CN112057615A - NiPS with tumor targeting function3Nano medicine and its preparing method and use - Google Patents

NiPS with tumor targeting function3Nano medicine and its preparing method and use Download PDF

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CN112057615A
CN112057615A CN202010432599.1A CN202010432599A CN112057615A CN 112057615 A CN112057615 A CN 112057615A CN 202010432599 A CN202010432599 A CN 202010432599A CN 112057615 A CN112057615 A CN 112057615A
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张家宜
吴宗泽
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Shenzhen Hanguang Technology Co ltd
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Abstract

The invention provides a NiPS with tumor targeting3Nano-drugs including two-dimensional NiPS3Nanosheets, and coating on the two-dimensional NiPS3Triphenylphosphine on the surface of the nanosheets. The invention has NiPS with tumor targeting3Nanoparticulates, in the anaerobic pathway, two-dimensional NiPS3The holes generated by the nano-flake after optical excitation can oxidize OH in water‑OH is formed, which represents the possibility of anoxic photodynamic action. Thus NiPS3The semiconductor photosensitive nano material can be applied to generate active free radicals under the anoxic condition, and further starts the apoptosis pathway. Two-dimensional NiPS3The nano-sheet has the advantages of obvious photodynamic effect, high chemical stability, low toxicity and good biocompatibility, and has wide application prospect in the fields of photothermal therapy, photodynamic therapy and drug-loaded therapy. The invention also discloses NiPS with tumor targeting3A preparation method and application of nano-drugs.

Description

NiPS with tumor targeting function3Nano medicine and its preparing method and use
Technical Field
The invention relates to the technical field of biological nano-medicine, in particular to NiPS with tumor targeting3The invention also relates to the NiPS with tumor targeting3The invention also relates to a preparation method of the nano-drug, and the NiPS with tumor targeting3Application of nanometer medicinal preparation is provided.
Background
Two-dimensional materials are materials in which electrons are free to move (planar motion) only in two dimensions, on a non-nanoscale (1-100 nm). In recent years, a series of quasi-two-dimensional materials with a thickness of only a single atomic layer, such as graphene, black phosphorus (phosphorus alkene), silylene, germanium alkene, stibene, and metal disulfide (such as titanium disulfide and molybdenum disulfide), are successively discovered, and have potential application prospects in the biomedical field (such as photothermal therapy, photodynamic therapy, drug-loaded therapy and photoacoustic imaging). However, few of these materials currently studied are capable of combining high stability, low toxicity, and the ability to produce active oxygen without relying on oxygen. However, one of the major factors that have severely limited photodynamic therapy in the field of antitumor therapy is: the tumor microenvironment is anoxic, and the existing photosensitizer is difficult to generate active oxygen in the tumor anoxic microenvironment, so that photodynamic therapy cannot be carried out under the anoxic condition. Therefore, the development of a novel photosensitive material capable of generating active oxygen by infrared light excitation under an oxygen-deficient condition is imperative.
Disclosure of Invention
In view of the above, the present invention provides a NiPS with tumor targeting3Nano medicine and NiPS with tumor targeting function3Preparation method of nano-drug, NiPS with tumor targeting3The nano-drug has both tumor cell targeting and mitochondrion targeting, and can be absentActive hydroxyl free radicals are generated in an oxygen environment to promote tumor cell apoptosis; the NiPS with tumor targeting3The nano-drug also has the advantages of obvious photodynamic effect, low toxicity, relatively simple preparation method and the like.
In a first aspect, the present invention provides a NiPS with tumor targeting3Nano-drugs including two-dimensional NiPS3Nanosheets, and coating on the two-dimensional NiPS3Triphenylphosphine on the surface of the nanosheets.
