CN115678545A - Near-infrared two-region luminescent gold nano material synthesized based on CB [7] and application thereof - Google Patents
Near-infrared two-region luminescent gold nano material synthesized based on CB [7] and application thereof Download PDFInfo
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Abstract
The invention discloses a near-infrared two-region luminescent gold nano material synthesized based on CB [7] and its application. The near-infrared two-region luminescent gold nano material is monodisperse nano particle with particle size of 1-3 nm, and has core of gold nano particle modified with polyethylene glycol modified CB 7 and linear chain targeting peptide. By changing the modification amount of the ligand and/or the type of the targeting peptide, a series of near-infrared two-region luminescent gold nano-materials with different surface ligand ratios are obtained. The difference of the surface ligands of the nanoparticles causes the particle size and the surface charge of the nanoparticles to be different, so that the nanoparticles have different organ targeting abilities, which provides conditions for drug delivery of different organs. In addition, the prepared luminescent gold nano material has good stability, combines the characteristics of high penetrating power, low tissue absorption and high resolution of the near-infrared two-region luminescent nano material, and has strong advantages in the aspects of tumor imaging and living body fluorescence imaging.
Description
Technical Field
The invention belongs to the field of functional nano material, in particular to a near-infrared two-region luminescent gold nano material synthesized based on CB [7] and an application thereof.
Background
At present, the defects of poor water solubility, low target enrichment efficiency, incapability of accurately reaching a focus part and the like exist in the treatment of diseases by using a single medicament. Researchers develop a plurality of nano drug-loaded carriers (such as liposomes, nanocapsules, nanospheres, metal nanoparticles and the like) to solve the problems of individual drugs in the treatment process, but the nano drug-loaded carriers have large sizes, which often cause long-term retention in RES organs such as liver and spleen, and are difficult to be thoroughly discharged out of the body, so that large biological toxicity exists, and the clinical application of the nano drug-loaded carriers is restrained. Secondly, because the nano-materials with larger sizes are easily enriched in organs such as liver and spleen, the nano-carriers with larger sizes have difficulty in realizing drug delivery to other organs (such as kidney) and related diseases. However, luminescent gold nanoparticles of ultra-small size (AuNPs,dless than 3 nm) as a novel nano material, has continuously adjustable optical property from a visible region to a near infrared region, easy surface functionalization modification and good biocompatibility, and has wide application in the fields of optical detection, cell imaging, disease diagnosis and the like. However, because of its small size, the luminescent AuNPs are rapidly excreted out of the body through the kidney during the circulation process in the body, and the reports of the luminescent AuNPs as a nano drug-loaded carrier are less. Therefore, it is important to develop a new method for preparing ultra-small gold nanoparticles with high biosecurity and prolonging the in vivo cycle time.
Cucurbituril (n) is used as a new generation of supramolecular host, has an upper hydrophilic carbonyl oxygen port, a lower hydrophilic carbonyl oxygen port and a hydrophobic cavity, can form an inclusion compound with guest molecules through ion dipole action or electrostatic interaction, and therefore has strong host-guest bonding capability, and the characteristics enable the cucurbituril to be widely concerned and researched in the aspects of molecular recognition, cell imaging, drug delivery, disease diagnosis and treatment and the like.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provides a near-infrared two-region luminescent gold nano material based on CB < 7 > synthesis and application thereof.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
the near-infrared two-region luminescent gold nano material synthesized based on CB [7] is monodisperse nano particles with the particle size of 1-3 nm, the core is gold nano particles, and the gold nano particles are modified with two ligands of CB [7] modified by polyethylene glycol and linear chain targeting peptide.
In some examples of near-infrared two-region luminescent gold nanomaterials, the molar ratio of CB [7] to linear targeting peptide is 1.8:0.2 to 0.2:1.8. the mixing ratio of CB 7 and linear chain targeting peptide and the modification amount on the gold nanometer material can be adjusted according to the requirements of different organs or tumor targeting.
In some examples of the near-infrared two-region luminescent gold nanomaterial, the terminal amino acid of the linear targeting peptide is cysteine, and the total number of amino acids of the linear targeting peptide is not more than 10. Cysteine has sulfydryl, and can be conveniently modified on the gold nano material. The linear targeting peptide may be an existing or designed targeting polypeptide. The near-infrared two-region luminescent gold nano material can have corresponding targeting capability by utilizing the targeting peptide.
