant advances in traditional tumor therapy (e.g., surgery, radiation therapy, and chemotherapy), cancer remains the first killer in humans. Novel tumor treatment means: photodynamic therapy (PDT) has attracted the attention of researchers. Compared with traditional tumor treatment approaches, PDT has unique advantages: the wound is small, the treatment can be repeated, the selectivity is high, and the treatment can be combined with other treatment means.
Briefly, PDT is a non-invasive local tumor treatment in tissues shared by photosensitizers, light and oxygen, where the photosensitizers are excited by light of a specific wavelength to convert from a ground state to an excited state and the surrounding oxygen is converted to Reactive Oxygen Species (ROS) by the energy released from the photosensitizers. ROS has certain cytotoxicity, can inhibit the growth of cells, promote apoptosis, destroy tumor blood vessels, stimulate the release of thromboxane and cause the infarction of tumor tissues. Therefore, PDT can selectively kill tumor cells due to the dependence on light and photosensitizers, so that specific sites of tumor growth can be laser-irradiated, killing tumor cells while protecting surrounding normal tissues.
PCN-224 is prepared by taking meso-tetra (4-carboxyphenyl) porphine as a ligand and Zr4+MOFs constructed through coordination have the characteristics of rigid structure, large specific surface area, high porosity, excellent photosensitivity, thermal stability and the like, and are often used for photodynamic therapy of tumors. At present, the research on applying the compound to tumor treatment and serving as a drug carrier has been carried out, but the difficulty that the compound is easy to aggregate and has high biological toxicity is always the difficulty which needs to be overcome in the field of photodynamic therapy.
Disclosure of Invention
The invention aims to overcome the technical defects, provides a PCN-224 composite material modified by poly-3-thiopheneacetic acid, and a preparation method and application thereof, and solves the technical problems of easy aggregation and high biological toxicity of PCN-224 in the prior art.
The invention provides a preparation method of a PCN-224 composite material modified by poly-3-thiopheneacetic acid, which comprises the following steps:
mixing and uniformly dispersing PCN-224 and poly 3-thiopheneacetic acid (PTAA) in a solvent, and then centrifuging, washing and drying the mixture to obtain the PCN-224 composite material modified by the poly 3-thiopheneacetic acid.
The second aspect of the invention provides a poly-3-thiopheneacetic acid modified PCN-224 composite material, and the poly-3-thiopheneacetic acid modified PCN-224 composite material is obtained by the preparation method of the poly-3-thiopheneacetic acid modified PCN-224 composite material provided by the first aspect of the invention.
The third aspect of the invention provides an application of a PCN-224 composite material modified by poly-3-thiopheneacetic acid, and the PCN-224 composite material modified by poly-3-thiopheneacetic acid is applied as a photosensitizer in tumor photodynamic therapy.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, the PCN-224 surface is coated with the poly-3-thiopheneacetic acid, so that the dispersibility can be improved, and the obtained PCN-224 composite material modified by the poly-3-thiopheneacetic acid has excellent biocompatibility, lower biotoxicity and high killing rate on tumor cells under the illumination condition.
Drawings
FIG. 1 shows absorption spectra at 300-800 nm of PTAA, PCN-224(P) and PCN-224@ PTAA (PP) obtained in example 1 of the present invention;
FIG. 2 is a graph showing the results of the cytotoxicity test assay of PCN-224(P) and PCN-224@ PTAA (PP) obtained in example 1 of the present invention; wherein, (1) is Hela cell, and (2) is U14 cell;
FIG. 3 is a graph showing the cytotoxicity test results of PCN-224(P) and PCN-224@ PTAA (PP) obtained in example 1 of the present invention; wherein, (1) is Hela cell, and (2) is U14 cell;
FIG. 4 is a photograph showing intracellular co-localization fluorescence images of cell membrane and cell nucleus of PCN-224(P) and PCN-224@ PTAA (PP) obtained in example 1 of the present invention;
FIG. 5 is a graph showing the effect of cellular uptake of PCN-224(P) and PCN-224@ PTAA (PP) obtained in example 1 of the present invention; wherein (A) is a bar graph and (B) is a line graph.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a preparation method of a PCN-224 composite material modified by poly-3-thiopheneacetic acid, which comprises the following steps:
mixing and uniformly dispersing PCN-224 and poly-3-thiopheneacetic acid (PTAA) in a solvent, and then centrifuging, washing and drying the mixture to obtain the PCN-224 composite material (marked as PCN-224@ PTAA or PP) modified by the poly-3-thiopheneacetic acid.
