CN111317819A - Application of novel material based on carbon nano tube and phycoerythrin - Google Patents
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Abstract
The invention discloses application of a novel material based on carbon nano tubes and phycoerythrin, namely the novel material is used as a photosensitizer for phototherapy tumor treatment. The chemical inertia of the single-walled carbon nanotube can be overcome, the light conversion efficiency of the single-walled carbon nanotube is improved, meanwhile, the stability of phycoerythrin is improved after the single-walled carbon nanotube and the phycoerythrin are connected, and the effect is more durable. The novel material not only has good stability, but also has excellent effect of treating cancers as a photosensitizer with photothermal effect for phototherapy cancer treatment.
Description
Technical Field
The invention belongs to the technical field of new materials, and particularly relates to application of a novel material based on carbon nanotubes and phycoerythrin.
Background
The single-walled carbon nanotube may be considered as an extension of a fullerene molecule having a cage structure in a one-dimensional direction, or may be considered as a closed tubular structure formed by rolling a graphite layer. The related drugs of the single-walled carbon nanotube firstly pass through the human circulation and metabolic system when entering the organism, and can reach the designated tissue part smoothly. Because the single-walled carbon nanotube is chemically inert and hardly reacts with other molecules, the modified single-walled carbon nanotube can be used as a carrier for coupling and transporting various biomolecules such as drugs, proteins, DNA, RNA and the like, and the biomolecules enter cells through endocytosis to obtain a better effect. Meanwhile, the carbon nano tube as an excellent photo-thermal material has stronger absorption in a near-infrared light region, and is suitable for being used as an effective component of a photo-thermal coupling agent. The carbon nano tube is activated by light with specific wavelength, so that light energy can be effectively converted into heat energy, space-time controllability is realized, local high temperature can be generated aiming at malignant tumor cells, necrosis or apoptosis of the malignant tumor cells is caused, and local photo-thermal killing on the malignant tumor cells is realized.
Phycobiliproteins are light harvesting phycobiliproteins with covalently linked open chain tetrapyrrole structures present in blue algae, red algae, cryptophyceae and a few dinoflagellates; the nanometer phycobiliprotein can transfer the captured light energy to the light reaction center in high efficiency, and is one kind of oligomer water soluble pigment protein with reduced carbon nanotube toxicity. Phycobiliproteins mainly include phycocyanin and phycoerythrin. Phycoerythrin is phycobiliprotein widely distributed in nature, and is abundant in most red algae species growing in natural environment, especially laver, a common species. The phycoerythrin containing red phycoerythrobilin can absorb visible light in the range from 500nm to 600nm and intensively emit red fluorescence in the range from 535nm to 615 nm.
In clinic, photodynamic therapy is used as a novel tumor therapy, which generates free radicals and active oxygen by means of photosensitizer enriched in a focal region after illumination, and carries out oxidative killing on tumor tissues. Therefore, screening of photosensitizer with high efficiency, low toxicity and good selectivity is the core of photodynamic therapy. Phycocyanin extracted from marine blue algae has been used for photodynamic therapy of skin cancer and other tumors abroad, has value of further popularization and application, and has been approved by the United states FDA for photodynamic therapy of cancer. Meanwhile, in Chinese patent publication No. CN1091976A and Chinese patent publication No. CN1325729A, phycocyanin and phycoerythrin are disclosed as photosensitizers for treating cancers. Researches on phycocyanin and phycoerythrin also show that the phycocyanin and phycoerythrin can cause tumor cell apoptosis through active oxygen substances generated in the process of photodynamic reaction, and meanwhile, the phycocyanin and the phycoerythrin also have the effect of inhibiting tumor growth, so that the aim of treating tumors can be achieved through the combined action in many aspects. However, phycocyanin has a less stable structure and a weak photosensitizing effect, and thus, the application of phycocyanin as a photosensitizer is greatly limited. Therefore, the novel high-efficiency novel biomedical material with photodynamic therapy prospect has important significance in treating tumor cells, particularly skin-related tumors.
