CARBON NANOPARTICLES SUSPENSION INJECTION-FE MIXTURE AS WELL AS PREPARATION METHOD

Abstract

The present disclosure claims a carbon nanoparticles suspension injection-Fe mixture. When the mixture is used in combination with near-infrared light for tumor low-temperature hyperthermia, the administration dosage can be decreased so as to reduce the occurrence of adverse reactions; the hyperthermia using the mixture has a low temperature, so that the surrounding normal tissues are not liable to be damaged, and the adverse reactions during the hyperthermia using carbon nanoparticles can also be reduced. Moreover, it is further proved that the CNSI-Fe mixture and the near-infrared irradiation are synergic. Based on the means and technical effects of low-temperature hyperthermia, the present disclosure has extensive clinical significance.

Claims

1. A carbon nanoparticles suspension injection (CNSI)-Fe mixture, wherein the CNSI-Fe mixture comprises a carbon nanoparticles suspension injection and ferrous sulfate for injection, and a mass ratio of the CNSI to the ferrous sulfate for injection is 20-100 : 3-300; the CNSI comprises carbon nanoparticles, a suspending agent, saline, a pH adjuster, sodium chloride and water for injection; further, each 1,000 mL of CNSI comprises 20-100 g of carbon nanoparticles, 17-30 g of suspending agent, 2-4 g of pH adjuster, 8-10 g of sodium chloride and a remaining amount of water for injection; the carbon nanoparticles suspension injection-Fe mixture is used in combination with near-infrared light to the treatment of cancer.

2. The carbon nanoparticles suspension injection-Fe mixture according to claim 1, wherein the use in combination with near-infrared light refers to heating a tissue using near-infrared light; the tissue was injected with the carbon nanoparticles suspension injection-Fe mixture.

3. The carbon nanoparticles suspension injection-Fe mixture according to claim 1, wherein a wavelength of the near-infrared light is 780-2,600 nm.

4. The carbon nanoparticles suspension injection-Fe mixture according to claim 2, wherein the heating temperature is 43-50° C.

5. The carbon nanoparticles suspension injection-Fe mixture according to claim 2, wherein the heating time is 5-60 minutes.

6. The carbon nanoparticles suspension injection-Fe mixture according to claim 1, wherein when the CNSI-Fe mixture is used, the CNSI and the ferrous sulfate for injection are uniformly mixed, and a pH value of the mixture is determined to be 2-7.

7. The carbon nanoparticles suspension injection-Fe mixture according to claim 6, wherein the carbon nanoparticles are any one or some of carbon nanoparticles, carbon nanotubes, carbon quantum dots, graphene, fullerene, carbon nanorods and carbon nanofibers.

8. The carbon nanoparticles suspension injection-Fe mixture according to claim 6, wherein the suspending agent is any one or some of poloxamer, polyvinylpyrrolidone C30 and Tween-80.

9. The carbon nanoparticles suspension injection-Fe mixture according to claim 1, wherein the pH adjuster is any one of sodium citrate, sodium acetate, sodium borate, sodium phosphate and sodium bicarbonate.

10. The carbon nanoparticles suspension injection-Fe mixture according to claim 1, wherein the ferrous sulfate for injection is a lyophilized powder of ferrous sulfate for injection; and preferably, the lyophilized powder of ferrous sulfate for injection is prepared from the following raw materials: ferrous sulfate heptahydrate, sulfuric acid and water for injection, wherein a mass ratio of ferrous sulfate heptahydrate : sulfuric acid : water for injection is 0.1-0.2 g : 0.4-0.85 .Math.g : 2 g.

11. The carbon nanoparticles suspension injection-Fe mixture according to claim 6, wherein the ferric salt is selected from any one or some of ferrous sulfate, ferric sulfate, ferrous chloride, ferric trichloride, ferrous gluconate, ferric sucrose, ferric ammonium citrate, ferrous succinate, ferric sorbitol and ferrous fumarate.

