A near-infrared fluorescent tetrahedron probe and its preparation method and application

Abstract

The present invention provides a near-infrared fluorescent tetrahedron probe and a preparation method thereof and application in tumor imaging at a near single-cell level. The preparation method comprises the following steps: 1) modifying a DNA tetrahedron nanostructure with a side length of 20 base pairs (bp) with a hydrophobic group to obtain a DNA nanocage with a specific hydrophobic cavity of precise morphology and size; 2) providing a hydrophobic fluorescent dye with near-infrared luminescence properties; and 3) mixing the DNA nanocage and the hydrophobic fluorescent dye by oscillation or ultrasound, so that the hydrophobic fluorescent dye enters the hydrophobic cavity of the DNA nanocage and is encapsulated therein, thereby obtaining a near-infrared fluorescent tetrahedron probe. The near-infrared fluorescent tetrahedron probe prepared according to the method of the present invention has high brightness, good stability and good biosafety, can image tumors with a sensitivity close to the single-cell level, and has good medical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically to a near-infrared fluorescent tetrahedron probe and a preparation method thereof, as well as an application in tumor imaging at a near single-cell level. Background Art

[0002] Microscopic lesions of malignant tumors usually do not cause obvious symptoms in the early stages. The size of microscopic tumor lesions is usually very small, which may be only a few millimeters or smaller. Traditional imaging techniques (such as CT scans or MRI) cannot accurately detect small lesions. In addition, microscopic lesions may be located in organ tissues without obvious boundaries from surrounding normal tissues, which makes it very difficult to distinguish them from surrounding tissues in imaging. Imaging of microscopic tumor lesions can help doctors develop more precise treatment plans, prevent cancer progression, improve cure rates, and reduce adverse reactions during treatment.

[0003] Among various imaging modalities, fluorescence imaging based on the near-infrared window (NIR, ~650-1700 nm) shows improved tissue penetration (mm to cm), low background autofluorescence and minimal photon scattering, thereby achieving a high signal-to-noise ratio, which shows great promise in in vivo imaging. However, the existing various NIR fluorescent probes designed using inorganic nanomaterials such as quantum dots (QDs), single-walled carbon nanotubes (SWNTs) and lanthanide down-conversion luminescent nanoparticles (DCNPs) remain in the body for a long time, resulting in unknown biological toxicity. Small molecule fluorophores that are easily metabolized usually show poor water solubility, which limits their direct use in biological environments. Therefore, it is necessary to develop tumor imaging probes with good biosafety, good water solubility and high sensitivity.

[0004] In the inventor's previous research (see Chinese patent application "201910589495.9"), we developed a customizable shape framework nucleic acid nanoluminescent body to achieve the encapsulation of hydrophobic drugs and fluorescent molecules. However, in this method, the hydrophobic group is modified at any position on the DNA single strand (including the 5' end, 3' end or any position in the middle), resulting in unstable drug / fluorescent molecule packaging effect; and the framework nucleic acid is of arbitrary shape, and the cellular uptake effect is low during further interaction with the living system, and it cannot be truly applied to in vivo imaging. Summary of the invention

[0005] The purpose of the present invention is to provide a near-infrared fluorescent tetrahedron probe and a preparation method thereof and its application in tumor imaging at the near single-cell level, so as to solve the problems of poor fluorescent dye encapsulation efficiency caused by arbitrary modification of the position of hydrophobic groups in the original probe and poor cell uptake effect caused by arbitrary shape and size of the framework nucleic acid.

[0006] To solve the above technical problems, the present invention adopts the following technical solutions:

[0007] According to a first aspect of the present invention, there is provided a method for preparing a near-infrared fluorescent tetrahedral probe, comprising the following steps: 1) Modifying a DNA tetrahedral nanostructure with a side length of 20 bp with a hydrophobic group, such that the hydrophobic group hangs towards the inside of the DNA tetrahedral nanostructure, thereby forming a hydrophobic confinement space inside, to obtain a DNA nanocage with precise morphological dimensions and a hydrophobic cavity; 2) Providing some hydrophobic fluorescent dyes with near-infrared luminescence properties; and 3) Mixing the DNA nanocage and the hydrophobic fluorescent dyes by shaking or sonication, so that the hydrophobic fluorescent dyes enter the hydrophobic cavity of the DNA nanocage and are encapsulated therein, thereby obtaining a near-infrared fluorescent tetrahedral probe.