In some examples of near-infrared two-region luminescent gold nanomaterials, the linear targeting peptide is CRGD.
In a second aspect of the present invention, there is provided:
the preparation method of the near-infrared two-region luminescent gold nano material comprises the following steps:
taking sulfhydryl polyethylene glycol modified CB [7], the tail end of which is provided with cysteine targeting peptide and chloroauric acid, and evenly stirring and mixing in a solvent;
adding a reducing agent, regulating and controlling the pH value to reduce the chloroauric acid into the gold nano material, stopping the reaction after the sufficient reaction, and purifying to obtain the near-infrared two-region luminescent gold nano material.
In some examples of the preparation method, the mole ratio of the thiol-polyethylene glycol modified CB [7] to the terminal cysteine-bearing targeting peptide is 1.8:0.2 to 0.2:1.8 and/or the molar ratio of chloroauric acid to mercapto ligand is 1: (1-6). The mixing ratio of CB [7], the linear chain targeting peptide and the chloroauric acid can be adjusted according to the targeting requirements of different organs and tumors.
In some examples of the preparation method, the reducing agent is sodium borohydride. The sodium borohydride has good safety and good reducibility.
In some examples of the preparation method, the initial molar ratio of chloroauric acid to sodium borohydride is 1: (2-10).
In some examples of the preparation method, the reduction reaction satisfies at least one of the following conditions:
the pH value of the reaction solution is 5-10;
the reaction temperature is 4-40 ℃;
the reaction time is 12-72 h.
In some examples of the preparation method, the reduction reaction satisfies the following condition:
the pH value of the reaction liquid is 5-10;
the reaction temperature is 4-40 ℃;
the reaction time is 12-72 h.
In some examples of preparation methods, excess ligand is removed by dialysis purification.
In some examples of the preparation method, the large particles are removed by centrifugation to obtain the near-infrared two-region luminescent gold nanomaterial.
In some examples of the preparation method, the number of revolutions of the centrifuge is 15000 to 21000 rpm and the time of the centrifuge is 10 to 30 min. Under the condition, the gold nano material with larger size and non-luminescence can be effectively removed.
In some examples of the preparation method, the change in fluorescence is monitored by a fluorescence spectrometer during the reduction reaction, and the reaction is substantially completed when the fluorescence remains stable.
In a third aspect of the present invention, there is provided:
the application of the near-infrared two-region luminescent gold nano material in the first aspect of the invention is at least one of the following applications:
preparing an organ or tumor targeted drug delivery carrier;
and preparing a living body fluorescence imaging probe.
The invention has the beneficial effects that:
the near-infrared two-region luminescent gold nano-materials of some examples of the invention can obtain a series of near-infrared two-region luminescent gold nano-materials with different surface ligand ratios simply by changing the modification amount of the ligand and/or the type of the targeting peptide. The difference of the surface ligands of the nanoparticles causes different particle sizes and surface charges, so that the nanoparticles have different organ targeting abilities, which provides conditions for drug delivery of different organs. In addition, the prepared luminescent gold nano material has good stability, combines the characteristics of high penetrating power, low tissue absorption and high resolution of the near-infrared two-region luminescent nano material, and has strong advantages in the aspects of tumor imaging and living body fluorescence imaging.
The near-infrared two-region luminescent gold nano-material of some examples of the invention has good biocompatibility, low toxicity and adjustable organ targeting capability, can perform fluorescence imaging on tumors and living organs by two-region fluorescence instrument imaging, has simple operation, short time consumption and high detection sensitivity, and rarely has the effect on the currently reported luminescent nano-carriers.
The near-infrared two-region luminescent gold nano material of some examples of the invention has the advantages of simple preparation method, low cost and easy industrial production.
According to the near-infrared two-region luminescent gold nanomaterial of some embodiments of the invention, the used gold nanomaterial can be discharged through the kidney or the liver, cannot be accumulated in the body, and has good biological safety.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the near-infrared two-region luminescent gold nano-material of the present invention.
