Nanocarbon-iron composite system as well as composition, preparation method and use thereof
11235976 · 2022-02-01
Assignee
Inventors
- Xiaohai Tang (Chongqing, CN)
- Yu Qiu (Chongqing, CN)
- Yuanfang Huang (Chongqing, CN)
- Pingfang Xia (Chongqing, CN)
Cpc classification
B82Y40/00
PERFORMING OPERATIONS; TRANSPORTING
A61K47/32
HUMAN NECESSITIES
A61K9/0019
HUMAN NECESSITIES
B82Y5/00
PERFORMING OPERATIONS; TRANSPORTING
B82Y30/00
PERFORMING OPERATIONS; TRANSPORTING
C01B32/174
CHEMISTRY; METALLURGY
International classification
C01B32/174
CHEMISTRY; METALLURGY
A61K9/00
HUMAN NECESSITIES
A61P35/00
HUMAN NECESSITIES
B82Y5/00
PERFORMING OPERATIONS; TRANSPORTING
B82Y30/00
PERFORMING OPERATIONS; TRANSPORTING
Abstract
The present invention provides a nanocarbon-iron composite system which is a composite structure formed by interaction of acid-treated nanocarbon serving as a carrier, with and ferrous ions and/or ferric ions in an iron salt. In an in-vitro experiment and an animal experiment, the nanocarbon-iron composite system of the present invention shows a very efficient inhibition effect on solid tumors containing liver cancer, breast cancer and cervical cancer and has an excellent targeting property. Accordingly, the present invention further provides a preparation method of the nanocarbon-iron composite system, use of the nanocarbon-iron composite system in preparation of a drug for treating solid tumors, and a suspension for injection based on the nanocarbon-iron composite system.
Claims
1. A nanocarbon-iron composite system being a composite structure which is formed by acid-treated nanocarbon serving as a carrier, and the nanocarbon contains a certain amount of carboxyl on the surface, and ferrous ions and/or ferric ions in an iron salt, wherein the carboxyl has a content range of 0.03 mmol/g-0.08 mmol/g, wherein the composite system has a particle size of 90-300 nm.
2. The composite system according to claim 1, wherein the ferrous ions and/or ferric ions in the iron salt have a concentration of 1.36-13.6 mg/mL.
3. The composite system according to claim 2, wherein the iron salt is selected from any one or more of ferrous sulfate, ferric sulfate, ferrous chloride, ferric chloride, ferrous gluconate, iron sucrose, ammonium ferric citrate, ferrous succinate, iron sorbitol and ferrous fumarate.
4. The composite system according to claim 3, wherein the mass ratio of the nanocarbon to an iron element in the iron salt is 40:1-3:1.
5. The nanocarbon-iron composite system according to claim 2, wherein the composite system has a pH of 3.0-6.0.
6. The composite system according to claim 2, wherein the nanocarbon has a carbon content of 86-98%, a hydrogen content of 0.5-2.5%, and an oxygen content of 1.0-10.0%.
7. The composite system according to claim 2, further comprising sodium citrate, wherein the mass ratio of the sodium citrate to the iron element in the iron salt is 0.1-3.
8. The nanocarbon-iron composite system according to claim 1, wherein the composite system has a pH of 3.0-6.0.
9. The composite system according to claim 1, wherein the mass ratio of the nanocarbon to an iron element in the iron salt is 40:1-3:1.
10. The composite system according to claim 1, wherein the nanocarbon has a carbon content of 86-98%, a hydrogen content of 0.5-2.5%, and an oxygen content of 1.0-10.0%.
11. The composite system according to claim 10, wherein the nanocarbon comprises at least one or more of carbon nanoparticles, carbon nanotubes, carbon quantum dots, graphene, fullerene, carbon nanorods, carbon nanofibres and nano-carbon-black C.sub.40.
12. The composite system according to claim 11, wherein the nanocarbon has a carboxyl content of 0.01-2.0 mmol/g.
13. The composite system according to claim 1, wherein the nanocarbon and the iron salt form a composite structure by a combination of electrostatic interaction, complexation and the Van der Waals force.
14. The composite system according to claim 1, further comprising sodium citrate, wherein the mass ratio of the sodium citrate to the iron element in the iron salt is 0.1-3.
15. The composite system according to claim 14, wherein the sodium citrate forms a complex with ferrous ions; and/or the sodium citrate forms a complex with iron ions.
16. The composite system according to claim 1, further comprising a suspending agent, wherein the suspending agent is selected from one or more of methylcellulose, sodium carboxymethylcellulose, hydroxypropyl cellulose, polyvinylpyrrolidone K.sub.30 and glucan.
17. The composite system according to claim 16, wherein the suspending agent has a concentration of 10-40 mg/ml.
18. The composite system according to claim 1, wherein-the nanocarbon-iron composite system is utilized in preparation of a drug for treating liver cancer, lung cancer, stomach cancer, colon cancer, breast cancer, cervical cancer, thyroid cancer or ovarian cancer.
19. A suspension for injection, comprising a nanocarbon-iron composite system being a composite structure which is formed by acid-treated nanocarbon serving as a carrier, and the nanocarbon contains a certain amount of carboxyl on the surface, and ferrous ions and/or ferric ions in an iron salt, wherein the carboxyl has a content range of 0.03 mmol/g-0.08 mmol/g, wherein the composite system has a particle size of 90-300 nm, wherein the nanocarbon-iron composite system is dispersed uniformly and stably in a mixed solution containing polyvinylpyrrolidone K30 and sodium citrate; in the composite system, the ferrous irons or/and ferric ions have a concentration of 1.36-13.6 mg/mL.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
(56) I. Preparation of Nanocarbon-iron Composite System
(57) The following samples are prepared and raw material compositions of samples are shown in tables 1-16 below in details, respectively.
