CN112933248B - Sentinel lymph node double-development tracer, preparation method and application - Google Patents
Sentinel lymph node double-development tracer, preparation method and application Download PDFInfo
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Abstract
The invention relates to a sentinel lymph node double-development tracer, a preparation method and application. The double-development tracer comprises an outer layer, a middle layer and an inner layer; the outer layer comprises chitosan and contrast agent adsorbed to each other, the contrast agent is nanocarbon or methylene blue, the middle layer comprises phosphatidylserine, and the inner layer comprises inert gas C 3 F 8 Or C 4 F 10 . The double-development tracer agent can resist the irradiation of high-frequency ultrasound, and meanwhile, phosphatidylserine can be phagocytized by macrophages in lymph nodes, so that the continuous development of sentinel lymph nodes before, during and after an operation is achieved; the possibility of different developing passages caused by different injection points of different developers is avoided, the ultrasonic contrast agent development can be detected again in the visible dyeing lymph nodes cut off after the operation, the problem of matching of the sentinel lymph nodes positioned before the operation of the ultrasonic contrast with the dye dyeing lymph nodes in the operation is solved, the bias caused by factors such as manual operation or cooperation proficiency and the like is avoided, the sentinel lymph node development with higher matching is realized, the positioning problem is solved by the help of the surgical operation, and the complications are reduced.
Description
Technical Field
The invention relates to the field of medical treatment, in particular to a sentinel lymph node double-development tracer, a preparation method and application.
Background
Sentinel Lymph Node (SLN) is the first node of tumor metastasis, and it is currently widely believed that mammary gland SLN can be used as an index for determining tumor axillary metastasis, and intraoperative axillary SLN biopsy (SLNB) is widely used. SLN localization studies of breast cancer have been a clinical hotspot and difficulty.
In the operation, dye such as methylene blue, radionuclide, fluorescent dye and the like is mostly adopted for positioning SLN (SLN), and biopsy is carried out in the operation according to the positioning, whether axillary lymph node cleaning is needed or not is determined after the pathological result in the operation, and the lymph node cleaning can be avoided for a patient with pathological SLN which is not transferred, so that the occurrence of postoperative complications is reduced. The nuclide tracing method utilizes radioactive isotopes, is expensive and radioactive, has influence on both patients and operators, needs special instruments and special preparation, and is difficult to popularize. The fluorescent dye needs a professional instrument for detection, the admission threshold is high, unsatisfactory lymph nodes are displayed, and the like, so that the fluorescent dye is difficult to popularize clinically. A Meilan micromolecule dye (Meilan and nanocarbon) tracing method is a commonly used intraoperative lymph node developing method in China surgery, Meilan/nanocarbon is injected subcutaneously or intradermally around a mammary areola region or a tumor, intraoperative exploration is carried out after 10-20min, the method is visual and easy to implement, the clinical acceptance is high, technical requirements on an operator are high, certain blindness and long operation time exist before an operation, and on the other hand, certain damage is caused to skin when the dye concentration is high, and local skin necrosis can be caused.
With the rapid development of ultrasonic contrast technology in recent years, ultrasonic contrast agents are widely applied to livers, thyroid glands, lymph nodes, mammary glands and the like, and are used for carrying out SLN ultrasonic contrast positioning on breast cancers at early stages before operation and then marking on body surfaces.
The number and the positions of the preoperative contrast agent positioning lymph nodes are marked on the body surface, a surgeon is informed of the depth of the SLN from the skin, and dye tracing positioning is carried out during the operation. However, the axilla is dissected into a three-dimensional structure, the number of lymph nodes is large, even if the lymph nodes are positioned before the operation, the stained lymph nodes can not be found locally in the operation, or the corresponding positions of the axilla can be detected to find out that the stained/unstained lymph nodes are the SLNs positioned before the operation, so that the dye-stained lymph nodes positioned in the operation are matched with the SLNs positioned before the operation by ultrasonic contrast, whether the removed SLNs are matched depends on the technologies of an ultrasonic doctor and an operating doctor and the cooperation between the ultrasonic doctor and the operating doctor, a long time is usually needed for the grinding period, and even though the SLNs cut after the operation still exist among the doctors with great cooperation, the matching with the SLNs positioned before the operation cannot be confirmed in real time.
