COMPOSITION FOR MAGNETIC ULTRASOUND CONTRAST AGENT, MAGNETIC ULTRASOUND CONTRAST AGENT, MAGNETIC MICROBUBBLE ULTRASOUND CONTRAST AGENT AND PREPARATION METHOD THEREOF

Abstract

Disclosed are a composition for magnetic ultrasound contrast agent, a magnetic ultrasound contrast agent, a magnetic microbubble ultrasound contrast agent, and a preparation method thereof. The composition for magnetic ultrasound contrast agent includes a lipid, a surfactant and a magnetic nanoparticle; the surfactant consists of a surfactant A and a surfactant B, the surfactant A is in a free state, and the surfactant B is in a bonding state and is modified on the surface of the magnetic nanoparticle; the HLB value of the surfactant A is greater than 8. The magnetic microbubble ultrasound contrast agent obtained from the composition for magnetic ultrasound contrast agent has higher stability and may meet requirements for in vivo cyclic targeted delivery, thereby achieving the local high-concentration targeted delivery of a medication.

Claims

1. A composition for magnetic ultrasound contrast agent, comprising a lipid, a surfactant and a magnetic nanoparticle; wherein: the surfactant consists of a surfactant A and a surfactant B, wherein the surfactant A is in a free state, and the surfactant B is in a bonding state and is modified on the surface of the magnetic nanoparticle; the surfactant A is a nonionic surfactant whose HBL value is greater than 8; the surfactant B is a citric acid-based compound; and relative to 1 mol of the lipid, a content of the surfactant A is 0.1 mol to 1 mol, a content of the surfactant B is 0.1 mol to 1 mol, and a content of the magnetic nanoparticle is 0.05 mol to 0.5 mol.

2. The composition for magnetic ultrasound contrast agent according to claim 1, wherein the surfactant A is a nonionic surfactant whose HBL value is greater than 15.

3. The composition for magnetic ultrasound contrast agent according to claim 2, wherein the surfactant A is a nonionic surfactant whose HBL value is 20 to 35.

4. The composition for magnetic ultrasound contrast agent according to claim 1, wherein the surfactant A is any one or more selected from the group consisting of: poloxamer, poly(isobutene-maleic anhydride), and poly(maleic anhydride-alt-1-octadecene).

5. The composition for magnetic ultrasound contrast agent according to claim 1, wherein the surfactant B is citrate and/or citrate ester.

6. The composition for magnetic ultrasound contrast agent according to claim 1, wherein the surfactant A is poloxamer and the surfactant B is citrate.

7. The composition for magnetic ultrasound contrast agent according to claim 1, wherein relative to 1 mol of the lipid, the content of the surfactant A is 0.2 mol to 0.6 mol, the content of the surfactant B is 0.2 mol to 0.5 mol, and the content of the magnetic nanoparticle is 0.1 mol to 0.3 mol.

8. The composition for magnetic ultrasound contrast agent according to claim 1, wherein the molar ratio of the magnetic particle to the surfactant B is 1:0.6 to 1:2.5.

9. The composition for magnetic ultrasound contrast agent according to claim 1, further comprising a drug, and relative to 100 parts by weight of a sum of the lipid, the surfactant and the magnetic nanoparticle, the content of the drug is 1 to 20 parts by weight.

10. The composition for magnetic ultrasound contrast agent according to claim 1, wherein the lipid consists of sorbitan monostearate and polysorbate 80 in a molar ratio of 1:0.5 to 1:2.

11. The composition for magnetic ultrasound contrast agent according to claim 1, wherein the magnetic nanoparticle is a superparamagnetic Fe.sub.3O.sub.4 nanoparticle with a particle size of 3 nm to 20 nm.

12. A magnetic ultrasound contrast agent, wherein the magnetic ultrasound contrast agent comprises, or is prepared from, the composition for magnetic ultrasound contrast agent according to claim 1.

