CN114931641B - Method for loading indocyanine green by nano-diamond and application of photothermal agent - Google Patents
Method for loading indocyanine green by nano-diamond and application of photothermal agent Download PDFInfo
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- CN114931641B CN114931641B CN202210762118.2A CN202210762118A CN114931641B CN 114931641 B CN114931641 B CN 114931641B CN 202210762118 A CN202210762118 A CN 202210762118A CN 114931641 B CN114931641 B CN 114931641B
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- MOFVSTNWEDAEEK-UHFFFAOYSA-M indocyanine green Chemical compound [Na+].[O-]S(=O)(=O)CCCCN1C2=CC=C3C=CC=CC3=C2C(C)(C)C1=CC=CC=CC=CC1=[N+](CCCCS([O-])(=O)=O)C2=CC=C(C=CC=C3)C3=C2C1(C)C MOFVSTNWEDAEEK-UHFFFAOYSA-M 0.000 title claims abstract description 150
- 238000000034 method Methods 0.000 title claims abstract description 32
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
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- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
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- A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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Abstract
The invention belongs to the technical field of nano diamond processing, and discloses a method for loading indocyanine green by nano diamond and application of a photothermal agent, wherein the method for loading indocyanine green by nano diamond comprises the following steps: directly aminating and modifying the oxidized nano diamond by using ethylenediamine, propylenediamine and ammonia water through a chemical method; and carrying indocyanine green on the three aminated modified nano diamonds through electrostatic interaction respectively to prepare composite nano particles, and testing the loading rate and the dispersibility of the composite nano particles. Aiming at the problems of poor green light thermal stability of indocyanine and weak photo-thermal effect of the nano diamond, the invention firstly uses three amination reagents of ethylenediamine, propylenediamine and ammonia water to aminate the oxidative detonation nano diamond, and enables the aminated nano diamond to directly load indocyanine green through electrostatic interaction, the photo-thermal heating effect of the aminated nano diamond is basically kept unchanged after tertiary circulation, and the cell uptake rate in HepG2 cells is highest.
Description
Technical Field
The invention belongs to the technical field of nano diamond processing, and particularly relates to a method for loading indocyanine green by nano diamond and application of a photo-thermal agent.
Background
Indocyanine green (ICG), a photothermal agent approved by the united states Food and Drug Administration (FDA), is a few photothermal agents that can be used for both photothermal and photodynamic therapy, and after absorbing NIR, indocyanine green not only can promote local warming for photothermal therapy, but also can release active oxygen for effective photodynamic therapy; indocyanine green, however, also has certain limitations such as concentration dependence, poor stability, easy non-specific binding to proteins, lack of targeting, etc., and its application is limited. In order to overcome such problems, indocyanine green is usually carried on a carrier to improve the stability, prolong the half-life of blood circulation, improve the accumulation rate of tumors and further increase the curative effect of photothermal therapy. The nano diamond has tunable surface chemistry as a carbon material with good biocompatibility, can generate strong adsorption or conjugation with biomolecules, and loads the biomolecules on the surface. Therefore, the surface groups of the nano diamond can be changed to be rich in positive charges, indocyanine green is loaded on the nano diamond through electrostatic interaction, so that the biotoxicity of the indocyanine green is reduced, and the photo-thermal stability of the indocyanine green is enhanced.
In the research situation at home and abroad, the synthesis of the nano-diamond and indocyanine green composite particles mainly comprises the steps of purifying the nano-diamond, coating the nano-diamond by using a polymer rich in positive charges, and finally adsorbing indocyanine green, so that a nano-platform for photothermal treatment is built. The polydopamine is used for coating the nano-diamond and then carrying indocyanine green, so that the composite nano-particle is prepared for photothermal treatment of glioblastoma cancer (Maziukiewicz D,B F,Coy E,et al.NDs@PDA@ICG conjugates for photothermal therapy of glioblastoma multiforme[J]biomimetics,2019,4 (1): 3.). The composite nano particle has high photo-thermal conversion efficiency of more than 40%, good photo-thermal treatment result and capability of eradicating glioblastoma cells more quickly. Harvey et al (Harvey S, raabe M, ermakova A, et al Transferrin-Coated Nanodiamond-Drug Conjugates for Milliwatt Photothermal Applications [ J)]Advanced Therapeutics,2019,2 (11): 1900067) polymerizing l-3, 4-dihydroxyphenylalanine on fluorescent nanodiamond, then combining with transferrin, and finally carrying indocyanine green to obtain a composite photothermal material, which has improved colloid stability and cell uptake rate, and has very low energy uptake (about 90mW/cm 2)) It can exhibit a strong photo-thermal effect. The application research of the nano diamond photo-thermal effect is also limited to a cell stage, and a tumor cell ablation experiment is not performed by implanting the nano diamond photo-thermal effect into a mouse body. Cui et al (Cui X Y, liang Z Y, lu J Q, et al A multifunctional nanodiamond-based nanoplatform for the enhanced mild-temperature photothermal/chemo combination therapy of triple negative breast cancer via an autophagy regulation strategy [ J)]Nanoscale,2021,13 (31): 13375-89.) more recently reported more biological uses of nanodiamond and indocyanine green complexes, which were first modified with protamine sulfate. And combining indocyanine green and a small molecular inhibitor with the nano diamond, assembling hyaluronic acid with negative charges on the outer surface, and carrying doxorubicin through electrostatic interaction to form the composite nano particle. The composite nano particles are found to have strong photo-thermal effect, can be taken up by cells, and can also perform fluorescence and photoacoustic imaging in vivo; the targeting agent has good targeting property, and realizes in vivo tumor treatment of mice; however, no intensive research has been conducted on the application of indocyanine green in photodynamic therapy.