Disclosure of Invention
In view of the above, the present invention provides a sentinel lymph node double development tracer, a preparation method and an application thereof, which at least partially solve the problems in the prior art.
The invention provides a double-development tracer, which comprises an outer layer, a middle layer and an inner layer; the material of the outer layer comprises chitosan and a contrast agent, the material of the middle layer comprises phosphatidylserine, and the inner layer comprises an inert gas.
Preferably, in the outer layer, the chitosan is in a strip structure, the contrast agent is in a solid spherical structure, and the chitosan with positive charges and the contrast agent with negative charges are adsorbed together; the phosphatidylserine in the composition structure of the middle layer is a hollow spherical structure with a linear tail, and is similar to a tadpole shape.
Preferably, the contrast agent comprises nanocarbon or methylene blue and the inert gas comprises C 3 F 8 Or C 4 F 10 。
The invention also provides a preparation method of the double-development tracer, which comprises the steps of preparing the micro-bubbles and assembling the nano carbon or the methylene blue on the surfaces of the micro-bubbles.
Wherein, the preparation of the microvesicle comprises the following steps: mixing phosphatidyl serine, Span60 and sodium chloride, grinding, dispersing in PBS solution, adding Tween80, and magnetically stirring to obtain suspension; treating the suspension at high temperature, stirring, cooling to room temperature, and introducing C 3 F 8 Or C 4 F 10 Continuously carrying out ultrasonic treatment on the gas by using an 1/2' probe of a Sonicator 4000 ultrasonic crusher under the maximum amplitude to obtain milky white suspension; standing and layering the milky white suspension, dividing bubbles with different sizes into three obvious layers due to the self buoyancy, and collecting the middle layer to obtain the microbubbles.
Preferably, the ratio of the mass of phosphatidylserine, the mass of Span60, the volume of sodium chloride and the volume of PBS solution is (1.45-1.50) g: (1.48-1.52) g: (45-55) mL: 1mL, preferably 1.48 g: 1.5 g: 50mL of: 1 mL.
Preferably, the treatment under the high temperature condition is carried out for 10-14min under the conditions of a sterilization kettle at the temperature of 110-; the ultrasonic treatment time is 2-4 min; further comprises rinsing the collected intermediate layer with PBS buffer solution repeatedly until the lower layer solution is clarifiedClearing; the preservation method of the microvesicles comprises the following steps: collecting the microbubbles in a penicillin bottle, mixing with PBS solution in equal volume, and filling C 3 F 8 Or C 4 F 10 Sealing with gas, and storing in a refrigerator at 4 deg.C.
The method for assembling the nano carbon or the methylene blue on the surface of the microbubble comprises the following steps: adding chitosan solution with positive charge into the microbubbles, adsorbing for 10-20min, and injecting C 3 F 8 Or C 4 F 10 Standing the gas in a refrigerator at 4 ℃ for layering, and removing the supernatant; washing with PBS buffer solution for 2 times to remove excessive chitosan; adding the nano carbon or methylene blue water solution with negative charge, adsorbing for 10-20min, and washing for 2 times with PBS buffer solution. After the assembly is finished, the obtained microbubbles coated with the nano carbon or the methylene blue are re-dispersed into a PBS solution, inflated and sealed, and stored in a refrigerator at 4 ℃. The Methylene blue (also called Methylene blue) adopted by the invention has a chemical formula as follows: C16H18N3ClSIUPAC, chinese name: 3, 7-bis (dimethylamino) phenothiazine-5-poly (p-phenylene) chloride, which is an aromatic heterocyclic compound, is used as a chemical indicator, a dye, a biological stain and a drug.
Preferably, the volume ratio of the microbubbles to the chitosan solution, the nanocarbon or the methylene blue aqueous solution is 1: 1: 1; the mass concentration of the chitosan solution is 0.9-1.1mg/mL, preferably 1 mg/mL; the mass concentration of the nano carbon or methylene blue aqueous solution is 0.9-1.1mg/mL, and preferably 1 mg/mL. The chitosan solution with positive charges adopted by the invention is prepared by 0.5M NaCl, and the nano-carbohydrate solution with negative charges and the methylene blue aqueous solution are prepared by 0.5M NaCl.
