IRON OXIDE/NANOKAOLIN COMPOSITE HEMOSTATIC AGENT AND PREPARATION METHOD THEREOF

Abstract

The present invention discloses an iron oxide/nanokaolin composite hemostatic agent and a preparation method thereof. The composite hemostatic agent is composed of nanokaolin and iron oxide, where the nanokaolin is used as a carrier, and the iron oxide is loaded on a surface of a nanokaolin flake. The composite hemostatic agent is obtained by a precipitation method. The composite hemostatic agent has the advantages of good hemostatic effect, rapid wound healing, no obvious cytotoxicity, no hemolysis, high biocompatibility, and the like.

Claims

1. An iron oxide/nanokaolin composite hemostatic agent, comprising an active ingredient of the composite hemostatic agent is a composite of a nanokaolin and an iron oxide.

2. The iron oxide/nanokaolin composite hemostatic agent according to claim 1, wherein the active ingredient is made by loading the iron oxide on a surface of the nanokaolin.

3. The iron oxide/nanokaolin composite hemostatic agent according to claim 2, wherein a mass percentage of the iron oxide is 15%-45%.

4. The iron oxide/nanokaolin composite hemostatic agent according to claim 1, wherein the iron oxide is iron oxyhydroxide, -ferric oxide, -ferric oxide and/or ferroferric oxide.

5. An iron oxide/nanokaolin composite hemostatic agent, comprising a composite of a nanokaolin and an iron oxide.

6. The iron oxide/nanokaolin composite hemostatic agent according to claim 5, wherein the iron oxide is loaded on a surface of the nanokaolin.

7. The iron oxide/nanokaolin composite hemostatic agent according to claim 6, wherein a mass percentage of the iron oxide is 15%-45%.

8. The iron oxide/nanokaolin composite hemostatic agent according to claim 5, wherein the iron oxide is iron oxyhydroxide, -ferric oxide, -ferric oxide and/or ferroferric oxide.

9. A preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 1, comprising: a preparation of an iron oxyhydroxide/nanokaolin composite hemostatic agent comprises following steps of: A, mixing nanokaolin with a polymerized hydroxy iron ion solution, and reacting under a system pH value of 3-7 and a temperature of 50 C. to 70 C. to obtain the iron oxyhydroxide/nanokaolin composite hemostatic agent; or a preparation of an -ferric oxide/nanokaolin composite hemostatic agent comprises following steps of: preparing the iron oxyhydroxide/nanokaolin composite hemostatic agent according to the step A; B, placing the iron oxyhydroxide/nanokaolin composite hemostatic agent in an air atmosphere, and calcining at a temperature of 500 C. to 550 C., to obtain the -ferric oxide/nanokaolin composite hemostatic agent; or a preparation of a ferroferric oxide/nanokaolin composite hemostatic agent comprises following steps of: preparing the -ferric oxide/nanokaolin composite hemostatic agent according to the steps A and B; C, placing the -ferric oxide/nanokaolin composite hemostatic agent under a reducing atmosphere, and a calcination is carried out at a temperature of 400 C. to 450 C. to obtain the ferroferric oxide/nanokaolin composite hemostatic agent; or a preparation of a -ferric oxide/nanokaolin composite hemostatic agent comprises following steps of: preparing the ferroferric oxide/nanokaolin composite hemostatic agent according to the steps A, B and C; and D, placing the ferroferric oxide/nanokaolin composite hemostatic agent in an air atmosphere and calcining at a temperature of 200 C. to 250 C. to obtain the -ferric oxide/nanokaolin composite hemostatic agent.

10. The preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 9, wherein the nanokaolin is obtained by intercalating and stripping a kaolin.

11. The preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 9, wherein the polymerized hydroxy iron ion solution is prepared by following method of: adding a sodium hydroxide solution to a ferric chloride solution dropwise under a condition of 40 C. to 80 C., and then aging at room temperature for 12 h to 24 h to obtain the polymerized hydroxy iron ion solution.

12. The preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 9, wherein a solid-liquid ratio of the nanokaolin to the polymerized hydroxy iron ion solution is (1-3)/50 g/mL, and a concentration of the polymerized hydroxy iron ion solution is 0.1-0.6 mol/L.

13. The preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 9, wherein in the step A, a reaction time is 24-72 h.

14. The preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 9, wherein in the step C, the calcination is performed in a hydrogen/argon mixed atmosphere with a hydrogen concentration of 5-12% by volume.