13. A method for preparing a magnetic microbubble ultrasound contrast agent, wherein raw materials comprise the composition for magnetic ultrasound contrast agent according to claim 1, wherein the method comprises the following steps: (1) making a first contact between the lipid and the surfactant A; (2) mixing the drug with the resulting material obtained by step (1), pumping gases into the mixed resulting material, and processing ultrasonic cavitation to form a first microbubble suspension; (3) processing a treatment for loading a positive charge on the first microbubble suspension to obtain a second microbubble suspension with a positive charge on the surface; and (4) making a second contact between the magnetic nanoparticle modified with the surfactant B on the surface and the obtained second microbubble suspension with a positive charge on the surface.

14. The method according to claim 13, wherein the first contact conditions comprise: a temperature of 110° C. to 130° C., and a time of 8 min to 16 min.

15. The method according to claim 13, wherein in step (2), the mixing is processed at a temperature of 30° C. to 50° C.

16. The method according to claim 13, wherein the ultrasonic cavitation conditions comprise: an ultrasonic power of 8 kW to 12 kW, and a time of 1 min to 8 min.

17. The method according to claim 13, wherein in step (3), the process of the treatment for loading a positive charge comprises: contacting the first microbubble suspension with an aqueous solution of cationic reagent.

18. The method according to claim 17, wherein the volume ratio of the aqueous solution of cationic reagent and the first microbubble suspension is 0.8:1 to 1.2:1, and the concentration of the cation in the aqueous solution of the cationic reagent is 0.5 mg/L to 2 mg/L.

19. The method according to claim 13, further comprising: leaving the resulting material, which have contacted with the cationic reagent, to stand for stratification, taking upper layer materials, and washing the upper layer materials with buffer solution.

20. The method according to claim 13, wherein in step (4), the second contact conditions comprise: processing the material with ultrasonic oscillation with an ultrasonic power of 170 W to 420 W, and a time of 15 min to 50 min.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0052] FIG. 1 is an image of the ultrasound contrast agent S1 obtained in Example 1 under an optical microscope.

[0053] (a) of FIG. 2 is an ultrasonic image of the enriched gray scale of the microbubble ultrasound contrast agent D4 obtained in Comparative Example 4.

[0054] (b) of FIG. 2 is an ultrasonic image of the enriched gray scale of the microbubble ultrasound contrast agent S1 obtained in Example 1.

DETAILED DESCRIPTION OF EMBODIMENTS

[0055] The present application will be described in detail below by the Examples. The described Examples of the present application are only a part of the examples of the present application, but not all of the examples. Based on the Examples of the present application, all other examples obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present application.

[0056] Unless otherwise specified, the materials used in the following Examples are all commercially available analytical grades.

[0057] The following preparation examples are used to prepare the magnetic nanoparticle modified with the surfactant B on the surface using in the Examples. The following preparation example is only a specific embodiment of the present application and not a limitation of the present application.

Preparation Example 1

[0058] (a) Triethylene glycol and ferric acetylacetonate are mixed in a molar ratio of 3:1 and heated at high temperature of 280° C. for 1 h, thereby obtaining a superparamagnetic Fe.sub.3O.sub.4 nanoparticle. And then the particle size is observed as 7 nm to 10 nm by a transmission electron microscope.

[0059] (b) The superparamagnetic nanoparticle is purified by magnetism.

[0060] (c) The superparamagnetic nanoparticle obtained by step (b) and the aqueous solution (sodium citric acid solution) of surfactant B are mixed in a molar ratio of 1:1.5, and then oscillated under the ultrasonic power of 300 kW. This process is repeated for 3 times.

[0061] A superparamagnetic Fe.sub.3O.sub.4 nanoparticle modified with the sodium citric acid on the surface is obtained.

Preparation Example 2

[0062] Referring to the method of Preparation Example 1, the difference is that the amount of the sodium citric acid is changed so that the molar ratio of the superparamagnetic nanoparticle to the surfactant B is 1:1.8.

Preparation Example 3

[0063] Referring to the method of Preparation Example 1, the difference is that the amount of the sodium citric acid is changed so that the molar ratio of the superparamagnetic nanoparticle to the surfactant B is 1:1.2.

Preparation Example 4

[0064] Referring to the method of Preparation Example 1, the difference is that the amount of the sodium citric acid is changed so that the molar ratio of the superparamagnetic nanoparticle to the surfactant B is 1:0.5.