(58) 1. Nanocarbon+Ferrous Sulfate
(59) TABLE-US-00001 TABLE 1 Nanocarbon Content (mg), Ferrous Carboxyl Content Sulfate Normal Homogenization (mmol/G) and Heptahydrate PVP Saline Sodium Pressure (mpa)/Number Number Particle Size (nm) (mg) (mg) (ml) Citrate of Times 1A 250, 0.01, 160 135.5 200 10 50 90/3 1B 500, 0.01, 160 271.0 200 10 50 90/3 2A 250, 0.07, 160 135.5 200 10 50 90/3 2B 500, 0.07, 160 271.0 200 10 50 90/3 3A 250, 2.00, 160 135.5 200 10 50 90/3 3B 500, 2.00, 160 271.0 200 10 50 90/3 4A 200, 0.07, 160 67.8 200 10 50 90/3 4B 400, 0.07, 160 135.5 200 10 50 90/3 5A 400, 0.07, 160 271 200 10 50 90/3 5B 800, 0.07, 160 542 200 10 50 90/3 6A 800, 0.07, 160 406.5 200 10 50 90/3 6B 1600, 0.07, 160 813 200 10 50 90/3 7A 1000, 0.07, 160 677.5 200 10 50 90/3 7B 2000, 0.07, 160 1355 200 10 50 90/3 8A 2500, 0.07, 160 1355 2000 100 500 90/3 8B 5000, 0.07, 160 2710 2000 100 500 90/3 9A 25000, 0.07, 160 13550 20000 1000 5000 90/3 9B 50000, 0.07, 160 27100 20000 1000 5000 90/3 10A 250, 0.07, 160 135.5 100 10 50 90/3 10B 500, 0.07, 160 271.0 100 10 50 90/3 11A 250, 0.07, 160 135.5 400 10 50 90/3 11B 500, 0.07, 160 271.0 400 10 50 90/3 12A 250, 0.07, 90 135.5 200 10 50 120/5 12B 500, 0.07, 90 271.0 200 10 50 120/5 13A 250, 0.07, 120 135.5 200 10 50 110/3 13B 500, 0.07, 120 271.0 200 10 50 110/3 14A 250, 0.07, 180 135.5 200 10 50 80/3 14B 500, 0.07, 180 271.0 200 10 50 80/3 15A 250, 0.07, 300 135.5 200 10 50 60/3 15B 500, 0.07, 300 271.0 200 10 50 60/3 16A 250, 0.07, 500 135.5 200 10 50 30/2 16B 500, 0.07, 500 271.0 200 10 50 30/2
(60) 2. Nanocarbon+Ferrous Chloride
(61) TABLE-US-00002 TABLE 2 Nanocarbon Content (mg), Homogenization Carboxyl Content Ferrous Normal Pressure (mmol/g) and Particle Size Chloride PVP Saline Sodium (mpa)/Number Number (nm) Tetrahydrate (mg) (mL) Citrate of Times 17 250, 0.07, 160 48.5 200 10 50 90/3 18 250, 0.07, 160 96.9 200 10 50 90/3 19 250, 0.07, 160 290.7 200 10 50 90/3
(62) 3. Nanocarbon+Ferric Chloride
(63) TABLE-US-00003 TABLE 3 Homogenization Pressure Nanocarbon Content (mg), Ferric Normal (mpa)/ Carboxyl Content (mmol/g) Chloride PVP Saline Sodium Number of Number and Particle Size (nm) (mg) (mg) (mL) Citrate Times 22 250, 0.07, 160 48.5 200 10 50 90/3 23 250, 0.07, 160 96.9 200 10 50 90/3 24 250, 0.07, 160 290.7 200 10 50 90/3
(64) 4. Nanocarbon+Ferric Sulfate
(65) TABLE-US-00004 TABLE 4 Homogenization Pressure Nanocarbon Content (mg), Ferric Normal (mpa)/ Carboxyl Content (mmol/g) Sulfate PVP Saline Sodium Number of Number and Particle Size (nm) (mg) (mg) (mL) Citrate Times 25 250, 0.07, 160 48.7 200 10 50 90/3 26 250, 0.07, 160 97.5 200 10 50 90/3 27 250, 0.07, 160 292.5 200 10 50 90/3
(66) 5. Carbon Nanotube+Ferrous Sulfate Heptahydrate
(67) TABLE-US-00005 TABLE 5 Homogenization Carbon Nanotube Ferrous Pressure Content (mg), Carboxyl Sulfate Normal (mpa)/ Content (mmol/g) and Heptahydrate PVP Saline Sodium Number of Number Particle Size (nm) (mg) (mg) (mL) Citrate Times 28 250, 0.07, 160 135.5 200 10 50 90/3
(68) 6. Graphene+Ferrous Sulfate Heptahydrate
(69) TABLE-US-00006 TABLE 6 Homogenization Graphene content (mg), Ferrous Pressure Carboxyl Content Sulfate Normal (mpa)/ (mmol/g) and Particle Heptahydrate PVP Saline Sodium Number of Number Size (nm) (mg) (mg) (mL) Citrate Times 29 250, 0.07, 160 135.5 200 10 50 90/3
(70) 7. Carbon Quantum Dots+Ferrous Sulfate Heptahydrate
(71) TABLE-US-00007 TABLE 7 Carbon Quantum Dot Ferrous Content (mg), Carboxyl Sulfate Normal Content (mmol/g) and Heptahydrate PVP Saline Sodium Homogenization Pressure Number Particle Size (nm) (mg) (mg) (mL) Citrate (mpa)/Number of Times 30 250, 0.07, 160 135.5 200 10 50 90/3
(72) 8. Fullerene+Ferrous Sulfate Heptahydrate
(73) TABLE-US-00008 TABLE 8 Fullerene Ferrous Content (mg), Carboxyl Sulfate Normal Content (mmol/g) and Heptahydrate PVP Saline Sodium Homogenization Pressure Number Particle Size (nm) (mg) (mg) (mL) Citrate (mpa)/Number of Times 31 250, 0.07, 160 135.5 200 10 50 90/3
(74) 9. Activated Carbon+Ferrous Sulfate Heptahydrate
(75) TABLE-US-00009 TABLE 9 Activated Carbon Ferrous Content (mg), Carboxyl Sulfate Normal Content (mmol/g) and Heptahydrate PVP Saline Sodium Homogenization Pressure Number Particle Size (nm) (mg) (mg) (mL) Citrate (mpa)/Number of Times 32 250, 0.07, 160 135.5 200 10 50 90/3
(76) 10. Nanocarbon+Ferric Hydroxide
(77) TABLE-US-00010 TABLE 10 Activated Carbon Content (mg), Carboxyl Ferric Normal Content (mmol/g) and Hydroxide PVP Saline Sodium Homogenization Pressure Number Particle Size (nm) (mg) (mg) (mL) Citrate (mpa)/Number of Times 33 500, 0.07, 160 25.9 200 10 50 90/3 34 500, 0.07, 160 51.9 200 10 50 90/3 35 500, 0.07, 160 155.7 200 10 50 90/3
(78) 11. Nanocarbon+Iron Sucrose
(79) TABLE-US-00011 TABLE 11 Nanocarbon Commercially- Content (mg), Carboxyl Available Iron Normal Content (mmol/g) and Sucrose PVP Saline Sodium Homogenization Pressure Number Particle Size (nm) (ml) (mg) (mL) Citrate (mpa)/Number of Times 36 250, 0.07, 160 0.7 200 9.3 50 90/3 37 250, 0.07, 160 1.4 200 8.6 50 90/3 38 250, 0.07, 160 4.2 200 5.8 50 90/3
(80) 12. Nanocarbon+Ferrous Succinate
(81) TABLE-US-00012 TABLE 12 Nanocarbon Commercially- Content (mg), Carboxyl Available Ferrous Normal Content (mmol/g) and Succinate PVP Saline Sodium Homogenization Pressure Number Particle Size (nm) (ml) (mg) (mL) Citrate (mpa)/Number of Times 39 250, 0.07, 160 0.25 200 9.7 50 90/3 40 250, 0.07, 160 0.5 200 9.5 50 90/3 41 250, 0.07, 160 1.5 200 8.5 50 90/3
(82) 13. Nanocarbon+Ferrous Gluconate
(83) TABLE-US-00013 TABLE 13 Nanocarbon Content (mg), Carboxyl Ferrous Normal Content (mmol/g) and Gluconate PVP Saline Sodium Homogenization Pressure Number Particle Size (nm) (mg) (mg) (mL) Citrate (mpa)/Number of Times 42 250, 0.07, 160 108.7 200 10 50 90/3 43 250, 0.07, 160 217.5 200 10 50 90/3 44 250, 0.07, 160 652.5 200 10 50 90/3
(84) 14. Nanocarbon+Iron Sorbitol
(85) TABLE-US-00014 TABLE 14 Nanocarbon Content (mg), Carboxyl Iron Normal Content (mmol/g) and Sorbitol PVP Saline Sodium Homogenization Pressure Number Particle Size (nm) (ml) (mg) (mL) Citrate (mpa)/Number of Times 45 250, 0.07, 160 0.55 200 9.4 50 90/3 46 250, 0.07, 160 1.1 200 8.9 50 90/3 47 250, 0.07, 160 3.3 200 6.7 50 90/3
(86) 15. Nanocarbon+Ferrous Fumarate
(87) TABLE-US-00015 TABLE 15 Nanocarbon Content (mg), Carboxyl Ferrous Normal Content (mmol/g) and Fumarate PVP Saline Sodium Homogenization Pressure Number Particle Size (nm) (mg) (mg) (mL) Citrate (mpa)/Number of Times 48 250, 0.07, 160 41.4 200 10 50 90/3 49 250, 0.07, 160 82.8 200 10 50 90/3 50 250, 0.07, 160 248.4 200 10 50 90/3
(88) 16. Nanocarbon+Ammonium Ferric Citrate
(89) TABLE-US-00016 TABLE 16 Canocarbon content (mg), Carboxyl Ammonium Normal Content (mmol/g) and Ferric Citrate PVP Saline Sodium Homogenization Pressure Number Particle Size (nm) (mg) (mg) (mL) Citrate (mpa)/Number of Times 51 250, 0.07, 160 118.9 200 10 50 90/3 52 250, 0.07, 160 237.9 200 10 50 90/3 53 250, 0.07, 160 713.7 200 10 50 90/3
(90) The specific preparation process of each sample is as follows (samples A are prepared according to method 1 and samples B are prepared according to method 2).
(91) 1. Nanocarbon+ferrous sulfate
(92) Sample 1A