FIG. 2 is a graph showing the fluorescence emission and ultraviolet absorption spectra of the near-infrared two-region luminescent gold nanomaterial synthesized in example 1, in which the ratio of the amounts of CB [7] to CRGD substances is 1.
FIG. 3 is a TEM image and a statistical analysis of particle size of a near-infrared two-region luminescent gold nanomaterial synthesized in example 1 in which the ratio of the amounts of CB [7] to CRGD substances is 1.
FIG. 4 is a graph of the fluorescence emission and ultraviolet absorption spectra of the synthesized near-infrared two-region luminescent gold nanomaterial of example 2 in which the ratio of the amount of CB [7] to the amount of CRGD substances is 0.6.
FIG. 5 is a TEM image and a statistical analysis of particle size of the synthesized NIR-two-region luminescent gold nanomaterial of example 2, wherein the ratio of the amount of CB [7] to CRGD is 0.6.
FIG. 6 is a graph showing fluorescence emission and ultraviolet absorption spectra of a near-infrared two-region luminescent gold nanomaterial synthesized in example 3 in which the ratio of the amounts of CB [7] to CRGD substances is 0.2.
FIG. 7 is a TEM image and a statistical analysis of particle size of the synthesized near-infrared two-region luminescent gold nanomaterial of example 3 in which the ratio of the amounts of CB [7] to CRGD substances is 0.2.
FIG. 8 is a FT-IR chart of the synthesized near-infrared two-region luminescent gold nanomaterial in examples 1, 2 and 3.
FIG. 9 shows Zeta potentials of the synthesized NIR two-region emitting Au nanomaterials of examples 1, 2 and 3 in a buffer at pH 7.4.
FIG. 10 is a fluorescence stability analysis of the synthesized NIR two-region luminescent gold nanomaterials of examples 1, 2 and 3 under DPBS conditions.
FIG. 11 is a fluorescence stability analysis of the synthesized near-infrared two-region luminescent gold nanomaterial of examples 1, 2 and 3 in the presence of 10% FBS.
FIG. 12 is a toxicity analysis of the synthesized NIR BIL-luminogold nanomaterials from examples 1, 2 and 3 on mouse breast cancer 4T1 cells.
FIG. 13 is the toxicity analysis of the synthesized NIR BIOLOGEN nanomaterials from examples 1, 2 and 3 on HK-2 cells of human renal tubule epithelium.
FIG. 14 is the pharmacokinetic profile of the NIR two-region luminescent gold nanoparticles in mice in example 4.
FIG. 15 is the biodistribution graph of the near-infrared two-region luminescent gold nano-materials in example 5 circulating in the mouse for different time.
FIG. 16 is an image of silver staining of the liver and kidney of the near-infrared two-region luminescent gold nanomaterial of example 5 after 1 h of in vivo circulation in mice.
FIG. 17 is a fluorescent image of the NIR-luminescent gold nanomaterial of example 6 injected via tail vein into mice inoculated with 4T1 subcutaneous tumors at various time points.
FIG. 18 is a graph showing the results of tumor targeting efficiency 24 h after injecting the NIR two-region Au nanomaterials into 4T1 mice inoculated with subcutaneous tumors via tail vein in example 7.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
In the following specific examples, reference is made to chloroauric acid (HAuCl) 4 ) Purchased from maire chemical technologies, inc; sodium borohydride (NaBH) 4 ) Purchased from shanghai rich chemicals, ltd; synthesizing linear chain polypeptide CRGD entrusted symbiotic bioengineering company; the 4T1 cells and HK-2 cells involved were purchased from ATCC; DMEM medium (Gibico, cat # LOT 8118192); fetal bovine serum (LONSA SCIENCE SRL, CAS number: S711-001S). DMEM complete medium is DMEM medium containing 10% FBS and 1% double antibody. The CCK-8 detection kit is purchased from Dongyun chemical industry. Characterizing the morphology of the nano material by using a JEM-2100F transmission electron microscope of Japan Electron Co., ltd; NIR-ii imaging picture acquisition using InGaAs camera (ZephIR 1.7, photon etc.) in canada; the gold content was measured using an inductively coupled plasma mass spectrometer (iCAPRQ) manufactured by Thermo Scientific, germany, or the like.