The invention provides an application of a double-development tracer in preparation of a product with functions of enhancing development and tracing operation.
The technical scheme provided by the invention has the following beneficial effects: the double-development tracer provided by the invention has a hard shell and can resist the irradiation of high-frequency ultrasound, and meanwhile, phosphatidylserine in the middle layer can be phagocytized by macrophages in lymph nodes, so that the double-development tracer can develop in an SLN for a long time to achieve the purpose of continuously developing the SLN before, during and after an operation; in addition, the possibility of different developing passages caused by different injection points of different developers is avoided due to the fact that the medicine is double developers after injection, the ultrasonic contrast development and dye dyeing are developed by a common lymph channel, and the characteristic of long-time development is added, the ultrasonic contrast development can be detected again in naked eye visible dyeing lymph nodes cut off after the operation, the problem that the positioning SLN before the ultrasonic contrast operation is matched with the dye dyeing lymph nodes in the operation is solved, the bias caused by factors such as manual operation or cooperation proficiency is avoided, the higher matching SLN development is realized, the positioning problem is solved by the aid of the surgical operation, and complications are reduced. In addition, the photo-thermal killing effect of the nano carbon on cancer cells under the near-infrared laser irradiation condition can provide possibility for local minimally invasive treatment of axillary lymph nodes.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a schematic structural diagram of a dual imaging tracer provided in accordance with the present invention;
FIG. 2 shows the axillary lymph nodes at the same time as the gray scale (left) and the contrast (right) of the present invention: the lymph node display is unsatisfactory under the gray scale condition, and sentinel lymph node double-development tracer development SLN and drainage lymphatic vessel running can be clearly displayed under the contrast condition;
FIG. 3 shows the present invention with gray scale (left) and contrast (right) showing post-ablation lymph node: SLN and small segment lymphatic vessels developed by a sentinel lymph node double-development tracer which is cut in physiological saline, and surrounding fat tissues are not developed by a contrast agent.
Detailed Description
Embodiments of the present invention are described in detail below with reference to the accompanying drawings.
It should be noted that, in the case of no conflict, the features in the following embodiments and examples may be combined with each other; moreover, based on the embodiments in the present disclosure, all other embodiments obtained by a person of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present disclosure.
It is noted that various aspects of the embodiments are described below within the scope of the appended claims. It should be apparent that the aspects described herein may be embodied in a wide variety of forms and that any specific structure and/or function described herein is merely illustrative. Based on the disclosure, one skilled in the art should appreciate that one aspect described herein may be implemented independently of any other aspects and that two or more of these aspects may be combined in various ways. For example, an apparatus may be implemented and/or a method practiced using any number of the aspects set forth herein. Additionally, such an apparatus may be implemented and/or such a method may be practiced using other structure and/or functionality in addition to one or more of the aspects set forth herein.
Example 1
The embodiment provides a sentinel lymph node double-development tracer, which is shown in a schematic structural diagram in fig. 1 and comprises an outer layer, a middle layer and an inner layer; wherein, the outer layer comprises chitosan and contrast agent nanocarbon which are mutually adsorbed together, the chitosan is in a strip structure, and the nanocarbon is in a solid spherical structure; the material of the middle layer (surrounding the gas) comprises phosphatidylserine, and is in a hollow spherical structure with a linear tail; the inner layer (central) comprising an inert gas C 3 F 8 。
Example 2
The embodiment provides a sentinel lymph node double-development tracer, which is shown in a schematic structural diagram in fig. 1 and comprises an outer layer, a middle layer and an inner layer; wherein, the outer layer comprises chitosan and contrast agent methylene blue which are mutually adsorbed together, the chitosan is in a strip structure, and the nano carbon is in a solid spherical structure; the material of the middle layer (surrounding the gas) comprises phosphatidylserine, and is in a hollow spherical structure with a linear tail; the inner layer (central) comprising an inert gas C 4 F 10 。
Example 3
The embodiment provides a preparation method of a sentinel lymph node double-development tracer, which comprises the following steps.