Comparative Preparation Example 1

[0065] Referring to the method of Preparation Example 1, the difference is that the sodium citric acid is replaced with the same molar weight of poloxamer, thereby obtaining a superparamagnetic Fe.sub.3O.sub.4 nanoparticle modified with the poloxamer on the surface.

Example 1

[0066] Processed by the Following Steps:

[0067] (1) Sorbitan monostearate (lipid), polysorbate 80 (lipid) and poloxamer F68 (Germany BASF, P21489-25G, surfactant A, HLB value=29) are mixed well in a molar ratio of 1:1:1.5, 10 mL of the obtained mixture is taken and put into a 120° C. sterilization pot under the high-temperature and high-pressure conditions for 12 min, and then continuously stirred and cooled until the temperature is 40° C.;

[0068] (2) The resulting material obtained by step (1) and paclitaxel (chemotherapy drug) are mixed in a weight ratio of 100:5, applying ultrasonic cavitation method, perfluorocarbon gases are pumped into the obtained mixture at room temperature, processing ultrasonic cavitation for 3 min under the power of 10 kW, and cooling down to room temperature to obtain a first microbubble suspension;

[0069] (3) The first microbubble suspension is stood for 12 h, and then centrifuged to remove the upper layer microbubbles with bigger particle size, measuring the particle size distribution of the microbubbles is in the range of 0.8 μm to 3 μm by a light microscope, and then 10 mL polyethyleneimine solution (concentration of 1 mg/L) is added slowly into the obtained microbubble suspension, oscillating under the ultrasonic power of 300 kW, after allowing to adsorb for 30 min, put into the 4° C. refrigerator to stand for stratification, discarding the subnatant, and then the upper layer solution is washed with PBS buffer repetitive for 3 times to remove the excess polyelectrolyte, thereby obtaining the second microbubble suspension with a positive charge on the surface;

[0070] (4) 10 mL of the aqueous solution of superparamagnetic nanoparticle modified with the sodium citric acid (wherein the molar ratio of the superparamagnetic nanoparticle to the lipid in the second microbubble suspension is 0.25:1) obtained by the Preparation Example 1 is added into the second microbubble suspension slowly, oscillating under the ultrasonic power of 300 kW, after allowing to adsorb for 30 min, put into the 4° C. refrigerator to stand for stratification, discarding the subnatant, and then the upper layer solution is washed with PBS buffer repetitive for 3 times to obtain a magnetic microbubble ultrasound contrast agent, denoted as S1.

[0071] The obtained magnetic microbubble ultrasound contrast agent S1 is observed by an optical microscope and the result is shown in FIG. 1. It may be seen from the FIG. 1 that the contrast agent is covered in dense microbubbles with particle sizes of about 1 μm, and the particle size distribution of the microbubbles is narrow, and there are no obvious impurities in the solution, which can meet the imaging requirement for the ultrasound contrast agent.

Example 2

[0072] Processed by the Following Steps:

[0073] (1) Sorbitan monostearate (lipid), polysorbate 80 (lipid) and poloxamer F127 (Sigma-Aldrich P2443-250G, surfactant A, HLB value=23) are mixed well in a molar ratio of 1:0.8:1.35, 10 mL of the obtained mixture is taken and put into a 110° C. sterilization pot under the high-temperature and high-pressure conditions for 16 min, and then continuously stirred and cooled until the temperature is 40° C.;

[0074] (2) The resulting material obtained by step (1) and paclitaxel (chemotherapy drug) are mixed in a weight ratio of 100:8, applying ultrasonic cavitation method, perfluorocarbon gases are pumped into the obtained mixture at room temperature, processing ultrasonic cavitation for 3 min under the power of 10 kW, and cooling down to room temperature to obtain a first microbubble suspension;

[0075] (3) The first microbubble suspension is stood for 12 h, and then centrifuged to remove the upper layer microbubbles with bigger particle size, measuring the particle size distribution of the microbubbles is in the range of 0.8 μm to 3 μm by a light microscope, and then 10 mL polyethyleneimine solution (concentration of 1.5 mg/L) is added slowly into the obtained microbubble suspension, oscillating under the ultrasonic power of 250 kW, after allowing to adsorb for 40 min, put into the 4° C. refrigerator to stand for stratification, discarding the subnatant, and then the upper layer solution is washed with PBS buffer repetitive for 3 times to remove the excess polyelectrolyte, thereby obtaining the second microbubble suspension with a positive charge on the surface;

