CN112007173B

CN112007173B - Application of fluorescent conjugated polymer nano probe in peripheral nerve imaging

Info

Publication number: CN112007173B
Application number: CN202010847922.1A
Authority: CN
Inventors: 张菊香; 唐诗逸; 熊丽琴
Original assignee: Shanghai Jiaotong University
Current assignee: Shanghai Jiaotong University
Priority date: 2020-08-21
Filing date: 2020-08-21
Publication date: 2021-07-30
Anticipated expiration: 2040-08-21
Also published as: CN112007173A

Abstract

The invention discloses an application of a fluorescent conjugated polymer nano probe in peripheral nerve imaging, which relates to the technical field of fluorescent conjugated polymer nano, and the application method comprises the following steps: preparing a fluorescent conjugated polymer nano probe, wherein the probe comprises a common fluorescent conjugated polymer nano probe and a near-infrared fluorescent conjugated polymer nano probe; preparing a fluorescent conjugated polymer nano probe into a colloidal aqueous solution of the fluorescent conjugated polymer nano probe; the imaging method is applied to imaging peripheral nerves of mammals, and comprises direct exposure imaging and intramuscular injection imaging. The conjugated polymer nanoprobe adopted by the invention has simple preparation process, is applied to peripheral nerve imaging, is concentrated on the nerve adventitia and the nerve bundle membrane to mark nerves, does not damage nerve cells, and has high biological safety; can realize the high-efficiency visualization of peripheral nerves of mammals, has no non-specific uptake to fat tissues and skin tissues around the nerves, and has strong clinical application value.

Description

Application of fluorescent conjugated polymer nano probe in peripheral nerve imaging

Technical Field

The invention relates to the technical field of fluorescent conjugated polymer nanometer, in particular to application of a fluorescent conjugated polymer nanometer probe in peripheral nerve imaging.

Background

Damage to the peripheral nervous system is a significant problem in surgery and is one of the most feared surgical complications. In particular, in malignant tumor resection, physiological aberrations around the malignant lesion increase the chance of peripheral nerve damage. There are 60 million cases of surgically created nerve damage in the united states alone each year. The damage of the peripheral nervous system greatly affects the life quality of the patients and has great influence on the physiology and the psychology of the patients. In thyroidectomy, for example, the laryngeal nerve is often injured, which can result in changes or loss of sound or respiratory disease in the patient. In head and neck cancer, peripheral nervous system injury is manifested as facial paralysis and abnormalities in the appearance of the patient, resulting in trauma to the patient at both emotional and physiological levels to varying degrees. A common side effect of prostatectomy is damage to the plexus nerve of the prostate, which may lead to partial or complete incontinence or erectile dysfunction. Iatrogenic nerve damage limits the quality of life of patients and increases medical expenditures, and while experienced surgeons can easily identify major nerves under normal anatomical conditions, previous trauma, radiation therapy, tumors, and previous surgery can lead to fibrotic tissue deposits and atypical surgical anatomical planes, making nerve discrimination challenging. Based on the above, direct nerve visualization is an effective strategy to avoid iatrogenic nerve injury. Currently, ultrasound, electromyography, optical coherence tomography and confocal endoscopy have been used to assist intraoperative nerve identification. However, the lack of specificity, resolution and wide area imaging capabilities of these techniques make real-time neurological detection difficult.

Fluorescence Guided Surgery (FGS) effectively achieves intraoperative visualization, is convenient to operate, can rapidly image, is easy to obtain imaging equipment, and has been successfully applied to clinic. Indocyanine green (ICG) and methylene blue are fluorescent contrast agents approved by the U.S. Food and Drug Administration (FDA) for use in FGS. However, ICG and methylene blue have poor tissue specificity, high non-specific uptake in peripheral muscle, fat and connective tissue, and no visualization of peripheral nerves with high signal-to-noise ratio. Meanwhile, ICG and methylene blue are easy to diffuse, can be diffused to peripheral tissues in a short time, pollute an operation window and cannot realize stable navigation in a long-time operation process.