Example 1
An embodiment of the present invention for producing a near infrared two-region luminescent gold nanomaterial (10CB [7 ]/CRGD-AuNPs) having a ratio of the amount of CB [7] to the amount of CRGD substances of 1 is as follows:
adding 1014 μ L to 5 mL EP tube at room temperature, adding 1500 μ L sulfhydryl polyethylene glycol modified CB [7]](2 mM) and 150. Mu.L of CRGD (20 mM) were stirred well at room temperature, followed by the addition of 186. Mu.L of HAuCl 4 (16.1 mM), after stirring well, 150. Mu.L of NaBH was added 4 (50 mM), adjusting the pH value of the reaction to 8.5, continuously stirring the reaction for 2 days at room temperature, stopping the reaction, dialyzing and purifying to remove redundant ligand, centrifuging at 21000 rpm to remove large particles, and finally performing ultrafiltration concentration to obtain the double-ligand CB [7]]And CRGD, a near infrared two-region luminescent gold nanomaterial 10CB [7]]/CRGD-AuNPs, and stored in a refrigerator at 4 ℃ for further use.
The near-infrared two-region luminescence 10CB 2 [ 10 ]/CRGD-AuNPs prepared in this example was examined for its fluorescence emission spectrum and ultraviolet absorption spectrum, and its morphology was characterized as follows:
FIG. 1 is a schematic diagram of a process for synthesizing a near-infrared two-region luminescent gold nano-material from supramolecular CB 7 and linear polypeptide CRGD.
FIG. 2 is a diagram showing the fluorescence emission and ultraviolet absorption spectra of a near-infrared two-region luminescent gold nanomaterial synthesized with the ratio of CB [7] to CRGD substance amount of 1. The fluorescence spectrogram shows that the luminescent gold nano material has a maximum emission peak at 1050 nm, and the ultraviolet absorption spectrum has no obvious absorption at 520 nm, which shows that the luminescent gold nano material has smaller size.
FIG. 3 is a TEM image of near-infrared two-region luminescence 10CB 2,/CRGD-AuNPs, and a statistical analysis of particle size. As is clear from the TEM image and statistical analysis, the luminescence 10CB [ 2 ]/CRGD-AuNPs are monodisperse nanoparticles having a size of 1.88. + -. 0.33 nm.
Example 2
An embodiment of the present invention for producing a near-infrared two-region luminescent gold nanomaterial (06CB [7 ]/CRGD-AuNPs) having a ratio of the amounts of CB [7] to CRGD species of 0.6 is as follows:
at room temperature, 1554 mu L of EP tube with 5 mL of the tube is taken, and 900 mu L of sulfhydryl polyethylene glycol modified CB [7] is added](2 mM) and 210. Mu.L of CRGD (20 mM) were stirred well at room temperature, followed by the addition of 186. Mu.L of HAuCl 4 (16.1 mM), stirred well, and then 150. Mu.L of NaB was addedH 4 (50 mM), adjusting the pH value of the reaction to 8.5, continuously stirring the reaction solution at room temperature for 2 days, stopping the reaction, dialyzing and purifying the reaction solution to remove excessive ligand, centrifuging the reaction solution at 21000 rpm to remove large particles, and finally performing ultrafiltration concentration to obtain the double-ligand CB [7]]And CRGD composed near infrared two-zone luminescent gold nano material 06CB [ 2 ], [7]]/CRGD-AuNPs, and stored in a refrigerator at 4 ℃ for further use.
The fluorescence emission spectrum and the ultraviolet absorption spectrum of the near-infrared two-region luminescence 06CB [ 2 ], [7]/CRGD-AuNPs prepared in the embodiment and the appearance thereof are examined and characterized:
FIG. 4 is a diagram showing the fluorescence emission and ultraviolet absorption spectra of a near-infrared two-region luminescent gold nanomaterial synthesized with a ratio of CB [7] to CRGD substance amounts of 0.6. As can be seen from a fluorescence spectrogram, the luminescent gold nano material has a maximum emission peak at 1050 nm, and an ultraviolet absorption spectrum has no obvious absorption at 520 nm, which shows that the luminescent gold nano material has a smaller size.