15. A preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 5, comprising: a preparation of an iron oxyhydroxide/nanokaolin composite hemostatic agent comprises following steps of: A, mixing nanokaolin with a polymerized hydroxy iron ion solution, and reacting under a system pH value of 3-7 and a temperature of 50 C. to 70 C. to obtain the iron oxyhydroxide/nanokaolin composite hemostatic agent; or a preparation of an -ferric oxide/nanokaolin composite hemostatic agent comprises following steps of: preparing the iron oxyhydroxide/nanokaolin composite hemostatic agent according to the step A; B, placing the iron oxyhydroxide/nanokaolin composite hemostatic agent in an air atmosphere, and calcining at a temperature of 500 C. to 550 C., to obtain the -ferric oxide/nanokaolin composite hemostatic agent; or a preparation of a ferroferric oxide/nanokaolin composite hemostatic agent comprises following steps of: preparing the -ferric oxide/nanokaolin composite hemostatic agent according to the steps A and B; C, placing the -ferric oxide/nanokaolin composite hemostatic agent under a reducing atmosphere, and a calcination is carried out at a temperature of 400 C. to 450 C. to obtain the ferroferric oxide/nanokaolin composite hemostatic agent; or a preparation of a -ferric oxide/nanokaolin composite hemostatic agent comprises following steps of: preparing the ferroferric oxide/nanokaolin composite hemostatic agent according to the steps A, B and C; and D, placing the ferroferric oxide/nanokaolin composite hemostatic agent in an air atmosphere and calcining at a temperature of 200 C. to 250 C. to obtain the -ferric oxide/nanokaolin composite hemostatic agent.

16. The preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 15, wherein the nanokaolin is obtained by intercalating and stripping a kaolin.

17. The preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 15, wherein the polymerized hydroxy iron ion solution is prepared by following method of: adding a sodium hydroxide solution to a ferric chloride solution dropwise under a condition of 40 C. to 80 C., and then aging at room temperature for 12 h to 24 h to obtain the polymerized hydroxy iron ion solution.

18. The preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 15, wherein a solid-liquid ratio of the nanokaolin to the polymerized hydroxy iron ion solution is (1-3)/50 g/mL, and a concentration of the polymerized hydroxy iron ion solution is 0.1-0.6 mol/L.

19. The preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 15, wherein in the step A, a reaction time is 24-72 h.

20. The preparation method of the iron oxide/nanokaolin composite hemostatic agent according to claim 15, wherein in the step C, the calcination is performed in a hydrogen/argon mixed atmosphere with a hydrogen concentration of 5-12% by volume.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

Description of the Drawings

[0051] FIG. 1 is the XRD patterns of composite hemostatic agents of the present invention.

[0052] FIG. 2 is the scanning electron micrographs of composite hemostatic agents of the present invention.

DETAILED DESCRIPTION OF THE EMBODIMENT

Best Mode of the Invention

[0053] To make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings and specific examples. It should be understood that the specific examples described herein are merely illustrative of the present invention and are not intended to limit the present invention.

[0054] A ferric oxide/nanokaolin composite of the present invention can be used for bleeding wounds, accelerates blood coagulation, and promotes wound healing.

[0055] The term kaolin in this specification has a chemical formula Al.sub.2O.sub.3.2SiO.sub.2.2H.sub.2O. In some forms, the kaolin includes a silica content of about 45.31%, an alumina content of about 37.21%, and a water content of about 14.1%.

[0056] The kaolin in the examples in this specification is a standard product of China Kaolin Clay Co., Ltd., and its origin is in Suzhou, Jiangsu.

EMBODIMENT 1

[0057] This embodiment provides preparation of nanokaolin, which was prepared by the following method:

[0058] 4 g of kaolin was weighed, 10 g of potassium acetate was added, the mixture was ground for about 30 min to paste, 10% deionized water was added, and the mixture was transferred to a beaker, and allowed to stand at room temperature for three days. The mixture was washed with ethanol, filtered, and dried at 60 C. for 24 h. Potassium acetate was intercalated into kaolin, and the mixture was added to deionized water, ultrasonically treated for 30 min, washed, filtered, dried, and marked as Kaolin.sub.KAc.

EMBODIMENT 2

[0059] This embodiment provides preparation of a polymerized hydroxy iron ion, which was prepared by the following method:

[0060] 2.4 g of sodium hydroxide was weighed, and 150 mL of water was added to formulate a 0.4 mol/L sodium hydroxide solution. 10.8 g of FeCl.sub.3.6H.sub.2O was weighed, and 100 mL of deionized water was added to formulate a 0.4 mol/L FeCl.sub.3 solution. 150 mL of a 0.4 mol/L NaOH solution was slowly added dropwise to 100 mL of 0.4 mol/L FeCl.sub.3 solution in a 70 C. water bath under vigorous stirring. After the completion of dropwise addition, the solution was removed and slowly cooled to obtain a stable reddish-brown transparent iron polymer solution for later use, which was marked as a polymerized hydroxy iron ion solution.