Through the above analysis, the problems and defects existing in the prior art are as follows:
(1) The prior art can not effectively solve the problems of poor green light thermal stability of indocyanine and weak photo-thermal effect of nano diamond.
(2) The photo-thermal heating effect of the composite nano particles obtained in the prior art is easy to change, and the cell uptake rate in cells is low.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention provides a method for loading indocyanine green by nano-diamond and application of a photothermal agent. In particular to a method for loading indocyanine green ICG by nano diamond and application of the indocyanine green ICG serving as a photo-thermal agent in the field of biological medicine.
The invention is realized in such a way that the method for loading indocyanine green by the nano diamond comprises the following steps:
directly aminating and modifying the oxidized nano diamond by using ethylenediamine, propylenediamine and ammonia water through a chemical method;
and carrying indocyanine green on the three aminated and modified nano diamonds through electrostatic interaction to prepare ND-EtNH 2 @ICG、ND-PrNH 2 @ ICG and ND-NH 2 @ ICG composite nanoparticles and testing the loading and dispersibility of the composite nanoparticles.
And further, placing the purified nano-diamond powder, ethylenediamine, propylenediamine and ammonia water in a grinding kettle for grinding at room temperature, centrifugally separating and filtering the ground mixture, and centrifuging for 10min at the centrifugal force of 8000g to obtain a clear and transparent black colloidal solution, and selecting the purified nano-diamond powder with proper particle size.
Further, adding an amination reagent into the purified nano-diamond solution uniformly dispersed in dimethyl sulfoxide, heating the solution to 105-250 ℃ slowly through oil bath heating, and preserving the temperature for a certain time; after the reaction is finished, cooling the solution to room temperature, and centrifuging under the condition that the centrifugal force is 8000g to obtain an aminated NDs crude product; 100mL of N, N-dimethylformamide solution was added to the crude product, followed by ultrasonic washing for 15min, and then centrifugation was repeated three times again under a centrifugal force of 8000g, to finally obtain aminated NDs.
Further, weighing a certain amount of indocyanine green, dissolving the indocyanine green into deionized water, and preparing a indocyanine green solution with a certain concentration; mixing the aminated nano-diamond colloid solution with indocyanine green solution; then the pH value of the solution is adjusted to be less than 6, and the solution is stirred and reacted for 24 hours under dark condition and room temperature environment after the magneton is added; centrifuging the solution after the reaction is finished, washing and centrifuging the solution with deionized water for three times, and collecting supernatant fluid of each centrifugation; obtaining a solution of aminated nano-diamond loaded indocyanine green composite nano-particles, and storing the solution under a light-shielding condition for subsequent load rate and dispersibility tests.
Further, in the direct amination modification of oxidized nanodiamond with ethylenediamine, propylenediamine and ammonia water by chemical method, the aminated nanodiamond colloidal solution is ND-EtNH 2 、ND-PrNH 2 And ND-NH 2 One or a mixture of three composite nano particles, wherein the concentration of the solution is 0.01-10%.
Further, the concentration of the indocyanine green solution is 0.01% -10%.
Further, the pH of the solution is adjusted to less than 6.
The invention also aims to provide the ICG-loaded aminated nano-diamond prepared by the method for loading indocyanine green by the nano-diamond.
The invention further aims to provide an application of the ICG-loaded aminated nano-diamond in preparing a diagnosis and treatment preparation for inhibiting cancer cells.
Further, in the apoptosis experiment of cancer cells, the concentration of the ICG-loaded aminated nanodiamond is 0.001-2 mg/ml.
In combination with the above technical solution and the technical problems to be solved, please analyze the following aspects to provide the following advantages and positive effects:
first, aiming at the technical problems in the prior art and the difficulty in solving the problems, the technical problems solved by the technical proposal of the invention are analyzed in detail and deeply by tightly combining the technical proposal to be protected, the results and data in the research and development process, and the like, and some technical effects brought after the problems are solved have creative technical effects. The specific description is as follows:
aiming at the problems of poor indocyanine green light thermal stability and weak photo-thermal effect of the nano diamond, the invention firstly uses three amination reagents of ethylenediamine, propylenediamine and ammonia water to aminate the oxidative detonation nano diamond, and enables the aminated nano diamond to directly load indocyanine green through electrostatic interaction, thus obtaining the composite nano particle with good photo-thermal stability; the photothermal warming effect of the cell is basically unchanged after three cycles, and the cell uptake rate in HepG2 cells is highest.
Secondly, the technical scheme is regarded as a whole or from the perspective of products, and the technical scheme to be protected has the following technical effects and advantages:
the invention relates to a preparation method of amination modified nano diamond by loading indocyanine green and a preparation method thereofThe application in cell labeling has the following beneficial effects: the oxidized nano diamond is subjected to amination surface modification by using ethylenediamine, propylenediamine and ammonia water through a chemical modification method, and indocyanine green is directly carried on the amination modified nano diamond to obtain ND-EtNH 2 @ICG、ND-PrNH 2 @ ICG and ND-NH 2 Three composite nano particles @ ICG are used for improving the photo-thermal stability and the cell uptake rate of indocyanine green. The fluorescence performance, the photo-thermal performance and the cell uptake rate of the particles are tested, and experiments prove that the fluorescence intensity and the photo-thermal effect of the particles are obviously improved, and the cell uptake rate is improved. Provides a new strategy for the research of the nano diamond in the aspects of fluorescent marking, photothermal treatment, drug delivery and the like.
