MAGNETIC, THERMOSENSITIVE, FLUORESCENT MICELLE AND METHOD FOR PREPARING THE SAME

Abstract

A magnetic, thermosensitive, fluorescent micelle includes a core, a carrier wrapping the core, and a plurality of water-soluble near-infrared CdHgTe quantum dots (QD) disposed on the carrier. The core includes dextran-magnetic layered double hydroxide-fluorouracil (DMF). The carrier includes a tripolymer of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-polylactic acid (PLA). N-isopropylacrylamide-co-N,N-dimethylacrylamide of the tripolymer includes a hydrophilic group and a hydrophobic carbon frame. The hydrophilic group is oriented outwards with respect to the and forms a shell. The hydrophobic carbon frame and polylactic acid are restrained to wrap the dextran-magnetic layered double hydroxide-fluorouracil to form the core.

Claims

1. A micelle, comprising: 1) a core, the core comprising dextran-magnetic layered double hydroxide-fluorouracil (DMF); 2) a carrier wrapping the core, the carrier comprises a tripolymer of poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-polylactic acid; and 3) a plurality of water-soluble near-infrared CdHgTe quantum dots (QD) disposed on the carrier; wherein: N-isopropylacrylamide-co-N,N-dimethylacrylamide of the tripolymer comprises a hydrophilic group and a hydrophobic carbon frame; the hydrophilic group is oriented outwards with respect to the and forms a shell, and the plurality of water-soluble near-infrared CdHgTe quantum dots is bonded to the shell; and the hydrophobic carbon frame and polylactic acid are restrained to wrap the core.

2. The micelle of claim 1, wherein a lower critical solution temperature of the micelle is 42 C.

3. The micelle of claim 1, wherein monomers for synthesizing the tripolymer are N-isopropylacrylamide (NIPAM), N,N-dimethylacrylamide (DMAM) and lactide (PLA).

4. A method of preparation of the micelle of claim 1, the method comprising: 1) copolymerizing N-isopropylacrylamide and N,N-dimethylacrylamide in the presence of 2,2-azobis(2-methylpropion amidine) dihydrochloride thereby forming a dipolymer of P(NIPAM-co-DMAM)-OH; polymerizing the dipolymer and lactide in the presence of stannous octanoate thereby yielding an amphiphilic tripolymer of P(NIPAM-co-DMAM)-b-PLA; 2) synthesizing dextran-magnetic layered double hydroxide-fluorouracil, and combining the amphiphilic tripolymer and the dextran-magnetic layered double hydroxide-fluorouracil to yield a magnetic thermosensitive precursor through synchronous hydration and dialysis; 3) synthesizing water-soluble near-infrared CdHgTe quantum dots by one-pot synthesis in an aqueous phase; and 4) attaching the water-soluble near-infrared CdHgTe quantum dots prepared in 3) to a surface layer of the magnetic thermosensitive precursor prepared in 2) by electrostatic bonding technology.

5. The method of claim 4, wherein preparing the dipolymer of P(NIPAM-co-DMAM)-OH comprises: mixing N-isopropylacrylamide and N,N-dimethylacrylamide in a mass ratio of 95-85:5-15 to form a mixture; dissolving the mixture in an organic solvent A; aerating the organic solvent A with nitrogen to remove oxygen, followed by an addition of 2,2-azobis(2-methylpropion amidine) dihydrochloride as an initiator; 10-12 h later at a constant temperature of 70-80 C., precipitating a resulting product with excess ether, filtering and drying the resulting product under vacuum.

6. The method of claim 5, wherein the organic solvent A is tetrahydrofuran or chloroform; and an addition amount of the 2,2-azobis(2-methylpropion amidine) dihydrochloride accounts for 1 to 2 wt. % of that of the N-isopropylacrylamide and N,N-dimethylacrylamide.

7. The method of claim 4, wherein preparing the amphiphilic tripolymer of P(NIPAM-co-DMAM)-b-PLA comprises: mixing the dipolymer of P(NIPAM-co-DMAM)-OH with the lactide in a mass ratio of 40-30:60-70 to form a mixture; dissolving the mixture in an organic solvent B, followed by an addition of stannous octanoate as a catalyst; aerating the organic solvent B with nitrogen to remove oxygen; 24-28 h later at a constant temperature of 120-140 C., precipitating a resulting product with excess ether, and drying under vacuum.