In the present invention, the particle size of PCN-224 is less than 500nm, more preferably 50 to 300nm, and still more preferably 50 to 150 nm.
In the invention, the mass ratio of the PTAA to the PCN-224 is 1: (10-100), further 1: (50-70), further 1: (55-60).
In the present invention, the solvent is a mixed solvent of water and an organic solvent. Further, the organic solvent is dimethyl sulfoxide (DMSO); furthermore, the volume ratio of the water to the organic solvent is (30-60): 1.
in the invention, the dosage ratio of the PCN-224 to the solvent is 1 mg: (5-20) mL.
In the invention, the step of mixing and uniformly dispersing PCN-224 and poly 3-thiopheneacetic acid in a solvent comprises the following steps:
adding PCN-224 into water and uniformly dispersing to obtain a PCN-224 solution; wherein the concentration of the PCN-224 solution is 0.1 to 10mM, further 0.5 to 2mM, further 1 mM;
dissolving the PTAA in an organic solvent to obtain a PTAA solution; wherein the concentration of the PTAA solution is 0.1-10 mg/mL, further 0.5-2 mg/mL, and further 1 mg/mL.
Uniformly mixing the PTAA solution and the PCN-224 solution to obtain a mixture; the PTAA solution and the PCN-224 solution are uniformly mixed in a stirring mode, and the stirring time is 2-24 hours, further 3-10 hours, and further 4-6 hours.
In the invention, the rotation speed of a centrifugal machine is 10000-15000 rpm, and further 12000rpm in the processes of centrifuging, washing and drying the mixture; centrifuging for 10-30 min, and further 20 min; the washing solvent was DMSO and water as 1: 99 volume ratio of the mixed solvent; the drying method is freeze drying.
In some preferred embodiments of the invention, PCN-224 is obtained by: subjecting meso-tetra (4-carboxyphenyl) porphin (H)2TCPP), zirconium oxychloride octahydrate (ZrOCl)2·8H2O) and Benzoic Acid (BA) are dissolved in Dimethylformamide (DMF), heated and stirred for 3-6 h at the temperature of 80-100 ℃, and then the PCN-224 is obtained through centrifugation, washing and drying. Wherein the dosage ratio of the meso-tetra (4-carboxyphenyl) porphin to the zirconium oxychloride octahydrate to the benzoic acid to the dimethylformamide is 0.1-5 mg: 1-5 mg: 10-50 mg: 1 mL; further 1 mg: 3 mg: 28 mg: 1 mL.
