Cancer cell classification imaging system and application thereof

Abstract

The invention discloses a cancer cell classification imaging system and application thereof. The cancer cell classification imaging system comprises a double-stranded DNA (deoxyribonucleic acid) structure S1 / S2 containing S1 and S2 single chains, and a target miRNA (micro Ribonucleic Acid)-started hybridization chain reaction system, the S2 single strand comprises a specific hybridization structural domain t1 and a specific hybridization structural domain t2; the specific hybridization structural domain t1 is complementary with the target miRNA; the expression level A of the target miRNA in cancer cells is higher than the expression level B of the target miRNA in normal cells; the expression level C of the non-target miRNA in cancer cells is lower than the expression level D of the non-target miRNA in normal cells. According to the method, the number of free target miRNA in normal cells is greatly reduced through a DNA molecular subtraction method; and the free target miRNA in the cancer cells is still maintained at a proper level to trigger the hybridization chain reaction, so that the problem of insufficient signal specificity of biological imaging of the cancer cells is solved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical engineering and molecular diagnostic technology, and particularly relates to a cancer cell classification imaging system and application thereof in identification and / or immunotherapy of cancer cells. BACKGROUND

[0002] MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs found in eukaryotes, with a length of about 20-25 nucleotides, and have specific regulatory functions [1,2] . Studies have shown that abnormal expression of miRNAs is closely related to the occurrence and development of various human cancers [3-5] . For example, down-regulation of miRNA-30 is associated with the occurrence of breast cancer [6,7] ; up-regulation of miRNA-21 and miRNA-182 increases the risk of cancer occurrence and metastasis [8-10] . Therefore, in clinical research, miRNAs can not only be used as potential biomarkers for disease diagnosis

[11] , but also as targets for treatment of malignant tumors [12,13] .

[0003] High-sensitivity detection of miRNAs in body fluids can be achieved by classical methods such as Northern blotting

[14] , microarray

[15] , and real-time quantitative PCR

[16] . However, intracellular miRNA imaging research is still in its infancy

[17] . At present, various technologies such as DNA nanomachines [18,19] , hybridization chain reactions [20,21] , DNA logic circuits [22-24] , catalytic hairpin assembly (CHA)

[25] , entropy-driven catalytic reactions (EDC)

[26] , rolling circle amplification (RCA)

[27] , CRISPR-Cas system [28,29] , and enzyme-activated signal amplification strategies [30,31] have been tried for intracellular miRNA imaging. Although these methods generally have the advantage of high sensitivity, they only use the concentration information of target miRNAs to distinguish cancer cells from normal cells. Since some cancer-related miRNAs are also expressed at low levels in normal cells, the biological imaging performance of these methods is affected.

[0004] In recent decades, significant progress has been made in the field of miRNA-triggered cancer treatment

[32] . For example, Wang et al. used antisense oligonucleotide shells to capture overexpressed miR-21 and miR-155 in tumor cells, promoted in situ generation of photothermal reagents and release of doxorubicin, and thus realized the combined treatment of photothermal-chemotherapy

[33] Zhang team developed a near-infrared light-regulated miRNA amplifier, which contains a hairpin chain called "photozip". Under 808 nm laser irradiation, "photozip" is released, exposing the miRNA-21 recognition region in the amplifier; overexpressed miRNA-21 in cancer cells triggers a cascade of hybridization reactions and activates photosensitizer to generate reactive oxygen species, thereby achieving cancer treatment

[34] Morihiro et al. reported a kind of oncolytic RNA hairpin pair (oHP), and miR-21 overexpressed in cancer cells can bind to oHP and trigger a hybridization chain reaction to generate long double-stranded DNA; this double-stranded DNA can activate the p53-p21 signaling pathway, leading to cell cycle arrest at the S phase and inducing cancer cell apoptosis

[35] However, existing miRNA-triggered cancer treatment methods are activated by target miRNAs, which may also be expressed at low levels in normal cells, so these therapies not only have cytotoxicity to cancer cells, but also damage normal cells. Therefore, there is an urgent need for a miRNA-triggered cancer treatment method that can selectively kill cancer cells.