(93) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.01 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(94) Sample 1B
(95) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 500 mg of nanocarbon powder (carboxyl content: 0.01 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 271.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(96) Sample 2A
(97) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(98) Sample 2B
(99) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 500 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 271.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(100) Sample 3A
(101) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 2.00 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(102) Sample 3B
(103) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 500 mg of nanocarbon powder (carboxyl content: 2.00 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 271.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(104) Sample 4A
(105) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 200 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 67.8 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(106) Sample 4B
(107) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 400 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 135.5 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(108) Sample 5A
(109) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 400 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 271.0 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(110) Sample 5B
(111) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 800 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 542.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(112) Sample 6A
(113) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 800 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 406.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(114) Sample 6B
(115) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 1600 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 813.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(116) Sample 7A
(117) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 1000 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm); complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 677.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(118) Sample 7B
(119) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 2000 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 1355.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(120) Sample 8A
(121) 2000 mg of PVP K30 is added to 100 ml of normal saline; after complete dissolution at the room temperature, 2500 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 500 mg of sodium citrate is added); 1355 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(122) Sample 8B
(123) 2000 mg of PVP K30 is added to 100 ml of normal saline; after complete dissolution at the room temperature, 5000 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 2710 mg of ferrous sulfate heptahydrate solid is dissolved in 1000 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(124) Sample 9A
(125) 20000 mg of PVP K30 is added to 1000 ml of normal saline; after complete dissolution at the room temperature, 25000 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 5000 mg of sodium citrate is added); 13550 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(126) Sample 9B
(127) 20000 mg of PVP K30 is added to 1000 ml of normal saline; after complete dissolution at the room temperature, 500 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 27100 mg of ferrous sulfate heptahydrate solid is dissolved in 10000 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(128) Sample 10A
(129) 100 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(130) Sample 10B
(131) 100 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 500 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 271.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(132) Sample 11A
(133) 400 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(134) Sample 11B
(135) 400 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 500 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 271.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(136) Sample 12A
(137) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 90 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 5 times (pressure: 120 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(138) Sample 12B
(139) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 500 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 90 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 5 times (pressure: 120 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 271.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(140) Sample 13A
(141) 200 mg of PVP K30 is added to 10 ml of normal saline: after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 120 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 110 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(142) Sample 13B
(143) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 500 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 120 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 110 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 271.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(144) Sample 14A
(145) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 180 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 80 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(146) Sample 14B
(147) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 500 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 180 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 80 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 271.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(148) Sample 15A
(149) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 300 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 60 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(150) Sample 15B
(151) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 500 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 300 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 60 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 271.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(152) Sample 16A
(153) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 500 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed twice (pressure: 30 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(154) Sample 16B
(155) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 500 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 500 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); after complete and uniform mixing at the room temperature, high-pressure homogenization is performed twice (pressure: 30 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation (component I); 271.0 mg of ferrous sulfate heptahydrate solid is dissolved in 10 ml of normal saline, bottling and freeze-drying are performed, and the bottle is sealed by filling the bottle with nitrogen for preservation (component II); and component I and component II are mixed during use.