Disclosure of Invention
The invention aims to provide a new application of a novel material based on carbon nano tubes and phycoerythrin, which improves the stability of the phycoerythrin and increases the photothermal effect of the phycoerythrin through the covalent combination of the carbon nano tubes and the phycoerythrin, and the phycoerythrin can reduce the toxicity of the carbon nano tubes and is used as a photosensitizer for treating tumors by phototherapy to realize the photosensitive photothermal dual effect.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of the novel material based on the carbon nano tube and the phycoerythrin is used as a photosensitizer for phototherapy tumor treatment, and the dual effects of light sensitivity and light heat are realized.
The novel material is formed by covalently connecting a single-walled carbon nanotube and phycoerythrin, and is prepared by the following method:
(1) preparing a carboxylated single-walled carbon nanotube dispersion liquid; preferably deionized water, and the prepared concentration is 1 mg/mL;
(2) ultrasonically dispersing the dispersion liquid obtained in the step (1) for 15-30 min, respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxy thiosuccinimide solution dissolved in PBS buffer solution, and magnetically stirring at room temperature for 8-24 h; the weight ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the carboxylated single-walled carbon nanotubes is 9-18: 1, and the weight ratio of the N-hydroxythiosuccinimide to the carboxylated single-walled carbon nanotubes is 5-10: 1; the PBS buffer is 50mM sodium phosphate buffer with pH of 7.0;
(3) then adding phycoerythrin into the reaction solution, and placing the mixture at room temperature for magnetic stirring for 8-24 h; the weight ratio of the phycoerythrin to the carboxylated single-wall carbon nanotube is 10-20: 1;
(4) and (3) removing redundant EDC, NHS and unconjugated phycoerythrin by a dialysis method at room temperature to obtain the sample.
Preferably, the room temperature is 20-25 ℃. When excessive EDC, NHS and unconjugated phycoerythrin were removed by dialysis, the dialysis time was 24 h.
The invention has the following advantages:
1. the phycoerythrin and the carboxylated carbon nanotube are directly connected, and the connection mode of the phycoerythrin and the carboxylated carbon nanotube is covalent connection, so that the phycoerythrin and the carboxylated carbon nanotube have stronger stability compared with non-covalent connection. Thereby the structure of the phycoerythrin connected with the light source is more stable and has better phototherapy effect.
2. The invention discloses an application of a single-walled carbon nanotube-phycoerythrin composite material as a photosensitizer for treating tumors, wherein the chemical inertness of the single-walled carbon nanotube is utilized to improve the light conversion efficiency and stability of phycoerythrin, and meanwhile, the single-walled carbon nanotube can be activated by light with specific wavelength after the single-walled carbon nanotube and the phycoerythrin are connected, and the photo-thermal effect is generated at the same time, so that the effect is more durable.
3. The novel material not only has good stability, but also has good effect of treating cancer as a photosensitizer for phototherapy for treating cancer.
Drawings
FIG. 1 is a schematic diagram of the reaction and linkage relationship between carboxylated single-walled carbon nanotubes and phycoerythrin according to the present invention;
FIG. 2 is a confocal laser microscopy image of carboxylated single-walled carbon nanotube-phycoerythrin covalent bonding material according to the present invention;
FIG. 3 is a scanning electron microscope image of a blank carboxylated single-walled carbon nanotube;
FIG. 4 is a scanning electron microscope image of a carboxylated single-walled carbon nanotube-phycoerythrin covalent attachment material in accordance with the present invention;
FIG. 5 is a Fourier infrared spectrum of a carboxylated single-walled carbon nanotube;
FIG. 6 is a Fourier infrared spectrum of a carboxylated single-walled carbon nanotube-phycoerythrin covalent attachment material according to the present invention;
FIG. 7 is a diagram of the effect of the new material of the present invention on temperature increase under laser irradiation and a confocal laser microscope image of the interaction with tumor cells;
FIG. 8 is a MTT detection map of the new material of the present invention showing both photodynamic and photothermal effects;
FIG. 9 is a graph showing the active oxygen generation performance (RNO) of the novel material of the present invention.
