CN104083771A - Fluorescence resonance energy transfer-based tumor imaging and therapeutic targeting system and construction method thereof - Google Patents

Abstract

The invention belongs to the technical field of medicine, in particular to a tumor imaging and treatment targeting system based on fluorescence resonance energy transfer and a construction method thereof. The invention adopts the electrochemical stripping method to synthesize luminescent carbon quantum dots, uses the EDC/NHS chemical coupling method to connect polyethylene glycol with amino groups at the end of the CDots, and then connects the tumor targeting drug folic acid, through carbon dots and anti-tumor The π-πstacking effect on the surface of the drug doxorubicin is connected to doxorubicin to obtain a two-photon imaging and targeted therapy tumor system based on fluorescence resonance energy transitions. The system is a composite nanomaterial, which can judge the release of drugs and the properties of energy resonance transfer through the intensity and color of the luminescence; the use of two-photon imaging can penetrate deep animal tissues, and can be developed into the field of tumor treatment. The invention has the advantages of stable reaction, simple and easy operation, low cost and no pollution, easy control of appearance and structure, uniform product distribution, difficult agglomeration, high purity and easy industrialization.

Description

Tumor imaging and therapeutic targeting system based on fluorescence resonance energy transfer and its construction method

technical field

The invention belongs to the technical field of medicine, and in particular relates to a two-photon imaging of fluorescence resonance energy transition and a targeted therapy tumor system and a construction method thereof.

Background technique

Carbon is the basis of all known life on earth. In recent years, research on carbon-based nanotechnology has become a research hotspot. Carbon nanostructure materials represented by carbon nanotubes are considered to be one of the focuses of current nanotechnology research due to their unique structure, physical and chemical properties, and have shown great potential in optics, electronics and informatics. Value. Conventional carbon materials are typical narrow-bandgap materials, so the conventional wisdom is that nanomaterials do not have abundant fluorescent properties like other semiconductor materials. Due to its various electron orbital characteristics (sp, sp2, sp3), many substances with peculiar structures and properties have been formed, among which fullerenes and carbon nanotubes are the most popular research objects in carbon nanostructures. In addition, Other members of the carbon nanomaterials family, such as carbon quantum dots, are also gradually receiving more and more attention.

It is noteworthy that recent studies have shown that carbon nanotubes can emit fluorescence under certain conditions, and the surface of carbon quantum dots can also produce strong fluorescence after being modified with polymers. The synthesis methods of carbon quantum dots are mainly divided into two categories: top-down and the bottom-up approach. The top-down approach refers to the formation or exfoliation of carbon quantum dots in relatively large carbon structural materials, which includes arc cutting, laser ablation, and electrochemical oxidation. The bottom-up pathway refers to the formation of carbon quantum dots.

Mainly from molecular precursors, it mainly includes calcination or heating method, carrier synthesis method, microwave synthesis method and ultrasonic synthesis method. Sun's research group prepared fluorescent carbon nanoparticles with a maximum emission wavelength of 400-694 nm by laser ablation method, which can be endowed with good water dispersibility through PEG modification, and PEG modification on the surface of carbon quantum dots successfully realized in vivo imaging of lymphatic circulation . ^[1] Liu et al prepared multicolor fluorescent carbon nanoparticles with a particle size of less than 2 nm and a maximum emission wavelength of 415-615 nm by treating the burned candle ash with oxidative acid and purifying it by gel electrophoresis. ^[2] The blue-emitting carbon nanoparticles prepared by Pang's research group by electrooxidation of graphite have good water dispersibility and light stability ^[3] Lee's research group recently developed an electrochemical method to prepare particles with a particle size of Fluorescent carbon nanoparticles of 1.2-3.8 nm can be prepared in one step by adjusting the current density, and carbon nanoparticles with emission colors ranging from blue to orange-red. This carbon nanoparticle also has good water dispersibility, strong fluorescence emission and up-conversion luminescence properties. ^[4] Due to the advantages of non-toxicity, low cost, and good stability, fluorescent carbon nanomaterials, especially fluorescent carbon nanoparticles with a clear structure, are widely used in nanobiomedical fields such as biological imaging, tumor detection and diagnosis, etc. It has important application value. Carbon quantum dots are a new type of carbon material discovered in recent years. Compared with traditional semiconductor quantum dots and organic dyes, this new member of the carbon family not only maintains the advantages of carbon materials such as good biocompatibility and low toxicity, Moreover, it also has the advantages of easy functionalization of two-photon, large absorption cross-section, adjustable luminous range, good photostability, no light flicker, low price, and easy large-scale synthesis. The non-quenching property of two-photon imaging is dynamic, real-time and accurate. The monitoring of biologically active substances in organisms provides a guarantee; two-photon imaging enhances light penetration and imaging depth through low-energy and long-wavelength light excitation, and effectively avoids background autofluorescence of organisms, thereby reducing photoinduced biological damage. Therefore, research on the emerging field of carbon quantum dots will have a significant impact on the development of materials science.