In some preferred embodiments of the invention, PTAA is obtained by the following steps: 3-thiopheneacetic acid and anhydrous FeCl3And anhydrous CH2Cl2After mixing, stirring and reacting for 3-5 h at 20-30 ℃ under the protection of nitrogen; then removing CH by distillation under reduced pressure2Cl2Adding ethanol, and stirring and reacting for 3-5 h at 20-30 ℃; filtering, removing filtrate, washing filter cakes with hydrochloric acid and ethanol respectively, and refluxing for 1-3 hours in ethanol at 70-90 ℃; filtering, and vacuum drying the filter cake overnight; dissolving the reddish brown powder in NaOH solution, stirring at 90-110 ℃ for 12-36 hours, and centrifugally separating to remove insoluble substances; adding concentrated HCl into the supernatant, stirring at normal temperature for 8-12 h, standing for 24-72 h for precipitation and aging, pouring out the supernatant, adding distilled water, performing microfiltration, washing a filter cake with water and ethanol, and performing vacuum drying to obtain the poly-3-thiopheneacetic acid. Wherein, anhydrous FeCl3The molar ratio of the 3-thiopheneacetic acid to the 3-thiopheneacetic acid is (4-6) 1 is further 5: 1; 3-Thiopheneacetic acid with anhydrous CH2Cl2The dosage ratio of the components is 1 g: (100-140) mL; in the stirring reaction process, the dosage ratio of the 3-thiopheneacetic acid to the ethanol is 1 g: (100-140) mL; in the reflux process, the dosage ratio of the 3-thiopheneacetic acid to the ethanol is 1 g: (100-120) mL;the concentration of the sodium hydroxide solution is 1-3 mol/L, and the dosage ratio of the 3-thiopheneacetic acid to the sodium hydroxide solution is 1 g: (100-140) mL.
The second aspect of the invention provides a poly-3-thiopheneacetic acid modified PCN-224 composite material, and the poly-3-thiopheneacetic acid modified PCN-224 composite material is obtained by the preparation method of the poly-3-thiopheneacetic acid modified PCN-224 composite material provided by the first aspect of the invention.
The third aspect of the invention provides an application of a PCN-224 composite material modified by poly-3-thiopheneacetic acid, and the PCN-224 composite material modified by poly-3-thiopheneacetic acid is applied as a photosensitizer in tumor photodynamic therapy.
Example 1
Preparation of PCN-224 (P): subjecting meso-tetra (4-carboxyphenyl) porphin (H)2TCPP)100mg, zirconium oxychloride octahydrate (ZrOCl)2·8H2O)300mg and Benzoic Acid (BA)2.8g were dissolved in a 250mL round bottom flask containing 100mL of DMF, heated and stirred at 300rpm in an oil bath at 90 ℃ for 5h, the PCN-224 nanoparticles (particle size about 70nm) were collected by centrifugation, washed 3 times with DMF and ethanol, and the final product was lyophilized to a powder and stored at 4 ℃ protected from light.
Preparation of PTAA: 0.71g (5mmol) of 3-thiopheneacetic acid and 4.06g (25mmol) of anhydrous FeCl are sequentially added into a 250mL eggplant-shaped bottle3(FeCl3Monomer molar ratio of 5:1) and 100mL of anhydrous CH2Cl2Stirring for 4 hours at normal temperature (25 ℃) under the protection of nitrogen; then, most of CH was removed by distillation under the reduced pressure2Cl2Adding 100mL of ethanol, and stirring for 4h at 25 ℃; filtering, discarding the filtrate, washing the filter cake with 100mL of 1mol/L hydrochloric acid and 100mL of ethanol respectively, and refluxing for 2 hours at 80 ℃ in 80mL of ethanol; filtering, and vacuum drying the filter cake at 30 ℃ overnight; dissolving the obtained reddish brown powder in 100mL of 2mol/L NaOH solution, stirring at 100 ℃ for 24 hours, and centrifuging to remove insoluble substances; adding 100mL of concentrated HCl into the supernatant, stirring at normal temperature for 10h, standing for 48h to precipitate and age, slightly pouring out the supernatant, adding 50mL of secondary distilled water, performing microfiltration, washing a filter cake with water and ethanol, and performing vacuum drying at 40 ℃ until the filter cake is dry to obtain bluish black poly (3-thiophene) acetic acid powder.