FIG. 5 is a TEM image of near-infrared two-region luminescence 06CB [ 2 ]/CRGD-AuNPs and a statistical analysis of particle size. As can be seen from the TEM image and statistical analysis, the luminescence 06CB 2 [ CB 7]/CRGD-AuNPs are monodisperse nanoparticles having a size of 1.70. + -. 0.33 nm.
Example 3
An embodiment of the present invention for producing a near-infrared two-region luminescent gold nanomaterial (02CB [7 ]/CRGD-AuNPs) having a ratio of the amounts of CB [7] to CRGD species of 0.2 is as follows:
at room temperature, 2094. Mu.L of the mixture was taken out of a 5 mL EP tube, and 300. Mu.L of thiol-polyethylene glycol-modified CB [7] was added](2 mM) and 270. Mu.L of CRGD (20 mM) were stirred well at room temperature, followed by the addition of 186. Mu.L of HAuCl 4 (16.1 mM), after stirring well, 150. Mu.L of NaBH was added 4 (50 mM), adjusting the pH value of the reaction to 8.5, continuously stirring the reaction solution at room temperature for 2 days, stopping the reaction, dialyzing and purifying the reaction solution to remove excessive ligand, centrifuging the reaction solution at 21000 rpm to remove large particles, and finally performing ultrafiltration concentration to obtain the double-ligand CB [7]]And CRGD constituent near infrared two-zone luminescent gold nano material 02CB [7]]/CRGD-AuNPs, and stored in a refrigerator at 4 ℃ for further use.
(1) The fluorescence emission spectrum and the ultraviolet absorption spectrum of the near-infrared two-region luminescence 02CB (2), 7/CRGD-AuNPs prepared by the embodiment and the appearance of the near-infrared two-region luminescence are inspected;
FIG. 6 is a diagram showing the fluorescence emission and ultraviolet absorption spectra of a near-infrared two-region luminescent gold nanomaterial synthesized with a ratio of CB [7] to CRGD species of 0.2. The luminescent gold nano material has strong fluorescence emission in a near infrared two-region according to a fluorescence spectrogram; and the ultraviolet absorption spectrum has no obvious absorption at 520 nm, which shows that the luminescent gold nano-material has smaller size.
FIG. 7 is a TEM image of near-infrared two-region luminescence 02CB [7]/CRGD-AuNPs and a statistical analysis of particle size. As can be seen from the TEM image and statistical analysis, the luminescence 02CB (2B, 7)/CRGD-AuNPs are monodisperse nanoparticles having a size of 1.63. + -. 0.27 nm.
(2) FT-IR test analysis and Zeta potential determination in a buffer at pH 7.4 were carried out on the near-infrared two-zone luminescent CB 7/CRGD-AuNPs prepared in examples 1, 2 and 3;
(3) Fluorescence stability analysis was performed on the near-infrared two-domain luminescence CB [7]/CRGD-AuNPs prepared in examples 1, 2 and 3 in DPBS and with 10% FBS;
(4) The cytotoxicity of the near-infrared two-region luminescent CB [7]/CRGD-AuNPs prepared in examples 1, 2 and 3 on mouse breast cancer 4T1 cells and human renal tubule epithelium HK-2 at different concentrations was examined and analyzed by a CCK8 detection kit.
FIG. 8 is a FT-IR chart of the near-infrared two-region luminescent gold nanomaterial prepared in examples 1, 2 and 3. The figure shows that the characteristic absorption peak v of polyethylene glycol exists in all three near-infrared two-region luminescent gold nano materials C-H C-H stretching vibration and v at 2871 nm C-O C-O stretching vibration at 1110 nm and CB [7] at 1731 nm]The characteristic absorption peak shows that the prepared three luminescent gold nano materials are all connected with more CB [7]]。
FIG. 9 shows Zeta potentials of the synthesized NIR two-region emitting Au nanomaterials of examples 1, 2 and 3 in a buffer at pH 7.4. As can be seen, the surface potential increases negatively with increasing linear polypeptide ligands. The surface potentials of the three near-infrared two-region luminescent gold nanomaterials of 10CB [ 2 ], [7]/CRGD-AuNPs ], 06CB [7]/CRGD-AuNPs, 02CB [7]/CRGD-AuNPs are-4.8 +/-1.1 mV, -9.1 +/-0.9 mV and-23.0 +/-1.1 mV, respectively.