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[0006] The purpose of this invention is to provide a cancer cell classification imaging system and its application in cancer cell identification and / or immunotherapy.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A cancer cell classification imaging system, comprising: A double-stranded DNA structure containing single strands S1 and S2; wherein the single strand S2 contains a hybridization-specific domain t1 and a hybridization-specific domain t2; wherein the hybridization-specific domain t1 is complementary to the target miRNA, and the hybridization-specific domain t2 is complementary to a non-target miRNA. The expression level of target miRNA in cancer cells is A higher than its expression level in normal cells; the expression level of non-target miRNA in cancer cells is C lower than its expression level in normal cells; A is greater than C. The hybridization chain reaction system consists of two single-stranded hairpin DNA strands, H1 and H2, in which H1 and H2 are partially complementary. The target miRNA opens the hairpin structure of H1 through base complementarity pairing, and the unpaired portion of H1 opens the hairpin structure of H2, triggering the hybridization chain reaction between the H1 and H2 strands to form a long double-stranded DNA.

[0008] In normal cells, highly expressed non-target miRNAs hybridize with the t2 domain of the S2 strand, while target miRNAs hybridize with the t1 domain of the S2 strand. This chain substitution reaction leads to the mutual consumption of target and non-target miRNAs, resulting in a DNA molecule subtraction reaction and a significant reduction in free target miRNAs in normal cells. In cancer cells, low-expressed non-target miRNAs consume only a small amount of target miRNAs, maintaining free target miRNAs at a moderate level capable of triggering a hybridization chain reaction (HCR). The remaining free target miRNAs in cancer cells trigger a hybridization chain reaction between the H1 and H2 strands, forming long double-stranded DNA (dsDNA) and inducing a strong FRET signal. In normal cells, due to the insufficient amount of free target miRNAs, the HCR reaction is weak, resulting in a weak FRET signal. By detecting and comparing the differences in FRET signals between the two cell types, selective fluorescence imaging of cancer cells can be achieved. In addition, the dsDNA generated by the HCR reaction can activate the intracellular cGAS-STING signaling pathway, promote the transcription of STING mRNA and IFN-β mRNA and the expression of IFN-β protein, thereby triggering immunogenic cytotoxicity and achieving selective killing of cancer cells.

[0009] According to embodiments of the present invention, the present invention can be further optimized, and the optimized technical solution is as follows: In one preferred embodiment, the cancer cells are breast cancer cells or non-small cell lung cancer cells.

[0010] In one preferred embodiment, when the cancer cells are breast cancer cells, the normal cells are normal breast cells MCF-10A.

[0011] In one preferred embodiment, when the cancer cells are non-small cell lung cancer, the normal cells are airway epithelial cells.

[0012] In one preferred embodiment, the cancer cells are breast cancer cells MCF-7, non-small cell lung cancer cells A549, non-small cell lung cancer cells H1299, non-small cell lung cancer cells H358, and non-small cell lung cancer cells PC9.

[0013] In one preferred embodiment, the target miRNA is miR-182 and the non-target miRNA is miR-30a.

[0014] miR-182 expression was significantly higher in breast cancer cells (MCF-7) than in normal breast cancer cells (MCF-10A), and also significantly higher in non-small cell lung cancer cells (A549, H1299, H358, and PC9) than in airway epithelial cells. miR-30a showed the opposite trend to miR-182. Therefore, in both breast cancer and non-small cell lung cancer cells, miR-182 is the target miRNA, and miR-30a is the non-target miRNA.

[0015] In one preferred embodiment, the specific hybridization domain t1 has a base length of 6-10; the specific hybridization domain t2 has a base length of 6-10.

[0016] In one preferred embodiment, when the target miRNA is miR-182, the length of the specific hybridization domain t1 is 8 bases; the length of the specific hybridization domain t2 is 8 bases.

[0017] In one preferred embodiment, H1 comprises a stem and a loop, the stem consisting of 16 complementary base pairs and n-14 prominent base sequences at the 5' end, and the loop consisting of 6 bases, for a total of n+38-14 bases, where n is the number of bases in the target miRNA.