1. Preparation of microbubbles
Preparation of surfactant (Span60, Tween80 and phosphatidylserine) coated C 3 F 8 Gaseous ST68 microbubble ultrasound contrast agents. The method comprises the following specific steps: phosphatidylserine, Span60(1.48g) and NaCl (1.50g) were thoroughly ground in a mortar, dispersed in 50mL PBS, and Tween80(1mL) was added thereto and magnetically stirred. Placing the mixed suspension in a sterilizing pot at 120 deg.C, heating under high pressure for 12min, and cooling to room temperature under continuous stirring. Introducing C into the solution 3 F 8 Gas, sonicated continuously for 3min at maximum amplitude with the 1/2 "probe of the Sonicator 4000 Sonicator. After shaking with sound, a uniform milky white suspension was obtained, which was collected in a 250mL separatory funnel and allowed to stand. After 35min, the bubbles with different sizes are divided into three obvious layers due to the self-buoyancy, and the microbubbles at the middle layer are collected and repeatedly rinsed by PBS buffer solution until the solution at the lower layer is clear. Finally, the microbubbles are collected in a penicillin bottle, mixed with PBS in equal volume and filled with C 3 F 8 Sealing with gas, and storing in a refrigerator at 4 deg.C.
ST68 micro-bubble surface assembled nano carbon
The method for assembling the nanocarbon on the surface of the ST68 microbubble by adopting the electrostatic attraction layer-by-layer self-assembly method comprises the following specific experimental steps: 10mL of prepared ST68 microbubble suspension was placed in a self-made centrifuge tube (with an opening at the lower end) and injected with C 3 F 8 Sealing, and standing in a refrigerator at 4 deg.C. After the microbubbles completely float to the upper layer of the liquid surface, discarding the supernatant, slowly adding 10mL of positively charged chitosan solution (1mg/mL, 0.5M NaCl) into the microbubbles, slightly shaking, adsorbing for 15min, and injecting C 3 F 8 The gas is placed in a refrigerator at 4 ℃ for standing and layering, and the supernatant is discarded. 20ml PBS buffer solution was added to the microbubbles, and washing was repeated 2 times in the same manner to remove excess chitosan. After the positively charged chitosan was assembled, 10mL of negatively charged nanocarbon solution (1mg/mL, 0.5M NaCl) was added to the microbubbles, which were gently shaken and adsorbed for 15 min. After assembly, the cells were washed 2 times with PBS buffer solution as well. After the assembly is finished, the obtained microbubbles coated with the nano carbon are redispersed in PBS, inflated and sealed, and placed inStoring in a refrigerator at 4 ℃ to obtain the sentinel lymph node double-development tracer.
Example 4
The embodiment provides a preparation method of a sentinel lymph node double-development tracer, which comprises the following steps.
1. Preparation of microbubbles
Preparation of surfactant (Span60, Tween80 and phosphatidylserine) coated C 4 F 10 Gaseous ST68 microbubble ultrasound contrast agents. The method comprises the following specific steps: phosphatidylserine and Span60(1.48g) and NaCl (1.50g) were thoroughly ground in a mortar and dispersed in 50mL PBS, and Tween80(1mL) was added thereto with magnetic stirring. Placing the mixed suspension in a sterilizing pot at 120 deg.C, heating under high pressure for 12min, and cooling to room temperature under continuous stirring. Introducing C into the solution 4 F 10 Gas, sonicated continuously for 3min at maximum amplitude with the 1/2 "probe of the Sonicator 4000 Sonicator. After sonication, a uniform milky white suspension was obtained, which was collected in a 250mL separatory funnel and allowed to stand. After 35min, the bubbles with different sizes are divided into three obvious layers due to the self-buoyancy, and the microbubbles at the middle layer are collected and repeatedly rinsed by PBS buffer solution until the solution at the lower layer is clear. Finally, the microbubbles are collected in a penicillin bottle, mixed with PBS in equal volume and filled with C 4 F 10 Sealing with gas, and storing in a refrigerator at 4 deg.C.