Two-photon has also become a research hotspot this year. Two-photon absorption/excitation is a process in which a molecule absorbs two photons at the same time under strong light excitation, and transitions from the ground state to an excited state with twice the energy of the photon. The two photons can be of the same wavelength or of different wavelengths, but they must be absorbed simultaneously (the time interval between the two photons reaching the excited molecule is less than 1 femtosecond). It can be understood in this way that the energy of a photon is absorbed to transition to a virtual intermediate state, and then the energy of a photon is absorbed to transition to an excited state.

Two-photon has the following advantages: Greater penetration depth with less photodamage; Less photobleaching and phototoxicity; Tunable excitation wavelength - polychromatic excitation; Imaging of nonlinear effects—SHG; better signal-to-noise ratio; Easier separation between excitation and emission wavelengths; UV and VIS can be excited by infrared (IR); dark field imaging can be realized with little background interference.

Fluorescence resonance energy transfer (Förster resonance energy transfer (FRET), is a mechanism to describe the energy transfer between two chromophores. It is required that the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor when they are spatially close to each other At a certain distance (1-10 nm), the fluorescence energy generated by exciting the donor is just absorbed by the nearby acceptor, so that the fluorescence intensity emitted by the donor attenuates, and the fluorescence intensity of the acceptor fluorescent molecules increases. The efficiency of energy transfer and The emission spectrum of the donor is related to the overlapping degree of the absorption spectrum of the acceptor, the relative orientation of the transition dipole of the donor and the acceptor, the distance between the donor and the acceptor, etc. The fluorescent substance must meet the following conditions: ① accept, The excitation light of the donor should be sufficiently separated; ②The emission spectrum of the donor should overlap with the excitation spectrum of the acceptor.

Contents of the invention

The purpose of the present invention is to provide a two-photon imaging and targeted therapy tumor system based on fluorescence resonance energy transition and its construction method.

The present invention proposes for the first time a carbon dot drug delivery system förster based on energy resonance transfer Resonance energy transfer (FRET)-based CDots drug delivery system (FRET-CDot-DDS), the synthesized carbon dots have good biocompatibility, between carbon dots and loaded fluorescent drug molecules such as anti-tumor drug doxorubicin There is an effective energy resonance transfer between them, which can promote drug delivery, cell imaging, and realize real-time dynamic monitoring of tumor drug effects. Furthermore, this energy resonance transfer-based carbon dot drug delivery platform exhibits two-photon imaging and drug tracking in deep tissues, which can be widely used in the field of bio-drug loading.

In the present invention, the luminescent carbon quantum dots (referred to as CDots) are firstly synthesized by the electrochemical stripping method, and the carbon dots are treated with concentrated nitric acid to bring hydroxyl and carboxyl groups on the surface, and the CDots are connected with amino groups at the end by EDC/NHS chemical coupling method Polyethylene glycol (PEG) (denoted as CDot-PEG), through EDC/NHS chemical coupling method, and then coupled with tumor targeting drug folic acid (denoted as CDot-PEG-FA), through carbon dots and anti-tumor drugs The π-πstacking effect on the surface of doxorubicin is coupled with doxorubicin to obtain a two-photon imaging and targeted therapy tumor system based on fluorescence resonance energy transition, which is denoted as CDot-FA-DOX. The CDot-FA-DOX is a composite nanomaterial.