[0018] In one preferred embodiment, H2 comprises a stem and a loop. The neck consists of 15 pairs of complementary base pairs and n-14 protruding base sequences at the 3' end, and the loop consists of 8 bases, for a total of n+38-14 bases, where n is the number of bases in the target miRNA.

[0019] In one preferred embodiment, the 3' end of H1 is labeled with a first fluorescent group, and the ring portion of H2 is labeled with a second fluorescent group near the 3' end, and the first fluorescent group and the second fluorescent group can generate Förster resonance energy transfer (FRET).

[0020] In one preferred embodiment, the first fluorescent group is Cy5 or TAMRA.

[0021] The fluorescent group Cy5 (Cyanine5) emits light at a wavelength of ~670 nm (near-infrared region), and its excitation wavelength is typically ~649 nm. The color is dark red to near-infrared fluorescence.

[0022] The fluorescent group TAMRA (tetramethylrhodamine) has an excitation wavelength of ~546nm and an emission wavelength of ~575-580nm.

[0023] In one preferred embodiment, the second fluorescent group is FAM.

[0024] The fluorescent group FAM (6-carboxyfluorescein) belongs to the fluorescein dye family and is one of the most commonly used green fluorescent groups. Its excitation wavelength is ~494 nm, and its emission wavelength is ~520 nm.

[0025] The FRET signal is obtained by detecting the fluorescence intensity ratio. When the first fluorescent group is Cy5, the detected fluorescence signal is F. Cy5 / F FAM (F) Cy5 F represents the fluorescence intensity of Cy5 at 670 nm. FAM (where FAM is the fluorescence intensity at 520 nm); when the first fluorophore is TAMRA, the detected fluorescence signal is F... TAMRA / F FAM (F) TAMRA (This represents the fluorescence intensity of TAMRA at 580 nm).

[0026] Based on the same inventive concept, the present invention also claims protection for the application of the above-mentioned cancer cell classification imaging system in identifying cancer cells.

[0027] Based on the same inventive concept, the present invention also claims protection for the application of the above-mentioned cancer cell classification imaging system in the preparation of reagents for treating cancer cells.

[0028] In one preferred embodiment, in the cancer cell classification imaging system described above, the amount of double-stranded DNA structure S1 / S2 containing single strands S1 and S2 added to cancer cells is higher than the amount of non-target miRNA; in normal cells, the amount of double-stranded DNA structure S1 / S2 containing single strands S1 and S2 added is higher than the amount of target miRNA.

[0029] In one preferred embodiment, the cancer cells are MCF-7.

[0030] Based on the same inventive concept, the present invention also claims protection for a liposome comprising the above-described cancer cell classification imaging system.

[0031] In one preferred embodiment, the liposomes further comprise, by weight: 50-60 parts of methyl 4-(N,N-dimethylamino)butyrate (dilinoleyl) ester, 30-45 parts of cholesterol, 5-15 parts of distearate phosphatidylcholine, and 3-8 parts of dimyristoylglycerol-polyethylene glycol.

[0032] Based on the same inventive concept, this invention also claims protection for a method for preparing the liposomes, comprising the following steps: S1. Dissolve 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, cholesterol, distearate, myristoyl glycerol, and polyethylene glycol in ethanol at a molar ratio of 50:37.5:7.5:5 to prepare solution #1; Dissolve S1 / S2, H1, and H2 in 10mM citrate buffer (pH=4) at a molar ratio of 1:1:1 to prepare solution #2. S2. Slowly inject 1 part of solution #1 into 10 parts of solution #2, and continuously stir the mixture at 37°C for 30 minutes. Then concentrate under reduced pressure to obtain the liposomes.

[0033] Based on the same inventive concept, the present invention also claims protection for the use of the liposomes in recognizing cancer cells.

[0034] Based on the same inventive concept, the present invention also claims protection for the use of the liposomes in the preparation of reagents for treating cancer cells.