FIG. 10 shows that the fluorescence stability of the near-infrared two-region luminescent gold nano-materials prepared in examples 1, 2 and 3 is good under the DPBS condition.
FIG. 11 shows that the near-infrared two-region luminescent gold nanomaterials prepared in examples 1, 2 and 3 have good fluorescence stability in the presence of 10% FBS.
FIG. 12 shows that the near-infrared two-region luminescent gold nanoparticles prepared in examples 1, 2 and 3 have less toxicity to mouse breast cancer 4T1 cells.
FIG. 13 shows that the near-infrared two-region luminescent gold nanomaterial prepared in examples 1, 2 and 3 is substantially non-toxic to human renal tubule epithelium HK-2 cells.
Example 4
The specific implementation scheme of the three near-infrared two-region luminescent gold nano materials 10CB [ 2 ], [7]/CRGD-AuNPs, 06CB [7]/CRGD-AuNPs and 02CB [7]/CRGD-AuNPs prepared by the invention for the mouse pharmacokinetic study is as follows: mu.L of the nanomaterial prepared above (1. Mu.M) was injected via tail vein into mice (Balb/c female mice, 5-7 weeks old) and blood samples at different time points (2, 5, 10, 30 min, 1, 3, 5, 8, 12, 24, 48 and 72 h) were obtained by orbital bleeding. The blood sample was placed in a 20 mL glass vial, 2-3 mL of freshly reconstituted aqua regia was added and allowed to digest overnight at room temperature. And heating the sample in an oil bath kettle at 120 ℃ until the aqua regia is completely volatilized, adding 5 mL of 10% aqua regia (v/v) to fix the volume, performing ultrasonic treatment for 20-30 min until the sample is completely dissolved, filtering by using a 0.22 mu m aqueous phase needle filter, and finally measuring the concentration of gold ions in the sample by using ICP-MS. The concentration of the nanomaterial in blood was calculated as% injected dose/g (% ID/g), and all animal experimental procedures were performed strictly in accordance with the provisions of the ethical committee on experimental animal research.
FIG. 14 is the pharmacokinetic profile of the synthesized near-infrared two-region luminescent gold nano-material in mice. The three materials 10CB, 7]/CRGD-AuNPs, 06 [ CB, 7]/CRGD-AuNPs, 02CB, 7]/CRGD-AuNPs had AUCs of 483.0% in the blood circulation time of 72 h, 253.1% ID h/g and 212.0% ID h/g, respectively, indicating that 10CB, [7]/CRGD-AuNPs had a longer blood circulation time.
Example 5
The embodiment of the three near-infrared two-region luminescent gold nano materials 10CB [ 2 ], [7]/CRGD-AuNPs, 06CB [7]/CRGD-AuNPs, 02CB [7]/CRGD-AuNPs used for mouse organ targeting is as follows: mu.L of the prepared nanomaterial (1. Mu.M) was injected into a mouse (Balb/c female mouse, 5-7 weeks old) via the tail vein, and organs and tissues such as heart (He), liver (Li), spleen (Sp), lung (Lu), kidney (Ki), skin (Sk), meat (Mu), blood (Bl), stomach (St), intestine (In), brain (Br), bone (Bo), tail (Ta) of the mouse were collected at 1 h, 12h, and 7 d after injection, respectively. Placing the obtained organ or tissue in a 20 mL glass bottle, adding 4-5 mL newly prepared aqua regia, and digesting at room temperature overnight. And heating the sample in an oil bath kettle at 120 ℃ until the aqua regia is completely volatilized, adding 10 mL of 20% aqua regia (v/v) to fix the volume, performing ultrasonic treatment for 20-30 min until the sample is completely dissolved, filtering by using a 0.22 mu m aqueous phase needle filter, and finally measuring the concentration of gold ions in the sample by using ICP-MS. In addition, mice 1 hour after tail vein injection of the near-infrared two-region luminescent gold nano-material (three) were euthanized, and the livers and kidneys thereof were taken for silver staining analysis.