Thirdly, as inventive supplementary evidence of the claims of the present invention, the following important aspects are also presented:
(1) The technical scheme of the invention fills the technical blank in the domestic and foreign industries:
indocyanine green is directly carried on aminated modified nano diamond to obtain ND-EtNH with the loading rate of 80 percent 2 @ICG、ND-PrNH 2 @ ICG and ND-NH 2 Three composite nano particles @ ICG are used for improving the photo-thermal stability and the cell uptake rate of indocyanine green.
(2) Whether the technical scheme of the invention solves the technical problems that people want to solve all the time but fail to obtain success all the time is solved:
composite nanoparticles were not seen with ICG loadings up to 80%.
Drawings
FIG. 1 is a flow chart of a method for loading indocyanine green with nano-diamond provided by the embodiment of the invention;
FIG. 2 is a graph of the standard curve of indocyanine green in deionized water provided by the example of the present invention;
FIG. 3 is a graph of the standard indocyanine green in phosphate buffer provided in the examples of the present invention;
FIG. 4 is a schematic illustration of a nano-diamond FTIR spectrum provided by an embodiment of the present invention;
FIG. 5 is a graph showing a particle size distribution of nanodiamond provided in an embodiment of the invention;
FIG. 6 is a graph showing a particle size distribution of composite nanoparticles provided in an embodiment of the present invention;
FIG. 7 is a photo-thermal heating graph of indocyanine green and composite nanoparticles provided by an embodiment of the present invention;
FIG. 8 is a graph of photo-thermal stability of indocyanine green and composite nanoparticles provided by embodiments of the present invention;
FIG. 9 is a photo-thermal stability diagram of indocyanine green and composite nanoparticles provided by an embodiment of the present invention;
FIG. 10 is a graph showing blue fluorescence of nuclei stained uniformly in an unlit cell line provided by an embodiment of the present invention;
FIG. 11 shows the apparent shrinkage of nuclei after irradiation with light according to the present invention.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
1. The embodiments are explained. In order to fully understand how the invention may be embodied by those skilled in the art, this section is an illustrative embodiment in which the claims are presented for purposes of illustration.
As shown in fig. 1, the method for loading indocyanine green by using nano diamond provided by the embodiment of the invention comprises the following steps:
s101, directly aminating and modifying the oxidized nano diamond by using ethylenediamine, propylenediamine and ammonia water through a chemical method.
S102, respectively carrying indocyanine green on three aminated and modified nano diamonds through electrostatic interaction to prepare ND-EtNH 2 @ICG、ND-PrNH 2 @ ICG and ND-NH 2 Three composite nanoparticles @ ICG and their loading and dispersibility were tested.
The photo-thermal test result shows that the photo-thermal effect of the aminated nano-diamond with low concentration is weak; the temperature rise curves of the three composite nano particles and the simple indocyanine green under the irradiation of near infrared light at the same time are relatively close, which indicates that indocyanine green is carried on the nano diamond and is not masked; the photo-thermal stability curves of three circulating lights show that the photo-thermal stability of the three composite nano particles is better than that of the pure indocyanine green; the cell uptake test of HepG2 shows that the cell uptake rate of the three composite nano particles after 4 hours is higher than that of the pure indocyanine green, so that the effect is improved.
In a preferred embodiment, purified nano-diamond powder with proper particle size is selected, the purified nano-diamond powder, ethylenediamine, propylenediamine and ammonia water are placed in a grinding kettle for grinding at room temperature, and the ground mixture is centrifugally separated, filtered and centrifuged for 10min at the centrifugal force of 8000g to obtain clear and transparent black colloid solution.
In a preferred embodiment, a solution of purified nanodiamond uniformly dispersed in dimethyl sulfoxide is added to an amination reagent, heated slowly to 105-250 ℃ by oil bath heating and incubated at this temperature for a period of time. After the reaction was completed, the solution was cooled to room temperature and centrifuged at a centrifugal force of 8000g to obtain an aminated NDs crude product. 100mL of N, N-dimethylformamide solution was added to the crude product, followed by ultrasonic washing for 15min, and then centrifugation was repeated three times again under a centrifugal force of 8000g, to finally obtain aminated NDs.
In a preferred embodiment, a quantity of indocyanine green is weighed into deionized water to prepare a concentration of indocyanine green solution. Mixing the aminated nano-diamond colloid solution with indocyanine green solution. Then the pH of the solution is adjusted to be less than 6, and the solution is stirred and reacted for 24 hours under dark condition and room temperature environment after the magneton is added. After the reaction is completed, the solution is centrifuged, and then washed and centrifuged by deionized water for three times, and the supernatant fluid of each centrifugation is collected. Obtaining a solution of aminated nano-diamond loaded indocyanine green composite nano-particles, and storing the solution under a light-shielding condition for subsequent performance test.
The loading rate of the aminated nano diamond to indocyanine green can reach 80%, the photo-thermal stability of the indocyanine green loaded composite nano particle is improved, the photo-thermal heating effect of the indocyanine green loaded composite nano particle is basically kept unchanged after three times of circulation, and the cell uptake rate in HepG2 cells reaches 68.54 +/-0.03%.
The purified nano diamond powder in the technical scheme of the invention can be a commercial product or a detonation synthesized purified nano diamond. In a preferred embodiment, the nano-diamond raw powder is an agglomerate obtained by a detonation method, the particle size of the agglomerate varies from tens of nanometers to tens of micrometers, and the nano-diamond content in the nano-diamond raw powder is 80-99 wt%.
In a preferred embodiment, the aminated nanodiamond colloidal solution is ND-EtNH 2 、ND-PrNH 2 And ND-NH 2 One or a mixture of three composite nano particles, wherein the concentration of the solution is 0.01-10%, preferably 0.05-5%.