12. The carbon nanoparticles suspension injection-Fe mixture according to claim 6, wherein a preparation method of the CNSI comprises the following steps: 1) taking 90% v/v of the formulation amount of water for injection, adding the formulation amount of sodium chloride, and stirring the mixture until the mixture is completely dissolved to obtain a salt solution; 2) adding the formulation amounts of pH adjuster and suspending agent into the salt solution obtained in step 1, and stirring the mixture until the mixture is completely dissolved to obtain an excipient solution; 3) adding the formulation amount of pretreated carbon nanoparticles into the excipient solution obtained in step 2, stirring the mixture evenly, and then adding the remaining amount of water for injection to a constant volume to obtain a constant volume solution; and 4) homogenizing the constant volume solution obtained in step 3 at a rate of 15,000-20,000 rpm for 3-10 min and then at a pressure of 15,000-30,000 psi for 1-5 times, then performing filling and capping, and finally performing steam sterilization at 115-130° C. for 10-30 min to obtain the CNSI.

13. The carbon nanoparticles suspension injection-Fe mixture according to claim 12, wherein the pretreatment in step 3 comprises the following steps: firstly washing the carbon nanoparticles degreased by ethyl acetate using a 8-15%v/v HNO.sub.3 aqueous solution, wherein a mass-to-volume ratio of the carbon nanoparticles to the HNO.sub.3 aqueous solution is 1 g : 3-5 /ml; then washing the carbon nanoparticles with water to be approximately neutral; then washing the carbon nanoparticles with a 0.08-0.15 mol/L NaOH aqueous solution, wherein a mass-to-volume ratio of the carbon nanoparticles to the NaOH aqueous solution is 1 g : 3-5 /ml; and washing the carbon nanoparticles with water to be approximately neutral.

14. The carbon nanoparticles suspension injection-Fe mixture according to claim 6, wherein a preparation method of the lyophilized powder of ferrous sulfate for injection comprises the following steps: S1: taking 90% of the formulation amount of water for injection, and continuously filling N.sub.2 below the liquid level during the preparation, with a nitrogen flow rate controlled at 4.0-5.0 m.sup.3/h; S2: then adding sulfuric acid to adjust the pH value to 2.4-2.8; S3: taking the formulation amount of ferrous sulfate heptahydrate, and fully stirring the ferrous sulfate heptahydrate for dissolution; S4: adding the remaining amount of sulfuric acid to adjust the pH value to 2.8-3.0, adding the remaining amount of purified water to fix the volume to a full volume, and stirring the mixture evenly to obtain a ferrous sulfate solution; and S5: filtering the obtained solution using a microporous filter membrane, then performing filling, lyophilization, vacuum plugging and capping to obtain the lyophilized powder of ferrous sulfate for injection.

15. The carbon nanoparticles suspension injection-Fe mixture according to claim 14, wherein in S5 of the preparation method of the lyophilized powder of ferrous sulfate for injection, the lyophilization step comprises the following specific operations: S51 pre-freezing: rapidly lowering the temperature to (-10 to -5) °C and keeping the temperature for 1-3 h, and then rapidly lowering the temperature to (-50 to -40) °C and keeping the temperature for 1-3 h; S52 drying: S521: gradually elevating the temperature from (-50 to -40) °C to (-20to -10) °C within 2-4 h, and keeping the temperature at (-20 to -10) °C and 80-120 mtorr for 2-4 h; S522: gradually elevating the temperature from (-20 to -10) °C to (-10 to 0) °C within 1-3 h, and keeping the temperature at 80-120 mtorr and (-10 to 0) °C for 1-3 h; and S523: gradually elevating the temperature from (0-20) °C within 3-6 h, and then keeping the temperature at 80-120 mtorr and 20° C. for 3-6 h.

16. The carbon nanoparticles suspension injection-Fe mixture according to claim 1, wherein the CNSI-Fe mixture comprises a carbon nanoparticles suspension injection and ferrous sulfate for injection, and a mass ratio of the CNSI to the ferrous sulfate for injection is 20-100 : 3-300; and preferably, the mass ratio is 50 : 7.5-180.

17. The carbon nanoparticles suspension injection-Fe mixture according to claim 1, wherein the CNSI-Fe mixture comprises a carbon nanoparticles suspension injection and ferrous sulfate for injection, and a mass ratio of the CNSI to the ferrous sulfate for injection is 20-100 : 3-300; and preferably, the mass ratio is 50 : 7.5-180.

