Near-infrared living body imaging method based on acupoint injection model and application of near-infrared living body imaging method

Abstract

The invention discloses a near-infrared living body imaging method based on an acupoint injection model, and the method comprises the steps: employing a near-infrared dye as a tracer agent, injecting the tracer agent to the body surface of an animal through an acupoint, carrying out the living body fluorescence imaging through a near-infrared imaging device, and achieving the long-time visualization of the transportation characteristics of an acupoint injection substance. The invention further discloses application of the near-infrared living body imaging method in research on transportation behaviors of substances on animal body surfaces. According to the method, the transmission behavior of the substance on the surface layer of the living animal can be visually studied, the transmission characteristics of the substance can be revealed, a new view angle is provided for understanding the dynamic transmission mechanism of the substance related to acupuncture points and meridians and collaterals in traditional Chinese medicine, and the potential of regulating and controlling the immune system by injecting the nano material into the acupuncture points is also revealed.

Description

Technical Field

[0001] The present invention relates to the field of biological imaging technology, and more specifically to a near-infrared in vivo imaging method based on an acupoint injection model and an application thereof. Background Art

[0002] In recent years, the research on the neuroanatomical basis and new transmission systems based on acupoints has received widespread attention. Acupoints refer to specific points or areas on the human meridians that have special physiological functions. These acupoints can be stimulated by acupuncture, massage, pressing, and moxibustion to regulate the body's physiological functions and achieve therapeutic effects. For example, in a set of sophisticated cross-genetic experiments, Ma Qiufu and his team found that low-intensity electroacupuncture stimulation of the "Zusanli (ST36)" acupoint can specifically inhibit systemic inflammatory responses by regulating the vagus-adrenal reflex, while similar stimulation is ineffective for the "Tianshu (ST25)" acupoint. This milestone study laid the neuroanatomical basis for the anti-inflammatory effect of electroacupuncture of the "ST36" acupoint. In similar studies, Kovacs et al. injected low-impedance acupoints in dogs. 99m After Tc, a specific path for rapid longitudinal migration was discovered. Another study showed that after the injection of fluorescent nanoparticles at the "ST36" acupoint in rats, the presence of a primitive vascular system was observed in the epididymis of the sciatic nerve of rats. Recently, Li Hongyi and his team used magnetic resonance imaging (MRI) and two-photon imaging techniques to discover an extracellular interstitial fluid transport system in the structural framework of human fibrous connective tissue, and also found similar fluid flow paths in rabbits. This system provides a new pathway for the transport of substances from the body surface to deep tissues. These studies show that acupoints are not only the basis of traditional therapies, but also have a unique material delivery system that can guide the flow and transport of substances in specific areas of the body. Therefore, it is necessary to develop methodologies to explore the transport behavior of substances on the surface of living animals, which can provide a new perspective for understanding the dynamic transport mechanisms associated with acupoints and meridians.

[0003] Near-infrared (NIR) imaging provides a versatile means for non-invasive, dynamic observation of living animals. Compared with visible light, near-infrared (NIR, 700-1700nm) imaging has stronger tissue penetration and significantly reduces the interference of biological background autofluorescence, thereby achieving higher imaging contrast and more sensitive and accurate in vivo detection. Therefore, near-infrared fluorophore dyes can be used as simplified material models to study local transport behavior on the surface of living animals. Summary of the invention

[0004] The purpose of the present invention is to provide a near-infrared in vivo imaging method based on an acupoint injection model and its application, so as to solve the problem that the existing methods cannot sensitively and accurately detect the transport behavior of substances on the surface of living animals.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] According to the first aspect of the present invention, there is provided a near-infrared in vivo imaging method based on an acupuncture point injection model, which is not used for the diagnosis and treatment of diseases. A near-infrared dye is used as a tracer, which is injected into the animal surface through the acupuncture points, and in vivo fluorescence imaging is performed using a near-infrared imaging device to achieve long-term visualization of the transport characteristics of acupuncture point injection substances.