[0076] (4) 10 mL of the aqueous solution of superparamagnetic nanoparticle modified with the sodium citric acid (wherein the molar ratio of the superparamagnetic nanoparticle to the lipid in the second microbubble suspension is 0.18:1) obtained by the Preparation Example 2 is added into the second microbubble suspension slowly, oscillating under the ultrasonic power of 350 kW, after allowing to adsorb for 20 min, put into the 4° C. refrigerator to stand for stratification, discarding the subnatant, and then the upper layer solution is washed with PBS buffer repetitive for 3 times to obtain a magnetic microbubble ultrasound contrast agent, denoted as S2.

Example 3

[0077] Processed by the Following Steps:

[0078] (1) Sorbitan monostearate (lipid), polysorbate 80 (lipid) and poloxamer F68 (Germany BASF, P21489-25G, surfactant A, HLB value=29) are mixed well in a molar ratio of 1:1.2:1.65, 10 mL of the obtained mixture is taken and put into a 130° C. sterilization pot under the high-temperature and high-pressure conditions for 8 min, and then continuously stirred and cooled until the temperature is 40° C.;

[0079] (2) The resulting material obtained by step (1) and paclitaxel (chemotherapy drug) are mixed in a weight ratio of 100:2, applying ultrasonic cavitation method, perfluorocarbon gases are pumped into the obtained mixture at room temperature, processing ultrasonic cavitation for 3 min under the power of 10 kW, and cooling down to room temperature to obtain a first microbubble suspension;

[0080] (3) The first microbubble suspension is stood for 12 h, and then centrifuged to remove the upper layer microbubbles with bigger particle size, measuring the particle size distribution of the microbubbles is in the range of 0.8 μm to 3 μm by a light microscope, and then 10 mL polyethyleneimine solution (concentration of 0.8 mg/L) is added slowly into the obtained microbubble suspension, oscillating under the ultrasonic power of 350 kW, after allowing to adsorb for 20 min, put into the 4° C. refrigerator to stand for stratification, discarding the subnatant, and then the upper layer solution is washed with PBS buffer repetitive for 3 times to remove the excess polyelectrolyte, thereby obtaining the second microbubble suspension with a positive charge on the surface;

[0081] (4) 10 mL of the aqueous solution of superparamagnetic nanoparticle modified with the sodium citric acid (wherein the molar ratio of the superparamagnetic nanoparticle to the lipid in the second microbubble suspension is 0.25:1) obtained by the Preparation Example 3 is added into the second microbubble suspension slowly, oscillating under the ultrasonic power of 250 kW, after allowing to adsorb for 40 min, put into the 4° C. refrigerator to stand for stratification, discarding the subnatant, and then the upper layer solution is washed with PBS buffer repetitive for 3 times to obtain a magnetic microbubble ultrasound contrast agent, denoted as S3.

Example 4

[0082] Referring to the method of Example 1, the difference is that the ratio of the two lipids is changed, specifically, the molar ratio of sorbitan monostearate to polysorbate 80 is 1:2.5. [92] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as S4.

Example 5

[0083] Referring to the method of Example 1, the difference is that the superparamagnetic nanoparticle modified with the sodium citric acid obtained by the Preparation Example 1 used in step (4) is replaced with the same weight of the superparamagnetic nanoparticle modified with the sodium citric acid obtained by the Preparation Example 4.

[0084] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as S5.

Example 6

[0085] Referring to the method of Example 1, the difference is that the poloxamer is replaced with the same molar weight of poly(isobutene-maleic anhydride) (Sigma-Aldrich, 531278-250G).

[0086] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as S6.

Example 7

[0087] Referring to the method of Example 1, the difference is that the poloxamer is replaced with the same molar weight of poly(maleic anhydride-alt-1-octadecene) (Sigma-Aldrich, 419117-250G).

[0088] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as S7.