Specifically, the synthesis steps of the composite nanomaterial CDot-FA-DOX are as follows:

(1) First, prepare 1.0-3.0 mg/ml CDots and 10-20 mg/ml polyethylene glycol 5000 with six amino groups, stirred for 5-15 min, then add 5-10 mM NHS, then sonicate for 60-120 min; then add 20-30 mM EDC and 5-10 mM NHS, stir together, react for 24-72 h; stop the reaction by adding mercaptoethanol; then centrifuge at high speed with 2×PBS for 1-2 hours to obtain the supernatant, which is CDot-PEG, and freeze Dry and store in the refrigerator for later use;

(2) Secondly, weigh 35-50 mg folic acid, add 15-30 mg EDC and 25-50 mg NHS at the same time, add 1-2 ml pH7.4-9.0 PBS, stirred at room temperature for 15-30 minutes; then added 20-40 mg CDot-PEG, stirred at room temperature for 24-48h, Wash and centrifuge and freeze-dry for later use;

(3) Finally, 1-3 mg/ml of CDot-PEG-FA and 2-5 mg/ml of DOX pH7.4-9.0 PBS stirred at room temperature overnight, polymer dialyzed through the dialysis bag for 48-72 hours in pure water, changing the water every 4-8 hours; the product in the dialysis bag was freeze-dried to obtain the final product CDot- FA-DOX.

The present invention found that through the characteristics of fluorescence energy resonance transfer between the luminescent carbon dots and the anti-tumor drug doxorubicin, when the two are a whole CDot-FA-DOX, excited by 405 nm laser, the emission wavelength is 495 nm , due to the effect of energy resonance transfer, it mainly emits the red light of doxorubicin. With the continuous release of the drug, the effect of energy resonance transfer slowly disappears; at this time, use 405 nm laser excitation, the emission wavelength is 498 nm, mainly emitting the green light of doxorubicin. Therefore, the release of the drug and the nature of the energy resonance transfer can be judged by the intensity and color of the luminescence. At the same time, CDot-FA-DOX, for the first time using two-photon imaging, can penetrate deep animal tissues, and then can look forward to the field of tumor treatment.

Cytotoxicity experiments showed that the average survival rate of cells was above 80%, and the cytotoxicity was grade 1. It can be considered that CDot-FA-DOX prepared by chemical coupling method has good cytocompatibility. When CDot-FA is compounded with DOX, the toxicity of simple DOX can be reduced. The experimental results can prove on the one hand that CDot-PEG is a safe carrier with low toxicity and good biocompatibility, and on the other hand can prove that CDot-FA-DOX nanocomplex is a safe drug delivery system for normal cells. The experimental results of the tumor inhibition rate of the cells showed that with the increase of the concentration of DOX, CDot-FA-DOX and CDot-PEG, the inhibitory effects of the three on Hela cells were significantly improved. Compared with DOX, the degree of inhibition of tumor cells has been improved to a certain extent, which is significantly greater than the inhibition rate of tumor cell activity of the drug molecule DOX itself. At the same time, the results of cell flow cytometry showed that CDot-FA-DOX not only has good sustained-release performance, but also has a higher tumor inhibition rate than pure DOX, thereby greatly improving the drug utilization of doxorubicin, while reducing a certain amount of normal Cytotoxicity, causing apoptosis of tumor cells thereby enhancing the antitumor properties of the drug.

The present invention has the following advantages:

1. This method is the first to propose a method for two-photon imaging of novel fluorescence resonance energy transitions and the construction of a targeted therapy tumor system. The product quantity is large, the particle size is small, the distribution is uniform, and the monodispersity is good. The invention fills in the blank of the two-photon imaging of the fluorescence resonance energy transition and the construction method of the targeted therapy tumor system.

2. The technology of the present invention is easy to operate, the reaction system is mild, stable, and less disturbed, and the product processing is easy to handle, which is convenient for large-scale synthesis of materials.

