Patent ID: 12220413

DETAILED DESCRIPTION OF THE EMBODIMENTS

In order to better understand the present disclosure, contents of the present disclosure will be further described with embodiments below, which are not limited by the following embodiments. Unless otherwise specified, materials, reagents, etc. used in the embodiments and test examples of the present disclosure may be obtained from commercial sources. Unless otherwise specified, the methods used in the embodiments and the test examples of the present disclosure are all conventional methods.

Embodiment 1

(1) Aminated ZnFe2O4hollow porous magnetic nano-particles are prepared as follows: 0.34 g of ZnCl2(2.5 mmol) and 1.35 g of FeCl3·6H2O (5 mmol) are dissolved in 40 mL of ethylene glycol, 3.6 g of NaAc and 1.0 g of polyethylene glycol (PEG) 20000 are added, magnetic stirring is conducted for 30 min, and then the mixture is sealed in a 50 mL polytetrafluoroethylene reaction kettle; a high-pressure reaction kettle is heated to 200° C. and kept for 8 h, and is cooled to a room temperature; PEG-NH2is added, and stirring is conducted for 2 h in a water bath at 50° C.; and a black product obtained is washed with ethanol 3 times and dried for 6 h at 60° C., such that the aminated ZnFe2O4hollow porous magnetic nano-particles ZnFe2O4@PEG-NH2are obtained.

(2) Silk fibroin modified ligustrazine hydrochloride (LTH-SF) nano-spheres are prepared as follows: 5 mL of silk fibroin (SF) solutions having concentrations of 10 mg/mL and 20 mg/mL are taken separately, 10 mL ligustrazine hydrochloride (LTH) aqueous solutions having concentrations of 0.625 mg/mL and 1.25 mg/mL are added separately, gent stirring is conducted for 20 min, and then 2 mL of anhydrous ethanol is dropped; the mixture is gently stirred for 3 min, and incubation is conducted in a refrigerator at −20° C. for 20 h; after unfreezing at a room temperature, a milky white emulsion is formed; and 5 mL of the prepared emulsion is taken, and dialysis is conducted with 2000 mL of deionized water for 4 h, such that most of LTH that is not wrapped with SF nano-particles or only adsorbed to a surface is removed. Then, the emulsion is freeze-dried, such that the ligustrazine hydrochloride nano-spheres LTH-SF are obtained.

(3) The magnetic nano-drug is prepared as follows: 20 mg of ZnFe2O4@PEG-NH2obtained in (1) and 3 mg of LTH-SF obtained in (2) are dissolved in 50 mL of 2-morpholinoethanesulphonic acid (MES) buffer solution (PH=5.0), excessive EDC/NHS (4 mg/2 mg) is added for activation, stirring and mixing reaction is conducted at the room temperature for 48 h, and freeze-drying is conducted after centrifugalization at 2000 rpm, such that the magnetic nano-drug required is obtained.

The magnetic nano-drug obtained in Embodiment 1 is scanned with a transmission electron microscope. A scanning result is shown inFIG.1. As can be seen fromFIG.1, the ligustrazine hydrochloride nano-spheres are well bound to the ZnFe2O4hollow porous magnetic nano-particles, such that the magnetic nano-drug having a porous structure can be presented.

Embodiment 2 Influences on Phenotypic Polarization of Co-Culture of Magnetic Nano-Particles Having Different Concentrations of ZnFe2O4and Macrophages

ZnFe2O4magnetic nano-particles prepared in Embodiment 1 are prepared to have concentrations of 10 μg/mL, 50 μg/mL, 100 μg/mL, and 500 μg/mL separately. After autoclaving, the particles are stored at 4° C. for later use. 3 6-well culture plates are prepared and placed for ultraviolet sterilization in a bio-safety cabinet for 1 h. Rat macrophages (NR8383) are prepared into a cell suspension, and inoculated into the sterilized 6-well cell culture plates, with an inoculation density of 1×105cells/well, and 2 mL of complete medium (90% DMEM+10% FBS+1% PS) is added. The cell culture plates are transferred to an incubator with 37° C. and 5% CO2, and incubation is conducted for 4 h until cells completely adhere to a wall. The cells are divided into 6 groups, with 3 parallel samples in each group. The six groups are: 20 ng/mL IL-4 (negative control), 100 ng/mL LPS (positive control), 10 μg/mL ZnFe2O4, 50 μg/mL ZnFe2O4, 100 μg/mL ZnFe2O4and 500 μg/mL ZnFe2O4. After incubation for 72 h, the culture medium is removed, and the cells are collected and detected with a flow cytometer.