(156) 2. Nanocarbon+Ferrous Chloride
(157) Sample 17
(158) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 48.5 mg of ferrous chloride is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(159) Sample 18
(160) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 96.9 mg of ferrous chloride is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(161) Sample 19
(162) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 290.7 mg of ferrous chloride is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(163) 3. Nanocarbon+Ferric Chloride
(164) Sample 20
(165) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 65.9 mg of ferric chloride is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(166) Sample 21
(167) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 131.8 mg of ferric chloride is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(168) Sample 22
(169) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 395.4 mg of ferric chloride is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(170) 4. Nanocarbon Ferric Sulfate
(171) Sample 23
(172) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 48.7 mg of ferrous sulfate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(173) Sample 24
(174) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 97.5 mg of ferrous sulfate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(175) Sample 25
(176) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon powder (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 292.5 mg of ferric sulfate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(177) 5. Carbon Nanotubes+Ferrous Sulfate Heptahydrate
(178) Sample 26
(179) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of carbon nanotubes (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(180) 6. Graphene+Ferrous Sulfate Heptahydrate
(181) Sample 27
(182) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of graphene (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(183) 7. Carbon Quantum Dots+Ferrous Sulfate Heptahydrate
(184) Sample 28
(185) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of carbon quantum dots (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(186) 8. Fullerene+Ferrous Sulfate Heptahydrate
(187) Sample 29
(188) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of fullerene (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(189) 9. Activated Carbon+Ferrous Sulfate Heptahydrate
(190) Sample 30
(191) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of activated carbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 135.5 mg of ferrous sulfate heptahydrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(192) 10. Nanocarbon+Ferric Hydroxide
(193) Sample 31
(194) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 51.9 mg of ferric hydroxide is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(195) Sample 32
(196) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 155.7 mg of ferric hydroxide is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(197) 11. Nanocarbon+Iron Sucrose
(198) Sample 33
(199) 200 mg of PVP K30 is added to 9.3 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 0.7 mL of iron sucrose is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(200) Sample 34
(201) 200 mg of PVP K30 is added to 8.6 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 1.4 mL of iron sucrose is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(202) Sample 35
(203) 200 mg of PVP K30 is added to 5.8 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 4.2 mL of iron sucrose is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(204) 12. Nanocarbon+Ferrous Succinate
(205) Sample 36
(206) 200 mg of PVP K30 is added to 9.75 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 0.25 mL of ferrous succinate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(207) Sample 37
(208) 200 mg of PVP K30 is added to 9.5 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 0.5 mL of ferrous succinate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(209) Sample 38
(210) 200 mg of PVP K30 is added to 8.5 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 1.5 mL of ferrous succinate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(211) 13. Nanocarbon+Ferrous Gluconate
(212) Sample 39
(213) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 108.7 mg of ferrous gluconate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(214) Sample 40
(215) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 217.5 mg of ferrous gluconate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(216) Sample 41
(217) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 652.5 mg of ferrous gluconate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(218) 14. Nanocarbon+Iron Sorbitol
(219) Sample 42
(220) 200 mg of PVP K30 is added to 9.45 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 0.55 mL of iron sorbitol is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(221) Sample 43
(222) 200 mg of PVP K30 is added to 8.9 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 1.1 mL of iron sorbitol is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(223) Sample 44
(224) 200 mg of PVP K30 is added to 6.7 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 3.3 mL of iron sorbitol is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(225) 15. Nanocarbon+Ferrous Fumarate
(226) Sample 45
(227) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 41.4 mg of ferrous fumarate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(228) Sample 46
(229) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 82.8 mg of ferrous fumarate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(230) Sample 47
(231) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 248.4 mg of ferrous fumarate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(232) 16. Nanocarbon+Ammonium Ferric Citrate
(233) Sample 48
(234) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 118.9 mg of ammonium ferric citrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(235) Sample 49
(236) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 237.9 mg of ammonium ferric citrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(237) Sample 50
(238) 200 mg of PVP K30 is added to 10 ml of normal saline; after complete dissolution at the room temperature, 250 mg of nanocarbon (carboxyl content: 0.07 mmol/g, and particle size: 160 nm) is added; complete stirring and uniform dispersion are performed, and sodium citrate is added to adjust the pH value to 6.8-7.2 (about 50 mg of sodium citrate is added); 713.7 mg of ammonium ferric citrate is added; after complete and uniform mixing at the room temperature, high-pressure homogenization is performed 3 times (pressure: 90 mpa); a suspension is collected into a penicillin bottle after completion of homogenization; and the penicillin bottle is sealed by filling the penicillin bottle with nitrogen for preservation.
(239) The above samples are prepared from combinations of different raw materials and different iron salts respectively through a similar process. Through the research on the structures and compositions of the nanocarbon-iron composite systems by XPS spectra and infrared spectra, respectively, it was found that the nanocarbon-iron composite systems have a fairly-consistent structure. Therefore, a variety of iron salts can implement the present invention and will not be enumerated here.
(240) As shown in
(241) It can be seen from the element analysis spectrum that, as shown in table 17, after nanocarbon adsorbs iron, there is less influence on the composition, and a small amount of water coordinates on Fe possibly because Fe is adsorbed on the surface of the nanocarbon, resulting in a slight increase in 0 content.
(242) TABLE-US-00017 TABLE 17 Comparison between element contents of nanocarbon and nanocarbon-iron composite Composition C (at %) O (at %) Fe (at %) N (at %) Nanocarbon 94.85 4.01 — 1.15 Nanocarbon-Iron 92.06 6.06 0.89 1.24 Composite
(243) As shown in the infrared spectra in
(244) By summarizing results of XPS, element analysis and the infrared spectrum analysis, it can be determined that the nanocarbon-iron composite is a composite formed by a combination of various interactions such as electrostatic interaction, complexation and Van der Waals force.
(245) For selection of the particle size and concentration, the nanocarbon used in an experiment is such nanocarbon that contains certain amount (0.01-0.10 mmol/g) of carboxyl on the surface after acid oxidation treatment. When the carboxyl content is lower than 0.03 mmol/g, the stability of a suspension system is reduced and deposition occurs easily. Thus, it is impossible to form a stable suspension. Since carboxyl is a hydrophilic group, the higher content of carboxyl facilitates the stability of the suspension system. Although an increase in the content of polyvinylpyrrolidone K30 (PVP K30) can also improve the stability of the suspension system to some extent, it also significantly increases the viscosity of the system, which is unfavourable for injection administration. When the carboxyl content is higher than 0.08 mmol/g, the colour of the suspension becomes light (change from black to light black), which is unfavourable for observation on nanocarbon tracing effects. Therefore, considering comprehensively, it is reasonable that the content range of carboxyl is 0.03-0.08 mmol/g.