Detailed Description
The present invention will be described in detail below with reference to specific examples. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. The description which follows is a preferred embodiment of the invention, but is made for the purpose of illustrating the general principles of the invention and not for the purpose of limiting the scope of the invention. The scope of the present invention is defined by the appended claims.
Unless otherwise specified, the various processes employed in the present invention are conventional and various materials and reagents are commercially available.
Before the following experiments, the invention shows that the purity of the phycoerythrin is high by detecting the purity of the phycoerythrin through non-denatured protein electrophoresis.
Embodiment 1 a method for preparing a novel material based on carbon nanotube-coupled phycoerythrin, comprising the steps of:
(1) preparing 1mg/mL carboxylated single-walled carbon nanotube dispersion liquid by deionized water, diluting 1mL carboxylated single-walled carbon nanotube dispersion liquid by 50 times, taking 10mL, and firstly carrying out ultrasonic treatment for 90 min;
(2) adding 0.2mL of a 9mg/mL 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) solution dissolved in PBS buffer and 0.2mL of a 5mg/mL N-hydroxythiosuccinimide (NHS) solution dissolved in PBS buffer (the specific weight ratios of the EDC and the NHS to the carboxylated single-walled carbon nanotubes are added at 9: 1 and 5: 1) to the dispersion, ultrasonically dispersing for 30min, and magnetically stirring at room temperature (25 ℃) for 18 h; the PBS buffer solution is a 50mM sodium phosphate buffer solution with the pH value of 7.0;
(3) then adding 4mg of phycoerythrin into the reaction solution, adding 20mg of phycoerythrin into each 1mg of carbon nano tube, and placing the mixture at room temperature for magnetic stirring for 24 hours;
(4) the sample was dialyzed at room temperature to remove excess EDC, NHS and unconjugated phycoerythrin for 24 h.
Embodiment 2 a method for preparing a novel material based on carbon nanotube-coupled phycoerythrin, comprising the steps of:
(1) preparing 1mg/mL carboxylated single-walled carbon nanotube dispersion liquid by deionized water, diluting 1mL solution of the carboxylated single-walled carbon nanotube dispersion liquid by 20 times, taking 10mL solution, and firstly performing ultrasonic treatment for 90 min;
(2) adding 0.5mL of a 9mg/mL 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) solution dispersed in PBS buffer and 0.5mL of a 5mg/mL N-hydroxythiosuccinimide (NHS) solution dispersed in PBS buffer (specific weight ratios of the EDC and the NHS to the single-walled carbon nanotube of 9: 1 and 5: 1 are added), ultrasonically dispersing for 30min, and magnetically stirring at room temperature (25 ℃) for 18 h; the PBS buffer solution is a 50mM sodium phosphate buffer solution with the pH value of 7.0;
(3) then adding 10mg of phycoerythrin into the reaction solution, adding 20mg of phycoerythrin into each 1mg of carbon nano tube, and placing the mixture at room temperature for magnetic stirring for 24 hours;
(4) the sample was dialyzed at room temperature to remove excess EDC, NHS and unconjugated phycoerythrin for 24 h. (the dialysate was changed once in the first 30 minutes and gradually extended thereafter).