In a preferred embodiment, the indocyanine green solution has a concentration of 0.01% to 10%, preferably 0.05% to 5%.
In a preferred embodiment, the pH of the solution is adjusted to less than 6; preferably pH1-4.
In a preferred embodiment, the concentration of the ICG-loaded aminated nano-diamond as a diagnosis and treatment preparation for inhibiting cancer cells is 0.001-2 mg/ml.
The particle size test of the nano diamond/oxidized nano diamond in each embodiment of the invention is carried out by using a Markov Zate potentiometer, specifically, a Dynamic Light Scattering (DLS) principle is adopted to prepare a sample into a dilute solution with a certain concentration for test, the time of the first 3 times is set to be 120s, the time of the last 3 times is set to be 30s, and the particle size measurement is carried out at normal temperature.
Example 1
The preparation method of the surface aminated nano-diamond capable of being monodisperse in the solution comprises the following steps:
350g of ball-milling beads with the diameter of 0.08-0.12 mm are put into a sealable ball-milling tank with the volume of 100mL, 0.5g of nano diamond (certain chemical Co., ltd.) is added with 10mL of deionized water, and ultrasonic vibration is carried out for 30min to uniformly disperse the nano diamond, the nano diamond suspension and 10mL of 25% ammonia water are put into the ball-milling tank, the rotating speed of a ball mill (model: QM-1SP2, certain university instrument factory) is set to be 580r/min, the ball-milling time is 2h, then the beads are removed, the solution is filtered, the product is washed by ethanol and dried, and black powder is obtained, and the infrared spectrum is shown as figure 4.
Example 2
The preparation method of the surface aminated nano-diamond capable of being monodisperse in the solution comprises the following steps:
first, 0.300g of oxidized NDs was weighed out, dispersed in 30mL of dimethyl sulfoxide solvent and dispersed by ultrasonic for 15min to uniformly disperse. On the basis, ethylenediamine and 1, 3-propylenediamine are adopted as amination reagents for amination, and the specific steps are as follows: the oxidized NDs solution, which was uniformly dispersed in dimethyl sulfoxide, obtained by ball milling was taken out and added to a 50mL flask. Thereafter 5mL of an amination reagent was added thereto, which was slowly heated to 150 ℃ by oil bath heating and incubated at this temperature for 5h. After the reaction was completed, the solution was cooled to room temperature and centrifuged at 8000rpm to obtain an aminated NDs crude product. 100mL of N, N-dimethylformamide solution was added to the crude product, followed by ultrasonic washing for 15min, and then centrifugation was repeated three times again at 8000rpm, to finally obtain aminated NDs.
In order to increase the yield of aminated nanodiamond, the nanodiamond of the examples may be oxidized to remove the graphite phase, and may be purified by, but not limited to, the following process, including the steps of:
reacting 10g of nano-diamond gray powder with 30-50 g of concentrated sulfuric acid and 10g of potassium permanganate in a reaction kettle, controlling the reaction temperature in the reaction kettle to be 220 ℃ and the reaction time to be 8 hours, oxidizing and purifying the nano-diamond gray powder in a concentrated sulfuric acid medium at a high temperature by using potassium permanganate, ultrasonically cleaning a product to be neutral by using deionized water, centrifuging and drying to obtain a purified nano-diamond powder; the nano diamond gray powder is a commercial product, and the diamond content in the nano diamond gray powder is more than 90%; the mass fraction of the concentrated sulfuric acid is 98%.
Example 3 aminated nanodiamond loaded indocyanine green composite nanoparticle.
Weighing 2.000mg of indocyanine green, dissolving in 8mL of deionized water, respectively diluting the aminated nano-diamond colloid solution to 1mg/mL, and adding 2mL into the indocyanine green solution to enable the mass ratio of reactants to be 1:1. Then, the pH of the solution was adjusted to 2.5 using 0.1mol/mL hydrochloric acid, and the reaction was stirred under dark conditions and room temperature for 24 hours after adding the magneton. After the reaction is completed, the solution is dialyzed for 48 hours to remove the non-adsorbed indocyanine green, and deionized water is replaced every 4 hours. And (3) freeze-drying and concentrating the solution after the dialysis is finished to obtain 200 mug/mL of solution of the indocyanine green composite nano particles loaded by the aminated nano diamond, and storing the solution under the light-proof condition for subsequent performance test.
2. Application example. In order to prove the inventive and technical value of the technical solution of the present invention, this section is an application example on specific products or related technologies of the claim technical solution.
The application of the ICG-loaded aminated nano diamond as a diagnosis and treatment preparation is provided based on the embodiment.
3. Evidence of the effect of the examples. The embodiment of the invention has a great advantage in the research and development or use process, and has the following description in combination with data, charts and the like of the test process.
One) Infrared Spectrometry test
Samples were tested using a TENSOR 27 type fourier transform infrared spectrometer (FTIR). Uniformly mixing each sample and KBr powder according to the mass ratio of 1:100, grinding, pressing into a film piece on a tablet press, and forming the film piece at 4000cm -1 ～400cm -1 Scanning test is performed in range.
(II) particle size test
The sample was subjected to particle size testing using a Zetasizer Advance Series-Pro nanoparticle size and Zeta potentiometer. Samples were formulated as 1mg/mL dispersion and tested at 25 ℃ with the test interval time set to 120s.