The invention has NiPS with tumor targeting3Nano-drugs including two-dimensional NiPS3Nanosheets, two-dimensional NiPS3The nano-sheet has good light absorption characteristics, which shows that the nano-sheet is an excellent photosensitizer and can be applied to the photodynamic treatment of tumors under the anoxic condition. Two-dimensional NiPS3The nano-sheet has better light absorption characteristic in the wave band of 300-900 nm, wherein the maximum is 638 nm. Indicating its potential as a good photocatalyst for photodynamic therapy. NiPS observations from Electron Spin Resonance (ESR) experiments3Under 660nm light irradiation, the separation of electron-hole pairs is obvious, and the basis is provided for the existence of OH. Further experiments show that stronger DMPO- & OH signals are observed under the conditions of normal oxygen and oxygen deficiency respectively, and the normal oxygen and oxygen deficiency ways can be used for preparing the & OH. In the ordinary oxygen pathway, oxygen is an effective electron acceptor, and is a main capture agent of photo-generated electrons absorbed by the surface of a catalyst, and related compounds can be oxidized to generate singlet oxygen, hydroxyl radicals and the like. In the hypoxic pathway, two-dimensional NiPS3The holes generated by the nano-flake after optical excitation can oxidize OH in water-OH is formed, which represents the possibility of anoxic photodynamic action. Thus NiPS3The semiconductor photosensitive nano material can be applied to generate active free radicals under the anoxic condition, and further starts the apoptosis pathway. Two-dimensional NiPS3The nano-sheet has the advantages of obvious photodynamic effect, high chemical stability, low toxicity and good biocompatibility, and has wide application prospect in the fields of photothermal therapy, photodynamic therapy and drug-loaded therapy.
The invention has NiPS with tumor targeting3The nano-drug also comprises a NiPS coating layer coated on the two-dimensional substrate3Triplet of nanosheet surfaceAnd (3) phenylphosphorus. NiPS3The nano-drug has stronger tumor cell targeting property by virtue of the active enrichment function of tumor cells. NiPS3The nanometer medicine is modified by triphenyl phosphorus (TPP) and has mitochondrion targeting property. The NiSP3 semiconductor nano-sheet is combined with triphenylphosphine through electrostatic interaction, and because three benzene rings of the triphenylphosphine form delocalized electrostatic charge, NiPS3 can be well mediated to target mitochondria, so that the mitochondrial targeting of tumors is improved. NiPS modified by triphenylphosphine and having tumor targeting function3After the nano-drug enters the tumor cells, NiPS3The nano material can target mitochondria more effectively, active oxygen, hydroxyl free radical and the like generated by the nano material under illumination better act on key organelles of tumors, and tumor cells are killed directly or apoptosis is induced.
Preferably, the two-dimensional NiPS3The length and width of the nano sheet are 10-500 nm, and the two-dimensional NiPS3The thickness of the nano sheet is 1-50 nm. The proper length and width dimensions can ensure that the biological activated carbon has a better passive enrichment effect on a tumor part in biological application, and simultaneously has a proper energy band structure, thereby being beneficial to generating a catalytic effect. In addition, the phenomenon that two-dimensional NiPS is caused by overlarge size can be avoided3The nano-sheet can not enter the tumor part, or the size is too small to cause two-dimensional NiPS3The nano-sheet is easy to leak from the tumor part. The selection of a thinner thickness increases the specific surface area and thus increases the photothermal effect and the loading rate.
The two-dimensional NiPS3The length and width of the nano sheet are 50-150 nm, and the two-dimensional NiPS3The thickness of the nanosheet is 1-35 nm.
More preferably, the two-dimensional NiPS3The length and width of the nano sheet are 70-100 nm, and the two-dimensional NiPS3The thickness of the nanosheet is 15-25 nm.
In a second aspect, the invention also provides a NiPS with tumor targeting3The preparation method of the nano-drug comprises the following steps:
providing two dimensional NiPS3Dissolving the nano-sheet and triphenylphosphine in absolute ethyl alcohol, performing ultrasonic treatment and centrifugation, and collectingAdding anhydrous ethanol solution of triphenylphosphine into the lower layer liquid, and performing ultrasonic dispersion to obtain NiPS with tumor targeting effect3A nanometer medicinal preparation.
The NiPS with tumor targeting provided by the second aspect of the invention3The nano-medicine has simple preparation method, strong controllability, rich raw materials and low production cost. NiPS with tumor targeting prepared by the method3The nano-drug can be applied to two-dimensional NiPS3The nano-sheet is better modified and coated, so that the targeting of the drug to tumor cells and mitochondria is effectively ensured.
Preferably, the two-dimensional NiPS3The mass ratio of the nanosheets to the triphenylphosphine is 1:1: 1;
the ultrasonic time is 1-10 h, the centrifugal rotating speed is 10000-15000 r/min, and the centrifugal time is 5-20 min.
