CpG oligodeoxynucleotide photoacoustic marker and application thereof in imaging

Abstract

The invention belongs to the field of immunology and biomedical imaging, and discloses a CpG oligodeoxynucleotide photoacoustic marker and an application thereof in imaging. The CpG oligodeoxynucleotide photoacoustic marker is obtained by coupling CpG oligodeoxynucleotide to gold nanoparticles, the photoacoustic marker has good biocompatibility, can be easily swallowed by cells and has a remarkable immunostimulation effect, and the concentration of cytokines generated by stimulation of the photoacoustic marker is increased by two orders of magnitude compared with that of naked CpG oligodeoxynucleotide, so that the CpG oligodeoxynucleotide photoacoustic marker has a good immunostimulation effect. The content is obviously higher than that of a commercial carried liposome reagent; the CpG oligodeoxynucleotide photoacoustic imaging device can remarkably increase the photoacoustic signal intensity and realize high-quality photoacoustic imaging, has the advantages of high resolution, real-time lossless imaging, high sensitivity and quantitative analysis, can accurately monitor and research the dynamic distribution of CpG oligodeoxynucleotide in cells, provides a powerful tool for immunological research, and has wide application prospects. The data reliability and the experiment specificity are obviously improved, and the method has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the field of immunology and biomedical imaging, and in particular relates to CpG oligodeoxynucleotide photoacoustic labeling and its application in imaging. Background Art

[0002] The interaction between adjuvants and cells and the activation of immune cells activate systemic immunity, which is an important prerequisite for the effectiveness of vaccines. In the immune system, macrophages have the ability to engulf and eliminate pathogens and regulate and maintain immune balance. In addition, they are also responsible for processing and presenting antigens to cause the activation of other immune cells.

[0003] The phagocytic and antigen-presenting abilities of macrophages ensure a rapid and effective immune response, improve the effectiveness of vaccines, and provide long-term protection. Therefore, exploring the dynamic process, distribution, and signal transduction of vaccine adjuvants in macrophages is helpful to understand and study the phagocytic function of macrophages, which is of great significance for optimizing vaccine design and improving vaccination effectiveness.

[0004] Adjuvant is a non-specific immunopotentiator that is mixed with antigens or injected into the human body before antigens to enhance the body's immune response to antigens or change the type of immune response. CpG-motif oligodeoxynucleotides (CpG) can increase the immunogenicity of antigens by improving antigen uptake by antigen-presenting cells, activating the functional maturation of antigen-presenting cells, and producing cytokines and chemokines. CpG oligodeoxynucleotides have the advantages of stability, low cost, easy synthesis, high efficiency and low toxicity, and are widely used adjuvants in vaccine research. However, CpG oligodeoxynucleotides are easily degraded by nucleases, and have low absorption efficiency and photoacoustic conversion efficiency in the near-infrared band, resulting in insufficient intensity of the generated photoacoustic signal, making it impossible to achieve high-resolution imaging.

[0005] Photoacoustic imaging is becoming more and more common in biomedical applications and can provide anatomical, functional and molecular information. The principle of this imaging method is that when a pulsed laser is irradiated onto biological tissue, the tissue inside absorbs the energy of the pulsed light, causing instantaneous heating and expansion, thereby generating ultrasonic waves. The ultrasonic transducer located on the surface of the tissue receives these ultrasonic waves and reconstructs an image of the light energy absorption distribution inside the tissue based on the detected photoacoustic signals.

[0006] Transmission photoacoustic microscopy focuses on optical excitation and acoustic detection, similar to confocal microscopy. The confocal configuration maximizes detection sensitivity, has high lateral resolution and rich optical contrast, and its non-destructive imaging properties make it suitable for living cell and in vivo research. It can observe and record the entire process of cell phagocytosis of drugs in detail, and dynamically detect the response of cells under different conditions in real time. At the same time, its resolution can study the distribution and function of materials inside cells, providing a powerful tool for in-depth understanding of cell phagocytosis mechanisms, optimizing adjuvant design, and promoting related biomedical research and clinical applications.