8. The method of claim 7, wherein the organic solvent B is anhydrous xylene or toluene.

9. The method of claim 4, wherein combining the amphiphilic tripolymer of P(NIPAM-co-DMAM)-b-PLA and dextran-magnetic layered double hydroxide-fluorouracil comprises: dissolving the dextran-magnetic layered double hydroxide-fluorouracil and the tripolymer in an organic solvent N,N-dimethylformamide; transferring a resulting mixture to a dialysis bag, dialyzing against distilled water and stirring at room temperature.

10. The method of claim 9, wherein the dialysis bag has a molecular-weight cut-off of 8000-14000 g.Math.mol.sup.1.

11. The method of claim 9, wherein a dialysis time is 48 h; the distilled water is renewed every 1 h for first 5 h, and then every 12 h.

12. The method of claim 4, wherein a mass ratio of the dextran-magnetic layered double hydroxide-fluorouracil to the tripolymer is 5-20:20.

13. The method of claim 9, wherein a mass ratio of the dextran-magnetic layered double hydroxide-fluorouracil to the tripolymer is 5-20:20.

14. The method of claim 4, wherein attaching the water-soluble near-infrared CdHgTe quantum dots to the surface layer of the magnetic, thermosensitive micelle comprises: mixing and grinding the water-soluble near-infrared CdHgTe quantum dots and the magnetic, thermosensitive micelle in a mass ratio of 1-3:1-1 to prepare a mixed powder; and suspending and dispersing the mixed powder in absolute ethanol thereby yielding a suspension, and ultrasonically dispersing the suspension; separating magnetic particles from the suspension using a magnet, centrifuging, washing a resulting product with absolute ethanol, and drying under vacuum.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0029] FIG. 1 shows morphology of DMF-TRM micelles (A) under transmission electron microscope and the fluorescence image of DMF-TRM-QD micelles (B);

[0030] FIG. 2 shows imaging results of MGC-803 cells after incubation with DMF-TRM-QD complex for 1 h (A), 3 h (B), 5 h (C) and 7 h (D); (wherein green fluorescence represents the nuclear signal of Hoechst 33342 labeled cells, red fluorescence represents the signal of DMF-TRM-QD labeled cells, yellow fluorescence represents overlap of these two colors, which are observed with a 40 objective lens);

[0031] FIG. 3 shows confocal imaging results of MGC-803 cells after incubation with DMF-TRM-QD under hyperthermia at 42 C. and different applied magnetic field gradient (the number of magnets are respectively 0, 5, 10, 15, 20, 25); (where green fluorescence represents the nuclear signal Hoechst 33342 labeled cells; red fluorescence represents the signal of fluorescent micelle particles in the cells; yellow fluorescence represents overlap of these two colors);

[0032] FIG. 4 shows linear relationship between the average optical density of red fluorescence in a cell and the applied magnetic field strength (T).

DETAILED DESCRIPTION OF EMBODIMENTS

[0033] The reagents used in the chemical synthesis of the invention are all conventional commercial reagents, and the materials used in the biological experiment are all commercial products. It should be noted that the following embodiments are intended to describe and not to limit the disclosure.

EXAMPLE 1

Synthesis of DMF-TRM-QD Micelles

1) Preparation of P(NIPAM-co-DMAM)-b-PLA tripolymer (poly(N isopropylacrylamide-co-N,N dimethylacrylamide)-b-polylactic acid)

[0034] 0.41 g of NIPAM and 0.046 g of DMAM were weighed as raw materials according to the mass ratio of NIPAM/DMAM=90:10, and they were mixed and put into a three-necked flask. 25 mL of freshly distilled tetrahydrofuran (THF) was added to dissolve the mixture to form a solution. 0.009 g of copolymerization initiator AMAD was then added to react with the solution at a constant temperature of 80 C. for 10 h after oxygen was removed under nitrogen protection for 30 min. The resulting product was precipitated with excess ether, filtered under vacuum, and dried under vacuum at room temperature for 12 h to obtain P(NIPAM-co-DMAM)-OH dipolymer (poly(N isopropylacrylamide)-co-N,N dimethylacrylamide)). Then the P(NIPAM-co-DMAM)-OH dipolymer was precipitated with excess ether, filtered under vacuum, and dried under vacuum at room temperature for 12 h to obtain a pure solid-phase dimer.