Preferably, the two-dimensional NiPS3The nano-sheet is prepared by adopting an electrochemical stripping method;
the electrochemical stripping method comprises the following steps: providing NiPS3The crystal is used as a working electrode, a platinum electrode is used as a counter electrode, DMF (dimethyl formamide) solution of tetrabutylammonium tetrafluoroborate is used as electrolyte, the distance between the working electrode and the counter electrode is 1-5 cm, a static bias voltage of-2-5V is applied to the working electrode, and the concentration of the tetrabutylammonium tetrafluoroborate is 0.01-0.1M;
after stripping, the obtained suspension is oscillated and centrifuged, the supernatant part is filtered by a filter, washed by DMF solution and filtered to prepare the two-dimensional NiPS3Nanosheets.
Preferably, the distance between the working electrode and the counter electrode is 1.5cm, a static bias voltage of-3V is applied to the working electrode, and the concentration of the tetrabutylammonium tetrafluoroborate is 0.05M;
the filter is a nylon membrane filter, and the pore size of the nylon membrane filter is 0.45 mu m.
In a third aspect, the present invention also provides a NiPS with tumor targeting according to the first aspect of the present invention3The application of nano medicine in preparing photodynamic medicine is disclosed.
The invention has a tumor targetOriented NiPS3The application of the nano-medicine in preparing photodynamic therapy medicines has the advantages of obvious photodynamic effect, good biocompatibility, no obvious acute toxicity, obvious tumor cell targeted killing effect and the like, and has great market value and potential value as a novel nano-medicine.
Advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of embodiments of the invention.
Drawings
In order to more clearly illustrate the contents of the present invention, a detailed description thereof will be given below with reference to the accompanying drawings and specific embodiments.
FIG. 1 shows NiPS according to an embodiment of the present invention3A representation of the nanoplatelets;
FIG. 2 is a schematic diagram of the present invention for analyzing NiPS by using the supercomputing method3Schematic diagram of TPP adsorption and NiPS3-map of tumor cell enrichment effect of TPP;
FIG. 3 shows the results of the biosafety photodynamic therapy test of the present invention;
FIG. 4 shows the NiPS of the present invention3Results of experiments targeting mitochondria for photodynamic therapy;
FIG. 5 shows the NiPS of the present invention3And NiPS3Test results of photodynamic therapy of solid tumors in mice with TPP.
Detailed Description
While the following is a description of the preferred embodiments of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
In a first aspect, the present invention provides a NiPS with tumor targeting3Nano-drugs including two-dimensional NiPS3Nanosheets, and coating on two-dimensional NiPS3Triphenylphosphine on the surface of the nanosheets. Coating on two-dimensional NiPS3The triphenyl phosphine on the surface of the nano sheet can be directly reacted with two-dimensional NiPS3Nanosheet covalent connection and electrostatic attractionAnd bonding means such as adhesion and van der waals force.
In a specific embodiment of the invention, the triphenylphosphine is coated on the two-dimensional NiPS in an electrostatic adsorption manner3And (3) the surface of the nanosheet.
Preferably, two-dimensional NiPS3The length and width of the nano sheet are 10-500 nm, specifically 10nm, 50nm, 100nm, 150nm, 200nm, 300nm, 400nm or 500 nm. Two-dimensional NiPS3The thickness of the nano sheet is 1-50 nm. Specifically, the wavelength may be 1nm, 5nm, 10nm, 20nm, 30nm, 40nm or 50 nm.
Preferably, two-dimensional NiPS3The length and width of the nano sheet are 50-150 nm, specifically 50nm, 100nm and 150 nm. Two-dimensional NiPS3The thickness of the nano-sheet is 1-35 nm, specifically 1nm, 5nm, 10nm, 15nm, 20nm and 35 nm.
Preferably, two-dimensional NiPS3The length and width of the nano-sheet are 70nm, 80nm, 90nm or 100 nm. Two-dimensional NiPS3The thickness of the nanosheets is 15nm, 20nm or 25 nm.
In a second aspect, the invention also provides a NiPS with tumor targeting3The preparation method of the nano-drug comprises the following steps:
providing two dimensional NiPS3Dissolving the nanosheet and triphenylphosphine in absolute ethyl alcohol, collecting lower-layer liquid after ultrasonic treatment and centrifugation, adding an absolute ethyl alcohol solution of triphenylphosphine into the lower-layer liquid, and performing ultrasonic treatment to obtain NiPS with tumor targeting property3A nanometer medicinal preparation.
Preferably, in the step of preparing the single-targeting nanosystem, the two-dimensional NiPS is added3Dissolving the semiconductor nano-particles and the triphenyl phosphine in absolute ethyl alcohol according to the mass ratio of 1:1, carrying out ultrasonic treatment for 3 hours, then 12000r/min, and centrifuging for 10 min.