(246) For the nanocarbon-iron composite system, a preparation has requirements for the particle size of the suspension in the aspects of both the stability and pharmacological efficacy. Since the blood capillary of a tumor tissue has a hole diameter of about 50 nm and a lymphatic vessel has a hole diameter of about 150 nm, when the nanocarbon-iron composite has a particle size of less than 50 nm, it is easy to enter the blood capillary, affecting the concentration of iron in blood. In addition, macrophages selectively phagocytose particles, and the larger the particle size is, the easier it is to be phagocytosed by macrophages. When the nanocarbon has the particle size of greater than 300 nm, the stability of the suspension becomes poor, and deposition and accumulation occur easily during still standing, which cannot meet the requirements on stability. Moreover, since the lymphatic vessel has an opening of about 150 nm, large carbon-iron nanoparticles having the particle size of garter than 300 nm may block the lymphatic vessel probably, which causes impossibility of subsequent carbon-iron nanoparticles to pass through the lymphatic vessel and thus reduces the tracing effect and treatment effect. Therefore, by taking the stability and pharmacological efficacy of the preparation into consideration, the particle size range of the nanocarbon-iron composite is controlled within 90-300 nm, preferably within 100-250 nm, and more preferably within 120-180 nm.
(247) In addition to adjusting the pH value of the suspension, sodium citrate added in the preparation process also uses as an anticoagulant to guarantee that the suspension can have certain fluidity after injection, thereby allowing the nanocarbon-iron composite to transfer effective components into cells.
(248) The composite contains ferrous irons and ferric irons, which are main active ingredients that exert an anti-cancer effect by “ferroptosis”, and apoptosis induced by polarization of macrophages M2 to macrophages M1. The iron in the composite may come from organic or inorganic iron salts, such as ferrous sulfate, ferric sulfate, ferrous chloride, ferric chloride, iron sucrose, ferrous succinate, ferrous gluconate, iron dextran, iron sorbitol, ferrous fumarate and ammonium ferric citrate, and preferably ferrous sulfate.
(249) In the nanocarbon-iron composite system, the nanocarbon has a larger specific area, a large number of voids and a relatively high adsorption capacity, and the Van der Waals force, complexation and electrostatic interaction exit between an oxygen-containing group on the surface of the nanocarbon and ferric ions. In addition, carbon-iron adsorption and binding strength is moderate, and ferrous ions undergo Fenton Reaction after the nanocarbon-iron composite is phagocytosed by macrophages of tumors. Therefore, the nanocarbon-iron composite is preferably divalent iron salt, and more preferably ferrous sulfate.
(250) The present invention also provides the range of a mass ratio of carbon nanoparticles to ferrous sulfate, which is a key factor for the nanocarbon-iron composite to exert the anti-cancer effect and is mainly obtained through pharmacological experiments. High doses of iron are directly cytotoxic to in-vitro and in-vivo tumor cells. Therefore, the dosage of iron needs to be selected reasonably. It has been proved by multiple experiments that a relatively high inhibition ratio for tumor cells is achieved when the mass ratio of nanocarbon to iron is 9.2:1 and thus the ratio of nanocarbon to iron is designed to be 3:1-40:1 in the pharmacological experiments. The results show that when the ratio of nanocarbon to iron is 5:1-30:1, the relatively high tumor inhibition ratio is achieved and is up to 50-80%, when the ratio of nanocarbon to iron is greater than 30:1, the tumor inhibition ratio is low; and when the ratio of nanocarbon to iron is less than 5:1, there is slight toxicity. Therefore, the range of the mass ratio of nanocarbon to iron in the composite is 5:1-30:1 and preferably 6:1-18:1.
(251) Under the guidance of the above theoretical analysis, a cell experiment and an animal experiment are performed on the above samples as follows.
(252) 1. Experimental Materials
(253) 1) Cell Strains
(254) SMMC7721 liver cancer cells, A549 lung cancer cells, SGC-7901 stomach cancer cells, HCT116 colon cancer cells, MDA-MB-231 breast cancer cells, Hela cervical cancer cells, TPC-1 thyroid cancer cells, SKOV3 ovarian cancer cells, and murine liver cancer H22 cells.
(255) 2) Cell Culture Media
(256) DMEM cell-culture medium, RMP11640 cell-culture medium, fetal bovine serum (FBS), a typsin-EDTA solution, a mixed solution of penicillin and streptomycin and phosphate buffered saline (PBS, pH 7.4).
(257) 3) Experimental Animals
(258) Female BalB/c-nu mice aged 4-6 weeks and with the body weight of 20±2 g are used and are allowed for free drinking and eating during the experiment. The mice are illuminated for 12 hours every day and are raised by mouse cages (5 mice/cage) which are independently ventilated and isolated.
(259) Female inbred strain Kunming mice of clean grade aged 6-7 weeks with the body weight of 20±2 g are used, allowed for free drinking and eating during the experiment, and illuminated for 12 hours every day. Mouse cages (5 mice/cage) are ventilated by a central ventilation system.
(260) 4) Experimental Drugs and Main Instruments
(261) A nanocarbon-iron suspension (the ratio of nanocarbon to ferric ions=9.2:1), a nanocarbon suspension, ferrous sulfate, ferrous gluconate, iron sucrose, ammonium ferric citrate, a cis-platinum injection, a 0.9% sodium chloride injection, a Prussian-blue staining kit, nuclear fast red staining liquor, xylene, anhydrous ethanol, hydrochloric acid, neutral gum, a dehydrator, an embedding machine, a pathological microtome, a tissue slicer, a high-speed centrifuge, a blast drying oven, a thermostat water bath, an inverted fluorescence microscope, a biological optical microscope, a constant-temperature incubator, a pure water filter, a high-pressure sterilizing pot, a super-clean bench, a microplate reader and an electronic scale.
(262) 2. Experimental Methods
(263) 1) Cell Experiment
(264) Cells growing in a log phase are collected, the concentration of the cell suspension is adjusted, 100 μL of cell suspension is added to each well and the cells are plated (edge wells are filled with sterile PBS) at a density of 1×10.sup.3-10.sup.4 cells/well. The cells are incubated for 24 hours at 37° C. under the condition of 5% CO2, a nanocarbon-iron solution having a concentration gradient (nanocarbon content: 125, 62.5, 15.63 and 3.91 μg/mL; and ferric ion content: 13.65, 6.83, 1.71 and 0.43 μg/mL) is added, and three sub-wells are set. Then the cells are incubated for 48 hours at 37° C. under the condition of 5% CO2. 10 μL of CCK8 solution is added to each well and then the cells are continuously incubated for 2 hours. The absorbance of each well is measured at OD=450 nm with the microplate reader. In addition, a negative control group, nanocarbon control groups having the same concentration and iron preparation control groups having the same concentration are set.