Detection of loading rate of aminated nano-diamond loaded indocyanine green composite nano-particles
10.000mg of indocyanine green was precisely weighed into a 100mL brown volumetric flask, and dissolved in deionized water to prepare a mother liquor with a concentration of 100. Mu.g/mL. Then a certain amount of mother solution is diluted by deionized water to the following concentration: 1.0,2.0,4.0,6.0,8.0, 10.0. Mu.g/mL, absorbance was measured using a Cary 60 ultraviolet spectrophotometer manufactured by Agilent corporation, U.S.A., linear regression was performed on indocyanine green concentration (C) with absorbance (A), and standard graph 2 of indocyanine green was plotted.
TABLE 1 UV absorbance of indocyanine green in deionized water
And then, taking the difference value between the absorbance of the indocyanine green mother solution before the reaction and the absorbance of the supernatant obtained by centrifugation after the reaction into a standard linear equation, calculating the concentration of the corresponding indocyanine green, and obtaining the concentration of the indocyanine green of the composite nano particle by difference. Finally, the drug loading rates of the three composite nanoparticles were calculated according to formula (1):
ICG loading capacity(％)＝(W 1 -W 2 )/W ND ×100 (1)
W 1 、W 2 and W is ND And respectively represents the mass of indocyanine green and nano-diamond of mother solution and supernatant. Wherein W is 1 And W is 2 Obtained by multiplying the calculated indocyanine green concentration by its corresponding volume.
(IV) photo-thermal performance test
Samples were dissolved in deionized water to make 200 μg/mL dispersion for photo-thermal performance testing, deionized water and 160 μg/mL indocyanine green solution were used as control groups. 100. Mu.L of the sample was pipetted into a 96-well plate using a pipette and then placed at 1W/cm using a 808nm laser 2 The sample was irradiated for 5min at 10s intervals with a near infrared thermal imager recording the temperature change of the sample.
(V) photo-thermal stability test
200. Mu.g/mL of the sample dispersion was pipetted into 100 using a pipettemu.L of indocyanine green solution (160. Mu.g/mL) was added to a 96-well plate as a control group. Then using 808nm laser at 1W/cm 2 The sample was irradiated for 5min, cooled for 5min, cycled 3 times while recording the temperature change of the sample with a near infrared thermal imager every 10 s.
Calculation of photo-thermal stability
In order to quantitatively evaluate the photo-thermal stability of the sample, equation (2) representing the degree of photo-thermal stability is now defined and calculated as follows:
Photothermal stability(％)＝T/T 0 ×100 (2)
wherein T is 0 Representing the highest temperature reached by the sample solution during the primary photothermal cycle; t represents the highest temperature reached by the sample solution during each cycle from the second photo-thermal cycle, and the photo-thermal stability of the sample is quantitatively analyzed according to the calculation result.
(seventh) cellular uptake
10.000mg of indocyanine green was precisely weighed into a 100mL brown volumetric flask, and dissolved in phosphate buffer to prepare a mother solution with a concentration of 100. Mu.g/mL. Then a certain amount of mother solution is diluted by phosphate buffer solution to the following concentration: 1.0,2.0,4.0,6.0,8.0, 10.0. Mu.g/mL, absorbance was measured using a Cary 60 ultraviolet spectrophotometer manufactured by Agilent corporation, U.S.A., linear regression was performed on indocyanine green concentration (C) with absorbance (A), and standard graph 3 of indocyanine green in phosphate buffer was plotted.
TABLE 2 UV absorbance of indocyanine green in phosphate buffer
The digested HepG2 cells are respectively inoculated into a 6-hole cell culture plate, after being cultured for 24 hours in a carbon dioxide incubator, 1mL of three composite nanoparticle dispersion solutions of 40 mug/mL and indocyanine green solution are added into each hole, and meanwhile, phosphate buffer solution with the same volume is used as a control group, and the three times are used. Then, after culturing in a carbon dioxide incubator for 4 hours, the culture plate was removed, and the supernatant in each well was aspirated and washed twice with 0.5mL of phosphate buffer solution to remove the sample solution attached to the cells. And measuring the absorbance in the supernatant by using an ultraviolet-visible spectrophotometer, and calculating the concentration of residual indocyanine green in the supernatant after cell ingestion, thereby obtaining the ingestion rate of the HepG2 cells on different samples.
(eight) surface chemistry of aminated nanodiamond as shown in FTIR spectrum of nanodiamond of fig. 4, (a) oxidized nanodiamond, (b) ethylenediamine aminated nanodiamond, (c) propylenediamine aminated nanodiamond, (d) ammonia aminated nanodiamond in fig. 5.
TABLE 3 Infrared Spectrometry analysis of oxidized nanodiamond and aminated nanodiamond
As can be seen from FIG. 4, for oxidized nanodiamond, at 3435cm -1 、1797cm -1 、1630cm -1 The infrared bands appearing at the positions are respectively the infrared bands of-OH stretching vibration, C=O stretching vibration and-OH bending vibration, which indicates that the carboxylic acid groups exist on the surface of the nano diamond after oxidation. At 1099cm -1 The infrared band appearing at the place is C-O-C stretching vibration, which shows that the surface of the infrared band contains ether bond, lactone or anhydride and other groups. Aminating nano diamond with ethylene diamine at 1652cm -1 There occurs C=O stretching vibration, i.e. "amide I" band, at 1530cm -1 The C-N stretching vibration, namely an 'amide II' band, occurs, and the C-N stretching vibration and the N-H bending vibration are coupled on a plane to generate the C-N stretching vibration, which shows that the oxidized nano-diamond and the ethylenediamine undergo amidation reaction in the ball milling process, so that an amide group is generated, and the ethylenediamine is successfully bonded to the surface of the nano-diamond. Para-propylene diamine aminated nanodiamond and ammonia aminated nanodiamond, which are also 1652cm -1 C=O stretching vibrations ("amide I" band) also occur nearby at 1530cm -1 The C-N stretching vibration (an amide II band) appears, which shows that the oxidized nano diamond can also successfully generate the reaction with the propylene diamine and the ammonia waterAmidation reaction and bonding to the nanodiamond surface.