[0007] According to a preferred embodiment of the present invention, the near-infrared in vivo imaging method comprises the following steps: 1) the near-infrared dye is dispersed in physiological saline and ultrasonically mixed; 2) a sterile microinjection pump is used to add the dispersed near-infrared dye solution and inject it into the animal body surface through acupuncture points; 3) a near-infrared in vivo imaging device is used to observe the transmission behavior of the dye on the animal body surface.

[0008] The acupoints include upper limb acupoints, such as Shenmen, Hegu, Neiguan, etc., and lower limb acupoints, such as Zusanli, Taixi, Yongquan, etc. The depth of acupuncture is 2 to 4 mm.

[0009] The near-infrared dyes include: fluorescent small molecule dyes, organic fluorescent nanoparticles and inorganic nano fluorescent quantum dots, and their emission bands are in the near-infrared region, with an emission wavelength of >700nm.

[0010] The near-infrared in vivo imaging method comprises: dissolving or dispersing the near-infrared dye in a physiological saline solution, wherein the concentration of the fluorescent small molecule dye and the organic fluorescent nanoparticles is 0.5 to 1.5 μmol / L, and the concentration of the inorganic nano fluorescent quantum dots is 0.5 to 1.5 mg / mL.

[0011] Preferably, the small molecule fluorescent dyes include Cy5 and Cy7.

[0012] Preferably, the organic fluorescent nanoparticles include tetrahedral framework nucleic acid nanoparticles labeled with Cy5 or Cy7, wherein the tetrahedral framework nucleic acid is self-assembled from three non-fluorescent DNA chains and one Cy5 or Cy7 labeled DNA chain.

[0013] Preferably, the DNA tetrahedron applicable to the present invention has a side length of 7-37 bp and a particle size of 3-13 nm.

[0014] Preferably, the inorganic nano fluorescent quantum dots include: quantum dots having emission bands in the near-infrared region II, such as Ag2S and PbS.

[0015] Preferably, the volume of the near-infrared dye injected into each acupoint is 10-50 μL, and the injection is performed using a sterile injection needle connected to a microinjection pump at an injection speed of 0.5-1 μL / second.

[0016] Preferably, the near-infrared in vivo imaging method comprises: performing near-infrared in vivo fluorescence imaging at different time points before and after injection.

[0017] Preferably, the tetrahedral nanostructures labeled with fluorescent small molecule dyes and fluorescent molecules are subjected to near-infrared imaging at a time point within a period of 0 to 7 hours after injection.

[0018] Preferably, the inorganic fluorescent nano-quantum dots are used for in vivo imaging of mice at a time point within a period of 0 to 28 days after injection.

[0019] Preferably, the near-infrared in vivo imaging method further comprises: 24 hours after the near-infrared dye is injected via acupoints, dissecting major organs such as the heart, liver, spleen, lungs, and kidneys for near-infrared two-zone fluorescence imaging.

[0020] According to a preferred embodiment of the present invention, a near-infrared in vivo imaging method based on an acupoint injection model is provided, and the near-infrared in vivo imaging method comprises the following steps:

[0021] S1: Near-infrared dyes were dispersed in physiological saline, the concentration of small molecules and organic fluorescent nanoparticles was 0.5-1.5 μmol / L, and the concentration of inorganic fluorescent nanoparticles was 0.5-1.5 mg / mL;

[0022] S2: The prepared fluorescent dye and fluorescent nanoparticles are injected at a speed of 0.5 to 1 μL / s using a syringe pump;

[0023] S3: Near-infrared in vivo imaging at different time points from 2 minutes to 28 days after acupoint injection;

[0024] S4: After in vivo imaging of mice, major organs such as the heart, liver, spleen, lungs, and kidneys were dissected for organ imaging.