Preferably, after adding an absolute ethyl alcohol solution of triphenylphosphine into the lower layer liquid and carrying out ultrasonic treatment for 3h, NiPS with tumor targeting function is prepared3A nanometer medicinal preparation.
Preferably, two-dimensional NiPS3The nano-sheet is prepared by adopting an electrochemical stripping method.
Preferably, the electrochemical stripping method comprises the following steps: providing NiPS3The crystal is used as a working electrode, a platinum electrode is used as a counter electrode, DMF solution of tetrabutylammonium tetrafluoroborate is used as electrolyte, the distance between the working electrode and the counter electrode is 1-5 cm, a static bias voltage of-2-5V is applied to the working electrode, and the concentration of the tetrabutylammonium tetrafluoroborate is 0.01-0.1M;
after stripping, the obtained suspension is oscillated and centrifuged, the supernatant part is filtered by a filter, washed by DMF solution and filtered to prepare the two-dimensional NiPS3Nanosheets.
Preferably, the distance between the working electrode and the counter electrode is 1.5cm, a static bias voltage of-3V is applied to the working electrode, and the concentration of tetrabutylammonium tetrafluoroborate is 0.05M.
Preferably, the filter is a nylon membrane filter having a pore size of 0.45 μm.
Two-dimensional NiPS is illustrated by the specific examples below3Preparation method of nanosheet and prepared two-dimensional NiPS3Nanosheets.
Example 1
NiPS with tumor targeting function3Preparation method of nano-drug and NiPS with tumor targeting prepared by nano-drug3The nano medicine includes the following steps.
(1) Preparation of bulk NiPS3Material
Preparing large block NiPS by CVT method3And (4) crystals. High-purity nickel, phosphorus and sulfur powder (the total mass is approximately equal to 2g) with a stoichiometric molar ratio (1:1:3) is fully mixed and ground in a glove box. Then, the powder was sealed in a vacuum quartz tube (length of the vacuum quartz tube 25 cm, outer diameter 13mm, wall thickness 1 mm) at 10-5An oxygen/hydrogen torch using a partulab apparatus was used under a vacuum of pa.
Next, the sealed tube was placed in a two-stage furnace. Then, the reaction zone and the growth zone were set to 700 ℃ and 650 ℃ at a heating rate of 1 ℃ min-1And the corresponding temperature was maintained for 7 days to generate a temperature gradient for volume crystal growth. Finally, naturally cooling the double-section heating furnace to room temperature, and collecting large blocks of NiPS3And (4) crystals.
(2) Electrochemical stripping preparation of two dimensionsNiPS3Nano-sheet
Electrochemical exfoliation of bulk NiPS3 crystals was performed using an electrochemical workstation. The bulk NiPS3 crystal obtained was sandwiched in a Pt clamp and used as the working electrode. A Pt paper (10 mm in length and 10mm in width) electrode as a counter electrode, mounted on a NiPS substrate3The crystal is about 1.5 cm. A DMF solution (60mL) containing 0.05M tetrabutylammonium tetrafluoroborate was used as an electrolyte. By applying a-3V static bias voltage to the working electrode, a bulk NiPS is achieved3And (4) electrochemical stripping of the crystal.
After the peeling process was completed, the resulting suspension was manually shaken for about 20s and then centrifuged to separate out undepeeled NiPS 3. Dispersing part of the top liquid in a nylon membrane filter, filtering (Agela, pore diameter of 0.45 μm), washing nylon membrane with DMF solution, filtering to remove residual salt, and collecting two-dimensional NiPS3Nanosheets.
Two-dimensional NiPS prepared in example 13Characterization of the nanoplates
As shown in the electron micrograph of FIG. 1A, the two-dimensional NiPS prepared in example 13The length and width of the nano sheet is 10-500 nm. As shown in FIG. 1B, for NiPS3Lattice analysis of (3), NiPS3Particle triclinic unit
Figure BDA0002501108270000061
Figure BDA0002501108270000062
Each atom forms an octahedron, S and P atoms are completely exposed, and in this model, Ni ions are fixed to [ P ]2S6]4-On the frame, the metal layers are sandwiched by distorted octahedral S layers, which are superimposed together by van der Waals forces to form a two-dimensional structure. This is an ideal catalytic hydrolysis structure.
As shown in FIGS. 1C and D, STEM-BF and STEM-HAADF image analysis showed that the two-dimensional NiPS prepared in example 13The nano-sheet has regular and complete crystal lattices, and shows that the material prepared by the method is excellent and has excellent light absorption performance.