[0035] 0.1879 g of lactide and 0.0909 g of dimer powder were weighed according to a mass ratio of D,L-lactide/P(NIPAM-co-DMAM)-OH=67:33, and they were mixed and placed into a three-necked flask. 20 mL of anhydrous xylene were added and stirred to dissolve the mixture. After 2-3 drops of stannous octoate were added and oxygen was removed under nitrogen protection for 30 min, the solution reacted at a constant temperature of 135 C. for 24 h to obtain P(NIPAM-co-DMAM)-b-PLA tripolymer (poly(N isopropylacrylamide-co-N,N dimethylacrylamide)-b-polylactic acid). The P(NIPAM-co-DMAM)-b-PLA tripolymer was precipitated with excess diethyl ether and dried to constant weight under vacuum at 30 C. for 48 h to obtain a pure solid-phase thermosensitive tripolymer.

2) Synthesis of Magnetic and Thermosensitive Micelles (DMF-TRM)

[0036] 20 mg of DMF powder and 20 mg of P(NIPAM-co-DMAM)-b-PLA tripolymer were weighed and dissolved with 10 mL of N,N-dimethylformamide to form a solution. The solution was transferred into a dialysis bag and dialyzed against 1000 mL of distilled water at room temperature with vigorous stirring for 24 h. The dialysis medium is replaced every 1 h for the first 5 h, and then every 12 h. A thermosensitive micelle solution was obtained after 2 days of dialysis, and then placed into a clean 100-mL beaker. After coagulating at 20 C., the micelle solution was quickly transferred to a pre-cooled vacuum freeze dryer to raise the temperature for preparation of micellar lyophilized powder which was stored at 4 C.

3) Synthesis of Water-Soluble Near-Infrared CdHgTe Quantum Dots

[0037] 5.3211 g of Cd(NO.sub.3).sub.2.4H.sub.2O solids were weighed and dissolved in excess water to give a final volume of 100 mL. 10 mL of the solution was transferred into a 1000 mL reactor, followed by dilution with 900 mL of water. The oxygen was removed with magnetic stirring at 300 rpm under nitrogen protection at room temperature. Then 5 L of Hg(NO.sub.3).sub.2.4H.sub.2O saturated solution was added and stirred for 30 min, followed by addition of 0.00517 mol of mercaptopropionic acid (MPA). 2.0 mol/L NaOH solution was then added dropwise under magnetic stirring at 300 rpm, and the pH value of the slurry was adjusted to 7.0 to yield a Cd.sup.2+Hg.sup.2+-MPA precursor.

[0038] 0.27 g Te powder and 0.16 g of solid-phase NaBH.sub.4 were weighed and placed into a 100 mL reactor, followed by addition of 10 mL of double-distilled water. The solution was magnetic stirred at 300 rpm in the presence of nitrogen at a constant temperature of 50 C. until Te powder disappeared, thus yielding NaHTe. The prepared slurry was injected into the precursor solution of Cd.sup.2Hg.sup.2+-mercaptopropionic acid, and the mixed slurry was magnetic stirred at 300 rpm at a constant temperature of 50 C. under nitrogen protection until fluorescence intensity of the liquid phase no longer increased. After static aging of the stirred slurry, ethanol was added to settle and separate; the supernatant was discarded, and the precipitate was centrifuged at 5000 rpm at room temperature; the obtained solid-phase sample was washed with absolute ethanol 2-3 times and dried in a vacuum dryer at 65 C. and 0.085 megapascal.