[0025] When Ag2S quantum dots are used, step S5 is also included: after organ imaging, the organ is weighed and digested, and the Ag element content in the organ and blood is analyzed by ICP-MS, and the in vivo distribution and metabolism of Ag2S nanoparticles after acupuncture injection are analyzed.

[0026] Preferably, in step S4, 24 hours after the injection of Ag2S nanoparticles, major organs such as the heart, liver, spleen, lungs, and kidneys are dissected for near-infrared second-zone fluorescence imaging.

[0027] Preferably, in step S5, the Ag element content is analyzed after the organs are weighed 24 hours after the injection of Ag2S nanoparticles, and the Ag element analysis in the blood is performed using ICP-MS at time points within the period of 0 to 28 days after the injection.

[0028] It should be understood that the method of the present invention is not limited to Ag2S nanoparticles, and therefore is not limited to checking the Ag element content. This is only used as a preferred embodiment rather than a limitation.

[0029] According to a second aspect of the present invention, there is provided a method for near-infrared in vivo imaging based on an acupoint injection model for use in the study of the transport behavior of substances on the body surface of living animals.

[0030] According to a third aspect of the present invention, there is provided an application of a near-infrared in vivo imaging method based on an acupoint injection model in the preparation of drugs.

[0031] The creativity of the present invention mainly lies in: 1) subcutaneous, intramuscular and intravenous injections are used as control injection sites to achieve a comparison of the transmission characteristics of acupoint injection and conventional injection; 2) a real-time, non-invasive near-infrared in vivo imaging method based on acupoint injection models such as Zusanli and Shenmen is developed; 3) based on the research findings of the present invention, most of the near-infrared dyes will remain in the transmission path, and the fluorescence intensity in the lymph nodes is relatively strong, so it is speculated that a part of them enters the lymphatic circulation, and only a small amount of near-infrared dyes enters the blood circulation or accumulates in the main organs, and its organ distribution amount is less than 1 / 50 of that of intravenous injection, which provides a new method for the future transmission of substances through living acupoints and meridians; 4) the present invention not only provides an acupoint injection model and method for the study of the transportation behavior of substances on the surface of living animals using near-infrared dyes, but also provides a near-infrared imaging preferred nanoparticle solution for the study of acupoints and meridians.

[0032] According to a near-infrared imaging method based on an acupoint injection model provided by the present invention, the positive effects compared with the prior art are as follows:

[0033] Acupuncture point injection of near-infrared dyes and nanoparticles shows a quasi-linear transmission feature. Compared with subcutaneous injection and intramuscular injection, which diffuse outward from the injection point, acupuncture point injection shows a quasi-linear transmission trajectory within a certain range.

[0034] The local transmission time of nanoparticles is significantly longer than that of fluorescent small molecule dyes. The duration of small molecule fluorescence is 30 to 60 minutes, and the duration of nanoparticle fluorescence is 6 hours to 28 days. It should be understood that different substances have different transmission speeds. Small molecules are transported faster, while nanoparticles are transported slower, so the fluorescence duration of nanoparticles is long. In the case of the same nanoparticles, the duration of local fluorescence is also related to the stability of the nanoparticles in the biological environment. Cy5-TDN is a type of DNA nanostructure. It is easily degraded in the living system due to the presence of multiple enzymes. The local fluorescence duration is ~6 hours, while Ag2S nanoparticles are relatively stable in the living system, so their fluorescence signals can still be detected 28 days after acupoint injection.

[0035] Ag2S nanoparticles in the near-infrared region II show better imaging effects, can be traced for a long time within 28 days, and can achieve long-term dynamic imaging observation. It should be understood that, on the one hand, the nanoparticles after acupoint injection are slower in transport than small molecules due to their nanoscale size. On the other hand, Ag2S is an inorganic nanoparticle, and the particle structure is stable in the living system and is not easy to degrade, so the fluorescence signal can still be detected 28 days after injection.