18. The carbon nanoparticles suspension injection-Fe mixture according to claim 7, wherein a preparation method of the CNSI comprises the following steps: 1) taking 90% v/v of the formulation amount of water for injection, adding the formulation amount of sodium chloride, and stirring the mixture until the mixture is completely dissolved to obtain a salt solution; 2) adding the formulation amounts of pH adjuster and suspending agent into the salt solution obtained in step 1, and stirring the mixture until the mixture is completely dissolved to obtain an excipient solution; 3) adding the formulation amount of pretreated carbon nanoparticles into the excipient solution obtained in step 2, stirring the mixture evenly, and then adding the remaining amount of water for injection to a constant volume to obtain a constant volume solution; and 4) homogenizing the constant volume solution obtained in step 3 at a rate of 15,000-20,000 rpm for 3-10 min and then at a pressure of 15,000-30,000 psi for 1-5 times, then performing filling and capping, and finally performing steam sterilization at 115-130° C. for 10-30 min to obtain the CNSI.

19. The carbon nanoparticles suspension injection-Fe mixture according to claim 8, wherein a preparation method of the CNSI comprises the following steps: 1) taking 90% v/v of the formulation amount of water for injection, adding the formulation amount of sodium chloride, and stirring the mixture until the mixture is completely dissolved to obtain a salt solution; 2) adding the formulation amounts of pH adjuster and suspending agent into the salt solution obtained in step 1, and stirring the mixture until the mixture is completely dissolved to obtain an excipient solution; 3) adding the formulation amount of pretreated carbon nanoparticles into the excipient solution obtained in step 2, stirring the mixture evenly, and then adding the remaining amount of water for injection to a constant volume to obtain a constant volume solution; and 4) homogenizing the constant volume solution obtained in step 3 at a rate of 15,000-20,000 rpm for 3-10 min and then at a pressure of 15,000-30,000 psi for 1-5 times, then performing filling and capping, and finally performing steam sterilization at 115-130° C. for 10--30 min to obtain the CNSI.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0040] FIG. 1 shows growth curves of gross tumor volumes of xenograft tumor of SMMC-7721 liver cancer nude mice treated in all groups.

[0041] FIG. 2 shows growth curves of gross tumor volumes of xenograft tumor of HCT-116 colon cancer nude mice treated in all groups.

[0042] FIG. 3 shows growth curves of gross tumor volumes of xenograft tumor of MDA-MB-231 breast cancer nude mice treated in all groups.

[0043] FIG. 4 is an oscillogram of hydroxyl radicals in tumor tissues of SMMC-7721 liver cancer.

[0044] FIG. 5 shows inhibitory effects on lymph node metastasis of H22 liver cancer cells in all groups.

DESCRIPTION OF EMBODIMENTS

(I) Examples

Example 1: Preparation of CNSI

[0045] For a CNSI, each 1,000 mL of CNSI included 50 g of carbon nanoparticles, 20 g of suspending agent, 3 g of pH adjuster, 9 g of sodium chloride and a remaining amount of water for injection; and a preparation method included the following steps:

[0046] 1) 90% v/v of the formulation amount of water for injection was taken, the formulation amount of sodium chloride was added, and the mixture was stirred until the mixture was completely dissolved to obtain a salt solution; 2) the formulation amounts of pH adjuster and suspending agent were added to the salt solution obtained in step 1), and the mixture was stirred until the mixture was completely dissolved to obtain an excipient solution; 3) the formulation amount of pretreated carbon nanoparticles was added to the excipient solution obtained in step 2), the mixture was stirred evenly, and then the remaining amount of water for injection was added to a constant volume to obtain a constant volume solution, wherein the pretreatment included the following steps: firstly the carbon nanoparticles degreased by ethyl acetate was washed using a 10%v/v HNO.sub.3 aqueous solution, wherein a mass-to-volume ratio of the carbon nanoparticles to the HNO.sub.3 aqueous solution was 1 g : 4 /ml, and then the carbon nanoparticles were washed with water to be approximately neutral; then the carbon nanoparticles were washed with a 0.10 mol/L NaOH aqueous solution, wherein a mass-to-volume ratio of the carbon nanoparticles to the NaOH aqueous solution was 1 g : 4 /ml, and then the carbon nanoparticles were washed with water to be approximately neutral; and 4) the constant volume solution obtained in step 3) was homogenized at a rate of 18,000 rpm for 5 min and then at a pressure of 20,000 psi 3 times, then filled and capped, and finally sterilized by steam at 121° C. for 15 min to obtain the CNSI.