EMBODIMENT 3

[0061] This embodiment provides an iron oxyhydroxide/nanokaolin composite hemostatic agent, which was prepared by the following method:

[0062] 5 g of kaolin intercalated with potassium acetate was weighed and added to 250 mL of a 0.4 M polymerized hydroxy iron ion solution. The pH of the reaction system was adjusted to 5 with a 5 mol/L NaOH solution. The reaction system was heated to 60 C. in a water bath, magnetically stirred for 72 h, washed, separated, and dried at 60 C. for 24 h. The product was sealed and stored in a drying vessel with allochroic silica gel at the bottom for later use, which was marked as FeOOH-Kaolin.sub.KAc.

EMBODIMENT 4

[0063] This embodiment provides an -ferric oxide/nanokaolin composite hemostatic agent, which was prepared by the following method:

[0064] 5 g of kaolin intercalated with potassium acetate was weighed and added to 250 mL of a 0.4 M polymerized hydroxy iron ion solution. The pH of the reaction system was adjusted to 5 with a 5 M NaOH solution. The reaction system was heated to 60 C. in a water bath, magnetically stirred for 72 h, washed, separated, and dried at 60 C. for 24 h. The reaction system was calcined under air atmosphere at 150 C. for 1 h, at 250 C. for 1 h, and at 550 C. for 4 h. The product was sealed and stored in a drying vessel with allochroic silica gel at the bottom for later use, which was marked as -Fe.sub.2O.sub.3-KaolinKAc.

EMBODIMENT 5

[0065] This embodiment provides an -ferric oxide/nanokaolin composite hemostatic agent, which was prepared by the following method:

[0066] 5 g of kaolin intercalated with potassium acetate was weighed and added to 250 mL of a 0.1 M polymerized hydroxy iron ion solution. The pH of the reaction system was adjusted to 3 with a 5 M NaOH solution. The reaction system was heated to 50 C. in a water bath, magnetically stirred for 24 h, washed, separated, and dried at 60 C. for 24 h. The reaction system was calcined under air atmosphere at 150 C. for 1 h, at 250 C. for 1 h, and at 550 C. for 4 h. The product was sealed and stored in a drying vessel with allochroic silica gel at the bottom for later use, which was marked as -Fe.sub.2O.sub.3-KaolinKAc-1.

EMBODIMENT 6

[0067] This embodiment provides an a-ferric oxide/nanokaolin composite hemostatic agent, which was prepared by the following method:

[0068] 5 g of kaolin intercalated with potassium acetate was weighed and added to 250 mL of a 0.2 M polymerized hydroxy iron ion solution. The pH of the reaction system was adjusted to 5 with a 5 M NaOH solution. The reaction system was heated to 60 C. in a water bath, magnetically stirred for 48 h, washed, separated, and dried at 60 C. for 24 h. The reaction system was calcined under air atmosphere at 150 C. for 1 h, at 250 C. for 1 h, and at 550 C. for 4 h. The product was sealed and stored in a drying vessel with allochroic silica gel at the bottom for later use, which was marked as -Fe.sub.2O.sub.3-KaolinKAc-2.

EMBODIMENT 7

[0069] This embodiment provides an -ferric oxide/nanokaolin composite hemostatic agent, which was prepared by the following method:

[0070] 5 g of kaolin intercalated with potassium acetate was weighed and added to 250 mL of a 0.6 M polymerized hydroxy iron ion solution. The pH of the reaction system was adjusted to 7 with a 5 M NaOH solution. The reaction system was heated to 70 C. in a water bath, magnetically stirred for 72 h, washed, separated, and dried at 60 C. for 24 h. The reaction system was calcined under air atmosphere at 150 C. for 1 h, at 250 C. for 1 h, and at 550 C. for 4 h. The product was sealed and stored in a drying vessel with allochroic silica gel at the bottom for later use, which was marked as -Fe.sub.2O.sub.3-KaolinKAc-6.