In addition, studies have found eight classes of neuro-specific contrast agents, stilbenes, coumarins, styrylpyridinium, Distyrylbenzene (DSB), tricarbocyanines, neuro-specific peptides, sodium channel selective peptides and oxazine fluorophores, however, these eight classes of contrast agents result in high non-specific adipose tissue and skin tissue uptake. Its neurofluorescence intensity is comparable to the non-specific uptake by fat, skin and muscle incised margins. Meanwhile, the toxicity research of the above various contrast agents is not complete, and the safety is unknown, for example, the contrast agent designed based on the sodium channel selective peptide may have a certain influence on the metabolism or the neuron function of the patient. Small molecule fluorophores may cross the blood-nerve barrier (BNB). A systematic and comprehensive toxicity study is necessary to determine the maximum tolerated dose of each type of contrast agent.

Therefore, those skilled in the art have made an effort to develop a method applicable to imaging peripheral nerves of mammals, which uses a contrast agent having a strong specificity, overcomes the problem that the existing neurofluorescence imaging contrast agent is taken up in nerves and peripheral tissues rather than the nerve and peripheral tissues, can stably navigate during surgery, and has high safety.

Disclosure of Invention

In view of the above-mentioned drawbacks of the prior art, the present invention is directed to a method for selecting a contrast agent having high specificity, stable navigation during surgery, and high safety, and applying the same to imaging of the peripheral nervous system.

The invention provides an application method of a fluorescent conjugated polymer nano probe in peripheral nerve imaging, which is designed and prepared, realizes high-efficiency fluorescent imaging on peripheral nerves of mammals by adopting a simple and convenient method, overcomes the problem that the existing neurofluorescent imaging contrast agent is taken up in the nerves and peripheral tissues rather than the nerves, can realize stable navigation on the peripheral nerves in the operation process, and has high safety.

The application of the fluorescent conjugated polymer nano probe in peripheral nerve imaging comprises the following steps:

step 1) preparing a fluorescent conjugated polymer nano probe, wherein the fluorescent conjugated polymer nano probe comprises a common fluorescent conjugated polymer nano probe and a near-infrared fluorescent conjugated polymer nano probe;

step 2) preparing the fluorescent conjugated polymer nano probe obtained in the step 1) into a colloidal aqueous solution of the fluorescent conjugated polymer nano probe;

and 3) imaging the peripheral nerves of the mammal with the fluorescent conjugated polymer nanoprobe colloidal aqueous solution obtained in the step 2), wherein the imaging mode comprises direct exposure imaging and intramuscular injection imaging.

Further, the preparation method of the general fluorescent conjugated polymer nanoprobe in the step 1) comprises the following steps:

step 1.1, respectively dissolving a fluorescent conjugated polymer and a surface ligand in tetrahydrofuran to prepare a conjugated polymer stock solution and a surface ligand stock solution;

step 1.2, sequentially adding stock solutions of the fluorescent conjugated polymer and the surface ligand in tetrahydrofuran to obtain a first solution system; the mass concentration ratio of the stock solution of the fluorescent conjugated polymer to the stock solution of the surface ligand is 0.8-1.2, and water bath ultrasound is carried out for 3-5 minutes to obtain a mixed solution A;

step 1.3, adding the mixed solution A into a water phase under the condition of probe ultrasound or water bath ultrasound to obtain mixed solution B;

and 1.4, introducing nitrogen into the mixed solution B, and removing the organic solvent in the mixed solution B to obtain the common fluorescent conjugated polymer nano probe.

Further, the preparation method of the near-infrared fluorescent conjugated polymer nanoprobe in the step 1) comprises the following steps:

step 1.5, respectively dissolving the fluorescent conjugated polymer, the surface ligand and the near-infrared dye in tetrahydrofuran to prepare a conjugated polymer stock solution, a surface ligand and a near-infrared dye stock solution;

step 1.6, sequentially adding a stock solution of the fluorescent conjugated polymer, a stock solution of the surface ligand and a stock solution of the near-infrared dye into tetrahydrofuran to obtain a second solution system, wherein the mass concentration ratio of the fluorescent conjugated polymer to the surface ligand is 0.8-1.2, and performing water bath ultrasound for 3-5 minutes to obtain a mixed solution C; (ii) a

Step 1.7, adding the mixed solution C into a water phase under the condition of probe ultrasound or water bath ultrasound to obtain mixed solution D;

and 1.8, introducing nitrogen into the mixed solution D, and removing the organic solvent in the mixed solution D to obtain the near-infrared fluorescent conjugated polymer nano probe.