[0036] Compared with subcutaneous and intramuscular injections, acupuncture point injections of nanomaterials have a shorter peak time in the blood and a lower peak concentration. The reason is that the drug transmission of intramuscular and subcutaneous injections usually diffuses outward from the injection point, while acupuncture point injections show a unique longitudinal linear transmission pattern. The structural characteristics of the acupuncture point are rich in peripheral blood vessels and lymphatic vessels, which can more effectively take substances into the blood. Compared with the local diffusion pattern of subcutaneous and intramuscular injections, acupuncture point injections significantly accelerate the transmission speed of nanoparticles, so their peak time in the blood is shorter. However, since nanoparticles are distributed in large quantities in the transmission path, their peak concentration in the blood is lower.

[0037] Compared with intravenous injection, acupuncture injection of nanomaterials has less distribution in major organs such as liver, spleen, heart, lungs, and kidneys, which is about 1 / 200 to 1 / 50 of intravenous injection, thus avoiding the side effects of nanoparticles in various organs.

[0038] Better yet, acupuncture-injected nanoparticles were relatively more distributed in the lymph nodes and spleen immune organs, revealing the potential of acupuncture-injected nanomaterials for regulating the immune system.

[0039] In summary, the present invention provides a near-infrared in vivo imaging method based on an acupoint injection model and its application. According to the method of the present invention, the transport behavior of substances in the surface of living animals can be visualized and its transport characteristics can be revealed. This provides a new perspective for understanding the dynamic transport mechanism of substances related to acupoints and meridians in traditional Chinese medicine, and also reveals the potential of acupoint injection of nanomaterials for regulating the immune system. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The results obtained in Example 1 include: the emission spectrum of the Cy5-labeled DNA tetrahedron (A), the hydrated particle size characterization (B) and the near-infrared emission spectrum of the silver sulfide quantum dots Ag2S QDs (C), the hydrated particle size characterization (D);

[0041] Figure 2 This is a near-infrared image of Cy5 and Cy5-TDN injected into Shenmen acupoint 15 minutes after Example 2;

[0042] Figure 3These are near-infrared images of different nanoparticles Cy5-TDN and Ag2S QDs at different time points after injection into the Shenmen acupoint in Example 3;

[0043] Figure 4 The near-infrared imaging images of Ag2S QDs injected at different acupoints in Example 4;

[0044] Figure 5 The near-infrared imaging images of Ag2S QDs injected by different injection methods in Example 5;

[0045] Figure 6 The ICP-MS analysis of the Ag content in the blood after the Ag2S QDs were injected by different injection methods in Example 5;

[0046] Figure 7 The in vivo imaging of Ag2S QDs after acupuncture and intravenous injection and the near-infrared imaging of their distribution in organs in Example 6;

[0047] Figure 8 ICP-MS analysis of Ag content in blood after acupuncture and intravenous injection of Ag2S QDs in Example 7. DETAILED DESCRIPTION

[0048] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0049] Example 1: Construction and characterization of near-infrared nanoparticles

[0050] Cy5, Cy5-labeled DNA tetrahedral nanoparticles and Ag2S nanoparticles were selected. The side length of the DNA tetrahedral nanoparticles was 20 bp and the original particle size was about 7 nm. 100 μL of the synthesized Cy5-TDN with a concentration of 1 μmol / L was taken, and its emission peak was measured at 670 nm. Then it was placed in a Malvern particle size analyzer and the particle size distribution was measured at 4°C. The hydrated particle size of the Cy5-TDN nanostructure was ~10 nm. The Ag2S nanoparticles were also characterized by fluorescence spectrum and hydrated particle size. It had a fluorescence emission peak at 1200 nm and its hydrated particle size was about 10 nm ( Figure 1 ).