4) Synthesis of DMF-TRM-QD Fluorescent Micelles by the Use of DMF-TRM Micellar Powder and Water-Soluble Near-Infrared CdHgTe Quantum Dots Via Electrostatic Bonding:

[0039] a. A certain amount of water-soluble near-infrared CdHgTe quantum dots and DMF-TRM micellar powder were weighed according to the mass ratio of QD:DMF-TRM=1:1, they were mixed and ground in an agate mortar for 10-50 min;

[0040] b. the mixed powder prepared in a) was suspended and dispersed with absolute ethanol, and the suspension was placed in a water bath at 30-50 C. and ultrasonically dispersed for 2 h;

[0041] c. the magnetic solid substance in the liquid-phase was attracted to a magnet, and the liquid-phase was discarded to remove the unbound CdHgTe quantum dots. The processes of dispersing, ultrasonic, and magnetic separation of the magnetic solid substance were repeated. Then the separated solid was centrifuged at 5000 rpm at room temperature, and the solid-phase was washed with anhydrous ethanol 2-3 times and dried in a vacuum dryer at 50-60 C. and 0.085 megapascal, thus yielding the final product.

EXAMPLE 2

Synthesis of DMF-TRM-QD Micelles

1) Preparation of P(NIPAM-co-DMAM)-b-PLA tripolymer (poly(N isopropylacrylamide-co-N,N dimethylacrylamide)-b-polylactic acid)

[0042] 0.41 g of NIPAM and 0.046 g of DMAM were weighed as raw materials according to the mass ratio of NIPAM/DMAM=90:10, and they were placed into a three-necked flask. 25 mL of freshly distilled THF was added to dissolve them to form a solution. 0.009 g of copolymerization initiator AMAD was added to react with the solution at a constant temperature of 80 C. for 10 h after oxygen was removed under nitrogen protection for 30 min, thus yielding P(NIPAM-co-DMAM)-OH) dipolymer (poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)). Then, P(NIPAM-co-DMAM)-OH dipolymer was precipitated with excess ether, filtered under vacuum, and dried under vacuum at room temperature for 12 h to obtain a pure solid-phase dimer.

[0043] 0.20 g of lactide and 0.13 g of dimer powder were weighed according to a mass ratio of D,L-lactide/P(NIPAM-co-DMAM)-OH=67:33, and they were mixed and placed into a three-necked flask. 20 mL of anhydrous xylene were added and stirred to dissolve the mixture; after 2-3 drops of stannous octoate were added and oxygen was removed under nitrogen protection for 30 min, the solution reacted at a constant temperature of 135 C. for 24 h to yield P(NIPAM-co-DMAM)-b-PLA tripolymer (poly(N isopropylacrylamide-co-N,N dimethylacrylamide)-b-polylactic acid). The P(NIPAM-co-DMAM)-b-PLA tripolymer was precipitated with excess diethyl ether and dried to constant weight under vacuum at 30 C. for 48 h to obtain a pure solid-phase tripolymer.

2) Preparation of Magnetic, Thermosensitive Micelles (DMF-TRM)

[0044] 5 mg DMF powder and 20 mg of lyophilized powder of thermosensitive polymer were weighed to prepared a DMF-TRM micellar lyophilized powder according to 2) in Example 1.

3) Preparation of Water-Soluble Near-Infrared CdHgTe Quantum Dots

[0045] The method is the same as 1) in Example 1.

4) Preparing of DMF-TRM-QD Fluorescent Micelles by the Use of DMF-TRM Micellar Powder and Water-Soluble Near-Infrared CdHgTe Quantum Dots Via Electrostatic Bonding

[0046] a. 8 mg of water-soluble near-infrared CdHgTe quantum dots and DMF-TRM micellar powder were weighed according to the mass ratio of QD:DMF-TRM=1:3, they were mixed and ground in an agate mortar for 10-50 min;

[0047] b. the mixed powder prepared in a) was suspended and dispersed with absolute ethanol, and the suspension was placed in a water bath at 30-50 C. and ultrasonically dispersed for 2 h;

[0048] c. the magnetic solid substance in the liquid-phase was attracted to a magnet, and the liquid-phase was discarded. The processes of dispersing, ultrasonic, and magnetic separation of the magnetic solid substance were repeated. Then the separated solid was centrifuged at 5000 rpm at room temperature, and the solid-phase was washed with anhydrous ethanol 2-3 times and dried in a vacuum dryer at 50-60 C. and 0.085 megapascal, thus yielding the final product.