Further, the fluorescent conjugated polymer comprises poly [ (9, 9-dioctylfluorenyl-2, 7 diyl) - (1, 4-benzo- {2,1', 3} -thiadiazole)]10% benzothiadiazole (y) (PFBT) or poly (9, 9-dioctylfluorene-co-benzothiadiazole) (F8BT) or poly [ 2-methoxy-5- (2-ethylhexyloxy) -1, 4-phenylacetylene](MEH-PPV); the surface ligand comprises amino-terminal polymethyl methacrylate (MMA-NH)₂) Or styrene-polyethylene glycol-carboxyl (PS-PEG-COOH);

further, the power of the probe ultrasound is set to be 10% -20%; the power of the water bath ultrasound is 80-100W.

Further, the near infrared dyes in step 1.5 include 2, 3-naphthalocyanine silicon bis- (trihexylsilyl oxide) (NIR 775); in the step 1.6, the content of the near-infrared dye in each 2mL of the second reaction system is 1-3 mu g.

Further, the step of imaging in the direct exposure mode in step 3) is as follows:

surgically substantially exposing the sciatic nerve of the mammal; dripping the colloidal aqueous solution of the fluorescent conjugated polymer nano probe obtained in the step 2) on the exposed sciatic nerve for incubation; after the incubation, the sciatic nerve of the mammal was thoroughly washed with PBS and the liquid was gently blotted with a dust-free paper.

Further, the concentration of the colloidal aqueous solution of the fluorescent conjugated polymer nanoprobe is 0.025 mg/mL-0.25 mg/mL; the dripping volume of the fluorescent conjugated polymer nano probe colloidal aqueous solution is 50-100 mu L; the incubation time of the aqueous solution of the fluorescent conjugated polymer nano probe dropped on sciatic nerve is 5 min-60 min.

Further, the step 3) of intramuscular injection mode imaging comprises the following steps: concentrating the fluorescent conjugated polymer nano probe colloidal aqueous solution by 2-50 times by using an ultrafiltration tube, anesthetizing the mammal, after anesthesia, injecting 25-50 mu L of probe solution into the calf muscle at one side of the mammal, and observing the clear development of the probe on the sciatic nerve after 30 min-24 h.

Furthermore, the concentration of the colloidal aqueous solution of the fluorescent conjugated polymer nanoprobe is 0.025 mg/mL-0.25 mg/mL.

Further, the concentration of the conjugated polymer stock solution and the surface ligand stock solution in the step 1.1 is 1 mg/mL-4 mg/mL.

Further, the concentration of the conjugated polymer stock solution and the surface ligand stock solution in the step 1.5 is 1 mg/mL-4 mg/mL.

Further, in the step 1.2, the volume of the first solution system is 2mL (the stock solution of tetrahydrofuran and the fluorescent conjugated polymer and the stock solution of the surface ligand), the power of water bath ultrasound is 80-100W, and the ultrasound time is 3-5 minutes.

Further, the volume of the second solution system in step 1.6 is 2mL (tetrahydrofuran, stock solution of the fluorescent conjugated polymer, stock solution of the surface ligand and near infrared fluorescent dye).

Further, the volume of the water phase in the step 1.3 is 10mL, the power of the probe ultrasonic wave is set to be 10% -20%, the ultrasonic wave stops for 2 seconds every 3 seconds, and the total ultrasonic time is 1 minute.

Further, the volume of the water phase in the step 1.7 is 10mL, the power of the probe ultrasonic wave is set to be 10% -20%, the ultrasonic wave stops for 2 seconds every 3 seconds, and the total ultrasonic time is 1 minute.

In a preferred embodiment of the present invention, example 1 details the preparation and performance characterization of fluorescent conjugated polymer nanoprobes;

in another preferred embodiment of the present invention, example 2 specifies the imaging of mammalian peripheral nerves with fluorescent conjugated polymer nanoprobes;

in another preferred embodiment of the present invention, example 3 specifies the determination of the binding site of the conjugated polymer nanoprobe to the sciatic nerve of mouse;

in another preferred embodiment of the present invention, example 4 details the procedure of surgical microscope imaging to simulate the navigation in clinical surgery.