Dispersibility of aminated nanodiamond
As shown in the particle size distribution of the nanodiamond of fig. 5, in fig. 5, (a) oxidized nanodiamond, (b) ethylenediamine aminated nanodiamond, (c) propylenediamine aminated nanodiamond, and (d) ammonia aminated nanodiamond.
As shown in fig. 6, the nano diamond subjected to amination modification by ethylenediamine and ammonia water has better dispersibility than the oxidized nano diamond, has average particle diameters of 89nm and 97nm respectively, has polydispersity coefficients of 0.192 and 0.243, and is still a clear and transparent black dispersion after long-term storage. The average grain diameter of the nano-diamond modified by the propylene diamine is 114nm, and the polydispersity coefficient is 0.237.
Load factor of (ten) composite nanoparticle
The three prepared composite nanoparticle dispersion solutions are diluted, and the loading rates of the three composite nanoparticles are obtained through ultraviolet spectrophotometry measurement and regression equation calculation as shown in the following table 4:
TABLE 4 load factor of composite nanoparticles
The loading rates of the three samples are respectively 80.14+/-0.23%, 79.86+/-0.19% and 79.98+/-0.11%, and the loading rates of the three aminated nano-diamonds on indocyanine green are not greatly different. Because the adsorption principle of the aminated nano-diamond to indocyanine green is electrostatic interaction, the content of amino on the surface of the aminated nano-diamond has a certain influence on the loading rate of indocyanine green. As shown by the thermogravimetric results, the ethylenediamine amination effect is best, and the quantitative value of amino groups on the surface of the ethylenediamine amination is 2.60mmol/g respectively, so that the loading rate of ethylenediamine amination nano-diamond to indocyanine green is highest; the amination effect of the ammonia water aminated nano-diamond is second, the quantitative value of the amino group on the surface of the ammonia water aminated nano-diamond is 1.83mmol/g, so that the load of the ammonia water aminated nano-diamond to indocyanine green is smaller than that of the ethylenediamine aminated nano-diamond; the amination effect of the propanediamine aminated nano-diamond is the worst, and the quantitative value of the amino group on the surface of the propanediamine aminated nano-diamond is 1.34mmol/g, so that the load of the propanediamine aminated nano-diamond to indocyanine green is the least.
(eleven) dispersibility of composite nanoparticles
As shown in the particle size distribution of the composite nanoparticle of FIG. 6, in FIG. 6, (a) ND-EtNH 2 @ICG，(b)ND-PrNH 2 @ICG，(c)ND-NH 2 @ICG。
The particle sizes of the three aminated nano-diamonds after being loaded with indocyanine green are obviously increased, namely 277nm, 330nm and 250nm, the polydispersity coefficients are respectively 0.256, 0.187 and 0.223, and the three aminated nano-diamonds are clear dark green dispersion liquid. The reason for the increase in particle size is mainly two: on the one hand, after the indocyanine green is carried on the aminated nano-diamond, the indocyanine green is stacked on the nano-diamond to increase the particle size; on the other hand, the combination of indocyanine green-loaded composite nanoparticles with aminated nanodiamonds that do not load indocyanine green causes aggregation of a plurality of nanocomposite particles, and also causes an increase in particle size. The ammonia aminated nano diamond has the advantages that the load rate on indocyanine green is low due to the shortest carbon chain on the surface, so that the increase of the particle size is minimum; the nano-diamond aminated by the propanediamine has the largest average particle diameter after adsorbing indocyanine green because the surface carbon chain is longest and the initial particle diameter is larger; the initial particle size of the ethylenediamine aminated nano-diamond is the smallest, but the loading rate of the ethylenediamine aminated nano-diamond to indocyanine green is higher, so that the average particle size increase range is larger than that of ammonia aminated nano-diamond.
Photo-thermal heating curve of (twelve) aminated nano-diamond loaded indocyanine green composite nano-particle
As shown in the photothermal temperature rise curve of indocyanine green and composite nanoparticle in fig. 7, a: temperature increase curve, B: temperature delta differential curve. (sample a: ND-EtNH) 2 @ ICG, sample b: ND-PrNH 2 @ ICG, sample c: ND-NH 2 @ ICG, sample d: ICG).
TABLE 5 temperature increment of composite nanoparticles
As shown in fig. 7 a, three aminated nanodiamonds loaded with indocyanine green were heated up greatly after being irradiated with near infrared laser at 808nm for 5min, the temperature increases were 53.1 ℃, 52.4 ℃ and 52.7 ℃, respectively, and the order of the temperature changes was consistent with the order of the load factor. The aminated nano diamond has strong photo-thermal effect even at low concentration (200 mug/mL) after adsorbing indocyanine green, and shows excellent photo-thermal conversion performance. And the photo-thermal effect is more obvious than that of the simple indocyanine green solution. The related research literature indicates that the heat resistance of tumor cells is lower than that of normal tissue cells. When the temperature of the tumor cells reaches 42-47 ℃, the tumor cells can be ablated. Meanwhile, the local high temperature can induce the property of tumor cell membrane to change, and increase the permeability and fluidity of the cell membrane, thereby enhancing the uptake of tumor cells to the carrier, and at the moment, if the nano carrier is loaded with the anticancer drug at the same time, the treatment effect of the drug on the tumor cells can be further improved. As can be seen from FIG. 7B, ND-EtNH 2 The @ ICG composite nanoparticle reaches the fastest heating rate when irradiated by near infrared light for 20 seconds, and the heating rate after 20 seconds is gradually reduced; ND-PrNH 2 @ ICG and ND-NH 2 The two composite nano particles of @ ICG reach the fastest temperature rising rate at 0s, then the temperature rising rate is rapidly reduced, and the temperature rising rate is slowly reduced after 30 s. The temperature rising rate of the simple indocyanine green solution reaches the fastest after the solution is irradiated by near infrared light for 10 seconds, and then the temperature rising rate is rapidly reduced. The indocyanine green and the three composite nano particles can be rapidly heated within 30 seconds after being irradiated by near infrared light, the temperature increment at the moment reaches about 15 ℃, and the temperature (45 ℃) for starting the ablation of tumor cells is reached. The method shows that the indocyanine green loaded on the aminated nano diamond has no obvious influence on the photo-thermal temperature rise condition of the indocyanine green.