FIG. 15 is a graph of the biodistribution of the near-infrared two-region luminescent gold nanoparticles in the circulation of mice for different periods of time and the analysis of different target enrichment efficiencies of liver and kidney. As can be seen from the figure, 10CB [ 2 ]/CRGD-AuNPs has a strong liver enrichment efficiency, while 02CB [7]/CRGD-AuNPs has a strong kidney targeting enrichment efficiency.
FIG. 16 is a silver staining image of liver and kidney of the near-infrared two-region luminescent gold nano-material after 1 h of in vivo circulation in mice. The results showed that 10CB [ 2 ], [7]/CRGD-AuNPs was more enriched in the liver during circulation for 1 h in vivo, while two materials, 06CB [7]/CRGD-AuNPs, 02CB [7]/CRGD-AuNPs, were relatively less enriched in the liver. However, the three materials are all enriched in the kidney, but the 02CB [7]/CRGD-AuNPs are obviously more enriched in the kidney and are distributed in the epithelial cells of the kidney, further showing that the three near-infrared two-region luminescent gold nano-materials prepared by the method have different organ targeting abilities.
Example 6
The embodiment of the three near-infrared two-region luminescent gold nanomaterials of 10CB [ 2 ]/CRGD-AuNPs, 06CB [7]/CRGD-AuNPs, 02CB [7]/CRGD-AuNPs prepared by the invention for in-vivo tumor imaging of mice is as follows: firstly, a 4T1 subcutaneous tumor mouse model (Balb/c female mouse, 4-5 weeks old) is constructed, after the mouse is depilated by depilatory cream, the surface of the mouse is lightly wiped by distilled water, and a two-region fluorescence living body imager is used for carrying out living body fluorescence imaging on the mouse, and the obtained picture is used as a background fluorescence picture of the mouse. Then injecting 300 mu L of the prepared nano material (1 mu M) into a mouse body through tail vein, and analyzing the fluorescence intensity change and the imaging effect of the tumor part of the mouse by using a two-region fluorescence living body imager at different time points. Mouse imaging conditions: the excitation wavelength was 808 nm and the exposure time was 5 s.
FIG. 17 is a fluorescent image of the near-infrared two-zone luminescent gold nanoparticles injected via tail vein into mice inoculated with 4T1 subcutaneous tumor at different time points. The results show that 10CB 7/CRGD-AuNPs are enriched more in tumor sites and livers, and the tumor targeting effect is obviously better than that of the other two materials.
Example 7
The embodiment of the three near-infrared two-region luminescent gold nano materials 10CB [ 2 ], [7]/CRGD-AuNPs, 06CB [7]/CRGD-AuNPs, 02CB [7]/CRGD-AuNPs used for mouse tumor targeted analysis is as follows:
mu.L of the nanomaterial (1. Mu.M) prepared above was injected via tail vein into mice inoculated with 4T1 subcutaneous tumors, and the mice were euthanized 24 h after injection, and tumor tissues thereof were taken. The obtained tumor tissue is placed in a 20 mL glass bottle, 4-5 mL of newly prepared aqua regia is added, and the mixture is digested overnight at room temperature. And heating the sample in an oil bath kettle at 120 ℃ until the aqua regia is completely volatilized, adding 10 mL of 20% aqua regia (v/v) to a constant volume, carrying out ultrasonic treatment for 20-30 min until the sample is completely dissolved, filtering by using a 0.22 mu m water phase needle type filter, finally measuring the concentration of gold ions in the sample by using ICP-MS, and calculating the tumor targeting efficiency of the three near-infrared two-region luminescent gold nano materials.
FIG. 18 is a graph showing the results of tumor targeting efficiency 24 h after the near-infrared two-region luminescent gold nanomaterial is injected into a 4T1 subcutaneous tumor-inoculated mouse via tail vein. The results showed that 10CB 7/CRGD-AuNPs have a significantly stronger efficiency of targeted enrichment at the tumor site than the other two materials because they have a longer blood circulation time.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
The foregoing is a further detailed description of the invention and is not to be taken in a limiting sense as the invention is defined by the appended claims. It will be apparent to those skilled in the art that various modifications, additions and substitutions can be made without departing from the spirit and scope of the invention.