[0008] In step 1), the hydrophobic group includes: alkyl alcohol {HO-(CH2) x -} y -, cholesterol, polycyclic aromatic hydrocarbon structure or other molecular structures with hydrophobic properties, wherein x is the length of the alkyl chain, 1 < x < 100, and y is the number of alkyl chains, 1 < y < 100. It should be understood that in the present invention, the hydrophobic group is not limited to the hydrophobic alkyl alcohol used in the examples, and actually molecular structures with hydrophobic properties are applicable to the present invention.

[0009] Preferably, in step 1), the DNA tetrahedral nanostructure is composed of four DNA single strands, and the hydrophobic group is modified at the end of the DNA single strand. By controlling the position of the double-stranded DNA opening formed during the synthesis of the DNA tetrahedral nanostructure, the modification position of the hydrophobic group on the edge is the 6th - 12th base in the 5'-3' direction.

[0010] It should be understood that the hydrophobic group is only modified on the four edges because the 20-bp DNA tetrahedron is composed of four DNA single strands. It can be understood that each face of the DNA tetrahedron with a side length of 20 bp has an alkyl chain, and this alkyl chain extends from the edge, and the hanging direction of the alkyl chain is towards the inside of the tetrahedron, thereby forming a hydrophobic cavity.

[0011] Most preferably, in step 1), the modification position of the hydrophobic group on the edge is the 9th base in the 5'-3' direction.

[0012] Preferably, in step 2), the hydrophobic fluorescent dyes with near-infrared luminescence properties include: hydrophobic fluorescent dye molecules selected from D-A-D type, cyanine type, rhodamine type, azabodipy type, with an emission peak in the range of 650 - 1700 nm.

[0013] Preferably, the near-infrared fluorescent dye Sq964 has an excitation wavelength of 808 nm and an emission wavelength of 964 nm. It should be understood that this fluorescent dye is only used as an example of a preferred embodiment and is not intended to be limiting. In fact, other conventional small molecule dyes with near-infrared luminescent properties having hydrophobic properties are suitable for the present invention.

[0014] According to a second aspect of the present invention, a near-infrared fluorescent tetrahedron probe prepared according to the above-mentioned preparation method is provided, wherein the near-infrared fluorescent tetrahedron probe is prepared by encapsulating a near-infrared fluorescent dye in a DNA nanocage with a hydrophobic cavity through hydrophilic-hydrophobic interaction, wherein the hydrophobic fluorescent dye with near-infrared luminescence properties has good deep tissue penetration ability, thereby imaging deep tissues.

[0015] According to a third aspect of the present invention, there is provided a use of a near-infrared fluorescent tetrahedron probe in tumor imaging at a near single-cell level.

[0016] Preferably, the tumor imaging includes: tumor imaging of breast cancer, melanoma, and basal cell carcinoma.

[0017] Preferably, the application further comprises: further connecting tumor targeting molecules, including antibodies, aptamers, and peptides, to the near-infrared fluorescent tetrahedron probe to achieve active targeted imaging of tumors.

[0018] According to the preparation method of the near-infrared fluorescent tetrahedral probe provided by the present invention, its working principle is: accurately control the position of the hydrophobic group on the tetrahedral edge, according to the characteristics of the DNA double helix, ensure that the hydrophobic group is suspended to the inside of the tetrahedron cavity to form a hydrophobic confined space, and the DNA skeleton itself is hydrophilic, so the tetrahedral framework nucleic acid is an amphiphilic structure with internal hydrophobicity and external hydrophilicity. It should be understood that when the dangling direction of the hydrophobic group is toward the inside of the tetrahedral cavity, a hydrophobic cavity is easily formed, otherwise tetrahedral aggregates will be formed outward. Near-infrared light has better tissue penetration and less tissue autofluorescence, and can achieve deep tissue imaging with a high signal-to-noise ratio, but fluorescent dyes are usually hydrophobic, and the hydrophobic dye can be encapsulated into the hydrophobic cavity of the DNA tetrahedron through hydrophilic-hydrophobic interactions, thereby obtaining a hydrophilic near-infrared imaging probe.