Preparation of PCN-224@ PTAA (PP): suspending 14.54mg of dried PCN-224 powder in 10mL of water, and sonicating for 30min to obtain a 1mM PCN-224 solution; dissolving 1mg of PTAA in 1mL of DMSO to obtain a PTAA solution with the concentration of 1 mg/mL; 0.25mL of the PTAA solution was added with stirring to the PCN-224 solution, stirring was continued for 5h, then the mixture was centrifuged (12000rpm, 20min) and washed three times with DMSO/water (v/v: 1/99) to eliminate excess PTAA ligand, and freeze dried to give PCN-224@ PTAA (PP).
Referring to FIG. 1, FIG. 1 shows the absorption spectra at 300-800 nm of PTAA, PCN-224(P) and PCN-224@ PTAA (PP) obtained in example 1 of the present invention. As can be seen from the graph in FIG. 1, although the PTAA has good biocompatibility and is nontoxic to cells, the PTAA has no absorption peak at 650-660 nm and cannot be excited by light, which indicates that the PTAA alone has no photodynamic effect; the PCN-224(P) has a weak absorption peak at 650-660 nm, which shows that the single PCN-224(P) has a certain photodynamic effect; the PCN-224@ PTAA (PP) has a stronger absorption peak at 650-660 nm, and can be better excited by light, so that the PCN-224@ PTAA (PP) has a better photodynamic effect.
Test examples
1. Cytotoxicity test
Hela cells and U14 cells were cultured in DMEM medium containing 10% fetal bovine serum by volume and 5% CO by volume, respectively2Cultured in an atmosphere at 37 ℃ and then the cultured cells were seeded in a 96-well plate at about 8000 cells per well and incubated for 20 hours. The drugs prepared in example 1 were added to 96-well plates at different concentrations, incubated for 4 hours, replaced with new medium, and incubated for 24 hours for MTT detection. The test results are shown in FIG. 2.
As can be seen from FIG. 2, the biocompatibility of PP is significantly better than that of P, which indicates that the modification of poly-3-thiopheneacetic acid is beneficial to improve the biocompatibility of PCN-224.
2. Cytotoxicity assays
Hela cells and U14 cells were cultured in DMEM medium containing 10% fetal bovine serum by volume and 5% CO by volume, respectively2Culturing at 37 deg.C in atmosphere, inoculating the cultured cells into 96-well plate at about 5000 cells per well, and incubating for 20 deg.Ch. The drug prepared in example 1 was added to a 96-well plate at different concentrations, incubated for 4 hours, replaced with a new medium, and subjected to light treatment at 658nm, and then serum was added to continue incubation for 24 hours, followed by MTT assay. The test results are shown in FIG. 3.
As can be seen from FIG. 3, PP has a cell killing power greater than that of P, indicating that modification with poly-3-thiopheneacetic acid is beneficial for increasing the phototoxicity of PCN-224.
3. Cell uptake effect test
PCN-224 has red fluorescence, so the nanoparticles can be used for fluorescence detection of cell uptake experiments. Cell membranes were labeled with DIO dye and nuclei were labeled with DAPI dye. In order to directly observe the uptake of the nanoparticles in the cells, a laser scanning confocal microscope (CLSM) is selected to observe the uptake of the nanoparticles by the cells. Meanwhile, the relation between the nanoparticle uptake capacity of cells in a certain concentration range and the nanoparticle concentration is detected by using a flow cytometer.
For CLSM imaging, first, a sterile cell culture slide was placed in a 24-well plate, and then Hela cells were cultured in the 24-well plate at a density of 8 ten thousand per well for 20 hours, so that the cells were adherently grown on the slide. After the culture was completed, the original medium was aspirated by a pipette gun, and then 5. mu.g/mL of PCN-224 and PTAA @ PCN-224 diluted with a serum-free medium were added, respectively, to continue the culture for 4 hours. The culture broth was then aspirated and the cells were washed three times with PBS to remove some of the nanoparticles not taken up by the cells. Adding 300 mu L of DIO working solution into each hole, continuing incubation for 15min, washing cells for three times by PBS after incubation is finished to remove redundant DIO staining solution, fixing the slide glass by 4% paraformaldehyde for 15min, taking out the slide glass, washing the cell slide glass for three times by PBS, reversely buckling the cell slide glass on a glass slide on which 10 mu L of DAPI-containing anti-fluorescence quenching mounting solution is dripped, and finally observing by laser scanning confocal microscope imaging, wherein the test result is shown in figure 4.