The beneficial technical effects of the invention are as follows:

(1) the invention provides an application of a fluorescent conjugated polymer nano probe in peripheral nerve imaging, and the adopted fluorescent conjugated polymer nano probe has simple preparation process;

(2) the fluorescent conjugated polymer nanoprobe adopted by the invention is concentrated on the nerve adventitia and the nerve bundle membrane to mark the nerve, so that nerve cells are not damaged, and the biological safety is high;

(3) the fluorescent conjugated polymer nanoprobe adopted by the invention can realize high-efficiency visualization of peripheral nerves, has no non-specific uptake to adipose tissues and skin tissues around the nerves, and can successfully describe nerve morphology in a complex operation environment;

(4) the invention provides an application of a fluorescent conjugated polymer nano probe in peripheral nerve imaging, which is used for imaging the peripheral nerve of a mouse in a direct exposure mode, and has the advantages of simple method of dripping, incubating and cleaning by adopting a colloidal aqueous solution of the fluorescent conjugated polymer nano probe, convenient operation and strong clinical application value;

(5) the invention provides application of a fluorescent conjugated polymer nano probe in peripheral nerve imaging, and peripheral nerve imaging of a mouse can be realized simply and efficiently by adopting an intramuscular injection mode.

(6) The invention provides an application of a fluorescent conjugated polymer nano probe in peripheral nerve imaging, which can simply and efficiently image peripheral nerves of a mouse and can be popularized and applied to general mammals and even peripheral nerves of human beings.

The conception, the specific structure and the technical effects of the present invention will be further described with reference to the accompanying drawings to fully understand the objects, the features and the effects of the present invention.

Drawings

FIG. 1 shows the PFBT-COOH nanoprobe and PFBT-NH in accordance with one preferred embodiment 1 of the present invention₂Ultraviolet absorption spectrum of the nanoprobe;

FIG. 2 shows the PFBT-COOH nanoprobe and PFBT-NH in accordance with one embodiment of the present invention₂Fluorescence emission spectra of the nanoprobes; (ii) a

FIG. 3 is a hydrated particle size distribution diagram of a PFBT-COOH fluorescent conjugated polymer nanoprobe according to a preferred embodiment 1 of the present invention;

FIG. 4 shows the PFBT-NH in accordance with the preferred embodiment 1 of the present invention₂A hydrated particle size distribution diagram of the fluorescent conjugated polymer nanoprobe;

FIG. 5 shows the PFBT-COOH fluorescent conjugated polymer nanoprobe and PFBT-NH in accordance with one preferred embodiment of the present invention₂Incubating the sciatic nerve of the mouse by the fluorescent conjugated polymer nano probe for different time to obtain a living body fluorescence imaging graph of the sciatic nerve of the mouse;

FIG. 6 shows the PFBT-COOH fluorescent conjugated polymer nanoprobe and PFBT-NH in accordance with one preferred embodiment of the present invention₂Fluorescent conjugated polymer sodiumImaging an in vitro fluorescence imaging graph of sciatic nerve of a mouse by a rice probe;

FIG. 7 shows the PFBT-COOH fluorescent conjugated polymer nanoprobe and PFBT-NH in accordance with one preferred embodiment of the present invention₂A fluorescence intensity comparison graph of sciatic nerve of a mouse is incubated by the fluorescent conjugated polymer nano probe for different times;

FIG. 8 is a cross-sectional view of a sciatic nerve of a mouse according to a preferred embodiment 3 of the present invention;

FIG. 9 is a longitudinal section of the sciatic nerve of a mouse according to a preferred embodiment 3 of the present invention;

FIG. 10 is a photograph of immunofluorescence staining of sciatic nerve of mouse according to a preferred embodiment 3 of the present invention;

FIG. 11 is an imaging view of a simulated surgical microscope of the preferred embodiment 4 of the present invention;

among them, 1-sciatic nerve, 2-muscle, 3-adventitia, 4-perineurium.