Example 8

[0089] Referring to the method of Example 1, the difference is that the dosage of the poloxamer is changed, specifically, sorbitan monostearate, polysorbate 80 and poloxamer F68 are mixed in the molar ratio of 1:1:1.2.

[0090] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as S8.

Example 9

[0091] Referring to the method of Example 1, the difference is that the dosage of the poloxamer is changed, specifically, sorbitan monostearate, polysorbate 80 and poloxamer F68 are mixed in the molar ratio of 1:1:1.0.

[0092] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as S9.

Example 10

[0093] Referring to the method of Example 1, the difference is that the dosage of the poloxamer is changed, specifically, sorbitan monostearate, polysorbate 80 and poloxamer F68 are mixed in the molar ratio of 1:1:0.8.

[0094] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as S10.

Example 11

[0095] Referring to the method of Example 1, the difference is that the dosage of the poloxamer is changed, specifically, sorbitan monostearate, polysorbate 80 and poloxamer F68 are mixed in the molar ratio of 1:1:0.6.

[0096] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as S11.

Example 12

[0097] Referring to the method of Example 1, the difference is that the dosage of the poloxamer is changed, specifically, sorbitan monostearate, polysorbate 80 and poloxamer F68 are mixed well in the molar ratio of 1:1:2.

[0098] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as S12.

Example 13

[0099] Referring to the method of Example 1, the difference is that the poloxamer is replaced with the same ratio weight of poloxamer P123 (Sigma-Aldrich, 435465-250ML, surfactant A, HLB value=9).

[0100] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as S13.

Comparative Example 1

[0101] Referring to the method of Example 1, the difference is that no poloxamer is added.

[0102] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as D1.

Comparative Example 2

[0103] Referring to the method of Example 1, the difference is that the surfactant A poloxamer used in the Example 1 is replaced with glycerol monostearate (TianWeiTaiDa RH30299-100g).

[0104] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as D2.

Comparative Example 3

[0105] Referring to the method of Example 1, the difference is that the superparamagnetic nanoparticle modified with the sodium citric acid obtained by the Preparation Example 1 is replaced with the same weight of superparamagnetic Fe.sub.3O.sub.4 nanoparticle modified with poloxamer on the surface obtained by the Comparative Preparation Example 1.

[0106] Finally, a magnetic microbubble ultrasound contrast agent is obtained, denoted as D3.

Comparative Example 4

[0107] Referring to the method of Example 1, the difference is that no superparamagnetic nanoparticle modified with the sodium citric acid is added, i.e., no step (3) or step (4) is performed, the first microbubble suspension with a positive charge on the surface obtained by step (2) is the final microbubble ultrasound contrast agent, denoted as D4.

Test Example

[0108] (1) Stability Test

[0109] The stability of the ultrasound contrast agent in vivo is reflected by the half-life of the ultrasound contrast agent, and the longer the half-life, the higher the stability. Particularly, the test method includes (taking a rabbit as the object):

[0110] Selecting a length of vascular area in the ultrasonic image randomly after the ultrasound contrast agent is injected under the record of real-time ultrasound imaging, and recording the change of the average grey value in the area. Recording the point-in-time as t1 when the average grey value reaches a maximum A, and recording the point-in-time as t2 when the average grey value drops to 50% of the A, so the half-life of this kind of microbubbles is 10421. Note: Ensuring the concentration and dosage of the ultrasound contrast agent are same for each injection.

[0111] The measured half-life results of the ultrasound contrast agents from the Examples and Comparative Examples are recorded in Table 1, respectively.

[0112] (2) Mechanical Index Test

[0113] In vitro enriched blasting experiment is performed. Particularly, the test method includes:

[0114] Injecting the ultrasound contrast agent of the Examples and Comparative Examples with the same concentration (10.sup.6 pcs/mL) into the cellulose hose (inner diameter is 1 mm) with ndfeb magnet (5000 Gs) placed on one side respectively, and performing an in-vitro enriched blasting experiment of magnetic microbubbles under the condition of physiological flow rate (100 mL/h). The ultrasonic imaging/blasting probe uses a linear array probe with 196 array elements, a center frequency of 12 MHz and a bandwidth of 6 MHz. Controlling the mechanical index (control range is MI=0.1-2.5, stepping is 0.1) of the ultrasonic wave emitted by the ultrasonic probe, and observing the blast of the microbubbles under different mechanical index and recording the mechanical indexes respectively when the blasting rates of the ultrasound contrast agent according to each Examples and Comparative Example are greater than 90% (time is less than 10 s) (blasting rate=|before blasting−after blasting|/before blasting*100%), and the results are recorded in Table 1.