[0035] Compared with the prior art, the beneficial effects of the present invention are:

[0036] This invention develops a DNA molecular subtraction controlled hybridization chain reaction (SHCR) for selective bioimaging and killing of cancer cells. This method utilizes not only the target miRNA but also a non-target miRNA expressed at higher levels in normal cells than in cancer cells. By simultaneously leveraging the expression difference between the target miRNA (highly expressed in cancer cells and lowly expressed in normal cells) and the non-target miRNA (highly expressed in normal cells and lowly expressed in cancer cells), this invention first consumes the low-level target miRNA in normal cells through DNA molecular subtraction, and then triggers the hybridization chain reaction (HCR) with the remaining target miRNA, achieving contrast in bioimaging signals between cancer cells and normal cells and selective killing of cancer cells.

[0037] Taking selective bioimaging and killing of human breast cancer cells MCF-7 as an example, compared with normal breast epithelial cells MCF-10A, miR-182 expression was upregulated in MCF-7 cells (the expression level of miRNA-182 in MCF-7 was 2.9 times that in MCF-10A), while miR-30a expression was downregulated (the expression level of miRNA-30a in MCF-7 was half that in MCF-10A). This invention fully utilizes this information and designs a partially complementary double-stranded DNA structure (S1 / S2) to achieve DNA molecule subtraction between miR-182 and miR-30a. In MCF-7 cells, the miR-182 remaining after DNA subtraction triggers a hybridization chain reaction between two hairpin DNA strands (i.e., H1-TAMRA / Cy5 and H2-FAM), generating a long double-stranded DNA structure and producing a strong Förster resonance energy transfer (FRET) effect between TAMRA / Cy5 and FAM. Conversely, due to the relatively high expression level of miR-30a and the relatively low expression level of miR-182 in MCF-10A cells, only a very small amount of free miR-182 remains after DNA subtraction, resulting in a very weak FRET effect. In this way, SHCR improves the imaging signal contrast of MCF-7 cells relative to MCF-10A cells. Furthermore, the long double-stranded DNA structure generated by SHCR can activate the cGAS-STING pathway, thereby triggering immunogenic cytotoxicity via downstream interferon-β (IFN-β). Based on the same principle, SHCR can efficiently kill MCF-7 cancer cells while maintaining a high survival rate of normal MCF-10A cells. Therefore, this invention designs a specific DNA molecule subtraction module and an HCR reaction module, utilizing the expression differences between the two to achieve precise differentiation and treatment of cancer cells.

[0038] This invention significantly improves the contrast of bioimaging signals from cancer cells, specifically as follows:

[0039] Traditional HCR (HLNP): F of MCF-7 and MCF-10A Cy5 / F FAM The ratio is only 1.5 times, the signal difference is so weak that it cannot be reliably distinguished.

[0040] This invention SHCR (SHLNP): F of MCF-7 and MCF-10A Cy5 / F FAM The ratio is increased to 3.5 times, the signal contrast is significantly enhanced, and the cancer cells can be accurately distinguished from normal cells.

[0041] This invention achieves highly selective killing of cancer cells, as detailed below:

[0042] The large amount of dsDNA generated by the SHCR of this invention can activate the intracellular cGAS-STING pathway, triggering immunogenic cytotoxicity through downstream interferon-β (IFN-β) and inducing apoptosis in cancer cells.

[0043] The results of the selective validation experiment showed that:

[0044] MCF-10A (normal cells): After SHLNP treatment, the expression levels of STING mRNA, IFN-β mRNA and IFN-β protein were significantly lower than those in the HLNP treatment group, and the cell apoptosis rate was only 17.9% (27.3% in the HLNP group), with a high survival rate of normal cells.

[0045] MCF-7 (cancer cells): After treatment with SHLNP, the expression level of STING / IFN-β was similar to that of the HLNP group, and the apoptosis rate reached 45.8% (51.0% in the HLNP group), still maintaining high killing efficiency.

[0046] The protective effect on normal cells becomes more significant as the concentration of DNA probes in SHLNP increases.

[0047] In summary, compared with the prior art, the present invention has the following advantages:

[0048] 1. High specificity: By utilizing the expression difference of two miRNAs through the DNA molecule subtraction module, the target miRNA is left in cancer cells to trigger the HCR response, thus solving the problem of "insufficient specificity caused by low expression of target miRNA in normal cells".

[0049] 2. High-contrast imaging: SHCR increases the imaging signal ratio of cancer cells to normal cells from 1.5 times to 3.5 times, enabling precise intracellular miRNA imaging and cancer cell identification.

