Photacoustic Labeling of CpG Oligodeoxynucleotides and Its Application in Imaging

Abstract

The present invention belongs to the fields of immunology and biomedical imaging, and discloses CpG oligodeoxynucleotide photoacoustic labeling and its application in imaging. By conjugating CpG oligodeoxynucleotides to gold nanoparticles, CpG oligodeoxynucleotide photoacoustic labeling is obtained. This photoacoustic labeling has good biocompatibility, is easily internalized by cells and produces a significant immune stimulation effect. The concentration of cytokines stimulated by it is increased by two orders of magnitude compared with naked CpG oligodeoxynucleotides, and is significantly higher than that of commercial delivery liposome reagents. It can significantly increase the photoacoustic signal intensity, 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, provide a powerful tool for immunological research, significantly improve the data reliability and experimental specificity, and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the fields of immunology and biomedical imaging, and particularly 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 the systemic immunity, which is an important prerequisite for the vaccine to play its role. In the immune system, macrophages have the ability to phagocytose and eliminate pathogens, as well as regulate and maintain immune balance. In addition, they are also responsible for processing and presenting antigens to trigger the activation of other immune cells.

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

[0004] An adjuvant is a non-specific immune enhancer that, when mixed with an antigen or injected into the body prior to the antigen, can enhance the body's immune response to the antigen or change the type of immune response. Oligodeoxynucleotides containing CpG motifs (CpG) can increase the immunogenicity of antigens by improving the antigen uptake of antigen-presenting cells and activating the functional maturation of antigen-presenting cells to produce 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 photoacoustic signal intensity and inability to achieve high-resolution imaging.

[0005] Photoacoustic imaging is becoming increasingly common in biomedical applications and can provide anatomical, functional, and molecular information. The principle of this imaging method is that when a pulsed laser irradiates biological tissue, the tissue will absorb the energy of the pulsed light, generating instantaneous heating and expansion, thereby generating ultrasonic waves. An ultrasonic transducer located on the tissue surface 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. Its non-destructive imaging property makes it suitable for live cell and in vivo studies. It can detailedly observe and record the whole process of cell phagocytosis of drugs, dynamically detect the responses 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 the cell phagocytosis mechanism, optimizing adjuvant design, and promoting related biomedical research and clinical applications.

[0007] There have been many studies on using fluorescent dyes to label and long-term observing the dynamic imaging of CpG oligodeoxynucleotides in cells by techniques such as laser confocal microscopy. However, long-term exposure may cause photobleaching and phototoxicity problems, significantly affecting the treatment of live cell imaging. Summary of the Invention

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

[0009] The technical solution adopted by the present invention is:

[0010] In the first aspect of the present invention, it provides:

[0011] A CpG oligodeoxynucleotide photoacoustic label, and its preparation method includes the following steps:

[0012] 1) Suspend gold nanoparticles in deionized water, add a thiolated CpG oligodeoxynucleotide solution, and incubate with oscillation to obtain a mixture S1;

[0013] 2) Add phosphate buffer to the mixture S1, and incubate with oscillation to obtain a mixture S2;

[0014] 3) Centrifuge the mixture S2 to obtain a precipitate, wash to remove unbound CpG oligodeoxynucleotides, and obtain the CpG oligodeoxynucleotide photoacoustic label.

[0015] In some examples of the CpG oligodeoxynucleotide photoacoustic label, the CpG oligodeoxynucleotide is a type B CpG oligodeoxynucleotide. Due to its open single-stranded structure and multiple CpG motifs, and being easy to modify and functionalize, it can more efficiently cover the surface of gold nanoparticles.

[0016] In some examples of the CpG oligodeoxynucleotide photoacoustic label, the particle size of the gold nanoparticles is 5 nm to 200 nm. When the size of the gold nanoparticles is small, after binding to the CpG oligodeoxynucleotide, it is easily phagocytosed by cells, and the spectrum is stable at 532 nm and is easily excited.

[0017] In some examples of the photoacoustic labeling of CpG oligodeoxynucleotides, in step 1), the time of oscillatory incubation is 10 to 14 h; and / or in step 2), the time of oscillatory incubation is 20 to 30 h.

[0018] The above features can be combined arbitrarily under the condition of non-conflict.