Example 2: Preparation of Ferrous Sulfate for Injection

[0047] A lyophilized powder of ferrous sulfate for injection was prepared from the following raw materials: ferrous sulfate heptahydrate, sulfuric acid and water for injection, wherein a mass ratio of ferrous sulfate heptahydrate : sulfuric acid : water for injection was 0.149 g : 0.4-0.85 .Math.g : 2 g; preferably, the sulfuric acid was 1% (by weight) sulfuric acid. A preparation method included the following steps:

[0048] S1: 90% of the formulation amount of water for injection was taken, N.sub.2 was continuously filled below the liquid level during the preparation, and a nitrogen flow rate was controlled at 4.0-5.0 m.sup.3/h; S2: sulfuric acid was then added to adjust the pH value to 2.4; S3: the formulation amount of ferrous sulfate heptahydrate was taken and fully stirred for dissolution; S4: the remaining amount of sulfuric acid was added to adjust the pH value to 2.8, the remaining amount of purified water was added to fix the volume to a full volume, and the mixture was stirred evenly to obtain a ferrous sulfate solution; and S5: the obtained solution was filtered with a 0.45 .Math.m microporous filter membrane, filled, lyophilized, vacuum plugged and capped to obtain the lyophilized powder; wherein the lyophilization step included the following specific operations:

[0049] S51 pre-freezing: the temperature was rapidly lowered to -5° C. and kept for 2 h, and then the temperature was rapidly lowered to -45° C. and kept for 2 h; S52 drying: S521: the temperature was gradually elevated from -45° C. to -15° C. within 3 h and kept at -15° C. and 100 mtorr for 3 h; S522: the temperature was gradually elevated to (-15 to -5)°C within 2 h and kept at -5° C. and 100 mtorr for 2 h; and S523: the temperature was gradually elevated to 0-20° C. within 4 h and kept at 20° C. and 100 mtorr for 4 h.

[0050] In examples 3-22, the CNSI and the ferrous sulfate for injection in different proportions and near-infrared light of different wavelengths were selected for hyperthermia of tumor.

TABLE-US-00001 CNSI Ferrous Sulfate for Injection Infrared Wavelength (nm) Example 3 50 mg 21 mg 780 Example 4 50 mg 21 mg 808 Example 5 50 mg 21 mg 1,064 Example 6 50 mg 21 mg 1,400 Example 7 50 mg 21 mg 2,600 Example 8 50 mg 15 mg 1,064 Example 9 50 mg 45 mg 808 Example 10 50 mg 7.5 mg 808 Example 11 50 mg 90 mg 808 Example 12 50 mg 180 mg 808 Example 13 50 mg 3 mg 808 Example 14 50 mg 300 mg 808 Example 15 20 mg 3 mg 808 Example 16 20 mg 30 mg 808 Example 17 20 mg 150 mg 808 Example 18 20 mg 300 mg 808 Example 19 100 mg 3 mg 808 Example 20 100 mg 30 mg 808 Example 21 100 mg 150 mg 808 Example 22 100 mg 300 mg 808

(II) Experimental Examples

1. Experimental Materials

1) Cell Strains

[0051] SMMC7721 liver cancer cells, HCT116 colon cancer cells, MDA-MB-231 breast cancer cells, and mouse derived liver cancer H22 cells

2) Cell Culture Media

[0052] DMEM cell medium, RMPI1640 medium, fetal bovine serum (FBS), trypsin for cell digestive solution, penicillin-streptomycin mixture, and phosphate buffer solution (PBS, pH 7.4)

3) Experimental Animals

[0053] BalB/c-nu mice, male, 5-7 weeks old, and weighing 20±2 g. The mice could drink and eat freely during the experiments. The mice were irradiated by light for 12 h every day, and fed in isolated cages with separate air supply, with 5 mice in each cage.