ST68 microbubble surface Assembly methylene blue
The method for assembling the nanocarbon on the surface of the ST68 microbubble by adopting the electrostatic attraction layer-by-layer self-assembly method comprises the following specific experimental steps: 10mL of prepared ST68 microbubble suspension was placed in a self-made centrifuge tube (with an opening at the lower end) and injected with C 4 F 10 Sealing, and standing in a refrigerator at 4 deg.C. After the microbubbles completely float to the upper layer of the liquid surface, the supernatant is discarded, 10mL of chitosan solution with positive charge (1mg/mL, 0.5M NaCl) is slowly added into the microbubbles, the mixture is slightly shaken and adsorbed for 15min, and then C is injected 4 F 10 The gas is placed in a refrigerator at 4 ℃ for standing and layering, and the supernatant is discarded. 20ml PBS buffer solution was added to the microbubbles, and washing was repeated 2 times in the same manner to remove excess chitosan. After assembling the chitosan with positive chargeThen, 10mL of negatively charged methylene blue aqueous solution (1mg/mL, 0.5M NaCl) was added to the microbubbles, and the mixture was gently shaken and adsorbed for 15 min. After assembly, the cells were washed 2 times with PBS buffer solution as well. And after the assembly is finished, re-dispersing the Meilan coated microbubbles into PBS, inflating and sealing, and storing in a refrigerator at 4 ℃ to obtain the sentinel lymph node double-developing tracer.
The present invention provides an effect study of the effect of the sentinel lymph node double imaging tracer prepared in example 4 in the localization of SLN in patients with early breast cancer. To better characterize the effect of the sentinel lymph node dual imaging tracer, the ultrasound contrast agent SonoVue was used as a control in this study. In recent years, along with the rapid development of ultrasonic contrast technology, SonoVue as a representative of the second-generation contrast agent has been widely applied to liver, thyroid, lymph node, mammary gland and the like, and SonoVue is a microbubble contrast agent formed by wrapping sulfur hexafluoride gas with a lipid membrane, and the efficiency of the SonoVue applied to early breast cancer SLN preoperative ultrasonic contrast positioning is equivalent to that of the intraoperative Meilan positioning method, and the combination of the two can improve the detection rate of SLN.
1. Study object
The clinical diagnosis of the hospital from 1/2018 to 30/1/2020 is 225 female patients and 1 male patient with breast cancer at T1-T2 stage. All patients underwent either ALND or SLNB to obtain pathological results.
1.1 inclusion criteria: clinically diagnosing early breast cancer patients with stage T1-2N0M 0; ② a T1-2 breast cancer patient confirmed by excisional biopsy or needle aspiration cytology.
1.2 exclusion criteria: firstly, the affected side has a history of breast surgery; multifocal lesions; clinically examining the axillary lymphadenectasis; radiotherapy is performed before the operation; fifth, there is history of axillary operation on the affected side; sixthly, pregnant women or patients allergic to the contrast agent.
2. Apparatus and method
The GE LOGIQ E9 real-time ultrasonic diagnostic apparatus and a matched probe are adopted, the frequency of the probe is 9MHz, a patient is in a horizontal position, two-dimensional ultrasonic examination and color Doppler ultrasonic examination are firstly carried out, then ultrasonic contrast agent SonoVue/sentinel lymph node double-development tracer agent is sequentially injected in equal quantity in the directions of 3, 6, 9 and 12 points in the skin around the mammary areola of the patient, and the total quantity is 2.0mL/0.4 mL.
Dividing the group cases into a SonoVue group and a sentinel lymph node double-development tracer group according to different ultrasonic contrast agent use conditions, dynamically observing and respectively recording the lymph node enhancement number, position and enhancement mode in real time, and performing body surface positioning. Injecting 2mL of 1% Mei blue subcutaneously around the areola after the SonoVue group radiography is finished, separating a lymphatic vessel and a lymph node according to the Mei blue tracing condition in the operation, comparing with the condition of an ultrasonic radiography positioning method SLN before the operation, observing and recording the running condition and the SLN condition of the lymphatic vessel; after the lymph node was surgically removed, the contrast-developed SLN-sustained development was observed at 5 minutes, 10 minutes, 60 minutes, and 120 minutes after the contrast injection, and the SLN-sustained development was compared with the different contrast agent groups according to the lymph node resection results after the surgery.