EXAMPLE 3

Synthesis of DMF-TRM-QD Micelles

1) Preparation of P(NIPAM-co-DMAM)-b-PLA tripolymer (poly(N isopropylacrylamide-co-N,N dimethylacrylamide)-b-polylactic acid).

[0049] 0.6 g of NIPAM and 0.032 g of DMAM were weighed as raw materials according to the mass ratio of NIPAM/DMAM=95:5. And P(NIPAM-co-DMAM)-b-PLA tripolymer was prepared according to the technical conditions and process of Example 1.

2) Preparation of Magnetic, Thermosensitive Micelles (DMF-TRM)

[0050] 15 mg of DMF powder and 20 mg of lyophilized powder of thermosensitive polymer were weighed and dissolved with 10 mL of N,N-dimethylformamide. The resulting mixture was transferred into a dialysis bag, and dialyzed against 1000 mL of distilled water at room temperature with vigorous stirring for 48 h. The distilled water was replaced every 1 h for the first 5 h, and then every 12 h. The thermosensitive micellar solution was obtained after 46 h of dialysis, and then placed into a clean 100-mL beaker. After coagulating at 20 C., the micelle solution was quickly transferred to a pre-cooled vacuum freeze dryer to increase the temperature for preparation of micellar lyophilized powders.

3) Preparation of Water-Soluble Near-Infrared CdHgTe Quantum Dots

[0051] The method is the same as 1) in Example 1.

4) Preparation of DMF-TRM-QD Fluorescent Micelles by the Use of DMF-TRM Micellar Powder and Water-Soluble Near-Infrared CdHgTe Quantum Dots Via Electrostatic Bonding

[0052] a. 16 mg of water-soluble near-infrared CdHgTe quantum dots and 32 mg of DMF-TRM micellar powder were weighed according to the mass ratio of QD:DMF-TRM=1:3, then they were mixed and ground in an agate mortar for 10-50 min;

[0053] b. the mixed powder prepared in a) was suspended and dispersed with absolute ethanol, and the suspension was placed in a water bath at 30-50 C. and ultrasonically dispersed for 2 h;

[0054] c. the magnetic solid substance in the liquid-phase was attracted to a magnet, and the liquid phase was discarded. The processes of dispersing, ultrasonic, and magnetic separation of the magnetic solid substance were repeated. Then the separated solid was centrifuged at 5000 rpm at room temperature, and the solid-phase was washed with anhydrous ethanol 2-3 times and dried in a vacuum dryer at 50-60 C. and 0.085 megapascal, to yield the final product.

[0055] Evaluation of the Implementation and Effect

[0056] The laser-scanning fluorescence confocal imaging technology was employed to verify the effects of cell transport and biological imaging of the DMF-TRM-QD fluorescent micelles. The experimental results showed that the fluorescent micelles formed by the combination of DMF magnetic, thermosensitive micelles and biological quantum dots, which can enter cells and reach nucleus regions. The micelles showed good fluorescent labeling and performance of magnetic and thermal targeting, and has great application prospects in magnetic and thermal targeted chemotherapy for tumors.

[0057] Experimental Process and Results

[0058] (1) Morphological Features of DMF-TRM-QD Fluorescent Micelles.

[0059] The morphological features of DMF-TRM micelles was determined by a transmission electron microscope (TEM, H-7560B, Hitachi, Tokyo). Two drops of micellar solution were added on the copper mesh, dried naturally at room temperature, and observed and photographed with an electron microscope under an accelerated voltage of 80 kV. As shown in the image A of FIG. 1, the dark hexagonal-layered appearance, which were located in the nuclear layer of micelles, are DMF nanoparticles with a side length of 58 nm. The diameter of internal cavity is 119 nm, with a shell thickness of 37 nm, and the micelle diameter is 198 nm. The morphological features of micelles demonstrated that the hexagonal magnetic DMF particles play a crucial guiding role in forming the core-shell structure of the complex micelles. The rigid magnetic DMF particles play a supporting role in the structure of the micelle cavity, and the hydroxyl groups on the surface of the DMF particles can form hydrogen bonds with the hydration layer of hydrophobic groups of the micelles, which make the core-shell structure of the micelles more contractive, stable and highly dispersible.