For flow cytometry assay, Hela cells were first cultured in 24-well plates at a density of 10 ten thousand per well for 20h, then the original medium was aspirated with a pipette gun, nanoparticles were diluted with serum-free medium at a range of different concentrations (5, 10, 20 μ g/mL) and added to the 24-well plates, after incubation for 4h the medium was aspirated, and the cells were washed three times with PBS to remove some of the nanoparticles that were not taken up by the cells, then 300 μ l of pancreatin was added per well, then the digestion was stopped with DMEM medium containing fetal bovine serum and aspirated into 1.5mL centrifuge tubes for centrifugation. After centrifugation, the supernatant was discarded, and then 1ml PBS was added for centrifugal washing. Then 200 microliters of PBS was added to each centrifuge tube and measured in ice bath. Finally, the intensity of the intracellular red fluorescence is detected by a flow cytometer, the mean ercp-A value is read, and the test result is shown in figure 5.
FIG. 4 is a photograph showing intracellular co-localization fluorescence images of cell membrane and nucleus of PCN-224(P) and PCN-224@ PTAA (PP) obtained in example 1 of the present invention. As can be seen from FIG. 4, when the PCN-224@ PTAA (PP) obtained in example 1 of the present invention is used for fluorescence detection of cell uptake experiments, the intracellular red fluorescence is obviously enhanced.
FIG. 5 is a graph showing the effect of cellular uptake of PCN-224(P) and PCN-224@ PTAA (PP) obtained in example 1 of the present invention. As can be seen from FIG. 5, the PCN-224@ PTAA (PP) of the present invention has better dispersibility and is easily swallowed by cells, and compared with the PCN-224, when the PCN-224@ PTAA is co-incubated with Hela cells under the same conditions, the cell-swallowing amount of the PCN-224@ PTAA is about twice that of the PCN-224.
Compared with the prior art, the invention has the beneficial effects that:
(1) the synthesized PCN-224@ PTAA has large specific surface area and particle size of about 110nm, and is very suitable for biological experiments;
(2) the PCN-224@ PTAA has better dispersibility and is easy to be swallowed by cells, and compared with the PCN-224, the PCN-224@ PTAA and Hela cells are co-incubated under the same condition, and the cell swallowing capacity of the PCN-224@ PTAA is about twice that of the PCN-224;
(3) the PCN-224@ PTAA has lower biological toxicity, and the toxicity of metal ions of the PCN-224 in normal tissues is reduced due to the coating of the PTAA;
(4) the results of the preparation and anti-tumor research of the PCN-224@ PTAA show that the PCN-224@ PTAA has good biocompatibility, the killing rate to the tumor cells under the condition of lower dose of light is more than 80%, and the killing rate under the dark condition is less than 10%.
(5) The invention provides a material with fluorescence effect, which is convenient for positioning and tracking in vivo fluorescence imaging;
(6) the PCN-224@ PTAA has good thermal stability and light stability, and is easy for less times of administration and repeated treatment of photodynamic therapy;
(7) the PCN-224@ PTAA has simple and safe synthesis steps, uses small dosage of organic reagents in the process, reduces the pollution to the environment, and is an effective method for synthesizing novel photosensitizer.
The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.
Claims (10)
1. A preparation method of a PCN-224 composite material modified by poly-3-thiopheneacetic acid is characterized by comprising the following steps:
mixing and uniformly dispersing PCN-224 and PTAA in a solvent, and then centrifuging, washing and drying the mixture to obtain the PCN-224 composite material modified by poly-3-thiopheneacetic acid.