3. Statistical method
All clinical and experimental data were processed using SPSS17.0 data statistics software. The Fisher's exact test was used to statistically analyze the data, and P < 0.05 was statistically significant for differences.
4. Comparison results
In 226 eligible cases, preoperative sonography localization, intraoperative melanism staining and surgical resection of patients were completed, excluding 18 preoperative chemotherapy patients, the data were incomplete in 4 cases, and a total of 204 cases were eventually entered into the study, of which males were 1 at age 22-75 years.
(1) The basic condition and SLN detection condition of two groups of cases (see Table 1)
After the two groups of contrast medium were completed, merlan mapping and surgical excision of SLNs were performed, the SonoVue group and sentinel lymph node double-imaging tracer group included 135 cases (male 1 case) and 69 cases, and the number of SLNs was 210 and 102 cases, among which those with metastasis were regarded as positive cases and those with metastatic lymph nodes as positive lymph nodes (see table 1).
TABLE 1 basic conditions of the Subjects
| SonoVue | Sentinel lymph node double-development tracer | |
| Group entry case | 149 | 77 |
| Chemotherapy | 11 | 8 |
| Incomplete data | 3 | 1 |
| Number of cases taken | 135 | 68 |
| Age (age) | 53.34±12.44 | 53.79±12.21 |
| Number of SLNs | 210 | 102 |
| Number of metastatic cases | 29 | 17 |
| Number of SLN transfers | 38 | 26 |
(2) SLN probability comparison of two ultrasound contrast agent detection
The SonoVue group had 1 case of unsuccessful mapping, all sentinel lymph node dual imaging tracer groups failed mapping, and the SonoVue group failed the blue-beauty mapping. The SonoVue group and sentinel lymph node double development tracer group are subjected to ultrasonic contrast detection and location SLN for Fisher accurate test, the p values are both greater than 0.05, and the difference of the results has no statistical significance.
(3) Comparison of two groups of contrast agents in SLN postoperative development
The SonoVue group SLN visualization time was observed to begin to clear at 5 minutes, contrast agent began to enter the second site lymph nodes, and clear was observed to be complete, i.e., not developable, at 10 minutes; the sentinel lymph node double development tracer group can be continuously developed when observed at 4 time points, and the latter does not enter the next lymph node within the operation time in the operation (see figure 2, the left image gray scale and the right image contrast simultaneously show the axillary lymph node condition, namely the lymph node is unsatisfied under the gray scale condition, the sentinel lymph node double development tracer development SLN and the drainage lymphatic vessel running can be clearly displayed under the contrast condition), and the sentinel lymph node double development tracer group can be developed in the SLN for a long time. Therefore, after all sentinel lymph node double development tracer groups are subjected to SLN resection, the patients are put into physiological saline for ultrasonic contrast to be probed again, lymph node development is consistent with the body number and shape, the SLN resection is considered to be satisfactory, and the developed lymph nodes are blue-stained with blue color by naked eyes (see figure 3, the gray scales of the left image and the right image simultaneously show the lymph nodes after resection, namely, the SLN and small sections of lymphatic vessels developed by sentinel lymph node double development tracer which is resected in the physiological saline, and the surrounding fat tissues are not developed by the contrast agent).
The sentinel lymph node double-visualization tracer applied to the SLN provided by the invention can enhance visualization under the condition of contrast of an ultrasonic instrument, can be used as a tracer for operation under the condition of visual indication in the operation of a surgeon, realizes complete matching of the two tracers, and provides possibility for future minimally invasive treatment.