Specific steps of detection with the flow cytometer are as follows: 1) 15 mL of cell staining buffer solution (PBS (PH=7.4)+1% FBS+0.09% NaN3) is added into the cells so as to resuspend, 350×g centrifuging is conducted for 5 min, and supernatant is discarded; 2) coupled fluorescent primary antibodies CD80 (12-0800-82, a mouse source, 1:1000, Thermo Fisher, USA) and incubation is conducted on ice for 15 min-20 min in the dark; 3) washing is conducted twice with 2 mL of cell staining buffer solution, and centrifuging is conducted (350×g, 5 min each time); 4) 0.5 mL of fixed solution is added to fix the cells at the room temperature in the dark for 20 min; 5) 350×g centrifuging is conducted for 5 min, and supernatant is discarded; 6) the fixed cells are resuspended in a cell permeable washing buffer solution, 350×g centrifuging is conducted for 5 min, and washing is conducted twice; 7) coupled fluorescent primary antibodies CD206 (PA5-114370, a rabbit source, 1:1000, Thermo Fisher, USA) are added, and incubation is conducted on ice for 20 min in the dark; 8) 350×g centrifuging is conducted with 2 mL of cell staining buffer solution for 5 min, and washing is conducted twice; and 9) data are collected and analyzed with a flow cytometer.

Detection results of the flow cytometer are shown inFIG.2. As can be seen fromFIG.2, ZnFe2O4having a lower concentration regulates M2 polarization of macrophages, while ZnFe2O4having a higher concentration regulates M1 polarization of macrophages, which may down-regulate expression of VEGF.

Embodiment 3 Influences on VEGF Expressions of Co-Culture of Magnetic Nano-Particles Having Different Concentrations of ZnFe2O4and Macrophages

ZnFe2O4magnetic nano-particles prepared in Embodiment 1 are prepared to have concentrations of 0 μg/mL, 10 μg/mL, 100 μg/mL, and 500 μg/mL separately. After autoclaving, the particles are stored at 4° C. for later use. A 12-well culture plate is placed for ultraviolet sterilization in a bio-safety cabinet for 1 h. Rat macrophages are prepared into a cell suspension, and inoculated into the sterilized culture plate, with an inoculation density of 1×105cells/well, and 1 mL of complete medium (90% DMEM+10% FBS+1% PS) is added.

The cell culture plate is transferred to an incubator with 37° C. and 5% CO2, and incubation is conducted for 4 h until cells completely adhere to a wall. The cells are divided into 4 groups, with 3 parallel samples in each group. The four groups are: 0 μg/mL ZnFe2O4, 10 μg/mL ZnFe2O4, 100 μg/mL ZnFe2O4and 500 μg/mL ZnFe2O4. After incubation for 24 h, supernatant is collected. The collected supernatant is transferred to a high-speed centrifuge, centrifuging is conducted at 12000 rpm for 5 min, and the supernatant is collected again. The VEGF is quantitatively detected with a rat vascular endothelial growth factor ELISA kit (MM-0179R1, Enzyme Immunosorbent Assay, China), and 3 parallel samples are set for each sample.

ELISA detection results are shown inFIG.3. As can be seen fromFIG.3, ZnFe2O4having a lower concentration can promote the VEGF expression, while ZnFe2O4having a higher concentration can significantly inhibit the VEGF expression, which show potential of angiogenesis inhibition.

Embodiment 4 Biocompatibility Detection of Co-Culture of Magnetic Nano-Particles Having Different Concentrations of ZnFe2O4and Macrophages

ZnFe2O4magnetic nano-particles prepared in Embodiment 1 are prepared to have concentrations of 0 μg/mL, 10 μg/mL, 100 μg/mL, and 500 μg/mL separately. After autoclaving, the particles are stored at, 4° C. for later use. A 24-well culture plate is placed for ultraviolet sterilization in a bio-safety cabinet for 1 h. Rat macrophages are prepared into a cell suspension, and then inoculated into the culture plate, with an inoculation density of 1×104cells/well, and 1 mL of complete medium (90% DMEM+10% FBS+1% PS) is added.

The cell culture plate is transferred to an incubator with 37° C. and 5% CO2, and incubation is conducted for 4 h until cells completely adhere to a wall. The cells are divided into 4 groups, with 3 parallel samples in each group. The four groups are: 0 μg/mL ZnFe2O4, 10 μg/mL ZnFe2O4, 100 μg/mL ZnFe2O4and 500 μg/mL ZnFe2O4. The four groups are incubated for 24 h and 48 h respectively, then a culture medium is removed, and non-adherent cells are washed with PBS (0.01 M, PH=7.4) three times.