As shown in FIG. 1E, TEM-EELS element analysis proves that prepared two-dimensional NiPS3The nanosheet material has three elements of Ni, P, S, consistent with assumptions.
As shown in FIG. 1F (NiPS from top to bottom respectively)3Crystalline, two-dimensional NiPS3Nanosheets and standard), X-ray diffraction analysis showed that NiPS3X-ray diffraction peaks and ultra-thin NiPS of nanoplatelets3The X-ray diffraction peaks of the nanoplatelets follow the standard XRD spectrum (JCPDS #01-78-0499) and no additional peaks are found. The results show that the method successfully synthesizes pure NiPS3No impurities are introduced during the electrochemical stripping process.
Two-dimensional NiPS prepared in example 13And (5) performing light absorption and catalytic verification on the nanosheets.
Ultraviolet visible spectrophotometer analysis: UV-Vis DRS UV-Vis spectral analysis by Hitachi U-3010, two-dimensional NiPS as shown in FIG. 1G3The nano-sheet absorbs in the visible light and near infrared range, the optimal absorption wavelength is 650nm, and the nano-sheet is suitable for the photocatalytic activity of PDT.
ESR analysis active oxygen analysis: the ESR test is mainly directed to OH analysis. The OH was detected and the whole test was carried out in aqueous solution.
First, 5mg of NiPS was weighed3Ultrasonic dispersion in 5ml water for 20 min. Argon gas was then bubbled through the solution for 20 minutes to remove oxygen from the solution. DMPO (100mM) solution (also with argon to remove oxygen) was used for the experiment. The 660nm NIR was used to detect OH formation in water and DMPO solutions. At the same time, the same experiment was also carried out in the presence of oxygen. As shown in FIG. 1H, the results indicate that NiPS is present in both the normoxic and anaerobic conditions3Can generate a large amount of OH.
Anoxic condition NiPS3ESR pattern in the dark and under illumination at 660nm for 5 min. As shown in FIG. 1I, the results confirm that NiPS3Separation of the mesoelectron-hole pairs and evidence for the presence of. OH. The peak signal intensity of the illumination is significantly stronger than the peak of the same sample in the dark, indicating the presence of a hole. The results demonstrate that NiPS3The nano material can be applied to photodynamic therapy under the anoxic condition.
NiPS with tumor targeting is specifically illustrated below by example 23Preparation method of nano-drug and NiPS with tumor targeting property prepared by preparation method3Nano medicine
Example 2
NiPS with tumor targeting function and capable of performing photodynamic tumor treatment under anaerobic condition3The preparation method of the nano-drug comprises the following steps:
(1) the prepared NiPS is added3Dissolving the semiconductor nano-sheet and the triphenylphosphine in absolute ethyl alcohol according to the mass ratio of 1:1, performing ultrasonic treatment for 3 hours, centrifuging at 12000r/min for 10 minutes, removing the supernatant, and collecting the lower-layer liquid.
(2) Providing TPP according to the mass ratio of NiPS3 to TPP of 1:1, dissolving the TPP in absolute ethyl alcohol, and fully dissolving under ultrasonic conditions.
(3) Adding the bottom liquid part of the collected bottom layer into the TPP absolute ethyl alcohol solution in the step (2), and continuing to perform ultrasonic treatment to form NiPS3TPP targeting nano system (NiPS for short in subsequent experiments)3TPP), i.e.NiPS with tumor targeting according to the invention3A nanometer medicinal preparation.
2A-D, are schematic atomic structure diagrams and electron clouds of Triphenylphosphine (TPP) and NiPS3 nanosheets adsorbing each other. Triphenylphosphine (TPP) mediates targeting of the nanoplatelets to mitochondria through delocalized electron action of three benzene rings. As shown in FIG. 2E, FIG. 2E verifies that NiPS has been modified by thermal imaging3TPP to NiPS3Better enrichment at the tumor site.
Effects of the embodiment
Effect example 1: NiPS with tumor targeting prepared in example 23And (5) carrying out in-vitro toxicity verification on the nano-drug.
NiPS3In vitro toxicity verification of liver cancer Huh-7 cells: huh-7 cells were cultured in DMEM cell culture medium containing 10% fetal bovine serum and placed in an incubator at 37 ℃ with 5% CO 2. The cells grown logarithmically were cultured in 96-well plates to a density of 80% and then NiPS at different concentrations3Incubation, NiPS3And (5) toxicity test.