Detailed Description

The technical contents of the preferred embodiments of the present invention will be more clearly and easily understood by referring to the drawings attached to the specification. The present invention may be embodied in many different forms of embodiments and the scope of the invention is not limited to the embodiments set forth herein.

Example 1 preparation and Performance characterization of fluorescent conjugated Polymer nanoprobes

0.25mL of 1mg/mL poly [ (9, 9-dioctylfluorenyl-2, 7 diyl) - (1, 4-benzo- {2,1', 3} -thiadiazole) was taken]10% benzothiadiazole (y) (PFBT) solution and 0.25mL of 1mg/mL amino-terminated polymethylmethacrylate (MMA-NH)₂) The solution was added to 1.5mL of Tetrahydrofuran (THF) with PFBT stock and MMA-NH₂The stock solutions were all prepared with THF as the solvent. The mixture was sonicated in a water bath for 5 minutes. Under the condition of probe ultrasonic treatment, the mixed solution is quickly transferred into 10mL of deionized water, and the probe ultrasonic treatment is carried out for 1 minute. Introducing N at the temperature of 45 DEG C₂The THF in the solution was removed in 40 minutes to prepare PFBT-NH at 0.025mg/mL₂Fluorescent conjugated polymer nanoprobes.

Taking the concentration of 0.25mLIs 1mg/mL of poly [ (9, 9-dioctylfluorenyl-2, 7 diyl) - (1, 4-benzo- {2,1', 3} -thiadiazole)]A10% benzothiadiazole (y) (PFBT) solution and 0.25mL of a 1mg/mL styrene-polyethylene glycol-carboxy (PS-PEG-COOH) solution were added to 1.5mL Tetrahydrofuran (THF), where both the PFBT stock and the PS-PEG-COOH stock were prepared using THF as the solvent. The mixture was sonicated in a water bath for 5 minutes. Under the condition of probe ultrasonic treatment, the mixed solution is quickly transferred into 10mL of deionized water, and the probe ultrasonic treatment is carried out for 1 minute. Then N is introduced at the temperature of 45 DEG C₂THF in the solution is removed in 40 minutes, and the PFBT-COOH fluorescent conjugated polymer nano-probe with the concentration of 0.025mg/mL is prepared.

FIG. 1 and FIG. 2 show a PFBT-COOH fluorescent conjugated polymer nanoprobe and a PFBT-NH, respectively₂The ultraviolet absorption spectrum and the fluorescence emission spectrum of the fluorescent conjugated polymer nanoprobe can be seen in the figure, and the PFBT-COOH fluorescent conjugated polymer nanoprobe and PFBT-NH can be seen in the figure₂The fluorescent conjugated polymer nano probe has maximum absorption at about 380nm and maximum emission at about 540nm, and PFBT-NH₂The maximum absorption intensity and the maximum fluorescence emission intensity of the fluorescent conjugated polymer nano probe are both higher than those of the PFBT-COOH fluorescent conjugated polymer nano probe.

FIG. 3 is a distribution diagram of hydrated particle size of a PFBT-COOH fluorescent conjugated polymer nanoprobe, and FIG. 4 is a diagram of PFBT-NH₂The hydrated particle size distribution diagram of the fluorescent conjugated polymer nanoprobe shows that the hydrated particle sizes of the two probes are about 60nm, the average polydispersity index (PDI) of the PFBT-COOH fluorescent conjugated polymer nanoprobe is 0.108, and the PFBT-NH is₂The PDI of the fluorescent conjugated polymer nano probe is 0.099, and the fluorescent conjugated polymer nano probe shows better colloidal stability.

Example 2 imaging of fluorescent conjugated Polymer nanoprobes on mammalian peripheral nerves

The imaging mode of the fluorescent conjugated polymer nanoprobe on peripheral nerves of mammals comprises a direct exposure mode imaging and an intramuscular injection mode imaging, which are respectively explained as follows:

1. direct exposure mode imaging

By removing the skin muscles and adipose tissues overlying the sciatic nerve,the sciatic nerve of the mice was surgically exposed. Respectively dripping 50 μ L of aqueous solution of fluorescent conjugated polymer nanoprobe on the exposed sites of left and right sciatic nerve to cover all tissues (nerve, muscle, fat and fascia) in the exposed region of operation, wherein the aqueous solution of PFBT-COOH fluorescent conjugated polymer nanoprobe is dripped on the left sciatic nerve, and the aqueous solution of PFBT-NH is dripped on the right sciatic nerve₂And (3) incubating the aqueous solution of the fluorescent conjugated polymer nanoprobes for a certain time, removing the aqueous solution of each probe, washing the whole dyeing area for more than ten times by using PBS (phosphate buffer solution) to remove any unbound fluorescent conjugated polymer nanoprobes, and slightly sucking the liquid at the sciatic nerve by using dust-free paper. After the staining was completed, a live fluorescence image of the entire stained area was acquired.

FIG. 5 shows a PFBT-COOH fluorescent conjugated polymer nanoprobe and PFBT-NH₂The fluorescence conjugated polymer nanoprobe is used for imaging a living body fluorescence image of sciatic nerve of a mouse, a control group (PBS incubation) sciatic nerve living body fluorescence imaging image is sequentially arranged from top to bottom, the fluorescence conjugated polymer nanoprobe is used for incubating for 5min, the fluorescence conjugated polymer nanoprobe is used for incubating for 30min, wherein the left sciatic nerve is incubated with a PFBT-COOH fluorescence conjugated polymer nanoprobe, the right sciatic nerve is incubated with a PFBT-NH₂Fluorescent conjugated polymer nanoprobes. As can be seen from fig. 5, the fluorescent conjugated polymer nanoprobe and the application method thereof in imaging of peripheral nerves have a good imaging effect on sciatic nerve 1, and have no significant non-specific uptake in peripheral tissues (muscle 2, fat, etc.).

FIG. 6 is an in vitro fluorescence imaging graph of sciatic nerve of mice in control group and experimental group at incubation time of 5min, 30min and 60min, wherein part I is an in vitro fluorescence imaging graph of sciatic nerve of control group after incubation in buffer PBS, left sciatic nerve in part I, and right sciatic nerve in part I; II, III and IV parts are in-vitro fluorescence imaging graphs of sciatic nerve at incubation time of 5min, 30min and 60min, the left side of the II, III and IV parts is PFBT-COOH fluorescence conjugated polymer nanoprobe incubated with left sciatic nerve, and the right side is PFBT-NH incubated with right sciatic nerve₂Fluorescent conjugated polymer nanoparticlesAnd (3) a probe. As can be seen in FIG. 6, the PFBT-COOH fluorescent conjugated polymer nanoprobe and PFBT-NH₂The fluorescent conjugated polymer nano-probe can be stably combined with sciatic nerve to realize the tracing function.

FIG. 7 is a quantification of the mean fluorescence intensity at the sciatic nerve of mice incubated for 5min, 30min and 60min, as can be seen from FIG. 7, PFBT-NH in the sciatic nerve of mice at each incubation period₂The average fluorescence intensity of the fluorescent conjugated polymer nano-probe is higher than that of the PFBT-COOH fluorescent conjugated polymer nano-probe. This is in contrast to PFBT-NH₂The nanoprobe is better related to lipid solubility. Meanwhile, with the increase of the incubation time of the sciatic nerve and the probe, the average fluorescence intensity of the two probes at the sciatic nerve of the mouse is increased.

2. Intramuscular mode imaging

And concentrating the aqueous solution of the fluorescent conjugated polymer nanoprobe by 2-50 times by using an ultrafiltration tube. And (3) anaesthetizing the mouse, after anaesthetizing, injecting 25-50 mu L of probe solution into the calf muscle on one side of the mouse by using an insulin needle, and observing the clear development of the probe on the sciatic nerve after 30 min-24 h.

Example 3 determination of binding site of conjugated Polymer nanoprobe to mouse sciatic nerve

And after the living body fluorescence imaging is finished, taking out the sciatic nerve of the mouse for an in vitro section experiment. OCT (water-soluble mixture of polyethylene glycol and polyvinyl alcohol) is embedded and then placed in a refrigerator at minus 80 ℃, frozen for more than 4 hours, and the tissue is cut into sections with the thickness of 10 mu m, then HE staining is carried out, and fluorescence imaging is carried out on the tissue after the sections are sealed.