Claims (10)
1. The near-infrared two-region luminescent gold nano material synthesized based on CB [7] is characterized in that the near-infrared two-region luminescent gold nano material is monodisperse nano particles with the particle size of 1-3 nm, the core is gold nano particles, and the gold nano particles are modified with two ligands of CB [7] modified by polyethylene glycol and linear chain targeting peptide.
2. The near-infrared two-region luminescent gold nanomaterial according to claim 1, wherein the molar ratio of CB [7] to linear targeting peptide is 1.8:0.2 to 0.2:1.8.
3. the near-infrared two-region luminescent gold nanomaterial according to claim 1, wherein the terminal amino acid of the linear targeting peptide is cysteine, and the total number of amino acids of the linear targeting peptide is not more than 10.
4. The near-infrared two-region luminescent gold nanomaterial according to claim 3, wherein the linear targeting peptide is CRGD.
5. The method for preparing the near-infrared two-region luminescent gold nano-material of any one of claims 1 to 4, comprising the following steps:
taking sulfhydryl polyethylene glycol modified CB [7], the tail end of which is provided with cysteine targeting peptide and chloroauric acid, and evenly stirring and mixing in a solvent;
adding a reducing agent, regulating and controlling the pH value to reduce the chloroauric acid into the gold nano material, stopping the reaction after the sufficient reaction, and purifying to obtain the near-infrared two-region luminescent gold nano material.
6. The preparation method according to claim 5, wherein the molar ratio of the thiol-polyethylene glycol-modified CB [7] to the targeting peptide having cysteine at the terminal is 1.8:0.2 to 0.2:1.8 and/or the molar ratio of chloroauric acid to mercapto ligand is 1: (1-6).
7. The production method according to claim 5 or 6, wherein the reducing agent is sodium borohydride.
8. The method of claim 7, wherein the molar ratio of starting chloroauric acid to sodium borohydride is 1: (2-10).
9. The production method according to claim 5, characterized in that the reduction reaction satisfies at least one of the following conditions:
the pH value of the reaction solution is 5-10;
the reaction temperature is 4-40 ℃;
the reaction time is 12-72 h.
10. The use of the near-infrared two-region luminescent gold nanomaterial of any one of claims 1 to 4, selected from at least one of the following applications:
preparing an organ or tumor targeted drug delivery carrier;
and preparing a living body fluorescence imaging probe.
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| CN105363043A (en) * | 2014-08-08 | 2016-03-02 | 屈晓超 | RGD-labeled fluorescent gold nano-cluster preparation method |
| CN109986090A (en) * | 2019-03-22 | 2019-07-09 | 华南理工大学 | A kind of double ligand gold nanoparticle aqueous solutions and its preparation method and application |
| CN110724519A (en) * | 2019-10-29 | 2020-01-24 | 西北工业大学深圳研究院 | Preparation method and application of fluorescence-enhanced gold nanocluster composite material based on supermolecule macrocycle |
| CN111303868A (en) * | 2018-12-11 | 2020-06-19 | 华南理工大学 | Near-infrared luminescent polypeptide self-assembled gold nano material and preparation method and application thereof |
| WO2022032871A1 (en) * | 2020-08-11 | 2022-02-17 | 苏州大学 | Infrared ii region fluorogold nanocluster, preparation and application thereof |
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| CN105363043A (en) * | 2014-08-08 | 2016-03-02 | 屈晓超 | RGD-labeled fluorescent gold nano-cluster preparation method |
| CN111303868A (en) * | 2018-12-11 | 2020-06-19 | 华南理工大学 | Near-infrared luminescent polypeptide self-assembled gold nano material and preparation method and application thereof |
| CN109986090A (en) * | 2019-03-22 | 2019-07-09 | 华南理工大学 | A kind of double ligand gold nanoparticle aqueous solutions and its preparation method and application |
| CN110724519A (en) * | 2019-10-29 | 2020-01-24 | 西北工业大学深圳研究院 | Preparation method and application of fluorescence-enhanced gold nanocluster composite material based on supermolecule macrocycle |
| WO2022032871A1 (en) * | 2020-08-11 | 2022-02-17 | 苏州大学 | Infrared ii region fluorogold nanocluster, preparation and application thereof |
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