Embodiment 3 a method for preparing a novel material based on carbon nanotube-coupled phycoerythrin, comprising the steps of:
(1) preparing 1mg/mL carboxylated single-walled carbon nanotube dispersion liquid by deionized water, diluting 1mL carboxylated single-walled carbon nanotube dispersion liquid by 10 times, and performing ultrasonic treatment on 10mL of carboxylated single-walled carbon nanotube dispersion liquid for 90 min;
(2) adding 1mL of a 9mg/mL 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) solution dissolved in a PBS buffer solution and 1mL of a 9mg/mL N-hydroxythiosuccinimide (NHS) solution dissolved in the PBS buffer solution (the specific weight ratio of the added EDC and NHS to the single-walled carbon nanotube is 9: 1 and 5: 1) into the dispersion solution, ultrasonically dispersing for 30min, and magnetically stirring at room temperature (25 ℃) for 18 h; the PBS buffer solution is a 50mM sodium phosphate buffer solution with the pH value of 7.0;
(3) then adding 15mg of phycoerythrin into the reaction solution, adding 15mg of phycoerythrin into each 1mg of carbon nano tube, and placing the mixture at room temperature for magnetic stirring for 24 hours;
(4) the sample was dialyzed at room temperature to remove excess EDC, NHS and unconjugated phycoerythrin for 24 h.
Embodiment 4 a method for preparing a novel material based on carbon nanotube-coupled phycoerythrin, comprising the steps of:
(1) preparing 1mg/mL carboxylated single-walled carbon nanotube dispersion liquid by deionized water, diluting 1mL carboxylated single-walled carbon nanotube dispersion liquid by 20 times, taking 10mL, and firstly performing ultrasonic treatment for 90 min;
(2) adding 1mL of a 9mg/mL 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) solution dissolved in a PBS buffer solution and 1mL of a 9mg/mL N-hydroxythiosuccinimide (NHS) solution dissolved in the PBS buffer solution (the specific weight ratio of the added EDC and NHS to the single-walled carbon nanotube is 18: 1 and 10: 1) into the dispersion solution, ultrasonically dispersing for 30min, and magnetically stirring at room temperature (25 ℃) for 18 h; the PBS buffer solution is a 50mM sodium phosphate buffer solution with the pH value of 7.0;
(3) then adding 5mg of phycoerythrin into the reaction solution, adding 10mg of phycoerythrin into each 1mg of carbon nano tube, and placing the mixture at room temperature for magnetic stirring for 24 hours;
(4) the sample was dialyzed at room temperature to remove excess EDC, NHS and unconjugated phycoerythrin for 24 h. (the dialysate was changed once in the first 30 minutes and gradually extended thereafter).
Embodiment 5 a method for preparing a novel material based on carbon nanotube-coupled phycoerythrin, comprising the steps of:
(1) preparing 1mg/mL carboxylated single-walled carbon nanotube dispersion liquid by deionized water, diluting 1mL carboxylated single-walled carbon nanotube dispersion liquid by 20 times, taking 10mL, and firstly performing ultrasonic treatment for 90 min;
(2) to the above dispersion was added 0.7mL of a 9mg/mL solution of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) dissolved in PBS buffer and 0.7mL of a 5mg/mL solution of N-hydroxythiosuccinimide (NHS) dissolved in PBS buffer (specific weight ratios of EDC and NHS to single-walled carbon nanotubes added of 12.5: 1 and 7: 1) and ultrasonically dispersed for 30min and placed under magnetic stirring at room temperature (25 ℃) for 18 h; the PBS buffer solution is a 50mM sodium phosphate buffer solution with the pH value of 7.0;
(3) then adding 5mg of phycoerythrin into the reaction solution, adding 10mg of phycoerythrin into each 1mg of carbon nano tube, and placing the mixture at room temperature for magnetic stirring for 24 hours;
(4) the sample was dialyzed at room temperature to remove excess EDC, NHS and unconjugated phycoerythrin for 24 h. (the dialysate was changed once in the first 30 minutes and gradually extended thereafter).