Fig. 8, 9 and 10 are section diagrams of sciatic nerve excised tissue of experimental groups, fig. 8 is a cross section fluorescence imaging and HE staining imaging diagram of sciatic nerve of mice, and fig. 9 is a longitudinal section fluorescence imaging and HE staining imaging diagram of sciatic nerve of mice, and it can be seen from the picture results that the probe mainly gathers in the nerve exomold 3 and the nerve bundle membrane 4 and does not enter into nerve cells. FIG. 10 is a photograph of ischiadic nerve immunofluorescence staining, wherein DAPI (4, 6-diamidine-2-phenylindole dihydrochloride) marks nuclei, MBP (myelin basic protein) marks nerve cells, PFBT is a fluorescence conjugated polymer nanoprobe signal channel, FIG. 10 shows the aggregation of fluorescence conjugated polymer nanoprobes on the nerve adventitia 3 and the nerve bundle membrane 4, and immunofluorescence imaging results further confirm that the probe does not enter the nerve cells, which suggests higher biosafety of the probe.

Example 4 surgical microscope imaging simulation of clinical intraoperative navigation

In order to confirm the feasibility of the fluorescent conjugated polymer nanoprobe in imaging peripheral nerves in a more clinically significant environment, a fluorescent stereomicroscope was used to test the ability of the fluorescent conjugated polymer nanoprobe to image peripheral nerves. The specific procedure was as described in example 2, wherein mice were incubated with PFBT-NH on both the left and right sciatic nerves₂And (3) carrying out 30min on the fluorescent conjugated polymer nanoprobe, and carrying out imaging test by using a fluorescent stereomicroscope after the related operation is finished.

Fig. 11 is an imaging diagram of a simulated surgical microscope, and fig. 11 shows a live imaging diagram of a control group (PBS incubation) stereo fluorescence microscope, a live imaging diagram of an experimental group (fluorescence conjugated polymer nanoprobe incubation) stereo fluorescence microscope, and an ex vivo sciatic nerve imaging diagram, respectively. As can be seen from FIG. 11, the fluorescent conjugated polymer nanoprobe in the surgical environment can clearly display the morphology and the boundary of sciatic nerve, is helpful for surgeons to visually and visually distinguish peripheral nerves, and has great clinical application value.

The foregoing detailed description of the preferred embodiments of the invention has been presented. It should be understood that numerous modifications and variations could be devised by those skilled in the art in light of the present teachings without departing from the inventive concepts. Therefore, the technical solutions available to those skilled in the art through logic analysis, reasoning and limited experiments based on the prior art according to the concept of the present invention should be within the scope of protection defined by the claims.

Claims

1. The application of a fluorescent conjugated polymer nano probe in the preparation of a peripheral nerve imaging agent is characterized in that the application method comprises the following steps:

step 1) preparing the fluorescent conjugated polymer nanoprobe, wherein the fluorescent conjugated polymer nanoprobe comprises a common fluorescent conjugated polymer nanoprobe and a near-infrared fluorescent conjugated polymer nanoprobe;

step 3) imaging the peripheral nerves of the mammal with the colloidal aqueous solution of the fluorescent conjugated polymer nanoprobe obtained in the step 2), wherein the imaging mode comprises direct exposure imaging and intramuscular injection imaging;

the general fluorescent conjugated polymer nano probe is prepared from a fluorescent conjugated polymer and a surface ligand, and the near-infrared fluorescent conjugated polymer nano probe is prepared from a fluorescent conjugated polymer, a surface ligand and a near-infrared dye; the fluorescent conjugated polymer is poly [ (9, 9-dioctyl fluorenyl-2, 7 diyl) - (1, 4-benzo- {2,1', 3} -thiadiazole)]10% benzothiadiazole (y) (PFBT); the surface ligand comprises amino-terminal polymethyl methacrylate (PMMA-NH)₂) Or styrene-polyethylene glycol-carboxyl (PS-PEG-COOH); the near infrared dye includes 2, 3-naphthalocyanine silicon bis- (trihexylsilyl oxide) (NIR 775).