[0050] 3. Low normal cell toxicity: The apoptosis rate of normal cells (MCF-10A) decreased from 27.3% to 17.9%, and the protective effect increased with increasing probe concentration, avoiding damage to normal tissues during treatment.

[0051] 4. Ease of implementation: The design of DNA probes (S1 / S2, H1, H2) is simple and can be optimized with the help of NUPACK software; the vector (LNP) preparation adopts the mature ethanol injection method, which is easy to scale up production.

[0052] 5. Versatility: The base sequence of the S1 / S2 base domain can be adjusted according to the expression profile of miRNAs in different cancer types (such as target miRNAs highly expressed in other cancers and non-target miRNAs highly expressed in normal cells), which can be extended to the imaging and treatment of various cancers and has a wide range of applications. Attached Figure Description

[0053] Figure 1 This is a schematic diagram of selective bioimaging and killing of cancer cells based on DNA molecular subtraction-controlled hybridization chain reactions.

[0054] Figure 2 It is based on the design of partially complementary double-stranded DNA structures (S1 / S2) in the DNA molecule subtraction system based on the identified target miRNA and non-target miRNA.

[0055] Figure 3 It is based on the design of a pair of hairpin DNA strands H1 and H2 in a hybridization chain reaction system based on the identified target miRNA.

[0056] Figure 4 This diagram illustrates the working mechanism of the DNA molecular subtraction module and the results of gel electrophoresis verification. Lane a is a schematic diagram of the strand substitution reaction between S1 / S and miR-182 / miR-30a; lane b shows agarose gel electrophoresis images of different reaction systems. Lane 1: miR-182; Lane 2: miR-30a; Lane 3: S1 / S2; Lane 4: S1 / S2 + miR-30a; Lanes 5-6: S1 / S2 + different concentrations of miR-182; Lane 7: S1 / S2 + equal amounts of miR-182 and miR-30a; Lane 8: S1 / S2 + miR-30a + excess miR-182.

[0057] Figure 5 These are the validation results of the HCR reaction module; where a is the gel electrophoresis image of HCR reaction triggered by different concentrations of miR-182; b is the F... Cy5 / F FAM Linear relationship between the ratio and miR-182 concentration; c is the specificity verification of HCR for different miRNAs; d is the fluorescence emission spectrum of different reaction systems; e is the F of different systems. Cy5 / F FAM Comparison of ratios.

[0058] Figure 6 These are the LNP vector characterization and cell imaging results; where a is the particle size distribution of BLPN, HLNP, and SHLNP; b is the TEM image of SHLNP; c is the zeta potential of the three LNPs; and d is the F of the two cell types after HLNP and SHLNP treatment. Cy5 / F FAM ratio.

[0059] Figure 7 This is a validation of the cell-killing effect; where a is the expression level of STING mRNA; b is the expression level of IFN-β mRNA; c is the expression level of IFN-β protein; d is the cell apoptosis rate; and e is the cell survival rate at different probe concentrations. Detailed Implementation

[0060] This invention is not limited to the specific embodiments listed below. Those skilled in the art can implement this invention using various other specific embodiments based on the content disclosed herein. Any modifications or alterations made to the design structure and concept of this invention fall within the protection scope of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0061] The technical solution of this invention consists of three parts: a DNA molecule subtraction module, a hybridization chain reaction (HCR) module, and a vector delivery module. A general schematic diagram is shown below. Figure 1 As shown, the specific structure and functions are as follows:

[0062] 1. DNA Molecular Subtraction Module

[0063] Core Structure: A partially complementary double-stranded DNA structure (S1 / S2) is designed, where the S2 strand contains two basement domains (t1 and t2), which are complementary to the corresponding regions of the target miRNA (miR-182) and the non-target miRNA (miR-30a), respectively (see schematic diagrams of the double-stranded DNA structure, target miRNA, and non-target miRNA). Figure 2 (As shown).