(265) Cells growing in a log phase are collected, the concentration of the cell suspension is adjusted, and 1 μL of cell suspension is added to each well of a 6-well plate at a density of 3×10.sup.4 cells/well. The cells are incubated for 24 hours at 37° C. under the condition of 5% CO2, a nanocarbon-iron solution having a concentration gradient (nanocarbon content: 125, 62.5, 15.63 and 3.91 μg/mL; and ferric ion content: 13.65, 6.83, 1.71, 0.43 μg/mL) is added, and three sub-wells are set. Then the cells are incubated for 48 hours at 37° C. under the condition of 5% CO2. The cells are trypsinized and counted. In addition, a negative control group, nanocarbon control groups having the same concentration and iron preparation control groups having the same concentration are set.
(266) 2) Tumor Growth Inhibition Experiments
(267) Cells growing in a log phase are collected, the concentration of the cell suspension is adjusted to 3×10.sup.7 cells/mL, and the cell suspension is inoculated subcutaneously into the right upper extremities of nude mice at the inoculation dosage of 0.1 mL (about 3×10.sup.6 cells) per mouse. When the average tumor volume of the inoculated mice reaches 100 mm.sup.3, the tumor-bearing mice are randomly divided into the following groups (8 nude mice per group): a negative control group (0.9% sodium chloride injection), a nanocarbon control group, an iron preparation control group, a nanocarbon-iron suspension experimental group, and a cis-platinum control group (intraperitoneal injection at the dosage of 5 mg/kg). The above various drugs are injected into the tumors. The tumor volume changes are recorded, and the formula for calculating the volume is Volume=(length×the square of width)/2.
(268) Milky white and thick ascites is extracted from the H22 tumor-bearing mice, the concentration of the cell suspension is adjusted to be 3×10.sup.7 cells/mL, 0.1 mL of cell suspension (about 3×10.sup.6 cells) is inoculated subcutaneously into the right upper extremity of each of Kunming mice. When the average tumor volume of the inoculated mice reaches 100 mm.sup.3, the tumor-bearing mice are randomly divided into the following groups (8 mice per group): a negative control group (0.9% sodium chloride injection), a nanocarbon control group, an iron preparation control group, a nanocarbon-iron suspension experimental group, and a cis-platinum control group (intraperitoneal injection at the dosage of 5 mg/kg). The above various drugs are injected into the tumors. The tumor volume changes are recorded, and the formula for calculating the volume is Volume=(length×the square of width)/2.
(269) 3) Lymph Node Metastasis Inhibition Experiment
(270) Cells growing in a log phase are collected, the concentration of the cell suspension is adjusted to 3×10.sup.7 cells/mL, the cell suspension is inoculated subcutaneously into the left hind foot pads of each nude mouse at the inoculation volume of 0.05 mL (about 1.5×10.sup.6 cells) and thus a lymph node metastasis mouse model is obtained. The mice are treated when the diameter of the tumor reaches 6-8 mm and there is no ulcer and necrosis. The mice are randomly divided into four groups (10 mice per group): a negative control group (0.9% sodium chloride injection), a nanocarbon control group, an iron preparation control group and a nanocarbon-iron suspension experimental group. 10 days after inoculation, the mice are killed, and popliteal lymph nodes are collected, weighed and fixed for pathological examination.
(271) Milky white and thick ascites is extracted from the H22 tumor-bearing mice, the concentration is adjusted to be 3×10.sup.7 cells/mL, 0.05 mL of cell suspension (about 1.5×10.sup.6 cells) is inoculated subcutaneously into the left hind foot pads of each Kunming mouse, and thus a lymph node metastasis mouse model is obtained. The mice are treated when the diameter of the tumor reaches 6-8 mm and there is no ulcer and necrosis. The mice are randomly divided into four groups (10 mice per group): a negative control group (0.9% sodium chloride injection), a nanocarbon control group, an iron preparation control group and a nanocarbon-iron suspension experimental group. 10 days after inoculation, the mice are killed, and popliteal lymph nodes are collected, weighed and fixed for pathological examination.
(272) 4) Intracellular Distribution Experiment about Ferric Ions
(273) Cells growing in a log phase are collected, the concentration of the cell suspension is adjusted, after cover glass is added to each well, and 1 mL of cell suspension is added to each well of a 6-well plate at a density of 3×10.sup.4 cells/well. The cells are incubated for 24 hours at 37° C. under the condition of 5% CO2, a nanocarbon-iron solution having a concentration of 125:13.65 μg/mL is added, and three sub-wells are set. Then the cells are incubated for 48 hours at 37° C. under the condition of 5% CO2. 1 mL of 4% paraformaldehyde solution is added to each well, the cells are fixed for 30 minutes, and Prussian-blue staining is performed.
(274) In the H22 subcutaneous tumor experiment, after observation for 3 weeks, tumors in the negative group, the nanocarbon group, the ferrous sulfate group and the nanocarbon-ferrous sulfate group are taken, fixed and subjected to Prussian blue staining, and then the ferric ions in the tumors are observed.
(275) 5) Mouse Lymph Node Tracing Experiment
(276) 50 ul of drug is injected into foot pads of each KM mouse, and 10 minutes later, the mice are killed, the popliteal lymph nodes, common iliac lymph nodes and paraaortic lymph nodes of mice are dissected, scored and photographed. The scoring standard is that the lymph node is completely stained with black for 1 point, is partially stained with black for 0.5 point and is not stained with black for 0 point.