[0054] SPF-class Balb/c mice, male, 4-6 weeks old, and weighing 20±2 g. The mice could drink and eat freely during the experiments. The mice were irradiated by light for 12 h every day, and fed in isolated cages with separate air supply, with 5 mice in each cage.

4) Experimental Drugs and Main Instruments

[0055] CNSI-Fe for example 4, CNSI-Fe for example 8, CNSI (with a concentration the same as those used in examples 4 and 8), 0.9% sodium chloride injection, blast drying oven, thermostatic water bath kettle, biological optical microscope, thermostatic incubator, water purifier, autoclave, ultra-clean workbench, electronic balance, 808 nm near-infrared hyperthermia instrument, 1,064 nm laser hyperthermia instrument, infrared thermograph, and electron spin resonance (ESR) spectrometer

2. Experimental Methods

(1) Cell Experiment

[0056] Cells in a logarithmic growth phase were collected and counted, a cell density was adjusted to 30,000 cells/ml, and 1 ml was added to a 25×25 mm glass dish and incubated with 5% CO.sub.2 at 37° C. for 24 h. The cells were divided into a negative group, a near-infrared irradiation group, a CNSI-Fe group, a CNSI + near-infrared irradiation group (37° C., 42° C., 45° C., 48° C. and 50° C.), and a CNSI-Fe + near-infrared irradiation group (37° C., 42° C., 45° C., 48° C. and 50° C.), with 3 duplicates for each group. The negative group and the near-infrared irradiation group were replaced with new culture solutions, and the CNSI + near-infrared irradiation group and the CNSI-Fe + near-infrared irradiation group were replaced with culture solutions containing CNSI or CNSI-Fe. The CNSI + near-infrared irradiation group and the CNSI-Fe + near-infrared irradiation group were irradiated to the required temperatures using the 808 nm near-infrared hyperthermia instrument, respectively, and the temperatures were maintained for 10 min. The irradiation time of the near-infrared radiation group was consistent with the longest irradiation time of the test group. After irradiation, the cells were cultured for another 48 h. The cells were digested with trypsin and counted, and a cell inhibition rate was calculated.

2) Experiment on Inhibiting Tumor Growth

[0057] Cells in a logarithmic growth phase were collected, a concentration of the cell suspension was adjusted to 3×10.sup.7 cells/mL, and the cells were inoculated subcutaneously in the upper right limb of each nude mouse at 0.1 mL/mouse (including about 3×10.sup.6 cells); when an average tumor volume of the inoculated mice reached 100 mm.sup.3, and the tumor bearing mice were randomly divided into a negative control group, a CNSI-Fe group, a CNSI + near-infrared irradiation group and a CNSI-Fe + near-infrared irradiation group (adopting the CNSI-Fe used in example 4). The method of administration was intratumoral injection, and a volume of administration was 50 .Math.L/time; the second administration was given after an interval of 2 days, and the mice were administrated twice in total. At 10 min after the intratumoral injection, tumors were irradiated by 808 nm near-infrared ray in the CNSI + near-infrared irradiation group and the CNSI-Fe + near-infrared irradiation group, respectively, wherein a power density was 0.5 W/cm.sup.2, an irradiation time was 30 min, and a temperature was maintained at about 45° C. The near-infrared radiation group was irradiated for the same period of time. The tumor volume of the mice were measured every week, and the volume was calculated by the following formula: volume=(length × width.sup.2)/2. The observation lasted for 21 days.

[0058] At 24 h after the near-infrared irradiation, the tumor tissue was taken from each mouse, 0.9% sodium chloride injection was added to prepare a 10% homogenate, a capture agent was added, and hydroxyl radicals were detected with ESR.