2. The use of the fluorescent conjugated polymer nanoprobe of claim 1 in the preparation of a peripheral nerve imaging agent, wherein the preparation method of the general fluorescent conjugated polymer nanoprobe in step 1) comprises the following steps:

1.1, respectively dissolving the fluorescent conjugated polymer and the surface ligand in tetrahydrofuran to prepare a conjugated polymer stock solution and a surface ligand stock solution;

step 1.2, sequentially adding a stock solution of the fluorescent conjugated polymer and a stock solution of the surface ligand into tetrahydrofuran to obtain a first solution system; the mass concentration ratio of the fluorescent conjugated polymer to the surface ligand is 0.8-1.2, and water bath ultrasound is carried out for 3-5 minutes to obtain a mixed solution A;

3. The use of the fluorescent conjugated polymer nanoprobe of claim 1 in the preparation of a peripheral nerve imaging agent, wherein the preparation method of the near-infrared fluorescent conjugated polymer nanoprobe in step 1) comprises the following steps:

step 1.5, dissolving the fluorescent conjugated polymer, the surface ligand and the near-infrared dye in tetrahydrofuran respectively to prepare a conjugated polymer stock solution, a surface ligand stock solution and a near-infrared dye stock solution;

step 1.6, sequentially adding a stock solution of the fluorescent conjugated polymer, a stock solution of the surface ligand and a stock solution of the near-infrared dye into tetrahydrofuran to obtain a second solution system, wherein the mass concentration ratio of the fluorescent conjugated polymer to the surface ligand is 0.8-1.2, and performing water bath ultrasound for 3-5 minutes to obtain a mixed solution C;

step 1.7, adding the mixed solution C into the water phase under the condition of probe ultrasound or water bath ultrasound to obtain mixed solution D;

and 1.8, introducing the nitrogen into the mixed solution D, and removing the organic solvent in the mixed solution D to obtain the near-infrared fluorescent conjugated polymer nano probe.

4. The use of the fluorescent conjugated polymer nanoprobe of claim 2 or 3 in the preparation of a peripheral nerve imaging agent, wherein the power of the probe ultrasound is set to 10% to 20%; the power of the water bath ultrasound is 80-100W.

5. The use of the fluorescent conjugated polymer nanoprobe in the preparation of a peripheral nerve imaging agent according to claim 3, wherein the content of the near-infrared dye in each 2mL of the second reaction system in the step 1.6 is 1-3 μ g.

6. The use of the fluorescent conjugated polymer nanoprobe of claim 1 in the preparation of a peripheral nerve imaging agent, wherein the step of direct exposure imaging in step 3) is as follows:

surgically substantially exposing the sciatic nerve of the mammal; dripping the colloidal aqueous solution of the fluorescent conjugated polymer nano probe obtained in the step 2) on the exposed sciatic nerve for incubation; after the incubation is finished, the sciatic nerve of the mammal is fully washed by PBS, and the liquid at the sciatic nerve is slightly sucked up by using a dust-free paper.

7. The use of the fluorescent conjugated polymer nanoprobe in the preparation of a peripheral nerve imaging agent according to claim 6, wherein the concentration of the colloidal aqueous solution of the fluorescent conjugated polymer nanoprobe is 0.025mg/mL to 0.25 mg/mL; the dripping volume of the fluorescent conjugated polymer nano probe colloidal aqueous solution is 50-100 mu L; the incubation time of the aqueous solution of the fluorescent conjugated polymer nano probe dropped on sciatic nerve is 5 min-60 min.

8. The use of the fluorescent conjugated polymer nanoprobe of claim 1 in the preparation of a peripheral nerve imaging agent, wherein the step of intramuscular injection imaging of step 3) comprises the following steps: concentrating the fluorescent conjugated polymer nano probe colloidal aqueous solution by 2-50 times by using an ultrafiltration tube, anesthetizing the mammal, after anesthesia, injecting 25-50 mu L of probe solution into the calf muscle on one side of the mammal, and observing the clear development of the probe on the sciatic nerve after 30 min-24 h.

9. The use of the fluorescent conjugated polymer nanoprobe in the preparation of a peripheral nerve imaging agent as claimed in claim 8, wherein the concentration of the colloidal aqueous solution of the fluorescent conjugated polymer nanoprobe is 0.025mg/mL to 0.25 mg/mL.