[0019] According to the present invention, the inventors prepared a variety of DNA nanostructures, among which the DNA tetrahedron with a side length of 20bp, the DNA cube with a side length of 20bp, and the DNA tetrahedron with a side length of 37bp were studied in detail. The research found that the DNA tetrahedron with a side length of 20bp had the best cellular uptake effect.

[0020] As described in the background technology section, Chinese patent application "201910589495.9" discloses a shape-customizable framework nucleic acid nanoluminescent body, but the framework nucleic acid nanoluminescent body has a low cellular uptake effect in the process of further interaction with the living system and cannot be truly applied to in vivo imaging. However, the creativity of the present invention relative to the closest prior art mainly lies in: 1) The prior patent published a universal preparation method, and in order to make the luminescent body into a biological probe, the present invention verified the modification position of the hydrophobic group, screened out the alkyl chain modification position with the best encapsulation efficiency, and provided a high-brightness probe for achieving high-sensitivity imaging of organisms; 2) The prior patent also designed DNA structures of different morphological sizes, but for biological imaging, efficient uptake of the probe by cells is also a prerequisite for imaging. Therefore, the present invention also screens the morphological size of DNA and determines the structure with the highest cellular uptake efficiency; 3) The present invention also demonstrates the high-sensitivity imaging of breast cancer in situ tumors at a near single-cell level by the probe.

[0021] The effect of the present invention compared with the existing technological progress is that the near-infrared fluorescent tetrahedron probe provided by the present invention has a high imaging brightness in tumor imaging and achieves imaging sensitivity close to the single-cell level. In practical applications, optimally, the probe achieves imaging of nearly 40 cells in a breast cancer in situ tumor. Therefore, the probe can be better used in living tumor imaging and can solve the problem of difficult detection of early tiny tumor lesions.

[0022] In summary, the near-infrared fluorescent tetrahedron probe prepared according to the method of the present invention has good biosafety, high imaging brightness and imaging sensitivity close to the single-cell level, and has good medical application prospects for the early diagnosis of tiny malignant tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The quantitative statistical results of 4T1 cell uptake of tetrahedrons and cubes of the same size but different morphologies in Example 1 are shown;

[0024] Figure 2 The agarose electrophoresis diagram of the probe prepared by modifying the alkyl chains at different positions on the tetrahedral edges in Example 2 is shown;

[0025] Figure 3 The quantitative statistical results of 4T1 cell uptake of tetrahedrons of the same morphology but different sizes in Example 3 are shown;

[0026] Figure 4A The fluorescence spectrum of the Sq964 fluorescent dye in Example 4 and the successful encapsulation of the dye in agarose are shown;

[0027] Figure 4BThe fluorescence spectrum of SiR650 fluorescent dye in Example 4 and the successful encapsulation of the dye by agarose are shown;

[0028] Figure 5 The quantitative results of the probe on cell division and proliferation in Example 5 are shown;

[0029] Figure 6 The results of imaging in situ breast cancer at the near single-cell level using the probe in Example 6 are shown. DETAILED DESCRIPTION

[0030] 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. The experimental methods for which specific conditions are not specified in the following examples are carried out according to conventional methods and conditions, or are selected according to the product specifications.

[0031] In the following specific implementation, the present invention mainly synthesizes a fluorescent small molecule Sq964 (emission wavelength 964nm) with near-infrared luminescence properties as the luminescent core, and a tetrahedron with a hydrophobic alkyl chain is a nanocage modified by the hydrophilicity of the fluorescent molecule to construct a tumor imaging probe, and the probe is used in tumor imaging to achieve sensitivity imaging close to the single cell level. The following examples specifically illustrate the implementation effect of the present invention.