Photo-thermal stability curve of thirteen aminated nano-diamond loaded indocyanine green composite nano-particle
As shown in fig. 8, the photo-thermal stability curves of indocyanine green and composite nanoparticles. In FIG. 9, the temperature profile of indocyanine green in the second cycle is observedThe temperature increment of the first cycle is 51.9 ℃, the temperature increment of the second cycle is reduced to 41.9 ℃, and the temperature increment of the third cycle is only 32.1 ℃. The results demonstrate that indocyanine green alone has poor photo-thermal stability. After the 808nm near infrared laser is continuously and repeatedly irradiated for 3 times, the highest temperature of the three composite nano particles is higher than the temperature of the free indocyanine green solution in each cycle irradiation, and the temperature increment loss of each cycle is also smaller than that of the simple indocyanine green, which indicates that the photo-thermal stability of the indocyanine green is obviously improved after the indocyanine green is loaded on the surface of the nano diamond. For three composite nano-particles of aminated nano-diamond loaded indocyanine green, their photo-thermal stability is ND-NH 2 Optimal @ ICG, ND-EtNH 2 @ICG second, ND-PrNH 2 @ ICG was worst. The reason for this phenomenon may be that the different carbon chain lengths of the amino groups on the surface of the aminated nanodiamond result in different binding tightness between the aminated nanodiamond and indocyanine green, thereby affecting the photo-thermal stability of the composite nanoparticle. The carbon chain on the surface of the ammonia aminated nano diamond is shortest, and the combination of the loaded indocyanine green and the nano diamond is the tightest, so ND-NH 2 The photo-thermal stability of @ ICG is best, and the maximum value of the temperature increment of three photo-thermal cycles is 52.7 ℃, 53.4 ℃ and 49.1 ℃ respectively; the surface carbon chain length of ethylenediamine aminated nano-diamond is longer than that of the former, and the bonding degree of the supported indocyanine green and the nano-diamond is not as close as that of the former, so ND-EtNH 2 The photo-thermal stability of @ ICG is weaker than that of the former, and the maximum value of the temperature increment of three photo-thermal cycles is 53.1 ℃, 51.6 ℃ and 45 ℃ respectively; because the carbon chain of the propylenediamine is longest, the indocyanine green loaded by the propylenediamine aminated nano-diamond is combined with the nano-diamond more loosely, and gradually falls off from the propylenediamine aminated nano-diamond in the process of circulating illumination and is subjected to photolysis, thus ND-PrNH is observed 2 The temperature increment of the @ ICG in the process of circulating illumination is gradually reduced, and the temperature increment of three photo-thermal cycles is 52.4 ℃, 47.5 ℃ and 39.1 ℃ respectivelyPhoto-thermal stability is the worst among the three composite nanoparticles.
Photo-thermal stability of (fourteen) aminated nano-diamond loaded indocyanine green composite nano-particles
As shown in fig. 9 the photo-thermal stability of indocyanine green and composite nanoparticles. As can be seen from FIG. 10, after 3 light cycles, ND-NH was found 2 The photo-thermal stability of @ ICG is optimal, and the photo-thermal stability values of the 2 nd and the 3 rd times are respectively 101.33% and 93.17%; ND-EtNH 2 Second, the 2 nd and 3 rd times photo-thermal stability values are 97.18% and 84.75%, respectively; ND-PrNH 2 The @ ICG was worst, and the photo-thermal stability values at times 2 and 3 were 90.65% and 74.62%, respectively. The light-heat stability attenuation degree of the simple indocyanine green is serious, and the light-heat stability values of the 2 nd and the 3 rd times are only 80.73% and 61.85% respectively. Therefore, the photo-thermal stability of the three composite nano particles is better than that of the simple indocyanine green, and the three composite nano particles can be repeatedly used for photo-thermal treatment. The reason for the improved photo-thermal stability of the composite nano-particles is probably that indocyanine green generates active oxygen by irradiation of near infrared light under the condition of oxygen, and the active oxygen is taken as an electron donor and can catalyze the photo-induced decomposition of indocyanine green [ The method comprises the steps of carrying out a first treatment on the surface of the The nano diamond plays a role in regulating and quenching the electron donor of active oxygen, can reduce the content of active oxygen in a system, reduces the phenomenon of light-induced indocyanine green decomposition, and further improves the photo-thermal stability of indocyanine green.