[0060] Quantum dot marker can be used for tracking the transport trajectory of DMF thermo-responsive micelles (TRM). The image B of FIG. 1 is a fluorescence image of DMF-TRM-QD complex particles observed and photographed with a 100 oil immersion objective lens. The prepared CdHgTe quantum dots with sizes below 10 nm, can emit red fluorescence which was excited by a 488 nm green helium-neon laser. The DMF-TRM-QD complex particles had a particle size of about 500 nm in aqueous solution at room temperature. As shown in the image B of FIG. 1, the spherical micelles had an external diameter of about 514 nm, which confirmed the size of the DMF-TRM-QD micelles.

[0061] The DMF particles, which comprised a plurality of hydroxyl groups on the surface thereof, were wrapped in the micelle core and capable of combining with QD. Therefore, the DMF-loaded micelles had a high fluorescence intensity in the micelle core, and an internal diameter of about 243 nm, which illustrated that the aggregated DMF particles were wrapped in the thermosensitive micelles. There were also a large number of hydrophilic groups on the surface of the composite micelles, with a shell thickness of about 135 nm, and the shell had a weak fluorescence intensity, which can reflect the thickness of the hydration layer of the micelles. The above results demonstrated that DMF-loaded micelles had combined successfully with QD to form a fluorescent complex.

[0062] (2) Cell Transport and Biological Imaging of DMF-TRM-QD Fluorescent Micelles

[0063] MGC-803 cells in exponential phase were digested with trypsin, and centrifuged to make a single cell suspension. The cells were seeded at 3105 cells per well in 6-well cell culture plates and cultured for 24 h until the cells adhere to the wall of the 6-well plates. The supernatant of the culture medium in each well was discarded and the cells were washed with PBS 3 times. 2 mL of drug-containing medium was added to each well. The room-temperature groups were taken out of the cell culture plates after incubation for 1 h, 3 h, 5 h, and 7 h, respectively. The hyperthermia groups were incubated in a constant-temperature box at 42 C. for 30 min and then quickly transferred to an incubator at 37 C. for 0.5 h, 2.5 h, 4.5 h, and 6.5 h, respectively. All the supernatant was aspirated carefully and discarded. After the cells were washed with PBS three times, 1 mL of Hoechst 33342 (10 g.Math.mL.sup.1, nucleating agent) was added and incubated for 30 min. Once again, all the supernatant was aspirated carefully and discarded, and then the cells were washed with PBS three times. 1 mL of 4% paraformaldehyde was added per well to fix the cells for 5 min and then washed with PBS three times. The cover slips were gently lifted with the tip of a burned syringe needle and removed quickly with a tweezer. The excess liquid on the slides was removed with filter paper, followed by an addition of 1-2 drops of anti-fade mounting medium. Then the mounted slides can be store at 20 C., protected from light. In the confocal imaging experiment, the cells in the field of view were observed with a 40 objective lens; the fluorescent micelles were observed with a 100 oil immersion objective lens; the fluorescence of QD was excited with a 488 nm green helium-neon laser; the emission wavelength was detected with a 560-660 nm bandpass filter. The excitation wavelength was 405 nm, and the fluorescence of the nuclear dye Hoechst 33342 was detected with a430-460 nm bandpass filter.