[0032] Example Comparison of cellular uptake efficiency between a tetrahedron and a cube with a side length of 20 bp

[0033] Cell culture. 4T1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences Typical Culture Collection Committee. RPMI1640 medium (containing 10% FBS, 100 units / mL penicillin, 100 μg / mL streptomycin, and 2 mM glutamine) was cultured in a constant temperature and humidity cell culture incubator at 37°C and 5% CO2. 7×10 4 A number of cells were seeded in a confocal dish and placed in a cell culture incubator overnight. After the cells attached to the wall, DNA tetrahedrons and cubes with a side length of 20 bp (final concentration 200 nM) were incubated with the cells for 6 hours. The cells were imaged using a laser confocal microscope (Leica sp8) to observe the uptake behavior of 4T1 cells towards these two nanostructures of the same size but different morphologies.

[0034] The sequences used in the DNA nanostructures (SEQ ID No. 1-9) are shown in Table 1 below:

[0035] Table 1

[0036]

[0037]

[0038]

[0039] D is a modified hydrophobic alkyl chain with the following structural formula:

[0040]

[0041] Synthesis of tetrahedron with side length of 20 bp: single-stranded DNA, S1-D, S2-D, S3-D, and S4-D accurately quantified by UV spectrophotometer were added to TM buffer at a stoichiometric ratio of 1:1:1:1, with a final concentration of each single strand of 1 μM. After thorough mixing, the mixture was placed in a PCR instrument for high temperature annealing to obtain a tetrahedron structure with side length of 20 bp modified with Cy5 fluorescence.

[0042] Synthesis of a cube with a side length of 20 bp: The single-stranded DNA, 1AA, 2AA, 3AA, 4AA, and A'-D, which were accurately quantified by UV spectrophotometer, were added to TAM buffer at a stoichiometric ratio of 1:1:1:1:8. The final concentrations of the four AA single strands were 1 μM, and the final concentration of the A'-D single strand was 8 μM. After thorough mixing, the mixture was placed in a PCR instrument for high-temperature annealing to obtain a cube structure with a side length of 20 bp modified with Cy5 fluorescence.

[0043] Results: Figure 1 The laser confocal imaging results showed that when incubated with 4T1 cells for the same time, although the side lengths of the two DNA nanostructures were the same, the uptake effect of the nanostructure with a tetrahedral morphology was significantly better than that of the cubic morphology, indicating that the morphology of the framework nucleic acid affects the cell uptake efficiency.

[0044] Example 2 Comparison of hydrophobic alkyl chains modified at different positions on the edges of DNA tetrahedron

[0045] In order to prove that the orientation of alkyl chains affects the formation of hydrophobic cavities in DNA nanostructures, four DNA tetrahedrons with different modification positions were designed by changing the position of alkyl chain modification on the tetrahedral edges, named TDF-3nt, TDF-6nt, TDF-9nt, and TDF-12nt, to represent the different directions in which the alkyl chains extend. The DNA double helix is ​​a double helix. In the TDF-9nt conformation, the alkyl chains are suspended toward the inside of the tetrahedral cavity, and in the TDF-3nt, TDF-6nt, and TDF-12nt conformations, the alkyl chains are all facing the outside of the tetrahedral cavity. The Sq964 fluorescent dye was incubated with the four tetrahedrons, and the samples were analyzed by agarose gel electrophoresis. By verifying whether the Sq964 fluorescent molecule can be encapsulated, it was proved whether the DNA tetrahedron with hydrophobic cavity was successfully synthesized.

[0046] Results: Figure 2Agarose gel electrophoresis showed that the structure of TDF-9nt had obvious bands in the near-infrared channel. The gel was further stained with GelRed and showed that the bands in the fluorescence channel co-localized with the DNA bands, indicating that the Sq964 molecule was wrapped in the hydrophobic cavity of the DNA tetrahedron. The fluorescence intensity of the fluorescence channels of the other conformations was weak, indicating that TDF-9nt was the most stable structure for forming a hydrophobic cavity. Unless otherwise specified, the alkyl chain was used to modify the position of the 9th base of the 5'-3' side length of the tetrahedron.