(277) 3. Experimental Results
(278) 1) Results about Cell Experiments
(279) By simultaneously examining the inhibition effects of mixtures of nanocarbon with ferrous sulfate, ferrous gluconate, ammonium ferric citrate and iron sucrose respectively, it can be seen from the results that in the four iron preparations, the nanocarbon-ferrous sulfate mixture has the strongest inhibition effect and has the best effect on Hela cells, SMMC-7721 liver cancer cells and H22 liver cancer cells, and the cell survival rate is 49.54%-61.26%, namely the inhibition ratio is 39.74%-50.46%. The results are shown in tables 18-21 and
(280) TABLE-US-00018 TABLE 18 The cell survival rates after nanocarbon-ferrous sulfate acts on various cancer cells Cell Survival Rate Concentration MDA- SMMC- SGC- Group (μg/mL) MB-231 Hela 7721 A549 7901 HCT116 TPC-1 SKOV3 H22 Nanocarbon 125 100 100 100 100 100 100 100 100 100 62.5 100 100 98.56 100 100 96.18 96.75 93.68 96.45 15.63 100 100 99.87 98.16 100 99.86 98.54 94.67 93.47 3.91 100 100 100 100 95.46 100 100 97.84 97.14 Ferrous 13.65 100 93.99 97.46 100 100 94.07 93.64 96.47 100 Sulfate 6.83 100 100 100 95.12 98.42 97.68 98.75 95.78 97.16 1.71 100 100 100 94.18 93.78 100 100 100 95.24 0.43 100 100 100 100 100 98.32 100 97.89 100 Nanocarbon- 125:13.65 78.1 49.54 61.26 84.65 80.14 75.46 70.69 76.98 56.97 Ferrous 62.5:6.83 99.83 60.18 74.52 89.35 87.31 83.61 79.86 82.69 70.12 Sulfate 15.63:1.71 100 64.56 88.69 91.78 92.87 89.78 84.13 89.41 86.35 3.91:0.43 100 62.48 95.78 93.67 95.86 96.34 90.46 94.36 87.14
(281) Table 19: The Cell Survival Rates after Nanocarbon-ferrous Gluconate acts on Various Cancer Cells
(282) TABLE-US-00019 TABLE 19 The cell survival rates after nanocarbon-ferrous gluconate acts on various cancer cells Cell Survival Rate Concentration MDA- SMMC- SGC- Group (μg/mL) MB-231 Hela 7721 A549 7901 HCT116 TPC-1 SKOV3 H22 Nanocarbon 125 100 100 100 100 100 100 100 100 100 62.5 98.46 100 100 100 100 100 100 100 100 15.63 95.76 100 100 100 100 100 96.89 100 100 3.91 100 100 100 97.65 97.61 100 100 98.47 100 Ferrous 13.65 100 97.86 100 97.31 97.43 98.67 98.34 97.11 96.14 Gluconate 6.83 100 100 97.68 100 99.48 100 100 96.47 100 1.71 100 100 96.12 100 100 100 100 100 100 0.43 100 100 100 100 100 100 100 100 100 Nanocarbon- 125:13.65 91.65 89.95 92.68 91.65 89.42 90.45 87.14 91.2 84.69 Ferrous 62.5:6.83 94.68 95.1 95.32 97.68 91.23 93.71 91.32 95.12 90.68 Gluconate 15.63:1.71 99.47 97.67 97.58 98.12 95.74 97.61 94.79 100 93.45 3.91:0.43 100 100 100 100 99.78 100 98.74 100 97.36
(283) Table 20: The Cell Survival Rates after Nanocarbon-iron Sucrose acts on Various Cancer Cells
(284) TABLE-US-00020 TABLE 20 The cell survival rates after nanocarbon-iron sucrose acts on various cancer cells Cell Survival Rate Concentration MDA- SMMC- SGC- Group (μg/mL) MB-231 Hela 7721 A549 7901 HTC116 TPC-1 SKOV3 H22 Nanocarbon 125 100 100 100 100 100 100 100 100 100 62.5 98.46 100 100 100 100 100 100 100 100 15.63 95.76 100 100 100 100 100 96.89 100 100 3.91 100 100 100 97.65 97.61 100 100 98.47 100 Iron 13.65 100 97.86 100 97.31 97.43 98.67 98.34 97.11 96.14 Sucrose 6.83 100 100 97.68 100 99.48 100 100 96.47 100 1.71 100 100 96.12 100 100 100 100 100 100 0.43 100 100 100 100 100 100 100 100 100 Nanocarbon- 125:13.65 91.65 89.95 92.68 91.65 89.42 90.45 87.14 91.2 84.69 Iron 62.5:6.83 94.68 95.1 95.32 97.68 91.23 93.71 91.32 95.12 90.68 Sucrose 15.63:1.71 99.47 97.67 97.58 98.12 95.74 97.61 94.79 100 93.45 3.91:0.43 100 100 100 100 99.78 100 98.74 100 97.36
(285) Table 21: The Cell Survival Rate after Nanocarbon-ammonium Ferric Citrate acts on Various Cancer Cells
(286) TABLE-US-00021 TABLE 21 The cell survival rate after nanocarbon-ammonium ferric citrate acts on various cancer cells Cell Survival Rate Concentration MDA- SMMC- SGC- Group (μg/mL) MB-231 Hela 7721 A549 7901 HCT116 TPC-1 SKOV3 H22 Nanocarbon 125 100 100 94.79 99.64 100 97.64 100 100 100 62.5 100 95.36 96.78 96.47 100 95.33 100 100 100 15.63 100 100 100 97.13 100 100 100 100 100 3.91 100 94.68 100 100 100 96.82 100 100 94.62 Ammonium 13.65 95.12 93.67 100 94.67 100 95.31 100 100 100 Ferric 6.83 97.28 97.46 94.19 95.76 96.41 94.03 100 94.36 95.12 Citrate 1.71 100 99.34 98.43 99.64 95.31 98.12 96.41 99.46 97.64 0.43 100 100 100 97.21 97.85 97.31 97.30 95.67 96.21 Nanocarbon- 125:13.65 88.67 86.14 83.96 88.74 86.14 90.36 87.30 90.25 84.76 Ammonium 62.5:6.83 93.46 89.75 87.91 90.13 89.31 97.16 91.38 94.67 89.67 Ferric 15.63:1.71 94.69 94.68 90.15 96.45 92.46 94.26 94.87 100 91.67 Citrate 3.91:0.43 97.68 100 94.78 96.78 96.74 99.43 96.71 98.46 96.78
(287) Tumor Growth Inhibition Results
(288) By simultaneously examining the inhibition effects of mixtures of nanocarbon with ferrous sulfate, ferrous gluconate, ammonium ferric citrate and iron sucrose respectively, the results show that in the four iron preparations, the nanocarbon-ferrous sulfate mixture has the strongest inhibition effect, and has a tumor inhibition ratio of 50-73% for various cancer cells, with the highest tumor inhibition ratio for H22 liver cancer cells that reaches 73, and the results are shown in
(289) Since there are two methods to prepare the nanocarbon-ferrous sulfate composite system, therapeutic effects of nanocarbon-ferrous sulfate composite systems, which are prepared through the two methods respectively, on H22 liver cancer cells, Hela cervical cancer cells and MDA-MB-231 breast cancer cells are compared and the results are shown in
(290) TABLE-US-00022 TABLE 22 Inhibition effects of nanocarbon-ferrous sulfate at different mass ratios on H22 tumor growth Mass Ratio 2:1 5:1 6:1 12:1 18:1 24:1 30:1 35:1 40:1 Tumor 80.93 81.73 80.06 76.17 73.10 68.13 52.14 31.23 26.87 Inhi- bition Ratio (%)
(291) By simultaneously examining the inhibition effects of mixtures of nanocarbon with ferrous sulfate, ferrous gluconate, ammonium ferric citrate and iron sucrose respectively on lymph node metastasis of various cancer cells, the results show that in the four iron preparations, the nanocarbon-ferrous sulfate mixture has the strongest inhibition effect, the weight of metastatic lymph nodes and the metastasis ratio are obviously reduced, and the inhibition effects of the mixture on 9 types of tumor cells including H22, A549, HCT, Hela, MDA-MB-231, SGC-7901, SKOV3, SMMC-7721 and TPC-1 in animals and detailed animal experiment comparison results are shown in table 23. The nanocarbon, ferrous sulfate, ferrous gluconate, ammonium ferric citrate and iron sucrose alone do not have an inhibition effect on the metastatic lymph nodes; nanocarbon-ferrous gluconate and nanocarbon-ammonium ferric citrate also do not have an inhibition effect on the lymph nodes, nanocarbon-iron sucrose has an inhibition effect on lymph node metastasis of H22 and TPC-1 (P<0.05), but nanocarbon-ferrous sulfate has an inhibition effect on metastasis of 9 types of lymph nodes.