Cell uptake of (fifteen) composite nanoparticles in cells
TABLE 6 cellular uptake of composite nanoparticles in HepG2 cells
As shown in Table 6, the cell uptake of indocyanine green alone in HepG2 cells was still the lowest, 46.06.+ -. 0.21%, while ND-NH 2 The highest cell uptake of @ ICG was 68.54.+ -. 0.03%, followed by ND-EtOH 2 At @ ICG, cell uptake was 63.63.+ -. 0.18%, ND-PrNH 2 @ ICG is larger in HepG2 cells due to its larger particle sizeThe uptake rate of the polymer is still lower than that of the other two composite nano particles, which is 56.53 plus or minus 0.05 percent. Compared with simple indocyanine green, the composite nanoparticle has the advantages that the cell uptake rate is greatly improved, the uptake rate of the tumor cells on the composite nanoparticle is higher, and the further analysis of the photothermal treatment of the tumor cells is expected.
Sixteen cell ablation experiments
(1) Samples were diluted to 40 μg/mL with PBS solution, 1mL of samples were added to each well in a 12 well plate, fresh medium was selected as control group, samples were added to both wells, and they were divided into light and non-light groups, and incubated in medium for 4h.
(2) After the incubation was completed, the drug-containing medium was discarded, and after three washes with pre-chilled PBS, the drug not taken in was removed, and replaced with fresh medium without blood and double antibody, and incubation was continued at 37 ℃ without light.
(3) And (3) light group treatment: and (3) irradiating the sample in the pore plate by using a 808nm near infrared laser, measuring the temperature by using a thermal infrared imager, suspending irradiation when the temperature of the solution reaches 47 ℃, continuing to irradiate to 47 ℃ when the temperature is reduced to 37 ℃, and placing the sample in an incubator after three times of circulation.
(4) If the temperature of the sample in the pore plate can not be raised to 47 ℃, the irradiation is stopped after the continuous irradiation is carried out for 5 min.
(5) After 24h of incubation, the cells were observed for ablation in the open field by fluorescence microscopy.
(6) Adding 70% ethanol, and fixing at 4deg.C for 10min; discarding ethanol, and rinsing with PBS once;
(7) Adding 50 mu L of 10 mu g/mL Hoechst 33258 dye, and dyeing for 15min at 4 ℃ in a dark place;
(8) Rinsing with PBS three times, and finally adding a small amount of PBS into the culture well; and (5) placing the materials under an inverted fluorescent microscope for observation, photographing and exciting the UV.
Hoechst 33258 is a commonly used nuclear dye that can be used for staining living cells and staining cells after fixation. It binds to DNA non-intercalating, mainly to the A-T base region of DNA. When excited by ultraviolet light, the fluorescent lamp emits bright blue fluorescence.
Cell nuclei of non-illuminated cell lines were stained with uniform blue fluorescence. As in fig. 10; after illumination, the cell nucleus is obviously condensed, as shown in figure 11, and obvious apoptosis bodies appear, the cell nucleus is in crescent shape, and a small amount of dispersed DNA exists outside the main nucleus.
The foregoing is merely illustrative of specific embodiments of the present invention, and the scope of the invention is not limited thereto, but any modifications, equivalents, improvements and alternatives falling within the spirit and principles of the present invention will be apparent to those skilled in the art within the scope of the present invention.
Claims (3)
1. The method for loading indocyanine green by the nano-diamond is characterized by comprising the following steps of:
(1) Directly aminating and modifying the oxidized nano diamond by using ethylenediamine, propylenediamine or ammonia water through a chemical method;
(2) And carrying indocyanine green on the three aminated and modified nano diamonds through electrostatic interaction to prepare ND-EtNH 2 @ICG、ND-PrNH 2 @ ICG or ND-NH 2 @ ICG composite nanoparticles and testing the loading and dispersibility of the composite nanoparticles;
the method comprises the steps of (1) grinding oxidized nano-diamond and ethylenediamine, propylenediamine or ammonia water in a grinding kettle at room temperature, centrifugally separating and suction-filtering the ground mixture, centrifuging for 10min at a centrifugal force of 8000g to obtain a clear and transparent black colloid solution, and selecting an aminated nano-diamond colloid solution with a proper particle size;
in the direct amination modification of oxidized nano diamond by using ethylenediamine, propylenediamine or ammonia water through a chemical method, the aminated nano diamond colloid solution is ND-EtNH 2 、ND-PrNH 2 Or ND-NH 2 One or a mixture of three composite nano particles, wherein the concentration of the solution is 0.01-10%;
step (2) is to weigh a certain amount of indocyanine green to be dissolved in deionized water, and prepare a indocyanine green solution with a certain concentration; mixing the aminated nano-diamond colloid solution with indocyanine green solution; then the pH value of the solution is adjusted to be less than 6, and the mixture is stirred and reacted under dark condition and room temperature environment for 24h after the magneton is added; centrifuging the solution after the reaction is finished, washing and centrifuging the solution with deionized water for three times, and collecting supernatant fluid of each centrifugation; obtaining a solution of aminated nano-diamond loaded indocyanine green composite nano-particles, and storing the solution under a light-shielding condition for subsequent load rate and dispersibility tests.
2. The method for loading indocyanine green by nano-diamond according to claim 1, wherein the concentration of indocyanine green solution is 0.01% -10%.
3. Indocyanine green-loaded nano-diamond prepared by the method for loading indocyanine green by nano-diamond according to any one of claims 1-2.
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| CN107162011A (en) * | 2017-05-04 | 2017-09-15 | 东华大学 | A kind of preparation method for the hectorite nano particle ICG/LAP for loading indocyanine green |
| CN109164074A (en) * | 2018-08-31 | 2019-01-08 | 华中师范大学 | The preparation method and its twice dispersing technique of a kind of monodisperse amination Nano diamond colloidal solution and the application in cell marking |
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| CN109164074A (en) * | 2018-08-31 | 2019-01-08 | 华中师范大学 | The preparation method and its twice dispersing technique of a kind of monodisperse amination Nano diamond colloidal solution and the application in cell marking |
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