(1) First useDifferent concentrations of unmodified NiPS3(12.5ppm, 50ppm, 100ppm, 200ppm) was incubated with Huh-7 for 12 and 24 hours, and then CCK-8 was used to test Huh-7 cell viability. The procedure does not use 660nm for photodynamic therapy. As shown in FIG. 3A (12 h, 24h for each group from left to right), the results show that NiPS is present3The nanometer material has little influence on the activity of the cells, which shows that NiPS3 has better biological safety.
(2) With different concentrations of NiPS3(12.5ppm, 25ppm, 50ppm, 100ppm and 200ppm) and Huh-7 cells were incubated for 6h, followed by photodynamic therapy with 660nm light at a light intensity of 0.3 W.cm-2And 10 min. As shown in FIG. 3B (each group is 12h, 24h, 48h, 72h from left to right), the results showed that the cell activity of all experimental groups was significantly reduced after photodynamic therapy, and NiPS was observed to increase with time3Increased concentration and decreased cellular activity, indicating that NiPS3 photodynamic therapy has a time and concentration dependence on tumor killing.
Effect example 2: verification of NiPS3Biosafety and photodynamic effects of TPP
The targeted system proved to kill cells as a result of photodynamic rather than toxic material itself. We have performed AO/PI experiments, Acridine Orange (AO) can pass through living cell membrane, DNA and RNA stain green fluorescence; propidium Iodide (PI) can only stain dead cells to red fluorescence. As shown in fig. 3C and D, the same dish had illuminated and non-illuminated areas, with only a few dead cells in the non-illuminated area, and almost all cells dead in the illuminated area. Because the culture environment is completely the same in the same culture dish, the only difference is whether the photodynamic therapy is carried out. The result proves that after TPP modification, the NiPS of the targeting system3TPP has perfect biological safety and obvious photodynamic treatment effect, and simultaneously has ideal photodynamic performance.
Effect example 3: NiPS3Detection of active oxygen production by TPP
To detect NiPS3Photodynamic effects of TPP, we used dichlorofluorescein diacetate (DCFH-DA) as a fluorescent probe to assess intracellular ROS levels. As shown in fig. 3E and FHuh-7 cells were seeded into 96-well culture plates and placed in 1% O2Cultured in an incubator at 37 ℃ to 80% confluence, and then 100. mu.L of DMEM medium (NiPS) was added3-TPP，NiPS3Equivalent 100ppm), 6 hours later, after adding DCFH-DA fluorescent solution for 30 minutes, the cells were exposed to NIR at 660nm at 0.3W cm-2And 10 min. The remaining material was then immediately washed thoroughly with PBS and washed three times in succession. Cells were then immediately photographed fluorescently with a laser Confocal (CLSM). As shown in FIGS. 3E and F, the results indicate that NiPS3Under the anoxic condition of TPP, a large amount of active oxygen can be generated by NIR excitation at 660nm, and NiPS is extracted3The generated active oxygen can be independent of oxygen in the environment, so that the problem that the tumor microenvironment is lack of oxygen and the photodynamic therapy cannot be carried out can be solved, and the volume basis of the clinical application of the photodynamic therapy is provided.
Effect example 4: NiPS3Effect of TPP photodynamic therapy on mitochondrial Membrane potential (. DELTA.. psi.m)
Cells were seeded in 24-well plates at a density of 5X 105Incubate to 80% density per well. With 100ppm of NiPS3Incubation of TPP nanoplates for 6 hours with 660nm NIR at 0.3W cm-2Followed by irradiation for 10 minutes. After 6 hours, the cells were washed with PBS and evaluated according to the instructions of the mitochondrial membrane potential detection kit (JC-1). Flow cytometry detects mitochondrial membrane potential. Respectively detecting the normal oxygen condition and the oxygen deficiency condition. As shown in FIG. 4, where A and B were run under normoxic conditions and C and D were run under anoxic conditions (1% O)2). A and C were not illuminated, and B and D were photodynamic treated. The result shows that the NiPS3 modified by Triphenylphosphine (TPP) can obviously reduce the mitochondrial membrane potential when being subjected to photodynamic therapy, and the modified NiPS is proved to have the same illumination intensity3TPP to NiPS3Better targeting to tumor cells. Also, since mitochondrial membrane potential is an irreversible event in apoptosis, NiPS is suggested3TPP can well solve the problem of photodynamic therapy under the condition of oxygen deficiency.