[0064] FIG. 2 showed the confocal microscopy fluorescence images after incubating human gastric cancer MGC-803 cells with DMF-TRM-QD micelles. The nucleus of the cells was stained with Hoechst 33342 to emit green fluorescence, but the DMF-TRM-QD micelles entered the cells and emitted red fluorescence inside the cells. The green fluorescence and the red fluorescence overlapped to yield yellow fluorescence. The image A of FIG. 2 confirmed that the nucleus had regular morphology and uniform size after the micelles interacted with cells for 1 h. But the red fluorescence was very weak in the cells, and only a few cells expressed the red fluorescence outside or near the nucleus. The results demonstrated that the fluorescent micelles had a low degree of cell internalization, and no significant effect on cell viability for the fluorescent micelle particles carrying nano-drugs. After the cells were incubated with the fluorescent complex for 3 h, the number of fluorescent micelles entering the cells was significantly increased relative to that after the incubation for 1 h, which were analyzed from the red fluorescence distribution. With regard to the nucleus morphology, the diameter distribution of the nucleus began to differentiate. Some nucleuses tended to be swelled and rounded, and the other nucleuses exhibited reduction in size and darkened in color. The results showed that after interaction for 3 h, the fluorescent micelles appeared in the central part of some nucleuses, which demonstrated that the cell internalization had reached significant level and the drug carried by the micelles also started to play an intervention role. The nucleus swelling may be related to the comprehensive biochemical response of the DMF-loaded micelles and the biological substances that constitute the organelles and nucleus, more than the effect of the released of the FU drugs. The nucleus pyknosis illustrated that the biochemical reactions between DMF-loaded micelles and nuclear materials may have terminated. After incubation for 5 h, there was no significant increase in number of the red fluorescent spots emitted from the cells compared with the image B of FIG. 2. However, the phenomenon still cannot determine whether the degree of cell internalization of fluorescent micelles increase with time. When the fluorescent micelles entered the cells in a smaller size and a more dispersed state, the imaging effect can be influenced by the imaging angle, organelle fusion, masking, etc. These questions caused the difficulty in clearly displaying the degree of cell internalization. The comparison of the hue in the pictures showed that the yellow component of the nucleus was significantly increased compared to that in the image B of FIG. 2, especially the yellow component of the nucleolus increased significantly, illustrating that the red fluorescent in the nucleus showed an increasing trend over time. With regard to the morphology of the nucleus, the increase in the density of the nucleus and the degree of nucleus pyknosis confirmed that the pharmacological effect of the fluorescent micelles in the cells became manifest over time. The image D of FIG. 2 showed that the changes in the state of aggregation and survival of the nucleus caused by fluorescent micelles are completely similar to those in the image C of FIG. 2. Although the number of macroscopically visible red aggregate particles decreased, the degree of cell coloration increased, which conformed that the small-sized and dispersed fluorescent micelles had increased in degree of cell internalization. The above results indicated that it takes just under 1 h for the fluorescent magnetic micelles of the disclosure to complete the process of cell internalization kinetics. The micelles reached the nucleus and induced the obvious differentiation of the nucleus morphology after administration for 3 h, and the pharmaceutical effect gradually increased with the prolonged action of the time.

[0065] The number of DMF-TRM-QD micelles entering the cell can be indirectly determined by calculating the average optical density of red fluorescence in the cell imaging. For four experimental groups carried out at normal temperature for 1 h, 3 h, 5 h and 7 h, respectively, the average optical density value (see Table 1) of red fluorescence was calculated with Image-Pro Plus analysis software. And a correlation analysis was performed to reveal the interrelationship between the length of intervention time and the average optical density value. Specifically, the analysis results were fitted to the following linear equation: Dmean=0.0409 t-0.0465 (where Dmean represented the average optical density value of red fluorescence emitted from the cells, t represented the intervention time of fluorescence complex, unit: hour). The correlation coefficient was 0.92, illustrating that the number of DMF-TRM-QD micelles entering the cell showed a linear growth trend over time. The confocal microscopy fluorescence images showed that the cell transport and pharmacodynamic process of the DMF-TRM-QD micelles increased with the intervention time, and the number of nano-scale fluorescent micelles wrapped by the cells also increased. The particles being transported to the nucleolus of the cells, can interact with nuclear biological substances to cause the swelling, contraction and solidification of the nucleus, thereby achieving the purpose of eliminating cancer cells and tumor tissues.

TABLE-US-00001 TABLE 1 Dmean of different intervention times (n = 3) t (h) 1 3 5 7 D.sub.mean 0.0089 0.0186 0.0092 0.1293 (X s) 0.0053 0.0009 0.0004 0.0980

[0066] (3) Effect of Applied Magnetic Responsiveness on Cell Transmission Efficiency of DMF-TRM-QD Fluorescent Micelles.

[0067] To investigate the effect of applied magnetic field on cell transmission efficiency of DMF-TRM-QD cells, the confocal imaging was photographed for MGC-803 cells after incubation with DMF-TRM-QD under hyperthermia at 42 C. for 7 h and different gradient of applied magnetic field.