FIG. 1 shows a schematic diagram of the reaction and connection relationship between single-walled carbon nanotube and phycoerythrin, and then the novel material synthesized by the method is detected by a laser confocal microscope, a scanning electron microscope and a Fourier infrared spectrum. Comparing laser confocal microscope pictures before and after loading protein, the pure single-walled carbon nanotube does not show red fluorescence, and the carbon nanotube (the invention) loaded with phycoerythrin shows strong red fluorescence (figure 2); the subsequent high resolution scanning electron microscope picture also shows that obvious attachments are formed on the surface of the original smooth carbon nano tube, the diameter of the single-walled carbon nano tube without the loaded protein is about 11-12 nanometers, and the loaded proteinThe increase of the rear diameter is about 18-19 nanometers (figures 3 and 4); fourier-infrared spectroscopy further showed a very significant difference between the two (fig. 5 and 6). The IR spectrum of the pure carboxylated single-walled carbon nanotube shown in FIG. 5 shows 1629cm-1The absorption peak at (b) corresponds to the stretching vibration of the C ═ O double bond in the carboxyl group (-COOH), which is attributed to the presence of carboxylate group (1582 cm) on the surface of carbon nanotube-1) C-OH bond (1078 cm)-1) And the like oxygen-containing functional groups. FIG. 6 shows the new appearance of the infrared spectrum of the new material (according to the invention) after loading with phycoerythrin at 3274cm-1Absorption peak of N-H stretching vibration of amide bond, and 1536cm of C-N stretching vibration and N-H in-plane bending vibration representing amide II and amide III-1And 1396cm-1Absorption peak. And 1632cm-1The absorption peak of (A) is the C ═ O stretching vibration of the amide I, is basically consistent with the corresponding absorption peak of the pure carboxylated single-walled carbon nanotube, and is only 3cm-1Is moved. This is caused by the interaction of phycoerythrin with the surface of the carbon nanotube. These absorption peaks are characteristic absorption peaks of protein molecules, and their appearance indicates that phycoerythrin molecules are successfully covalently coupled on the surface of the single-wall carbon nanotube.
Example 6
Use of novel materials based on carbon nanotubes and phycoerythrin as photosensitizers for phototherapy of neoplasia. The inventors have conducted the following tests thereon:
(1) dark toxicity and phototoxicity assay for in vitro tumor cells
To measure phototoxicity of various materials, RAW264 tumor cells were cultured in culture media containing 10% fetal bovine serum and 1% penicillin-streptomycin, respectively, under conditions of 37 ℃ and 5% carbon dioxide atmosphere in an incubator, and the RAW264 tumor cells were seeded into 96-well cell plates, each well being 2 × 104The cells were cultured at 37 ℃ for 24 hours. Solutions of novel materials based on single-walled carbon nanotubes and phycoerythrin at a range of concentrations (0-40 μ g mL)-1Phycoerythrin equivalents) were treated separately and incubated for 24 hours. After washing the cells three times with PBS, the cells were cultured in fresh medium. Irradiating two kinds of cells with 580nm laser and 808nm laser for 10min, and performing in vitroPhotodynamic and photothermal combined therapy experiments. To verify the synergistic therapeutic effect, cell viability was measured using the MTT assay and calculated using the following equation:
Cell Viability[％]＝(ODtreated/ODcontrol)×100％
fig. 7 is a confocal picture of the interaction of the new material of the present invention with RAW264 cells. The insertion part at the upper left corner shows that under the synergistic action of the two lasers, the novel material shows good photothermal effect, and the temperature rises to 52 ℃ within ten minutes, so that the novel material can kill tumor cells. Meanwhile, pictures show that the novel material is obviously attached to the surface of cells, and RAW264 is particularly obvious; the scavengerreceiver-A receptor which is over-expressed on the cell surface has specific affinity effect with phycoerythrin, which is caused by the interaction of positive and negative charges. The specific targeting causes the specific enrichment of the novel material on the surface of the cancer cell membrane, thereby being capable of increasing the combined treatment effect of photodynamic and photothermal.