Effect example 5: NiPS3Effect of TPP photodynamic therapy on apoptosis
Cells were seeded in 24-well plates and sealedDegree of 5X 105Incubate to 80% density per well. With 100ppm of NiPS3-TPP nanomaterials incubated for 6 hours with 660nm NIR at 0.3W cm-2And 10 min. After 12h, cells were washed with PBS and evaluated according to the instructions of the early and late apoptosis detection kit (Annexin V-FITC). And detecting the apoptosis rate by using a flow cytometer. As shown in FIG. 4, where E and F were run under normoxic conditions and G and H were run under anoxic conditions (1% O)2) (ii) a E and G were not illuminated, and F and H were photodynamic treated. The results show that the NiPS modified by Triphenylphosphine (TPP)3The photodynamic therapy can obviously cause cell apoptosis. The results are consistent with the results of the mitochondrial membrane potential experiments, indicating that NiPS3TPP can well solve the problem of photodynamic therapy under the condition of oxygen deficiency.
Effect example 6: animal experiments
Female Balb/c (15-20g) was purchased from the institute for model animals, university of Nanjing, and all mouse experiments were performed according to the national regulations on Care and use of laboratory animals. First, human hepatoma cell line Huh-7 cells were injected into the right anterior axilla of each female mouse for tumor modeling. When the tumor diameter is 4-6 mm, tumor-bearing mice are randomly divided into six groups (n is 5. the 1 st group of normal saline treatment group, the second group of pure 660nm photodynamic treatment group and the third group of two-dimensional NiPS3Nano sheet (NiPS for short in the following)3) Treatment, group IV NiPS3+660nm photodynamic therapy, NiPS3-TPP in the fifth group and NiPS3-TPP +660nm photodynamic therapy in the sixth group respectively. The injection dose is 3 mg/kg. After 10h of injection, the injection time is 0.3 W.cm-2660nm for 10 minutes. Notably, each mouse was photodynamic treated every 2d three times consecutively, and mouse tumor volume and body weight were recorded every 2d until the end of the experiment. The vertical diameter of the tumor was measured with a caliper and the growth of the tumor was observed. Tumor volume (mm)3) 0.5 × length × width.
As shown in FIG. 5A, NiPS3Does not cause damage or inflammation of organs such as heart, liver, spleen, lung, kidney and the like. In addition, as shown in FIG. 5B (the curves corresponding to 14 days are, from top to bottom, the third group, the sixth group, the first group, the fifth group and the fourth groupGroup II), NiPS3The body weight of the treated nude mice was almost the same as that of the untreated control group. Indicating that NiPS3It is a promising biocompatible photosensitizer with little toxicity at the tested doses, and can be used in vivo biomedical applications. Thus, we evaluated NiPS3Photodynamic effects of TPP targeting systems on solid tumors. As shown in FIG. 5C (the curves corresponding to 14 days are from top to bottom, respectively, the second group, the first group, the fifth group, the third group, the fourth group and the sixth group) and D, the nano NiPS3Tumor volume in mice after TPP targeting system and photodynamic therapy was significantly smaller than in untreated group. In particular, NiPS group six3TPP +660nm photodynamic therapy, two mice had completely disappeared tumors and were much smaller in volume than the other groups. Of note, NiPS of the fourth group3Compared with the +660nm photodynamic therapy, the method has significant difference, thereby indicating that the targeting system modified by TPP is more effective than NiPS without target modification3The photodynamic effect of the treatment is much better, so that the TPP is proved to play a role in targeted enhancement of NiPS3The function of (1).
We developed a NiPS3TPP semiconductor photosensitizers, which can generate ROS independently of oxygen and selectively target mitochondria in tumor cells through TPP. The design combines the photocatalysis and targeting ability independent of oxygen into a simple nano system, thereby providing a theoretical basis for developing photodynamic therapy means applied to clinic. NiPS3TPP produces a cavitation effect through an efficient band transition, producing high levels of OH under hypoxic conditions, achieving good photodynamic therapy, while triggering mitochondrial apoptotic pathways. In the liver cancer model, based on NiPS3Systemic photodynamic therapy significantly inhibited tumor growth without affecting normal tissues. Thus, NiPS3Is a promising next-generation photosensitizer, NiPS3TPP is a nano-Zengmin system which can target mitochondria to perform hypoxic photodynamic therapy.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (9)

1. NiPS with tumor targeting function3The nano-drug is characterized by comprising two-dimensional NiPS3Nanosheets, and coating on the two-dimensional NiPS3Triphenylphosphine on the surface of the nanosheets.