3. The CDot-FA-DOX prepared by the present invention has the characteristics of high drug loading, stable drug loading and can be absorbed and degraded by the body. It is a kind of effective drug carrier and can be used as an effective drug carrier in the field of medicine.

4. The process of preparing CDot-FA-DOX by direct surface chemical coupling not only has mild reaction, but also avoids environmental pollution, and the production cost is low. It is an ideal green material preparation method.

5. The CDot-FA-DOX prepared by the present invention can realize the real-time dynamic monitoring of the release of anti-tumor drugs by fluorescence energy transfer, and at the same time perform two-photon live cell imaging and tissue imaging for the first time, and penetrate deep into the tissue.

Description of drawings

The carbon quantum dots synthesized in Fig. 1 Example 1. The size of carbon quantum dots is about 5 nm, and the thickness is also 5 nm. The lattice conforms to the 002 crystal plane of graphene.

Figure 2 is a comparison of the sustained release of CDot-FA-DOX and DOX at different pHs.

Figure 3 shows the inhibition rate of CDot-FA-DOX and DOX on tumor cells.

Figure 4 shows the apoptosis rate of CDot-FA-DOX and DOX on tumor cells.

Figure 5 shows the changes in the doxorubicin channel of CDot-FA-DOX at 0.5 hours.

Detailed ways

Example 1:

In the first step, the preparation of fluorescent carbon quantum dots mainly includes the following steps: Electrolyte solution configuration: ethanol/water (V/V=99.5:0.5) slowly add 0.2-0.4 g NaOH to the above solution, and stir evenly with a glass rod. Electrochemical electrolysis preparation process: Two graphite rods (diameter 0.5 cm) are used as cathode and anode respectively, the current density is controlled at 10-200mAcm ^-2 , and the reaction takes 2 hours. Purification of luminescent quantum dots: add MgSO ₄ (5-7 wt%) to the above crude product and stir for 20 min, then seal and store overnight to remove salt and water, then separate and purify on a silica gel column to obtain carbon quantum dots with uniform size.

In the second step, prepare 1.0-3.0 mg/ml CDots and 20-40 mg/ml polyethylene glycol 5000 (PEG-6NH ₂ ) with six amino groups, stir for 5-10 min, then add 5 mM NHS, and then Sonicate for 60-120 min, then add 20-30 mM EDC and 5-10 mM NHS and stir for 24-72 h, the reaction is terminated by adding mercaptoethanol, and then centrifuged with 2×PBS at high speed (15000 rpm) for 1-2 hours The obtained supernatant is CDot-PEG, freeze-dried and stored in the refrigerator for later use. Weigh 35-50 mg folic acid, add 15-30 mg EDC and 25-50 mg NHS at the same time, add 1-2 ml pH7.4-9.0 PBS, stir at room temperature for 15-30 minutes, then add 20-40 mg CDot-PEG, room temperature Stir for 24-48h, wash, centrifuge and freeze-dry for later use. Finally, 1-3 mg/ml of CDot-PEG-FA and 2-5 mg/ml of DOX pH7.4-9.0 PBS were stirred overnight at room temperature, and the polymer was dialyzed through a dialysis bag (molecular weight cut-off 3500) for 48-72 hours in pure In the water, change the water every 4-8 hours. The product in the dialysis bag was freeze-dried to obtain the final product CDot-FA-DOX, which was stored in a refrigerator at 4°C until use. (All reagents above are of analytical grade)

Results of Example 1: The carbon quantum dots synthesized as shown in Figure 1 are relatively uniformly dispersed, with a size of about 5 nm and a thickness of 5 nm, and the lattice conforms to the 002 crystal plane of graphene.

Example 2:

Maximum absorption wavelength: Weigh 0.003-0.005 g doxorubicin (DOX), put it in a 10mL volumetric flask, dissolve it with ddH ₂ O and make to volume. In the range of 200-800 nm, the ultraviolet-visible spectrophotometer was used to scan, and 480 nm was the maximum absorption wavelength of doxorubicin, so the experiment chose to test the concentration of doxorubicin in the solution at this wavelength.