(292) TABLE-US-00023 TABLE 23 The weight of lymph nodes after nanocarbon-iron acts on metastatic lymph nodes of various cancer cells Weight of Lymph Node (mg) Group H22 SKOV3 SGC-7901 SMMC-7721 Hela Negative 53.38 ± 13.21 33.66 ± 4.21 47.13 ± 18.46 55.00 ± 18.41 30.14 ± 7.69 Nanocarbon 58.41 ± 15.97 .sup. 32.53 ±13.79 45.97 ± 13.29 52.45 ± 6.64 33.98 ± 15.74 Ferrous Sulfate 44.27 ± 20.69 35.62 ± 6.94 50.12 ± 20.94 50.19 ± 18.73 29.48 ± 6.46 Nanocarbon- 14.67 ± 12.08** 13.68 ± 7.9** 18.34 ± 9.76** 20.65 ± 11.29** 12.94 ± 8.93** Ferrous Sulfate Ferrous 57.19 ± 15.92 33.57 ± 5.94 52.14 ± 17.43 58.97 ± 19.63 32.14 ± 21.36 Gluconate Nanocarbon- 50.31 ± 26.74 37.42 ± 12.11 49.68 ± 12.94 51.23 ± 27.91 27.61 ± 20.18 Ferrous Gluconate Ammonium 53.94 ± 11.58 32.98 ± 6.42 43.69 ± 8.97 56.98 ± 17.59 30.94 ± 10.68 Ferric Citrate Nanocarbon- 47.11 ± 14.84 34.25 ± 9.16 40.19 ± 13.75 53.06 ± 23.27 31.28 ± 14.75 Ammonium Ferric Citrate Iron Sucrose 62.26 ± 24.06 37.94 ± 6.78 50.98 ± 21.69 49.68 ± 18.67 34.26 ± 14.25 Nanocarbon-Iron 39.29 ± 12.10* 35.17 ± 8.94 40.67 ± 18.37 54.68 ± 13.24 28.94 ± 13.68 Sucrose Weight of Lymph Node (mg) Group MDA-MB-231 TPC-1 HCT116 A549 Negative 43.15 ± 12.39 38.49 ± 4.81 52.17 ± 9.02 42.24 ± 10.95 Nanocarbon 47.98 ± 18.92 36.19 ± 10.64 49.15 ± 17.67 40.19 ± 12.68 Ferrous Sulfate 40.18 ± 11.14 35.71 ± 13.45 48.12 ± 10.58 44.97 ± 16.53 Nanocarbon- 17.68 ± 10.36** 28.91 ± 4.56* 19.26 ± 6.05** 15.62 ± 10.75** Ferrous Sulfate Ferrous 40.19 ± 20.69 40.16 ± 9.03 55.18 ± 20.12 47.58 ± 20.34 Gluconate Nanocarbon- 44.18 ± 13.45 42.98 ± 14.39 50.16 ± 14.54 41.36 ± 13.78 Ferrous Gluconate Ammonium 46.97 ± 10.36 41.97 ± 20.12 49.57 ± 20.67 45.67 ± 10.68 Ferric Citrate Nanocarbon- 42.19 ± 20.36 39.47 ± 7.56 54.25 ± 13.49 35.97 ± 16.48 Ammonium Ferric Citrate Iron Sucrose 45.69 ± 16.45 35.68 ± 10.38 53.47 ± 10.87 43.35 ± 11.68 Nanocarbon-Iron 40.39 ± 15.67 30.19± 8.03* 45.87 ± 21.89 38.97 ± 12.43 Sucrose
(293) Notes: the symbol “*” denotes P<0.05 in comparison with the negative group; and the symbol “**” denotes P<0.01 in comparison with the negative group.
(294) In the cell experiments, there is no stained ferric ion in the negative group (a1), nanocarbon group (b1) and ferrous sulfate group (c1), and there are more stained ferric ions visible in the nanocarbon-ferrous sulfate group (d1). In the animal tumors, no ferric ion can be observed in the negative group (a2) and nanocarbon group (b2); few ferric ions can be observed in the ferrous sulfate group (c1); and a large number of ferric ions can be observed in the nanocarbon-ferrous sulfate group (d2). The results are shown in
(295) In order to screen a proper carrier, we compare the tracing effect of nanocarbon-ferrous sulfate and carbon nanotube-ferrous sulfate on mouse lymph nodes. In the tracing results of mouse lymph nodes, the nanocarbon and nanocarbon-ferrous sulfate have better tracing effects, while the carbon nanotube and carbon nanotube-ferrous sulfate have a poor tracing effect. The results are shown in
(296) TABLE-US-00024 TABLE 24 Tracing and scoring results of nanocarbon, carbon nanotubes, nanocarbon-ferrous sulfate and carbon nanotube-ferrous sulfate Lymph Node Score Popliteal Common Iliac Paraaortic Sample Name Lymph Node Lymph Node Lymph Node Nanocarbon 1 1 1 Carbon Nanotubes 0.5 0 0 Nanocarbon-Ferrous Sulfate 1 1 1 Carbon Nanotube-Ferrous 0 0 0 Sulfate
(297) It should be noted that the above description only aims to illustrate the technical solution of the present invention without limitation. Although the present invention is described in detail with reference to the above embodiments, it should be understood by an ordinary person skilled in the art that modifications or equivalent replacements may be made to the present invention, and any modifications or partial replacements made without departing from the spirit and scope of the present invention should be comprised within the protection scope of the present invention.