2. NiPS with tumor targeting according to claim 13A nano-drug characterized in that said two-dimensional NiPS3The length and width of the nano sheet are 10-500 nm, and the two-dimensional NiPS3The thickness of the nano sheet is 1-50 nm.
3. NiPS with tumor targeting according to claim 23A nano-drug characterized in that said two-dimensional NiPS3The length and width of the nano sheet are 50-150 nm, and the two-dimensional NiPS3The thickness of the nanosheet is 1-35 nm.
4. NiPS with tumor targeting according to claim 33A nano-drug characterized in that said two-dimensional NiPS3The length and width of the nano sheet are 70-100 nm, and the two-dimensional NiPS3The thickness of the nanosheet is 15-25 nm.
5. NiPS with tumor targeting function3The preparation method of the nano-drug is characterized by comprising the following steps:
providing two dimensional NiPS3Dissolving the nanosheet and triphenylphosphine in absolute ethyl alcohol, collecting lower-layer liquid after ultrasonic treatment and centrifugation, adding an absolute ethyl alcohol solution of triphenylphosphine into the lower-layer liquid, and performing ultrasonic treatment to obtain NiPS with tumor targeting property3A nanometer medicinal preparation.
6. NiPS with tumor targeting according to claim 53The preparation method of the nano-drug is characterized in that the two-dimensional NiPS is3The mass ratio of the nanosheets to the triphenylphosphine is 1: 1;
the ultrasonic time is 1-10 h, the centrifugal rotating speed is 10000-15000 r/min, and the centrifugal time is 5-20 min.
7. NiPS with tumor targeting according to claim 53The preparation method of the nano-drug is characterized in that the two-dimensional NiPS is3The nano-sheet is prepared by adopting an electrochemical stripping method;
the electrochemical stripping method comprises the following steps: providing NiPS3The crystal is used as a working electrode, a platinum electrode is used as a counter electrode, DMF (dimethyl formamide) solution of tetrabutylammonium tetrafluoroborate is used as electrolyte, the distance between the working electrode and the counter electrode is 1-5 cm, a static bias voltage of-2-5V is applied to the working electrode, and the concentration of the tetrabutylammonium tetrafluoroborate is 0.01-0.1M;
after stripping, the obtained suspension is oscillated and centrifuged, the supernatant part is filtered by a filter, washed by DMF solution and filtered to prepare the two-dimensional NiPS3Nanosheets.
8. NiPS with tumor targeting according to claim 73The preparation method of the nano-drug is characterized in that the distance between the working electrode and the counter electrode is 1.5cm, a static bias voltage of-3V is applied to the working electrode, and the concentration of the tetrabutylammonium tetrafluoroborate is 0.05M;
the filter is a nylon membrane filter, and the pore size of the nylon membrane filter is 0.45 mu m.
9. NiPS with tumor targeting according to any of claims 1-43The application of nano medicine in preparing photodynamic medicine is disclosed.
CN202010432599.1A 2020-05-20 2020-05-20 NiPS with tumor targeting function3Nano medicine and its preparing method and use Withdrawn CN112057615A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114836766A (en) * 2022-04-12 2022-08-02 西安交通大学 MoS 2 /NiPS 3 Composite heterostructure nanosheet electrocatalyst and preparation method and application thereof
CN114890385A (en) * 2021-07-01 2022-08-12 中国科学院上海硅酸盐研究所 Efficient antioxidant two-dimensional hydrogermanium alkene nanosheet and preparation method and application thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114890385A (en) * 2021-07-01 2022-08-12 中国科学院上海硅酸盐研究所 Efficient antioxidant two-dimensional hydrogermanium alkene nanosheet and preparation method and application thereof
CN114890385B (en) * 2021-07-01 2023-09-08 中国科学院上海硅酸盐研究所 Efficient antioxidant two-dimensional hydrogen germanium alkene nano-sheet and preparation method and application thereof
CN114836766A (en) * 2022-04-12 2022-08-02 西安交通大学 MoS 2 /NiPS 3 Composite heterostructure nanosheet electrocatalyst and preparation method and application thereof
CN114836766B (en) * 2022-04-12 2024-04-02 西安交通大学 MoS (MoS) 2 /NiPS 3 Composite heterostructure nanosheet electrocatalyst and preparation method and application thereof

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