[0051] Example 2: Dynamic imaging characteristics of Cy5 small molecule dye and Cy5-labeled DNA tetrahedral nanoparticles after acupuncture injection

[0052] Cy5-TDN is constructed from four parent chains (one of which is connected to Cy5 fluorescence). The four parent chains are fully mixed in TM buffer (10mmol / L Tris-HCl, 5mmol / L MgCl2, pH 8.0) with a final concentration of 1μmol / L. The total reaction volume is controlled to be 100μL. After a brief centrifugation, it is placed in a BIO-RAD PCR instrument and heated to 95℃ for denaturation for 10 minutes, and then quickly cooled to 4℃ to obtain the DNA tetrahedral structure Cy5-TDN.

[0053] BALB / c mice, female, 6-8 weeks old, weighing 25±2g, fluorescent small molecule dye Cy5 and Cy5-TDN nanostructures were formulated into a concentration of 1μM and injected through the Shenmen acupoint with an injection volume of 25μL. In vivo imaging was performed at 2min, 5min, 10min, 15min, 30min, 1h, 3h, 6h, and 7h after injection. The excitation wavelength was 630nm and the exposure time was 1s. When the local fluorescence at the injection site disappeared, imaging was stopped to obtain the transmission characteristics of small molecule dyes and nanoparticles after acupoint injection. The transmission paths of the two were similar, and the nanoparticles stayed in the local area for a longer time ( Figure 2 ).

[0054] Example 3: Comparison of different types of nanoparticles delivered via acupoint injection

[0055] Take 1 μM concentration of Cy5-TDN and 1 mg / mL Ag2S nanoparticles. The preparation of Cy5-TDN is the same as Example 2.

[0056] BALB / c mice, female, 6-8 weeks old, weighing 25±2g, were injected with 1μM concentration of Cy5-TDN and 1mg / mL of Ag2S nanoparticles through Shenmen acupoint, with an injection volume of 25μL. Near-infrared zone I and zone II live imaging was performed at different time points after injection, 2min, 15min, 1h, 6h, 1d, 7d, and 28d, respectively, to compare the transmission of different nanoparticles through acupoints. The transmission paths of the two nanoparticles were similar. The local fluorescence signal of Cy5-TDN lasted for 6h, and the near-infrared zone II fluorescence signal of Ag2S nanoparticles could still be detected on the 28th day after injection ( Figure 3 ).

[0057] Example 4: Transport characteristics after injection of Ag2S QDs at different acupoints

[0058] BALB / c mice, female, 6-8 weeks old, weighing 25±2g, were injected with 1mg / mL Ag2S nanoparticles via Zusanli and Taixi acupoints, and near-infrared zone II images were taken at different time points. The local transmission paths of injections at different acupoints were all along the local tissues, showing linear transmission characteristics ( Figure 3-5 ).

[0059] Example 5: Effects of different injection methods on the transport characteristics of Ag2S QDs and their metabolism in blood

[0060] BALB / c mice, female, 6-8 weeks old, weighing 25±2g, were injected with 1mg / mL Ag2S nanoparticles via Zusanli, intramuscular, and subcutaneous injections, with an injection volume of 25μL. Near-infrared zone II fluorescence imaging was performed at different time points. Acupoint injection showed linear transmission characteristics, while subcutaneous and intramuscular injections showed diffusion characteristics from the injection site to the surrounding tissues ( Figure 5 After imaging, blood was collected and digested. The Ag content in the blood was detected by ICP-MS. The metabolic dynamics in the blood were analyzed. The peak concentration was reached after 3 days of acupuncture injection, while it was 14 days and 7 days for subcutaneous injection and intramuscular injection, respectively. The peak concentration of acupuncture injection was 17.1 ng / mL, which was much lower than the peak concentration of subcutaneous injection (24.1 ng / mL) and intramuscular injection (25.6 ng / mL). Figure 6 ). The amount of acupuncture injection that enters the blood circulation is smaller, avoiding systemic toxic side effects.