EMBODIMENT 8

[0071] This embodiment provides a -ferric oxide/nanokaolin composite hemostatic agent, which was prepared by the following method:

[0072] 5 g of kaolin intercalated with potassium acetate was weighed and added to 250 mL of a 0.4 M polymerized hydroxy iron ion solution. The pH of the reaction system was adjusted to 5 with a 5 M NaOH solution. The reaction system was heated to 60 C. in a water bath, magnetically stirred for 72 h, washed, separated, and dried at 60 C. for 24 h. The reaction system was calcined under air atmosphere at 150 C. for 1 h, at 250 C. for 1 h, and at 550 C. for 4 h. The reaction system was calcined in high purity argon containing 10% hydrogen at 450 C. for 1 h and calcined in an air atmosphere at 250 C. for 1 h. The product was sealed and stored in a drying vessel with allochroic silica gel at the bottom for later use, which was marked as -Fe.sub.2O.sub.3-KaolinKAc.

EMBODIMENT 9

[0073] This embodiment provides a ferroferric oxide/nanokaolin composite hemostatic agent, which was prepared by the following method:

[0074] 5 g of kaolin intercalated with potassium acetate was weighed and added to 250 mL of a 0.4 M polymerized hydroxy iron ion solution. The pH of the reaction system was adjusted to 5 with a 5 M NaOH solution. The reaction system was heated to 60 C. in a water bath, magnetically stirred for 72 h, washed, separated, and dried at 60 C. for 24 h. The reaction system was calcined under air atmosphere at 150 C. for 1 h, at 250 C. for 1 h, and at 550 C. for 4 h. The reaction system was calcined in high purity argon containing 10% hydrogen at 450 C. for 1 h. The product was sealed and stored in a drying vessel with allochroic silica gel at the bottom for later use, which was marked as Fe.sub.3O.sub.4-Kaolin.sub.KAc.

EMBODIMENT 10

[0075] This embodiment provides an -ferric oxide particle, which was prepared by the following method:

[0076] 250 mL of a 0.4 mol/L polymerized hydroxy iron ion solution was added to a conical flask. The pH of the reaction system was adjusted to 5 with a 5 M NaOH solution. The reaction system was heated to 60 C. in a water bath, magnetically stirred for 72 h, washed, separated, and dried at 60 C. for 24 h. The reaction system was calcined under air atmosphere at 150 C. for 1 h, at 250 C. for 1 h, and at 550 C. for 4 h. The product was sealed and stored in a drying vessel with allochroic silica gel at the bottom for later use, which was marked as -Fe.sub.2O.sub.3.

Application Embodiment

[0077] Hemostasis experiment: Sprague-Dawley rat (4-6 weeks old) were selected as experimental animal subjects. The rats were randomly divided into groups, each group including six rats, and the rats used for experiment were fed normally without restriction. A surgical scissor was used to make an 1 cm long wound at the tail vein. After the bleeding, a sample was applied to cover the wound sufficiently. When a medicine was applied, the bleeding wound was slightly pressed, and the bleeding time was recorded with a stopwatch. The hemostasis standard was that there was no ejection and oozing of blood on a wound surface, namely, the blood was clotted and no longer re-oozed.

[0078] Wound healing: Before a wound treatment experiment, an experimental rat was first anesthetized by intramuscular injection of 35.0 mg kg.sup.1 ketamine and 5.0 mg kg.sup.1 xylazine, and fixed on a rat fixation plate. The back of the rat was subjected to depilation treatment, and the effective area of depilation was about (2 cm2 cm), and the depilated back of the rat was washed with ethanol.

[0079] The surgical scissor and forceps were used to make a wound (1.0 cm1.0 cm) on the depilated and cleaned back of the rat. The blood flowing out of the wound was wiped off, composite hemostatic sample powder was immediately spread on the wound, and the wound was wrapped with bandages and gauze in sequence. The wound healing process of each composite hemostatic agent was recorded using a digital camera. The medicine (composite hemostatic agent) was changed every 2-3 days, and the healing of the wound was observed.

[0080] Histological analysis of healed wounds: After 10 days, the wound was basically healed. The tissue at the wound was cut, fixed by directly immersing in 4% paraformaldehyde, embedded, sliced, and stained with a hematoxylin-eosin reagent (H&E), Masson, and Sirius Red (SR), respectively to observe the tissue healing in each group.

[0081] Cytotoxicity of composite hemostatic agent: Fibroblast L929 cells were selected to evaluate the biocompatibility of the composite hemostatic agent. Fibroblasts were cultured using Dulbecco's Modified Eagle Medium (specifically, 10% fetal bovine serum, 2 mM glutamine, 4.5 g L.sup.1 glucose, and 1% antibiotic/antimycotic solution). The fibroblasts were cultured at 37 C. in a 5% CO.sub.2 sterile environment. The medium was replaced with a fresh medium every two days until the cells reach a level of aggregation.