3) Experiment on Inhibiting Lymph Node Metastasis

[0059] Milky white ascites was extracted from each tumor bearing mouse with H22 liver cancer and added to normal saline, and the mixture was centrifugated and re-suspended; the number of cells was adjusted to 3×10.sup.7 cells/mL, the cells were inoculated subcutaneously on the foot pad of the left hind limb of each Balb/c mouse, and each mouse was inoculated with 50 .Math.L to obtain a mouse model of cancer lymph node metastasis. The mice were treated when a tumor diameter reached 6-8 mm and there was neither ulcer nor necrosis. The mice were randomly divided into 5 groups, with 7 mice in each group, that is, a negative control group, a CNSI-Fe group, a near-infrared irradiation group, a CNSI + near-infrared irradiation group and a CNSI-Fe + near-infrared irradiation group (adopting the CNSI-Fe used in example 8). The method of administration was intratumoral injection, and a volume of administration was 50 .Math.L/time; the second administration was given after an interval of 2 days, and the mice were administrated twice in total. At 10 min after the intratumoral injection of drugs, each mouse was irradiated in the position of axillary lymph nodes in the left hind limb by 1,064 nm near-infrared ray in the CNSI + near-infrared irradiation group and the CNSI-Fe + near-infrared irradiation group, respectively, wherein a power density was 0.5 W/cm.sup.2, an irradiation time was 30 min, and a temperature was maintained at about 45° C. The near-infrared radiation group was irradiated for the same period of time. At 2 weeks after the irradiation, the mice were killed, and the axillary lymph nodes were collected and weighed.

3. Experimental Results

(1) Cell Experiment

[0060] In the near-infrared irradiation group, there was no obvious killing effect on the SMMC7721 liver cancer cells, the HCT116 colon cancer cells and the MDA-MB-231 breast cancer cells, and the inhibition rates were all less than 10%. The inhibition rates of the CNSI-Fe acting alone to the SMMC7721 liver cancer cells, the HCT116 colon cancer cells and the MDA-MB-231 breast cancer cells were 40.15±2.98%, 34.97±1.67% and 32.85±3.07%, respectively. The inhibition rates to the three kinds of cells in the CNSI + near-infrared irradiation group and the CNSI-Fe + near-infrared irradiation group are as shown in Tables 1-3. When the temperature was 37° C. and 40° C., there was basically no effect on the cells, and the inhibition rates gradually increased as the temperature was elevated. When the temperature was ≥ 42° C., the inhibition rate in the CNSI-Fe + near-infrared irradiation group was greatly increased and was better than those in the CNSI-Fe group and the CNSI + near-infrared irradiation group at the same temperature. In the CNSI + near-infrared irradiation group, when the temperature reached 50° C., the inhibition rate to the three kinds of cells reached above 90%; but in the CNSI-Fe + near-infrared irradiation group, the inhibition rate to the three kinds of cells reached above 90% when the temperature was 45° C.

TABLE-US-00002 Inhibition Rates to SMMC7721 Liver Cancer Cells in the CNSI + Near-infrared Irradiation Group and the CNSI-Fe + Near-infrared Irradiation Group Temperature Inhibition Rate (%) CNSI + Near-infrated CNSI-Fe + Near-infrared Irradiation Group Irradiation Group 37° C. 4.92±2.94 38.95±3.68 40° C. 6.79±1.57 41.92±2.06 42° C. 39.03± 4.11 70.31± 1.53 45° C. 57.18± 2.67 92.77±1.14 48° C. 76.18± 3.41 98.46±2.64 50° C. 91.14±1.90 99.58±2.47

TABLE-US-00003 Inhibition Rates to HCT116 Colon Cancer Cells in the CNSI + Near-infrared Irradiation Group and the CNSI-Fe + Near-infrared Irradiation Group Temperature Inhibition Rate (%) CNSI + Near-infrared CNSI-Fe + Near-infrared Irradiation Group Irradiation Group 37° C. 6.49±3.75 35.89±4.12 40° C. 10.98±2.85 37.15±3.48 42° C. 32.44±1.72 65.78±2.45 45° C. 60.54±4.82 90.64±3.56 48° C. 70.15±3.47 97.22±1.68 50° C. 92.86±3.86 99.69±2.76

TABLE-US-00004 Inhibition Rates to MDA-MB-231 Breast Cancer Cells in the CNSI + Near-infrared Irradiation Group and the CNSI-Fe + Near-infrared Irradiation Group Temperature Inhibition Rate (%) CNSI + Near-infrared CNSI-Fe + Near-infrared Irradiation Group Irradiation Group 37° C. 8.10±4.45 37.66±3.28 40° C. 15.46±2.75 45.52±2.79 42° C. 42.38±3.27 73.91±1.05 45° C. 55.43±1.41 90.55±3.72 48° C. 75.25±2.72 98.47±3.61 50° C. 93.75±2.76 99.74±4.04