Use the same method to detect the full ultraviolet spectrum of DOX/CDot-FA-DOX nanomaterials, observe the absorption of the ultraviolet spectrum, and determine whether there is interference at the DOX sensitive absorption wavelength of this material.

Drawing of DOX working curve: Prepare 1, 2, 3, 4, 5, 6, 8, 10 μg/ml DOX standard solutions respectively, and measure their absorbance at 480 nm. Fit the working curve.

Determination of drug loading capacity of DOX/CDot-FA-DOX composite nanomaterials: Prepare samples, put 0.003-0.005 g of DOX/CDot-FA-DOX dry powder in 10 ml Into the volumetric flask, add 100 μL of 6 M hydrochloric acid solution dropwise, and adjust the volume to 10 ml with 0.02 M, pH 7.45 phosphate buffer solution. After ultrasonication for 30 minutes, place in a 37 °C water bath overnight. The next day, measure its UV absorbance value at 480 nm, and compare it with the previously drawn DOX working curve to obtain the concentration of podophyllum in the sample. Calculated using the following formula:

Drug loading of CDot-FA-DOX = (mass of drug in CDot-FA-DOX / total mass of composite material) × 100%

Results of Example 2: The average drug loading of DOX/CDot-FA-DOX composite nanomaterials was 37.6%.

Example 3:

Step 1: Use the normal cell line HEK 293T to detect its cytotoxicity, and select Hela for the inhibitory effect on tumor cells tumor cells.

Step 2: After pre-cultured for 24 hours, the cells adhered to the wall, and the cell culture medium was used as the negative control group. Different concentrations of CDot-PEG, CDot-FA, DOX solution, and CDot-FA-DOX suspension were added to 3 wells in each group. solution and suspension (concentrations were 0.5, 1.0, 2.5, 5.0 μg/ml).

Step 3: After culturing for 24 h, add 20 μl of MTT solution to each well, discard the supernatant after incubation for 4 h, and add 150 μl of DMSO to each well to terminate the reaction.

Step 4: Shake the culture plate horizontally for 30 min, and use an enzyme-linked detector at 480 The absorbance was measured at nm, and the cell viability was calculated according to the formula:

Cell viability% = A _{480 ( sample )} / A _{480 ( control )} × 100 %

A _{570 (sample)} : the absorbance value of the added sample group; A _{570 (control)} : the absorbance value of the blank medium cells in the control group.

1.0×106 cells per well were inoculated in a 24-well plate, and the volume of each well was 500 μl. The culture plate was moved to a CO2 incubator, and cultured for 24 hours at 37 ºC, 5 % CO2 and saturated humidity. If the wall is firm, add CDot-FA-DOX diluted to 10 μg/ml in 1640 culture medium, set 1 blank cell in each plate, set 3 duplicate wells in each group, and continue to culture for 48 hours respectively.

Dyeing process:

(1) Wash the cells fully with PBS, and take a total of about 5-2.5×104 cells for testing;

(2) Resuspend the cells with 250 µl binding buffer and make the concentration 2~5×105/ml;

(3) Take 195 µl of cell suspension and add 5 µl Annexin V/PI;

(4) After mixing, incubate at room temperature in the dark for 10 minutes;

(5) Wash the cells once with 190 µl of binding buffer;

(6) Resuspend the cells with 190 µl of binding buffer;

(7) Add 10 µl of 20 µg/ml propidium iodide solution (final concentration: 1 µg/ml; flow cytometry FACS analysis.

Results of Example 3: As shown in Figure 2, Figure 3 and Figure 4, CDot-FA-DOX not only has good sustained-release performance than pure DOX, but also has a higher tumor inhibition rate, thereby greatly improving the drug utilization of doxorubicin At the same time, it reduces certain normal cytotoxicity, causes apoptosis of tumor cells, and improves the antitumor properties of the drug.