[0007] There have been many studies on the long-term observation of dynamic imaging of CpG oligodeoxynucleotides in cells using fluorescent dye labeling and laser confocal microscopy. However, long-term exposure may cause photobleaching and phototoxicity problems, which significantly affect the treatment of live cell imaging. Summary of the invention

[0008] The object of the present invention is to overcome at least one disadvantage of the prior art and to provide a CpG oligodeoxynucleotide photoacoustic label and its application in imaging.

[0009] The technical solution adopted by the present invention is: The first aspect of the present invention provides: The preparation method of CpG oligodeoxynucleotide photoacoustic labeling comprises the following steps: 1) suspending gold nanoparticles in deionized water, adding thiolated CpG oligodeoxynucleotide solution, and incubating with shaking to obtain a mixture S1; 2) adding phosphate buffer to the mixture S1, and incubating with shaking to obtain a mixture S2; 3) The mixture S2 is centrifuged to obtain a precipitate, and the unbound CpG oligodeoxynucleotide is removed by washing to obtain the CpG oligodeoxynucleotide photoacoustic label.

[0010] In some examples of CpG oligodeoxynucleotide photoacoustic labeling, the CpG oligodeoxynucleotide is a type B CpG oligodeoxynucleotide, which can be more efficiently covered on the surface of gold nanoparticles due to its open single-stranded structure and multiple CpG motifs, and is easy to modify and functionalize.

[0011] In some examples of CpG oligodeoxynucleotide photoacoustic labeling, the particle size of the gold nanoparticles is 5nm to 200nm. When the gold nanoparticles are small in size, they are easily engulfed by cells after binding with CpG oligodeoxynucleotides, and the spectrum is stable at 532nm, making them easily excited.

[0012] In some examples of CpG oligodeoxynucleotide photoacoustic labeling, in step 1), the shaking incubation time is 10 to 14 h; and / or in step 2), the shaking incubation time is 20 to 30 h.

[0013] The above features can be combined arbitrarily unless they conflict with each other.

[0014] The second aspect of the present invention provides: The first aspect of the present invention relates to the use of the CpG oligodeoxynucleotide photoacoustic labeling in the preparation of a living cell photoacoustic microscopy imaging enhancer.

[0015] The third aspect of the present invention provides: A method for photoacoustic microscopy of living cells comprises the following steps: Fix the living cells on the confocal dish and move a single cell to the center of the field of view; Adding the CpG oligodeoxynucleotide photoacoustic labeling described in the first aspect of the present invention to the confocal dish, adjusting the step length and scanning range of the two-dimensional galvanometer so that the scanning range of the light beam covers the field of view of the cell of interest; Adjust the nano-objective lens stage to the calibrated cell level position; The computer gives pulse light source instructions, two-dimensional galvanometer scanning instructions and acquisition card sampling timing, and processes the two-dimensional distribution photoacoustic image of cells and CpG oligodeoxynucleotide photoacoustic labeling, and repeatedly obtains multiple photoacoustic images.

[0016] In some examples of photoacoustic microscopy, the energy of the pulsed light source is 35 to 45 nJ. If the energy is too strong, it will cause cell damage, and vice versa, the photoacoustic signal of CpG-Au will be significantly reduced, and the imaging quality will deteriorate.

[0017] In some examples of photoacoustic microscopy methods, the signals are filtered through wavelet filtering and SVD filtering, and then the collected photoacoustic signals are reconstructed into photoacoustic images through a maximum projection method.

[0018] In some examples of photoacoustic microscopy, the output wavelength of the pulsed light source is 532 nm. Gold nanoparticles have a good absorption peak near 532 nm. After combining with gold nanoparticles, the UV-visible absorption spectrum of CpG-Au changes, and a strong peak is also generated at 532 nm.

[0019] In some examples of the photoacoustic microscopy method, the photoacoustic microscopy system includes a light source, an optical transmission module, a laser scanning module, a signal acquisition module, and a data processing module. The optical transmission module is used to transmit the laser to the laser scanning module; The laser scanning module is used to focus the laser beam on the target in a point and line scanning manner. Imaging area; The signal acquisition module is used to collect the photoacoustic signal excited by the laser; The data processing module is connected to the signal acquisition module, and projects the acquired photoacoustic signals to reconstruct images.

[0020] The beneficial effects of the present invention are: The CpG oligodeoxynucleotide photoacoustic labels of some examples of the present invention have good biocompatibility and are easily internalized by cells to produce significant immunostimulatory effects. The concentration of cytokines stimulated by them is two orders of magnitude higher than that of naked CpG oligodeoxynucleotides and is significantly higher than that of commercially available liposome-based reagents.