[0082] Evaluation of cytotoxicity by MTT assay: The composite hemostatic agent was added to a 24-well plate, and each well was inoculated with 1 mL of cell suspension at a concentration of 2104 cells mL.sup.1, and the concentration of the composite hemostatic agent was 0-2 mg/mL. A 100 L MTT solution was injected into each well, and then the cells were cultured for 4 h. Once the culture was completed, supernatant was removed, and 200 L of DMSO was added to give a blue reduction product, formazan. The dissolved solution was then transferred to a 96-well plate and the absorbance was measured with a microplate reader (570 nm). Three parallel experiments were performed. The group only with a culture solution was used as a blank group, and the group with the cells and the culture solution was used as a control group to correct the cell survival rate.

[0083] Hemolysis test: 80 mg of the composite hemostatic agent was mixed with 0.4 mL of a 10% red blood cell solution, and allowed to stand at room temperature for 1 h. 150 L was taken into a 96-well plate, and the absorbance was measured with a microplate reader (540 nm). Water and PBS were used as positive and negative controls, respectively.

[0084] Hemolysis rate (%)=(sample absorbance-negative control absorbance)/(positive control absorbance-negative control absorbance)100%.

Experimental Results

[0085] Preparation of iron oxide/nanokaolin composite hemostatic agent: upon identification by an X-ray diffraction technique, as shown in FIG. 1, the samples of iron oxyhydroxide/nanokaolin, -ferric oxide/nanokaolin, -ferric oxide/nanokaolin, and ferroferric oxide/nanokaolin obtained in Embodiment 3, Embodiment 4, Embodiment 8, and Embodiment 9 have characteristic diffraction peak of iron oxyhydroxide, -ferric oxide, -ferric oxide, and ferroferric oxide, respectively. As shown in FIG. 2, the kaolin retains a complete pseudo-hexagonal flake structure, and the iron oxyhydroxide in example 3 has a spindle-like morphology and a particle size of about 100 nm. The iron oxides of Embodiment 4, Embodiment 8, and Embodiment 9 are polyhedrals, and small particles are supported on the surface and sides of the kaolin flake. In addition, some of the particles form large particles having a particle size of about 300 nm during calcination.

[0086] Hemostasis experiment: The hemostatic time of the blank control group, kaolin and the samples of iron oxyhydroxide/nanokaolin, -ferric oxide/nanokaolin, -ferric oxide/nanokaolin, and ferroferric oxide/nanokaolin obtained in Embodiment 3, Embodiment 4, Embodiment 8, and Embodiment 9 are 31323, 22717, 29814, 18316, 21211, and 21815 s, respectively. The samples have a better hemostatic effect than the blank control group and kaolin alone.

[0087] The hemostatic time of kaolin and the -ferric oxide/nanokaolin composites with different -ferric oxide mass fractions prepared in Embodiment 5, Embodiment 6, Embodiment 4, Embodiment 7, and Embodiment 10 are 22717, 15821, 13213, 18316, 21613, and 1939 s, respectively. The corresponding -ferric oxide mass fractions are 0, 15.7%, 23.9%, 43.6%, 49.4%, and 100%, respectively.

[0088] Wound healing: The kaolin and the samples of iron oxyhydroxide/nanokaolin, -ferric oxide/nanokaolin, -ferric oxide/nanokaolin, ferroferric oxide/nanokaolin and -ferric oxide particle obtained in Embodiment 3, Embodiment 4, Embodiment 8, Embodiment 9, and Embodiment 10 accelerate wound healing, reduce inflammation, and promote tissue remodeling.

[0089] Cytotoxicity: The cytotoxicity of each composite hemostatic agent was evaluated for fibroblasts as an object. The kaolin and the samples of iron oxyhydroxide/nanokaolin, -ferric oxide/nanokaolin, -ferric oxide/nanokaolin, ferroferric oxide/nanokaolin and -ferric oxide particle hemostatic agent obtained in Embodiment 3, Embodiment 4, Embodiment 8, Embodiment 9, and Embodiment 10 have almost no toxicity to cells and have good biocompatibility.

[0090] Hemolysis: Kaolin has a high hemolysis rate of 30% and hemolysis. The hemolysis rates of the samples of iron oxyhydroxide/nanokaolin, -ferric oxide/nanokaolin, -ferric oxide/nanokaolin, ferroferric oxide/nanokaolin and -ferric oxide particle obtained in Embodiment 3, Embodiment 4, Embodiment 8, Embodiment 9, and Embodiment 10 are all less than 5%, indicating that no hemolysis is caused.

[0091] The foregoing descriptions are merely preferred embodiments of the present invention and is not intended to limit the present invention. Any modifications, equivalent replacement, or improvement made within the spirit of the present invention shall fall with the protection scope of the present invention.