[0068] The small sterilized magnets were stuck to the bottom of the external surface of the 6 well cell plates with a sterile white tape prior to the experiments. The clean cover slips were placed at the bottom of the wells. MGC-803 cells were inoculated in the exponential phase and cultured for 24 h until the cells adhere to the wall of the 6-well plates. The supernatant of the culture medium in each well was discarded and the cells were washed with PBS 3 times. 2 mL of drug-containing medium was added to each well. The room-temperature groups were taken out of the cell culture plate after incubation for 1 h, 3 h, 5 h, and 7 h, respectively. The hyperthermia groups were incubated in a constant-temperature box at 42 C. for 30 min and then quickly transferred to an incubator at 37 C. for 0.5 h, 2.5 h, 4.5 h, and 6.5 h, respectively. Subsequently, the processes for mounting and confocal imaging were as the same as in (2).

[0069] The cells imaging results were shown in FIG. 3. The image A of FIG. 3 showed when there is no magnetic field hyperthermia, only a few cells can be observed with a red fluorescent signal. A brightly dyed cell was observed and the entire cell was red and had a clear cell outline. Fluorescent magnetic micelles reached the nucleolus of the nucleus, and then quantum dots fluoresced and illuminated all areas within the cell at the moment when the cell was fixed and blackened. As shown in the images B-E of FIG. 3, the number of cells emitting red fluorescence increased significantly with increasing magnetic field strength, and the fluorescence density increased accordingly.

[0070] Table 2 showed the average optical density of the red fluorescence in six hyperthermia experimental groups at a steady state temperature of 42 C. And the six magnetic field gradients were designed by Image-Pro Plus software. For the magnets the number were 0, 5, 10, 15, 20 and 25, respectively. For each micro magnetic the strength of was 5770 Gauss or 0.577 T. FIG. 4 showed the linear relationship between the intracellular mean fluorescence density and the applied magnetic field gradient, which fitted the linear equation: Dmean=0.00536 B+0.00254 (Dmean represented the average optical density of red fluorescence emitted from the cells; B represented the applied magnetic field strength; the unit for the magnetic field strength was Tesla T; and the linear correlation coefficient is 0.91). The results showed that the amount of DMF-TRM-QD entering the cell goes up linearly with the magnetic field gradient, which illustrated the intracellular magnetic targeting transport kinetics of fluorescent magnetic micelle particles.

[0071] As shown in the images A-F of FIG. 3, the relationship between the nuclear density N and the applied magnetic field gradient fitted the linear equation: N=2.6457 n+20.429 and R2=0.6528, indicating that the nuclear density N was also regularly distributed with the magnetic field gradient.

TABLE-US-00002 TABLE 2 Dmean of different magnetic field strength (X s) (T) 0 2.885 5.77 8.655 11.54 14.425 D.sub.mean 0.0164 0.0064 0.0077 0.0007 0.0256 0.0074 0.0461 0.0009 0.0662 0.0343 0.0855 0.0135

[0072] The above results showed that DMF-TRM-QD micelles were transported into cells soon after contacting the cells. The micelles were excited by laser beam and emitted a specific wavelength of red fluorescence in cells. An increase in the degree of cell internalization of micelles and the magnetic response of nuclear distribution were achieved by exposing the cells to the applied magnetic fields. Therefore, it was confirmed that the DMF-TRM-QD micelles had special application value for in vivo diagnostic imaging and cancer treatment.

[0073] The combination of DMF-loaded micelles and the biological quantum dots can produce DMF-TRM-QD composite particles with multiple functions of fluorescent labeling, thermal sensitivity and magnetic targeting. The cell internalization and transport efficiency of DMF-TRM-QD micelles goes up linearly with the magnetic field strength. The density of nucleus and the degree of pyknosis both showed a regular change with the magnetic field gradient, which made it was possible to improve the effects of intracellular imaging and chemotherapy only by the use of magnetic fields. The successful recombination of DMF-TRM with biological quantum dots and the ideal effect of cell transport demonstrated that DMF-TRM-QD micelles would be an intelligent and multifunctional nano-system with great development prospects and application value.

[0074] It will be obvious to those skilled in the art that changes and modifications may be made, and therefore, the aim in the appended claims is to cover all such changes and modifications.