2) Experiment on Inhibiting Tumor Growth

[0061] The inhibitory effects of CNSI-Fe, CNSI + near-infrared irradiation, CNSI-Fe + near-infrared irradiation, and near-infrared irradiation on the growth of subcutaneous xenograft tumor of the nude mice with SMMC7721 liver cancer cells, HCT116 colon cancer cells and MDA-MB-231 breast cancer cells were observed (FIGS. 1-3). The results showed that at the end of the observation, the near-infrared irradiation alone had no obvious inhibitory effect on the subcutaneous xenograft tumor of the three kinds of cells. The inhibition rates of the CNSI-Fe to the subcutaneous xenograft tumor of the three kinds of cells were 48.57%, 52.03% and 45.79%, respectively; the inhibition rates of the CNSI + near-infrared irradiation to the subcutaneous xenograft tumor of the three kinds of cells were 53.81%, 48.06% and 49.35%, respectively; and the inhibition rates of the CNSI-Fe + near-infrared irradiation to the subcutaneous xenograft tumor of the three kinds of cells were 90.78%, 90.14% and 93.3%, respectively. Comparing the CNSI-Fe + near-infrared irradiation with the CNSI-Fe alone or the CNSI + near-infrared irradiation, there were significant differences. According to the calculation using the Jin’s formula method, the q value of the CNSI + near-infrared irradiation was greater than 1, indicating that CNSI-Fe and near-infrared irradiation were synergic and could enhance the anti-tumor effect of hyperthermia using CNSI-Fe and CNSI.

[0062] At the same time, the content of hydroxyl radicals in tumors of animals was compared among the groups (FIG. 4). The results respectively showed that FIG. 4a represented that the samples were not treated by any method; FIG. 4b and FIG. 4d represented that there were not significant hydroxyl radicals produced by the CNSI + near-infrared irradiation and the near-infrared irradiation, and the CNSI-Fe group (FIG. 4c) and the CNSI-Fe + near-infrared irradiation group (FIG. 4e) had the highest contents of hydroxyl radicals in tumor, which were about 2 times that of the CNSI-Fe group. It indicates that the CNSI-Fe after near-infrared irradiation can effectively elevate the temperature in tumor, improve the efficiency of the Fenton reaction and produce more hydroxyl radicals.

3) Experiment on Inhibiting Lymph Node Metastasis

[0063] The inhibitory effects of the CNSI-Fe, CNSI + near-infrared irradiation, CNSI-Fe + near-infrared irradiation, and near-infrared irradiation on the lymph node metastasis of H22 liver cancer cells were tested (FIG. 5). The results showed that compared with the negative group (“*” represented p<0.05, and “**” represented p<0.01), the weight of lymph nodes in the near-infrared irradiation group was not changed obviously; and the weight of lymph nodes in the CNSI-Fe group, the CNSI + near-infrared irradiation group and the CNSI-Fe + near-infrared irradiation group was significantly decreased (p<0.01). The CNSI-Fe + near-infrared irradiation had the greatest inhibitory effect on the lymph node metastasis, the weight of the transferred lymph nodes was the lightest, and there was significant difference (p<0.05) as compared with the CNSI-Fe alone or the CNSI + near-infrared irradiation. According to the calculation using the Jin’s formula method, the q value of the CNSI-Fe + near-infrared irradiation was greater than 1, indicating that CNSI-Fe and near-infrared irradiation were synergic and could enhance the inhibitory effects of hyperthermia using CNSI-Fe and CNSI on lymph node metastasis.

[0064] It should be stated that the above-mentioned embodiments are only used for explaining, rather than limiting, the technical solutions of the present disclosure. Although the present disclosure is explained in detail by reference to the above-mentioned embodiments, those of ordinary skill in the art should understand that modifications or equivalent substitutions may be made to the present disclosure still, and any modification or local substitution without deviating from the spirit and scope of the present disclosure should fall into the scope of the claims of the present disclosure.