[0115] (3) Magnetism Test

[0116] In vitro enrichment experiment is performed. Particularly, the test method includes:

[0117] Injecting the ultrasound contrast agent of the Examples and Comparative Examples with the same concentration (10.sup.6 pcs/mL) into the cellulose hose (inner diameter is 1 mm) with ndfeb magnet (5000 Gs) placed on one side respectively, and performing an in-vitro enriched blasting experiment of magnetic microbubbles under the condition of static flow rate (0 mL/h). The ultrasonic imaging probe uses a linear array probe with 196 array elements, a center frequency of 12 MHz and a bandwidth of 6 MHz. Controlling the mechanical index (control range is MI<0.1) of the ultrasonic wave emitted by the ultrasonic probe, and observing the magnetism of the magnetic microbubbles under different mechanical index.

[0118] Within 15 min, if almost all the magnetic microbubbles are enriched on the magnet side (ultrasonic image shows that the gray characteristic of the magnetic microbubbles on the magnet side is larger than 95% of all the microbubbles signal), it is considered that the magnetism is normal.

[0119] Within 15 min, if more than half of the magnetic microbubbles are enriched on the magnet side (ultrasonic image shows that the gray characteristic of the magnetic microbubbles on the magnet side is larger than 50% of all the microbubbles signal), it is considered that the magnetism is weak.

[0120] Within 15 min, if the magnetic microbubbles are uniformly distributed and have no adsorption effect (ultrasonic image shows that the gray characteristic of the magnetic microbubbles on the magnet side is distributed in the cellulose hose uniformly), it is considered as no magnetism.

[0121] (4) Taking the magnetic microbubble ultrasound contrast agent S1 of Example 1 and the ultrasound contrast agent D4 of Comparative Example 4 as examples, after 10 enriched blasting experiment, the enriched gray scales of S1 and D4 are compared, as shown in (a) of FIG. 2 and (b) of FIG. 2. It can be significantly seen from FIG. 2 that S1 has an obvious local enrichment while D4 has no enrichment.

TABLE-US-00001 TABLE 1 Half-life Blasting mechanical (min) index MI Magnetism S1 6.3 1.1 normal S2 5.8 1.1 normal S3 5.6 1.0 normal S4 5.3 0.9 normal S5 5.6 1.0 weak S6 5.1 0.8 normal S7 5.0 0.9 normal S8 6.2 1 normal S9 6 0.9 normal S10 5.6 0.9 normal S11 5.5 0.8 normal S12 5.5 0.8 normal S13 5.0 0.8 normal D1 2 0.3 normal D2 2.5 0.3 normal D3 6.5 1.1 no magnetism D4 6.8 1.2 no magnetism

[0122] It can be seen from Table 1, the composition for ultrasound contrast agent and the ultrasound contrast agent according to the present application have both longer half-life and higher blasting mechanical index, as well as good magnetism, thereby the requirements for in vivo cyclic targeted delivery may be met by the magnetic microbubble ultrasound contrast agent.

[0123] And it can be seen from Example 1, Example 8 to Example 11, when the amount of emulsifier is in a proper range, the higher the content of emulsifier, the longer half-life of the magnetic ultrasound contrast agent and the higher threshold of blasting mechanical index; It can be seen from Example 12, when the content of the emulsifier is too high, the half-life of the magnetic ultrasound contrast agent and the threshold of blasting mechanical index are decreased. Therefore, the performance of the ultrasound contrast agent is controllable and is of great significance for practical application.

[0124] The preferred embodiments of the present application have been described above in detail; however, the present application is not limited thereto. Within the scope of the technical concept of the present application, a variety of simple modifications may be made to the technical solutions of the present application, including combining various technical features in any other suitable manner. These simple modifications and combinations should also be regarded as the content disclosed in the present application, and they all belong to the protection scope of the present application.