Example 4:

Step 1: Inoculate 1.0×106 cells per well on a 6-well plate, with a volume of 1000 μl per well, move the culture plate to a CO ₂ incubator, under the conditions of 37 ºC, 5 % CO ₂ and saturated humidity, After culturing for 24 h, the cells were firmly attached to the wall, and CDot-PEG, CDot-FA, DOX solution, and CDot-FA-DOX suspension diluted to 10 μg/ml were added, and a blank cell control group was set on each plate, and the culture was continued. 30 min.

Step 2: Wash the cells added with the material three times with PBS, and finally add 0.5 mL of serum-free medium to observe the cells directly on laser confocal.

Results of Example 4: As shown in Figure 5, when CDot-FA-DOX is at 0.5 hours, when there is FRET, the doxorubicin channel is very bright. The mycin channel slowly becomes weaker, while the carbon dot channel gradually becomes stronger, at which point FRET is turned off. Therefore, the drug release can be monitored dynamically in real time by fluorescence energy resonance transfer.

references

[1] L. Cao, M. J. Meziani, S. Sahu, Y.-P. Sun, Accounts of chemical research 2012, 46, 171-180.

[2] S. N. Baker, G. A. Baker, Angewandte Chemie International Edition 2010, 49, 6726-6744.

[3] Q.-L. Zhao, Z.-L. Zhang, B.-H. Huang, J. Peng, M. Zhang, D.-W. Pang, Chemical Communications 2008, 5116-5118.

[4] H. Li, X. He, Z. Kang, H. Huang, Y. Liu, J. Liu, S. Lian, C. H. A. Tsang, X. Yang, S. T. Lee, Angewandte Chemie International Edition 2010, 49, 4430-4434..

Claims (3)

1. a construction method for the tumor imaging based on FRET (fluorescence resonance energy transfer) and treatment target system, is characterized in that concrete steps are:

First adopt electrochemical stripping method to synthesize luminous carbon quantum dot, be designated as CDots, process carbon point with concentrated nitric acid and make hydroxyl and carboxyl on its surface band;

Connect end with amino Polyethylene Glycol (PEG) at CDots by EDC/NHS chemical coupling method, product is designated as CDot-PEG;

By EDC/NHS chemical coupling method, then connect tumor-targeting drug folic acid (FA), product is designated as CDot-PEG-FA);

Put with π-π stacking effect on antitumor drug amycin surface and connected amycin by carbon, obtain two-photon imaging and targeting therapy on tumor system based on fluorescence resonance energy transition, product is designated as CDot-FA-DOX; This CDot-FA-DOX is a kind of composite nano materials.

2. the construction method of the tumor imaging based on FRET (fluorescence resonance energy transfer) according to claim 1 and treatment target system, is characterized in that concrete operations flow process is as follows:

(1) first, preparation 1.0-3.0 mg/ml CDots and 10-20 mg/ml, with six amino Polyethylene Glycol 5000, stir 5-15 min, then add 5-10 mM NHS, more ultrasonic 60-120 min; Add again the NHS of 20-30 mM EDC and 5-10 mM, stir together, reaction 24-72 h; By adding mercaptoethanol cessation reaction; Then using 2 × PBS high speed centrifugation 1-2 hour, obtain supernatant, is CDot-PEG, and it is stand-by that lyophilization is put in refrigerator;

(2) secondly, take 35-50 mg folic acid, add 15-30 mg EDC and 25-50 mg NHS simultaneously, add 1-2 ml pH7.4-9.0 PBS, under room temperature, stir 15-30 minute; Then add 20-40 mg CDot-PEG, under room temperature, stir 24-48h, clean centrifugal lyophilization stand-by;

(3) last, the DOX pH7.4-9.0 PBS stirred overnight at room temperature of the CDot-PEG-FA of 1-3 mg/ml and 2-5 mg/ml o, polymer is dialysed 48-72 hour in pure water by bag filter, changes water once every 4-8 hour; Product in bag filter, by lyophilization, obtains end product CDot-FA-DOX.

3. construction method builds the tumor imaging based on FRET (fluorescence resonance energy transfer) and the treatment target system that obtain as claimed in claim 1 or 2.