[0021] The CpG oligodeoxynucleotide photoacoustic labeling of some examples of the present invention can significantly increase the photoacoustic signal intensity and achieve high-quality photoacoustic imaging, and has the advantages of high resolution, real-time non-destructive imaging, high sensitivity and quantitative analysis. It can accurately monitor and study the dynamic distribution of CpG oligodeoxynucleotides in cells, and provides a powerful tool for immunological research, significantly improves data reliability and experimental specificity, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The figures are characterization results of photoacoustically labeled CpG oligodeoxynucleotides provided in some embodiments of the present invention; Figure A is the transmission electron microscopy result of gold nanoparticles and CpG-Au; Figure B is the ultraviolet absorption spectrum of CpG-Au.

[0023] Figure 2 It is a schematic diagram of the structure of a photoacoustic microscopy imaging system provided in some embodiments of the present invention.

[0024] Figure numerals: 1. Light source assembly; 1-1. Pulse light source; 1-2. Fiber optic collimator; 2. Optical path transmission module; 2-1. Two-dimensional scanning galvanometer; 2-2. Semi-transparent and semi-reflective mirror; 2-3. Nano-objective lens displacement stage; 2-4. Flat-field objective lens; 3-1. Microscope and camera with adjustable magnification; 4-1. Cell sample stage; 5. Signal processing assembly; 5-1. Ultrasonic transducer; 5-2. Signal amplifier; 6-1. Data acquisition component, including data acquisition card and computer; 7-1. Synchronous control component.

[0025] Figure 3 It is a schematic flow chart of a photoacoustic microscopy method provided in some embodiments of the present invention.

[0026] Figure 4 This is a result diagram of a photoacoustic microscopy imaging observation process of cell phagocytosis of CpG-Au provided by some embodiments of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention. It should also be noted that, for ease of description, only parts related to the present invention, rather than all structures, are shown in the accompanying drawings.

[0028] Example 1 The preparation method of CpG oligodeoxynucleotide photoacoustic labeling comprises the following steps: S1) 5 mL of the gold nanoparticle solution dissolved in citrate was extracted, centrifuged at 1000 g for 20 min to obtain pure gold nanoparticles, and resuspended in 1 mL of deionized water; S2) then adding the thiolated CpG oligodeoxynucleotide solution to a final concentration of 30 μg / mL; S3) After the mixture was incubated at room temperature on a shaker at 120 rpm for 12 h, 100 mM phosphate buffered saline (PBS) (pH 7.4) was added to the mixture to a final concentration of 10 mM, and the mixture was further incubated for 24 h under continuous shaking; S4) The mixture was then centrifuged at 1000 g for 20 minutes at 4°C to obtain a successfully labeled CpG-Au precipitate; the mixture was then washed three times with PBS buffer to remove unbound CpG, and the CpG-Au was finally resuspended in PBS buffer and stored at 4°C for further use.

[0029] The following is the characterization test of the photoacoustically labeled CpG oligodeoxynucleotide in Example 1: (1) Transmission electron microscopy images of isolated single gold nanoparticles Figure 1 As shown in (A), the results show that the separated gold nanoparticles are in the form of well-dispersed spheres with good dispersion; (2) The transmission electron microscopy image of the photoacoustically labeled CpG oligodeoxynucleotide obtained in Example 1 is as follows Figure 1 As shown in (A), from the transmission electron microscopy results, it can be seen that after binding with CpG, the gold nanoparticles are wrapped by a layer of soft material (DNA chain) and the particle size increases; (3) The three materials in Example 1: gold nanoparticle solution, CpG oligodeoxynucleotide solution and CpG-Au solution were subjected to ultraviolet absorption spectroscopy detection. The results are as follows: Figure 1 As shown in (B), it can be seen that gold nanoparticles have a good absorption peak near 532nm. After binding with gold nanoparticles, the UV-visible absorption spectrum of CpG-Au changes, and a strong peak is also produced at 532nm, indicating successful binding with gold nanoparticles.

[0030] Example 2 Figure 2 It is a schematic diagram of the structure of a photoacoustic microscopy imaging provided by an embodiment of the present invention. For specific implementation methods, please refer to the following embodiments.