[0019] The second aspect of the present invention provides:

[0020] The application of the CpG oligodeoxynucleotide photoacoustic labeling described in the first aspect of the present invention in the preparation of a live cell photoacoustic microscopy imaging enhancer.

[0021] The third aspect of the present invention provides:

[0022] A photoacoustic microscopy imaging method for live cells, comprising the following steps:

[0023] Fix the live cells in a confocal dish and move a single cell to the center of the field of view;

[0024] Add the CpG oligodeoxynucleotide photoacoustic labeling described in the first aspect of the present invention to the confocal dish, adjust the step size and scanning range of the two-dimensional galvanometer, and the scanning range of the light beam covers the cell field of view of interest;

[0025] Adjust the nano-objective displacement stage to the calibrated cell layer position;

[0026] Give instructions to the pulsed light source, two-dimensional galvanometer scanning instructions and the timing of the acquisition card through a computer, and process to obtain a two-dimensional distribution photoacoustic image of the cells and the CpG oligodeoxynucleotide photoacoustic labeling, and repeat to obtain multiple photoacoustic images.

[0027] In some examples of the photoacoustic microscopy imaging method, the energy of the pulsed light source is 35 to 45 nJ. If the energy is too strong, it will cause cell damage. On the contrary, the photoacoustic signal of CpG-Au will be significantly reduced and the imaging quality will deteriorate.

[0028] In some examples of the photoacoustic microscopy imaging method, the signal is filtered by wavelet filtering and SVD filtering, and then the acquired photoacoustic signal is reconstructed into a photoacoustic image by the maximum projection method.

[0029] In some examples of the photoacoustic microscopy imaging method, the output wavelength of the pulsed light source is 532 nm. The gold nanoparticles have a good absorption peak near 532 nm. After binding with the gold nanoparticles, the ultraviolet-visible absorption spectrum of CpG-Au changes and a strong peak also appears at 532 nm.

[0030] In some examples of the photoacoustic microscopy imaging method, the photoacoustic microscopy imaging system includes a light source, an optical path transmission module, a laser scanning module, a signal acquisition module and a data processing module.

[0031] The optical path transmission module is used to conduct the laser to the laser scanning module;

[0032] The laser scanning module is used to focus the laser beam on the target in the form of point and line scanning

[0033] imaging area;

[0034] The signal acquisition module is used to acquire the photoacoustic signals excited by the laser;

[0035] The data processing module is connected to the signal acquisition module to perform projection reconstruction on the acquired photoacoustic signals to obtain images.

[0036] The beneficial effects of the present invention are:

[0037] The CpG oligodeoxynucleotide photoacoustic labeling in some examples of the present invention has good biocompatibility, is easily internalized by cells and produces a significant immune stimulation effect. The concentration of cytokines stimulated by it is increased by two orders of magnitude compared with naked CpG oligodeoxynucleotides, and is significantly higher than that of commercial liposome reagents for delivery.

[0038] The CpG oligodeoxynucleotide photoacoustic labeling in some examples of the present invention can significantly increase the photoacoustic signal intensity, 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, provide a powerful tool for immunological research, significantly improve the data reliability and experimental specificity, and has broad application prospects. Description of the Drawings

[0039] Figure 1 It is a characterization result diagram of the photoacoustic-labeled CpG oligodeoxynucleotides provided by some embodiments of the present invention; wherein FIG. A is the result of a transmission electron microscope of gold nanoparticles and CpG-Au; FIG. B is the ultraviolet absorption spectrum diagram of CpG-Au.

[0040] Figure 2 It is a schematic structural diagram of a photoacoustic microscopy imaging system provided by some embodiments of the present invention.

[0041] Reference numerals: 1, light source assembly; 1-1, pulsed light source; 1-2, fiber collimator; 2, optical path transmission module; 2-1, two-dimensional scanning galvanometer; 2-2, semi-transmissive semi-reflective mirror; 2-3, nano-objective displacement stage; 2-4, flat-field objective; 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 components, including data acquisition card and computer; 7-1, synchronous control component.

[0042] Figure 3 It is a schematic flowchart of a photoacoustic microscopy imaging method provided by some embodiments of the present invention.

[0043] Figure 4 It is a result diagram of the observation process of a photoacoustic microscopy imaging of cells phagocytosing CpG-Au provided by some embodiments of the present invention. Detailed implementation manners

[0044] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It can be understood that the specific embodiments described herein are only used to explain the present invention, rather than limiting the present invention. Additionally, it should be noted that for the convenience of description, only parts related to the present invention rather than all structures are shown in the accompanying drawings.