[0047] Example 3 Comparison of cellular uptake efficiency of tetrahedrons with side lengths of 20 bp and 37 bp

[0048] Cell culture. The 4T1 cell culture method was the same as in Example 1. After the cells adhered to the wall, tetrahedrons with side lengths of 20 bp and 37 bp (final concentration 200 nM) were co-incubated with the cells for 6 hours. The cells were imaged using a laser confocal microscope (Leica sp8) to observe the uptake behavior of 4T1 cells for these two DNA nanostructures of the same morphology but different sizes.

[0049] The sequences and preparation methods used for the DNA tetrahedron with a side length of 20 bp are the same as those in Example 1, and the sequences (SEQ ID No. 10-17) used for the DNA tetrahedron with a side length of 37 bp are shown in Table 2 below:

[0050] Table 2

[0051]

[0052]

[0053] Synthesis of DNA tetrahedron with a side length of 37 bp: The single-stranded DNAs A3, A5-D, B3, B5-D, C3, C5-D, D3, and D5-D accurately quantified by UV spectrophotometer were added to TM buffer at a stoichiometric ratio of 1:1:1:1:1:1:1:1, with a final concentration of each single strand of 1 μM. After thorough mixing, the DNA was placed in a PCR instrument for high-temperature annealing to obtain a tetrahedral structure with a side length of 37 bp modified with Cy5 fluorescence.

[0054] Results: Figure 3 The laser confocal imaging results show that when incubated with 4T1 cells for the same time, although the morphology of the two DNA nanostructures is tetrahedron, the uptake effect of the tetrahedron with a side length of 20 bp is significantly better than that of the tetrahedron with a side length of 37 bp, indicating that cells internalize less DNA nanostructures with larger sizes. Therefore, according to the present invention, a near-infrared fluorescent tetrahedron probe is provided, preferably a DNA tetrahedron with a side length of 20 bp.

[0055] Example 4 Comparison of packaging of dyes with different luminescent properties

[0056] Two near-infrared fluorescent dyes, DAD-type Sq964 fluorescent dye and rhodamine-type SiR650 fluorescent dye, were selected, and their luminescence properties were characterized by fluorescence spectrometer. They were respectively encapsulated with tetrahedrons with hydrophobic cavities, and agarose gel electrophoresis was used to verify whether the dyes were successfully encapsulated.

[0057] Results: The fluorescence spectrum showed that the emission peak of Sq964 was at 964nm. Figure 4A , the emission peak of SiR650 is at 650nm, Figure 4B The two fluorescent dyes have obvious bands in the near-infrared channel, and further staining of the gel with GelRed shows that the bands in the fluorescent channel co-localize with the DNA bands, indicating that both near-infrared fluorescent dyes can be encapsulated in the hydrophobic cavity of the DNA tetrahedron.

[0058] Example 5 Biosafety of Near-Infrared Fluorescent Tetrahedron Probe

[0059] Cell growth curve. Cell proliferation was measured using the Cell Counting Kit-8 (CCK-8). 4T1 cells were plated at 3×10 3 The density of cells was seeded in a 6-well plate and incubated for 12 hours to allow cell adhesion. The near-infrared fluorescent tetrahedron probe was then incubated with 4T-1 cells for 12 hours, and then washed repeatedly with PBS to remove unbound FDF spots and fresh cell culture medium was added to the plate wells. After the desired incubation time (24, 48, 72 hours), 10 μL CCK-8 solution was added to each well, and the plate was incubated for another 2 hours at 37 ° C. The absorbance was measured at 450 nm using a microplate reader.

[0060] Results: After culturing the cells for 72 hours, the growth rate of cells labeled with DNA nanoprobes was compared with that of unlabeled cells. It was found that there was no significant difference in the number of cells whether the probes were incubated with the cells or not. Figure 5 This indicates that the probe will not affect cell growth, division and proliferation.