The above description is only for the specific embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
1. A dual imaging tracer, characterized by: the double-development tracer comprises an outer layer, a middle layer and an inner layer; the material of the outer layer comprises chitosan and a contrast agent, the material of the middle layer comprises phosphatidylserine, and the inner layer comprises an inert gas; in the outer layer, the chitosan is in a strip structure, the contrast agent is in a solid spherical structure, and the chitosan and the contrast agent are mutually adsorbed; the contrast agent comprises nanocarbon or methylene blue, and the inert gas comprises C3F8 or C4F 10; the preparation method of the double-development tracer comprises the steps of preparing micro-bubbles and assembling nano carbon or methylene blue on the surfaces of the micro-bubbles; the preparation of the microvesicles comprises the steps of: mixing phosphatidyl serine, Span60 and sodium chloride, grinding, dispersing in PBS solution, adding Tween80, and stirring to obtain suspension; treating the suspension at high temperature, stirring, cooling to room temperature, and introducing C3F8 or C4F10 gas for ultrasonic treatment to obtain milky suspension; standing and layering the milky white suspension, and collecting the middle layer to obtain the microvesicles; the high temperature condition is 110-130 ℃; assembling nano carbon or methylene blue on the surface of the microbubble comprises the following steps: adding a chitosan solution with positive charges into the micro-bubbles, adsorbing for 10-20min, injecting C3F8 or C4F10 gas, standing and layering in a refrigerator at 4 ℃, and removing the lower clear liquid; then washing with PBS buffer solution to remove excessive chitosan; adding the nano carbon or methylene blue water solution with negative charges, adsorbing for 10-20min, and washing with PBS buffer solution.
2. The dual imaging tracer of claim 1, wherein: the composition material structure of the middle layer is a hollow spherical structure with a linear tail.
3. A method for preparing a dual-contrast tracer according to claim 1 or 2, comprising the steps of preparing microbubbles and assembling nanocarbon or methylene blue on the surfaces of the microbubbles.
4. The method of preparing a dual imaging tracer of claim 3, wherein the preparation of the microbubbles comprises the steps of: mixing phosphatidyl serine, Span60 and sodium chloride, grinding, dispersing in a PBS solution, adding Tween80, and stirring to obtain a suspension; treating the suspension at high temperature, stirring, cooling to room temperature, and introducing C3F8 or C4F10 gas for ultrasonic treatment to obtain milky suspension; and standing and layering the milky white suspension, and collecting the middle layer to obtain the microvesicle.
5. The method of preparing a dual imaging tracer according to claim 4, wherein: the ratio of the mass of phosphatidylserine, the mass of Span60, the volume of sodium chloride, and the volume of PBS solution was (1.45-1.50) g: (1.48-1.52) g: (45-55) mL: 1 mL.
6. The method of preparing a dual imaging tracer according to claim 4, wherein: the treatment under the high temperature condition is carried out for 10-14min at the temperature of 110-130 ℃; the ultrasonic treatment time is 2-4 min; the collected middle layer is repeatedly rinsed by PBS buffer solution until the lower layer solution is clear; the preservation method of the microvesicles comprises the following steps: collecting the microbubbles in a bottle, mixing with PBS solution in the same volume, filling C3F8 or C4F10 gas, sealing, and storing in a refrigerator at 4 deg.C.
7. The method for preparing dual-imaging tracer according to claim 3, wherein the assembling of nanocarbon or methylene blue on the surface of the microbubble comprises the steps of: adding a chitosan solution with positive charges into the microbubbles, adsorbing for 10-20min, injecting C3F8 or C4F10 gas, standing in a refrigerator at 4 ℃ for layering, and removing the supernatant; then washing with PBS buffer solution to remove excessive chitosan; adding the nano carbon or methylene blue water solution with negative charges, adsorbing for 10-20min, and washing with PBS buffer solution.
8. The method of preparing a dual imaging tracer according to claim 7, wherein: the volume ratio of the micro bubbles to the chitosan solution and the nano carbon or methylene blue aqueous solution is 1: 1: 1; the mass concentration of the chitosan solution is 0.9-1.1 mg/mL; the mass concentration of the nano carbon or methylene blue water solution is 0.9-1.1 mg/mL.
9. Use of the dual imaging tracer of claim 1 or 2 in the manufacture of a product having both enhanced imaging and surgical tracing capabilities.
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