[0045] Example 1

[0046] Photoacoustic labeling of CpG oligodeoxynucleotides, and its preparation method includes the following steps:

[0047] S1) Extract 5 mL of the gold nanoparticle solution dissolved in citrate, centrifuge at a speed of 1000 g for 20 minutes to obtain pure gold nanoparticles, and resuspend them in 1 mL of deionized water;

[0048] S2) Subsequently, add and mix with the thiolated CpG oligodeoxynucleotide solution, and add it according to a final concentration of 30 μg / mL;

[0049] S3) After incubating the mixture at room temperature on a shaker at a speed of 120 rpm for 12 h, add 100 mM phosphate buffer solution (PBS) (pH 7.4) to the mixture, with a final concentration of 10 mM, and further incubate for 24 h while continuously shaking;

[0050] S4) Then centrifuge the mixture at 4°C at a speed of 1000 g for 20 minutes to obtain the successfully labeled CpG-Au precipitate; continue to wash it three times with PBS buffer solution to remove unbound CpG, and finally resuspend CpG-Au in PBS buffer solution and store it at 4°C for further use.

[0051] The following is the characterization test of the photoacoustic-labeled CpG oligodeoxynucleotides in Example 1:

[0052] (1) The transmission electron microscope image of the isolated single gold nanoparticles is as shown in Figure 1 (A), and the results show that the isolated gold nanoparticles are in a well-dispersed spherical shape with good dispersibility;

[0053] (2) The transmission electron microscope image of the photoacoustic-labeled CpG oligodeoxynucleotides obtained from Example 1 is as shown in Figure 1As shown in (A), it can be seen from the transmission electron microscope results that after binding to CpG, the gold nanoparticles are wrapped by a soft material (DNA strand), and the particle size increases;

[0054] (3) The three materials in Example 1: gold nanoparticle solution, CpG oligodeoxynucleotide solution, and CpG-Au solution were subjected to ultraviolet absorption spectroscopy detection, and the results are as Figure 1 shown in (B). It can be seen that the gold nanoparticles have a good absorption peak near 532 nm. After binding with the gold nanoparticles, the ultraviolet-visible absorption spectrum of CpG-Au changes, and a strong peak also appears at 532 nm, indicating successful binding with the gold nanoparticles.

[0055] Example 2

[0056] Figure 2 It is a schematic structural diagram of photoacoustic microscopy provided by an embodiment of the present invention. For specific implementation manners, please refer to the following embodiments.

[0057] The pulsed light source 1-1 emits pulsed laser as an excitation source. The light beam is reflected by a silver mirror and then coupled to the fiber collimator 1-2. The light beam is transmitted through the optical fiber to obtain better control and flexibility;

[0058] Then the other end shoots from the same type of collimator to the two-dimensional scanning galvanometer 2-1, and is transmitted to the next step through the semi-transmissive and semi-reflective mirror 2-2, and propagates in two beams of light. One beam passes through the nano-objective displacement stage 2-3 and is transmitted to the flat-field objective 2-4;

[0059] The other beam of light is transmitted to the microscope and camera 3-1 with adjustable magnification;

[0060] The objective lens focuses the light beam onto the cell sample stage 4-1;

[0061] The photoacoustic signal generated by the pulsed laser excitation is received by the signal processing component 5;

[0062] The ultrasonic transducer 5-1 of the signal processing component collects the photoacoustic signal and transmits it to the signal amplifier 5-2;

[0063] The signal is transmitted to the data acquisition component after amplification and filtering, including a data acquisition card and a computer 6-1;

[0064] The synchronous control component 7-1 is respectively connected to the pulsed light source, the two-dimensional scanning galvanometer, and the data acquisition component for realizing timing synchronization.

[0065] Furthermore, the pulsed light source 1-1 outputs a wavelength of 532 nm and a repetition frequency of 50 kHz, and the fiber collimator 1-1 is a collimator dedicated to a wavelength of 532 nm.

[0066] Furthermore, the standard input light spot of the lens carried by the two-dimensional scanning galvanometer 2-1 is 5 mm, and the effective scanning angle is ±20°; the semi-transmissive and semi-reflective mirror 2-2 has a 50% passing and transmitting efficiency for the laser, and the effective displacement of the nano-objective displacement stage 2-3 is 0 to 200 μm, with a minimum stroke of 10 nm, which is used to drive the objective lens to move axially to better focus the light beam onto the cell plane. The flat-field objective lens 2-4 is a 40x objective lens.