[0061] Example 6 Imaging of Breast Cancer Tumor in Situ

[0062] Construction of 4T1 orthotopic tumor nude mouse model: A large number of 4T1 cells were cultured and labeled with near-infrared fluorescent tetrahedron probes (final concentration 200nM), the labeled cells in the logarithmic growth phase were digested, resuspended with sterile saline after centrifugation and counted three times to obtain the average value. In order to verify the high imaging sensitivity, high brightness and deep tissue penetration performance of near-infrared fluorescent tetrahedron probes in vivo, different numbers of probe-labeled cells (20, 40 cells) were inoculated in situ in the second mammary fat pad on the right side of 18-20g nude mice (male, Shanghai Qianquan Biotechnology Co., Ltd.), and the nude mice were imaged in vivo using a small animal imager to observe near-infrared fluorescence signals.

[0063] Results: Under 808nm laser irradiation, fluorescent spots were observed around the injection site of mice. Figure 6 It is noteworthy that only 40 labeled cells can produce clear fluorescent spots in vivo, indicating that the DNA nanoprobe has a sensitivity close to the single-cell level in detecting breast cancer in situ tumors, which may be attributed to the ultra-high fluorescence brightness of the DNA nanoprobe in a physiological environment.

[0064] 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 embodiment of the present invention can also be modified in various ways. That is, 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 method for preparing a near-infrared fluorescent tetrahedron probe that can be used for tumor imaging at the near single-cell level, characterized in that: The following steps are involved: 1) A DNA tetrahedral nanostructure with a side length of 20 bp is modified with a hydrophobic group so that the hydrophobic group is suspended toward the interior of the DNA tetrahedral nanostructure, thereby forming a hydrophobic confined space inside, and obtaining a DNA nanocage with precise morphology and size and a hydrophobic cavity, wherein the DNA tetrahedral nanostructure is composed of four DNA single strands, and the hydrophobic group is modified at the end of the DNA single strand, and by controlling the position of the double-stranded DNA opening formed during the synthesis of the DNA tetrahedral nanostructure, the modification position of the hydrophobic group on the edge is the 9th base from 5' to 3'; 2) providing a hydrophobic fluorescent dye with near-infrared luminescence properties, wherein the hydrophobic fluorescent dye with near-infrared luminescence properties comprises: a hydrophobic fluorescent dye molecule selected from donor-acceptor-donor (DAD), cyanine, rhodamine, and aza-BODIPY, with an emission peak at 650-1700 nm; and 3) The DNA nanocage and the hydrophobic fluorescent dye are mixed by oscillation or ultrasound, so that the hydrophobic fluorescent dye enters the hydrophobic cavity of the DNA nanocage and is encapsulated therein, thereby obtaining a near-infrared fluorescent tetrahedron probe that can be used for tumor imaging at the near single-cell level.

2. The preparation method according to claim 1, characterized in that: In step 1), the hydrophobic group includes: alkyl alcohol {HO-(CH2) y -} y -, cholesterol, polycyclic aromatic hydrocarbon structure, where x is the length of the alkyl chain, 1 < x < 100, and y is the number of alkyl chains, 1 < y < 100.

3. A near-infrared fluorescent tetrahedron probe for near single-cell level tumor imaging prepared according to the preparation method according to any one of claims 1-2, characterized in that: The near-infrared fluorescent tetrahedral probe is prepared by encapsulating a hydrophobic fluorescent dye with near-infrared luminescence properties in a DNA nanocage with a hydrophobic cavity through hydrophilic-hydrophobic interaction, wherein the hydrophobic fluorescent dye with near-infrared luminescence properties has good deep tissue penetration ability, thereby imaging tumor tissue at a near single-cell level.

4. Use of the near-infrared fluorescent tetrahedron probe according to claim 3 as a tumor imaging agent at the near single-cell level.

5. The use according to claim 4, characterized in that: The tumor imaging includes: tumor imaging of breast cancer, melanoma, and basal cell carcinoma.

6. The use according to claim 5, characterized in that: The application also includes: further connecting tumor targeting molecules, including antibodies, aptamers, and peptides, to the near-infrared fluorescent tetrahedron probe to achieve active targeted imaging of the tumor.

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