2. The method for preparing the PCN-224 composite material modified by the poly-3-thiopheneacetic acid according to the claim 1, wherein the particle size of the PCN-224 is less than 500 nm.
3. The preparation method of the poly (3-thiopheneacetic acid) -modified PCN-224 composite material according to claim 1, wherein the mass ratio of the PTAA to the PCN-224 is 1: (10-100).
4. The method for preparing the PCN-224 composite material modified by poly-3-thiopheneacetic acid according to claim 1, wherein the solvent is a mixed solvent of water and dimethyl sulfoxide.
5. The preparation method of the PCN-224 composite material modified by the poly 3-thiopheneacetic acid according to the claim 1, wherein the step of mixing and uniformly dispersing the PCN-224 and the poly 3-thiopheneacetic acid in the solvent comprises the following steps:
adding PCN-224 into water and uniformly dispersing to obtain a PCN-224 solution;
dissolving the PTAA in dimethyl sulfoxide to obtain a PTAA solution;
the PTAA solution and the PCN-224 solution were mixed uniformly to obtain a mixture.
6. The preparation method of the PCN-224 composite material modified by the poly-3-thiopheneacetic acid as claimed in claim 5, wherein the PTAA solution and the PCN-224 solution are uniformly mixed by stirring for 2-24 h.
7. The method for preparing the PCN-224 composite material modified by the poly-3-thiopheneacetic acid according to the claim 1, wherein the PCN-224 is obtained by the following steps: dissolving meso-tetra (4-carboxyphenyl) porphin, zirconium oxychloride octahydrate and benzoic acid in dimethylformamide, heating and stirring at 80-100 ℃ for 3-6 h, and then centrifuging, washing and drying to obtain PCN-224.
8. The method for preparing a poly 3-thiopheneacetic acid-modified PCN-224 composite according to claim 1, wherein the PTAA is obtained by the steps of: 3-thiopheneacetic acid and anhydrous FeCl3And anhydrous CH2Cl2After mixing, stirring and reacting for 3-5 h at 20-30 ℃ under the protection of nitrogen; then removing CH by distillation under reduced pressure2Cl2Adding ethanol, and stirring and reacting for 3-5 h at 20-30 ℃; filtering, removing filtrate, washing filter cakes with hydrochloric acid and ethanol respectively, and refluxing for 1-3 hours in ethanol at 70-90 ℃; filtering, and vacuum drying the filter cake overnight; dissolving the obtained reddish brown powder in NaOH solution, stirring at 90-110 ℃ for 12-36 hours, and centrifugally separating to remove insoluble substances; adding concentrated HCl into the supernatant, stirring at normal temperature for 8-12 h, standing for 24-72 h for precipitation and aging, pouring out the supernatant, adding distilled water, performing microfiltration, washing the filter cake with water and ethanol, and performing vacuum drying to obtain PTAA。
9. A poly 3-thiopheneacetic acid modified PCN-224 composite material, characterized in that the poly 3-thiopheneacetic acid modified PCN-224 composite material is obtained by the method for preparing the poly 3-thiopheneacetic acid modified PCN-224 composite material according to any one of claims 1 to 8.
10. Use of the PCN-224 composite modified with poly-3-thiopheneacetic acid according to claim 9, wherein the PCN-224 composite modified with poly-3-thiopheneacetic acid is used as a photosensitizer in photodynamic therapy of tumors.
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| US20210236651A1 (en) * | 2017-07-13 | 2021-08-05 | Northwestern University | General and Direct Method for Preparing Oligonucleotide-Functionalized Metal-Organic Framework Nanoparticles |
| US20210162369A1 (en) * | 2018-01-02 | 2021-06-03 | Daegi Gyeongbuk Institute Of Science And Technololgy | Method for preparing, in situ, metal-organic framework by using control of diffusion within ionic polymer |
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