[0031] A pulse light source 1-1 emits a pulse laser as an excitation source, the light beam is reflected by a silver mirror and then coupled to a fiber collimator 1-2, and the light beam is transmitted through an optical fiber to obtain better control and flexibility; Then the other end is emitted from the same type of collimator to the two-dimensional scanning galvanometer 2-1, and transmitted to the next step through the semi-transparent and semi-reflective mirror 2-2, and propagated in two beams, one of which passes through the nano-objective lens displacement stage 2-3 and is transmitted to the flat field objective lens 2-4; Another beam of light is transmitted to the microscope with adjustable magnification and camera 3-1; The objective lens focuses the light beam onto the cell sample stage 4-1; The photoacoustic signal generated by the pulsed laser excitation is received by the signal processing component 5; The ultrasonic transducer 5-1 of the signal processing component collects the photoacoustic signal and transmits it to the signal amplifier 5-2; After being amplified and filtered, the signal is transmitted to the data acquisition component, including a data acquisition card and a computer 6-1; The synchronization control component 7 - 1 is respectively connected to the pulse light source, the two-dimensional scanning galvanometer and the data acquisition component to achieve timing synchronization.

[0032] Furthermore, the output wavelength of the pulse light source 1-1 is 532nm, the repetition frequency is 50kHz, and the optical fiber collimator 1-1 is a collimator dedicated to the 532nm wavelength.

[0033] Furthermore, the two-dimensional scanning galvanometer 2-1 can carry a lens with a standard input spot of 5mm and an effective scanning angle of ±20°; the semi-transparent and semi-reflective mirror 2-2 has a 50% efficiency for the passage and transmission of the laser respectively, and the effective displacement of the nano-objective lens displacement stage 2-3 is 0 to 200μm, with a minimum stroke of 10nm, which is used to drive the axial movement of the objective lens to better focus the light beam onto the cell plane, and the flat-field objective lens 2-4 is a 40x objective lens.

[0034] Furthermore, the magnification of the microscope 3-1 is adjustable between 200 and 2000 times, and the camera 3-1 is used to calibrate the cell position and observe whether the cell is alive.

[0035] Furthermore, the ultrasonic transducer 5 - 1 of the signal processing component 5 is a focused transducer with a center frequency of 25 MHz, wherein the gain of the signal amplifier 5 - 2 is 50 dB, which further amplifies the collected electrical signal.

[0036] Furthermore, the sampling rate of the data acquisition card 6-1 is 125MHz, and it has two acquisition channels. The computer 6-1 controls the system operation and signal acquisition through the Labview program, and then processes the signal through the matlab program and projects it into a photoacoustic image through the maximum value.

[0037] Furthermore, the synchronization control component 7 - 1 is controlled by FPGA and is used to control the timing synchronization of the pulse light source emitting light, the two-dimensional galvanometer scanning and the acquisition card acquisition.

[0038] Example 3 like Figure 3 As shown, this embodiment provides a method for observing the transcellular membrane process of CpG-Au by photoacoustic microscopy, comprising the following steps: S1. Fix the adherent cells in good condition on the photoacoustic microscopy imaging system, find the appropriate cell field of view under the bright field microscope, and move the single cell to the center of the field of view; S2. Add pre-heated colorless DMEM medium and photoacoustically labeled CpG oligodeoxynucleotide solution into the confocal dish; S3, adjust the step size and scanning range of the two-dimensional galvanometer so that the scanning step size of the two axes is controlled at 125nm, the entire field of view is 50μm×50μm, and the scanning range of the light beam covers the field of view of the cells of interest; S4. Adjust the nano-objective lens stage to the calibrated cell level. If the position is lower or higher than this interface, it may cause defocusing, resulting in the laser focusing level not being at the cell position. S5, giving FPGA instructions to the pulse light source, two-dimensional galvanometer scanning instructions and acquisition card sampling timing through the computer; S6. Repeat S5 every 10 minutes and obtain multiple two-dimensional distribution images of cells and CpG-Au through MATLAB processing.

[0039] Preferably, the signal processing method in step S6 includes: The signal was filtered by wavelet and SVD, and then the collected photoacoustic signal was reconstructed into a photoacoustic image by the maximum projection method.