The mixture of a fresh complete culture medium and a cell-counting-kit (CCK)-8 reagent with a volume ratio of 10:1 is added into the well plate, and transferred to a cell incubator for incubation for 2 h. A suspension is transferred to a 96-well plate (200 L/well), and absorbance is measured at 450 nm with an enzyme-labeled instrument.

Detection results of a CCK-8 cell viability assay of co-culture of magnetic nano-particles having different concentrations of ZnFe2O4and macrophages are as shown inFIG.4. As can be seen fromFIG.4, 10 μg/mL and 100 μg/mL experimental groups have slightly lower cell viability than a blank control group at 24 h, a difference between which is not significant (P>0.05), and even a 500 μg/mL experimental group has higher cell viability. At 48 h, each experimental group has slightly lower cell viability than the blank control group, without significant difference (P>0.05). It is indicated that the ZnFe2O4magnetic nano-particles have high biocompatibility.

Embodiment 5 Influences on Inhibiting VEGF-VEGFR Expressions of Co-Culture of a Magnetic Nano-Drug, Macrophages, and Endothelial Progenitor Cells

The magnetic nano-drug prepared in Embodiment 1 is prepared to have a concentration of 400 μg/mL, and is placed in a bio-safety cabinet for ultraviolet sterilization, and then stored at 4° C. for later use. 2 24-well culture plates are placed for ultraviolet sterilization in the bio-safety cabinet for 1 h. Rat macrophages and vascular endothelial cells are prepared into a cell suspension, and inoculated into the culture plates respectively, with an inoculation density of 5×104cells/well, and 1.5 mL of complete media (rat macrophage complete medium: 80% F12+20% FBS+1% PS; and vascular endothelial cell complete medium: 90% DMEM+10% FBS+1% PS) are added.

The cell culture plates are transferred to an incubator with 37° C. and 5% CO2, and incubation is conducted for 4 h until cells become stable or adhere to a wall. The cells are divided into 4 groups, with 6 parallel samples in each group. The four groups are: blank control, 400 μg/mL aminated ZnFe2O4magnetic nano-particles, 400 μg/mL LTH-SF nano-spheres, and 400 μg/mL magnetic nano-drug. After incubation for 48 h, supernatant is collected with a macrophage culture plate for ELISA detection, and supernatant is removed with an endothelial cell culture plate. The cells are fixed for immunohistochemical staining.

ELISA detection: the collected supernatant is transferred to a high-speed centrifuge, centrifuging is conducted at 12000 rpm for 5 min, and the supernatant is collected again. An ELISA kit is used to detect influences of nano-drugs on secretion of VEGF cytokines by macrophages, where 6 parallel samples are determined for each sample. The ELISA detection results are shown inFIG.5A. As can be seen fromFIG.5A, the magnetic nano-drug significantly reduces the VEGF expression, which proves potential of down-regulating VEGF to M1 polarization by regulating macrophage reprogramming to inhibit angiogenesis.

Immunofluorescence staining: a 4% paraformaldehyde fixed solution is removed, washing is conducted with PBS 3 times, each time of which lasts 5 min, and primary antibodies VEGFR2 are added dropwise at 4° C. overnight. The primary antibodies are poured out, and washing is conducted with PBS 3 times, each time of which lasts 5 min. A secondary antibody reagent (SA00003-2, Fluorescein (FITC)-conjugated affinipure Goat Anti-Rabbit IgG (H+L), Proteintech, China) is added dropwise, and incubation is conducted at a room temperature for 2 h. PBS washing is conducted for the first time, DAPI counterstaining is conducted for 10 min, and PBS washing is conducted twice, each time of which lasts 5 min. Finally, pictures are observed and collected under a microscope. Immunofluorescence staining results are shown inFIG.5B. As can be seen fromFIG.5B, both LTH-SF having a concentration of 400 μg/mL and the magnetic nano-drug show significant inhibition of VEGFR2 expression, which show potential of preventing the VEGF ligand from activating a receptor, so as to inhibit vascularized pain transmission and relieve pain.

To sum up, the magnetic nano-drug with double targeting VEGF-VEGFR according to the present disclosure has the potential of inhibiting vascularization in degenerated intervertebral discs and reducing innervation, and has a wide application prospect in treatment of discogenic pain.

What are described above are merely preferred implementations of the present disclosure. It should be noted that those of ordinary skill in the art can also make some improvements and modifications without departing from the principle of the present disclosure, and these improvements and modifications should also fall within the protection scope of the present disclosure.