MTT test results show that the new material has good cell killing effect, as shown in figure 8, wherein the new material is prepared according to the preparation method of the invention (as in examples 1-5), and the cell killing effect is 10 mu gmL-1To 40. mu.gmL-1The new material with concentration gradient phycoerythrin equivalent causes 45.3% to 86.5% cell death respectively under the illumination condition determined by the experiment; and the killing effect has positive correlation with the concentration gradient. 40 mu g mL in the absence of light-1Compared with the new material with phycoerythrin equivalent, the cytotoxicity caused by the pure carbon nano tube with the same quantity is obviously reduced, which is about 42% of the toxicity of the pure carbon nano tube with the same quantity, and this shows that the cytotoxicity of the carbon nano tube can be effectively reduced by the loading of the surface protein. Under the condition of illumination, the reaction solution is mixed with 40 mu gmL-1The new material is compared with the pure carbon nano tube with the same amount and the larger dosage of pure phycoerythrin and phycocyanin reported in the prior art, and the dosage is from 20 mu gmL-1The new material shows better cell killing effect at the beginning of concentration; and 40. mu.gmL-1The new material at phycoerythrin equivalent concentration resulted in the highest cell death rate of 86.5%. MTT experimental results fully show that the new materialLow cell dark toxicity and good photo-thermal killing effect.
(2) And detecting the active oxygen accumulation amount.
1) RNO preparation: 5mg of RNO was weighed out and dissolved in 2.200ml of ethanol, and finally mixed to form a 15mM RNO solution, followed by gradient dilution to the desired 15. mu.M RNO solution.
2) Preparing imidazole: 5mg of imidazole was weighed into 4.896ml of water and finally mixed to form a 15mM imidazole solution, which was then diluted in a gradient to the desired 15. mu.M imidazole solution.
3) Dialyzing material (40. mu.gmL)-1Protein concentration) was uniformly mixed with 15. mu.M RNO solution, 15. mu.M imidazole solution, and PBS buffer solution of pH 7.410mM 1: 1 in light-irradiated vials (250. mu.L each was added), followed by 10min of synergistic laser irradiation as in the cell experiments.
4) OD values at 440nm, the maximum absorption peak of RNO, were measured immediately after irradiation for each group at 0, 3, 6, 9, 12, 15min as detection time points. The decrease in the absorbance peak of RNO at 440nm may reflect an increase in the amount of active oxygen accumulated (see FIG. 9). The accumulation of active oxygen, which represents the photosensitizing property, increases with time, indicating that this novel material has an excellent ability as a photosensitizer.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (4)
1. The application of the novel material based on the carbon nano tube and the phycoerythrin is characterized in that the novel material is used as a photosensitizer with photo-thermal effect for phototherapy tumor treatment.
2. The use of the novel material based on carbon nanotubes and phycoerythrin according to claim 1, wherein the novel material is formed by covalently linking single-walled carbon nanotubes and phycoerythrin, and is prepared by the following method:
(1) preparing a carboxylated single-walled carbon nanotube dispersion liquid;
(2) ultrasonically dispersing the dispersion liquid obtained in the step (1) for 15-30 min, respectively adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxy thiosuccinimide solution dissolved in PBS buffer solution, and magnetically stirring at room temperature for 8-24 h; the weight ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide to the carboxylated single-walled carbon nanotubes is 9-18: 1, and the weight ratio of the N-hydroxythiosuccinimide to the carboxylated single-walled carbon nanotubes is 5-10: 1; the PBS buffer is 50mM sodium phosphate buffer with pH of 7.0;
(3) then adding phycoerythrin into the reaction solution, and placing the mixture at room temperature for magnetic stirring for 8-24 h; the weight ratio of the phycoerythrin to the carboxylated single-walled carbon nanotube is 10-20: 1;
(4) and (3) removing redundant EDC, NHS and unconjugated phycoerythrin by a dialysis method at room temperature to obtain the sample.
3. The novel material based on carbon nanotube-conjugated phycoerythrin according to claim 1, wherein the room temperature is 20-25 ℃.
4. The novel material based on carbon nanotube-coupled phycoerythrin according to claim 1, wherein the dialysis time is 24 hours.
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