[0040] like Figure 4 Shown is a result diagram of a photoacoustic microscopy imaging observation process of cell phagocytosis of CpG-Au provided in an embodiment of the present application. Figure 4 (A) The process of undifferentiated monocyte macrophages phagocytosing CpG-Au within 120 minutes; Figure 4(B) shows the process of activated macrophages phagocytizing CpG-Au within 120 minutes. It can be seen that CpG-Au has a uniform particle size and good dispersibility. CpG-Au quickly diffuses around the two groups of cells and contacts the cell membrane; then, a gradual endocytosis process begins. At 60 minutes, the cells have successfully phagocytosed some CpG-Au; at 120 minutes, the activated macrophages show significantly stronger phagocytic ability than undifferentiated monocyte macrophages. The imaging results show that the photoacoustically labeled CpG oligodeoxynucleotides can be captured by high-resolution photoacoustic microscopy, thereby achieving the purpose of cell phagocytosis of CpG-Au.

[0041] The above is a further detailed description of the present invention, which should not be regarded as a limitation on the specific implementation of the present invention. For ordinary technicians in the technical field to which the present invention belongs, simple deduction or replacement without departing from the concept of the present invention is within the protection scope of the present invention.

Claims

1. CpG oligodeoxynucleotide photoacoustic labeling, the preparation method thereof comprises the following steps: 1) suspending gold nanoparticles in deionized water, adding thiolated CpG oligodeoxynucleotide solution, and incubating with shaking to obtain a mixture S1; 2) adding phosphate buffer to the mixture S1, and incubating with shaking to obtain a mixture S2; 3) The mixture S2 is centrifuged to obtain a precipitate, and the unbound CpG oligodeoxynucleotide is removed by washing to obtain the CpG oligodeoxynucleotide photoacoustic label.

2. The CpG oligodeoxynucleotide photoacoustic labeling according to claim 1, characterized in that: The CpG oligodeoxynucleotide is a B-type CpG oligodeoxynucleotide.

3. The CpG oligodeoxynucleotide photoacoustic labeling according to claim 1, characterized in that: The particle size of gold nanoparticles is 5nm to 200nm.

4. The CpG oligodeoxynucleotide photoacoustic labeling according to claim 1, characterized in that: In step 1), the shaking incubation time is 10 to 14 h; and / or in step 2), the shaking incubation time is 20 to 30 h.

5. Use of the CpG oligodeoxynucleotide photoacoustic label according to any one of claims 1 to 4 in the preparation of a living cell photoacoustic microscopy imaging enhancer.

6. A method for photoacoustic microscopy of living cells, comprising the following steps: Fix the living cells on the confocal dish and move a single cell to the center of the field of view; Adding the CpG oligodeoxynucleotide photoacoustic label according to any one of claims 1 to 4 to the confocal dish, adjusting the step length and scanning range of the two-dimensional galvanometer so that the scanning range of the light beam covers the field of view of the cell of interest; Adjust the nano-objective lens stage to the calibrated cell level position; The computer gives pulse light source instructions, two-dimensional galvanometer scanning instructions and acquisition card sampling timing, and processes the two-dimensional distribution photoacoustic image of cells and CpG oligodeoxynucleotide photoacoustic labeling, and repeatedly obtains multiple photoacoustic images.

7. The photoacoustic microscopy method according to claim 6, characterized in that: The energy of the pulse light source is 35 to 45 nJ.

8. The photoacoustic microscopy method according to claim 6, characterized in that: The signal was filtered by wavelet and SVD, and then the collected photoacoustic signal was reconstructed into a photoacoustic image by the maximum projection method.

9. The photoacoustic microscopy method according to claim 6, characterized in that: The output wavelength of the pulse light source is 532nm.

10. The photoacoustic microscopy method according to claim 6, characterized in that: The photoacoustic microscopy imaging system includes a light source, an optical transmission module, a laser scanning module, a signal acquisition module and a data processing module. The optical transmission module is used to transmit the laser to the laser scanning module; The laser scanning module is used to focus the laser beam on the target in a point and line scanning manner. Imaging area; The signal acquisition module is used to collect the photoacoustic signal excited by the laser; The data processing module is connected to the signal acquisition module, and projects the acquired photoacoustic signals to reconstruct images.

Images