[0067] Further, the microscope 3-1 with adjustable magnification magnifies at a magnification of 200 to 2000 times, and the camera 3-1 is used to calibrate the cell position and observe whether the cells survive.

[0068] Further, the ultrasonic transducer 5-1 of the signal processing component 5 uses a focused transducer with a center frequency of 25 MHz, and the gain of the signal amplifier 5-2 is 50 dB to further amplify the collected electrical signal.

[0069] Furthermore, the sampling rate of the data acquisition card 6-1 is 125 MHz, 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 as a photoacoustic image through the maximum projection.

[0070] Further, the synchronization control component 7-1 is controlled by an FPGA and is used to control the timing synchronization of the pulsed light source emission, two-dimensional galvanometer scanning, and data acquisition card acquisition.

[0071] Example 3

[0072] As Figure 3 shown, this example provides a method for observing the transmembrane process of CpG-Au through photoacoustic microscopy imaging, including the following steps:

[0073] S1. Fix the well-conditioned adherent cells in the photoacoustic microscopy imaging system, find a suitable cell field of view under the bright-field microscope, and move a single cell to the center of the field of view;

[0074] S2. Add preheated colorless DMEM culture medium and photoacoustically labeled CpG oligodeoxynucleotide solution into the confocal dish;

[0075] 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 125 nm, the entire field of view is 50 μm × 50 μm, and the scanning range of the light beam covers the cell field of view of interest;

[0076] S4. Adjust the nano-objective displacement stage to the calibrated cell layer position. Being lower or higher than this interface may cause defocusing, resulting in the laser focusing layer not being at the cell position;

[0077] S5. Give instructions to the pulsed light source, two-dimensional galvanometer scanning instructions, and the timing of the acquisition card through a computer to the FPGA;

[0078] S6. Repeat the operation of S5 every 10 minutes, and after being processed by Matlab, obtain multiple two-dimensional distribution images of cells and CpG-Au.

[0079] Preferably, the signal processing method in step S6 includes:

[0080] Filter the signal through wavelet filtering and SVD filtering, and then reconstruct the acquired photoacoustic signal into a photoacoustic image through the maximum projection method.

[0081] As Figure 4 shown is the result diagram of the observation process of a photoacoustic microscopy for cell phagocytosis of CpG-Au provided by an embodiment of the present application. Figure 4 (A) is the process of undifferentiated monocyte macrophages phagocytosing CpG-Au within 120 minutes; Figure 4 (B) is the process of activated macrophages phagocytosing 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; afterwards, a gradual endocytosis process begins. At 60 minutes, the cells have successfully phagocytosed some CpG-Au; at 120 minutes, the activated macrophages significantly show stronger phagocytic ability than the undifferentiated monocyte macrophages. The imaging results show that the CpG oligodeoxynucleotides labeled by photoacoustic can be captured by high-resolution photoacoustic microscopy, so as to achieve the purpose of observing the process of cell phagocytosis of CpG-Au.

[0082] The above is a further detailed description of the present invention, and it should not be regarded as a limitation to the specific implementation of the present invention. For those of ordinary skill in the technical field to which the present invention belongs, simple deductions or substitutions without departing from the concept of the present invention are within the protection scope of the present invention.

Claims

1. Application of CpG oligodeoxynucleotide photoacoustic labeling in the preparation of a living cell photoacoustic microscopy imaging enhancer, the preparation method of the 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.

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

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

4. The use 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. 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, the signal is filtered by wavelet filtering and SVD filtering, and then the collected photoacoustic signal is reconstructed into a photoacoustic image by the maximum projection method. The two-dimensional distribution photoacoustic image of cells and CpG oligodeoxynucleotide photoacoustic labeling is obtained by processing, and multiple photoacoustic images are obtained repeatedly.

6. The photoacoustic microscopy method according to claim 5, characterized in that: The energy of the pulse light source is 35 to 45 nJ.

7. The photoacoustic microscopy method according to claim 5, characterized in that: The output wavelength of the pulse light source is 532nm.

8. The photoacoustic microscopy method according to claim 5, characterized in that: 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.

Images