[0061] Example 6: Comparison of organ distribution of nanoparticles via acupoint injection and intravenous injection

[0062] BALB / c mice, female, 6-8 weeks old, weighing 25±2g, were injected with 1mg / mL Ag2S nanoparticles via acupuncture and intravenous injection, respectively, with an injection volume of 25μL. In vivo imaging was performed 24h after injection. After imaging, the organs were dissected and near-infrared zone II imaging of the main organs was performed. The differences in organ distribution of nanoparticles via acupuncture and intravenous injection were compared. The distribution of acupuncture injection in the organs is about 1 / 200-1 / 50 of that of intravenous injection, which is much lower than that of intravenous injection. It can avoid toxicity to major organs, especially the distribution in lymph nodes and spleen, showing the potential of acupuncture injection substances to regulate the immune system ( Figure 7 ).

[0063] Example 7: Comparison of blood metabolism of nanoparticles injected via acupoints and intravenously

[0064] Ag2S injection was performed according to the injection method of Example 6. Blood was collected at 0min, 2min, 5min, 10min, 15min, 30min, 1h, 3h, 6h, 12h, 1d, 4d, 7d, 14d, 21d, and 28d after injection, and digested with nitric acid. Then, the content of Ag in the blood was detected by ICP-MS. The amount of nanoparticles entering the blood was very small. Even at the peak concentration of acupoint injection, the amount of Ag in the blood after intravenous injection was still ~8 times that of acupoint injection ( Figure 8 ).

[0065] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A near-infrared in vivo imaging method based on an acupoint injection model, not used for diagnosis and treatment of diseases, characterized in that: Near-infrared dyes are used as tracers and injected into the animal's body surface through acupuncture points. In vivo fluorescence imaging is performed using near-infrared imaging equipment to achieve long-term visualization of the transport characteristics of acupuncture point injected substances.

2. The near-infrared in vivo imaging method according to claim 1, characterized in that: The acupoints include: Shenmen, Hegu, Neiguan, Zusanli, Taixi and Yongquan, and the depth of acupuncture is 2 to 4 mm.

3. The near-infrared in vivo imaging method according to claim 1, characterized in that: The near-infrared dyes include: fluorescent small molecule dyes, organic fluorescent nanoparticles and inorganic nano fluorescent quantum dots, and their emission bands are in the near-infrared region, with an emission wavelength of >700nm.

4. The near-infrared in vivo imaging method according to claim 3, characterized in that: The near-infrared in vivo imaging method comprises: dissolving or dispersing the near-infrared dye in a physiological saline solution, wherein the concentration of the fluorescent small molecule dye and the organic fluorescent nanoparticles is 0.5 to 1.5 μmol / L, and the concentration of the inorganic nano fluorescent quantum dots is 0.5 to 1.5 mg / mL.

5. The near-infrared in vivo imaging method according to claim 3, characterized in that: The small molecule fluorescent dyes include Cy5 and Cy7; the organic fluorescent nanoparticles include tetrahedral framework nucleic acid nanoparticles labeled with Cy5 or Cy7; and the inorganic nano fluorescent quantum dots include: Ag2S and PbS quantum dots.

6. The near-infrared in vivo imaging method according to claim 1, characterized in that: The volume of near-infrared dye injected into each acupoint is 10 to 50 μL, and the injection is performed using a sterile injection needle connected to a microinjection pump at an injection speed of 0.5 to 1 μL / second.

7. The near-infrared in vivo imaging method according to claim 1, characterized in that: The near-infrared in vivo imaging method comprises: performing near-infrared in vivo fluorescence imaging at different time points before and after injection.

8. The near-infrared in vivo imaging method according to claim 7, characterized in that: The near-infrared in vivo imaging method further comprises: 24 hours after the near-infrared dye is injected through the acupoints, dissecting the heart, liver, spleen, lungs, and kidneys for near-infrared two-zone fluorescence imaging.

9. Application of the near-infrared in vivo imaging method based on the acupoint injection model according to any one of claims 1 to 8 in the study of the transport behavior of substances on the body surface of living animals.

10. Use of the near-infrared in vivo imaging method based on an acupoint injection model according to any one of claims 1 to 8 in the preparation of drugs.

Images