[0064] Working mechanism: The t1 domain of the S2 chain hybridizes with miR-182 and the t2 domain hybridizes with miR-30a, triggering a chain displacement reaction that causes the S1 chain to dissociate from the S1 / S2 double strand. In this reaction, miR-182 and miR-30a consume each other in a 1:1 ratio. Only when the concentration of miR-182 is higher than that of miR-30a will there be free miR-182 remaining.

[0065] For normal cells MCF-10A (high expression of miR-30a and low expression of miR-182): miR-30a can completely consume the low level of miR-182, so there is almost no free miR-182 residue;

[0066] For cancer cells MCF-7 (high expression of miR-182 and low expression of miR-30a): miR-30a consumes only a small amount of miR-182, leaving a large amount of free miR-182 to trigger subsequent HCR responses.

[0067] 2. Hybridization Chain Reaction (HCR) Module

[0068] Core structure: Two hairpin DNA strands (H1 and H2) were designed and labeled with fluorescent groups (H1 labeled Cy5 / TAMRA, H2 labeled FAM). The stem-loop structures of H1 and H2 can be hybridized stepwise through complementary sequences to form long double-stranded DNA (dsDNA) (the structural diagrams of the target miRNA, H1, and H2 are shown in the figure). Figure 3 (As shown).

[0069] Working mechanism: Residual free miR-182 acts as a trigger, binding complementaryly to the hairpin loop of H1, opening the stem-loop structure of H1 and exposing the complementary sequence of H1 and H2; the opened H1 further hybridizes with H2 and opens the stem-loop structure of H2, forming the H1-H2 double-stranded intermediate; this intermediate continues to hybridize with unreacted H1 and H2, initiating a chain reaction to generate a long double-stranded DNA structure (dsDNA); at the same time, the Cy5 / TAMRA on H1 and the FAM on H2 generate Förster resonance energy transfer (FRET) due to the shortening distance, and cell imaging is achieved by detecting the FRET signal (fluorescence intensity ratio of FCy5 / FFAM or FTAMRA / FFAM).

[0070] Specificity: The HCR module responds only to miR-182 and has no cross-reactivity with other cancer-related miRNAs such as miR-21, miR-101, and miR-143, ensuring detection specificity.

[0071] 3. Carrier delivery module

[0072] Carrier selection: Lipid nanoparticles (LNPs) were used as delivery carriers and prepared by ethanol injection method. S1 / S2 (500 nM), H1 (1 μM), and H2 (1 μM) were encapsulated in LNPs to form "SHLNP". At the same time, "HLNP" encapsulated only H1 and H2 was prepared as a control, as well as blank LNP (BLPN).

[0073] Carrier performance ( Figure 6 a~6c):

[0074] Size and morphology: SHLNP, HLNP, and BLPN have an average particle size of 120-150 nm, are spherical, and are conducive to phagocytosis by cells;

[0075] Stability: When the nitrogen-to-phosphorus ratio (N / P) is ≥4, LNP can completely encapsulate the DNA probe without significant leakage;

[0076] Surface potential: The zeta potential is approximately 20 mV, which can promote cellular uptake and enable lysosomal escape, ensuring the activity of the probe within the cell;

[0077] Nuclease resistance: S1 / S2, H1, and H2 remain intact after incubation at 37°C for 24 hours in a buffer containing fetal bovine serum (FBS), which can meet the requirements for SHCR within 1 hour.

[0078] The DNA and miRNA sequences involved in the embodiments of the present invention are shown in Table 1. '^' indicates that the two bases are connected by a thiophosphate bond.

[0079] Example 1

[0080] 1. DNA probe design and synthesis

[0081] The S1, S2, H1, and H2 sequences were designed using NUPACK software (https: / / nupack.org / ) (Table 1) to ensure partial complementarity of S1 / S2, hairpin structure stability of H1 and H2, and specific binding of S2 to miR-182 and miR-30a.

[0082] The S1, S2, H1, and H2 sequences were synthesized by a biotechnology company, with H1 labeled with Cy5 (or TAMRA) and H2 labeled with FAM, with a purity ≥99% (HPLC grade).

[0083] 2. Validation by in vitro DNA molecule subtraction and HCR reaction

[0084] Gel electrophoresis verification: reaction systems containing different concentrations of miR-182, miR-30a and S1 / S2 were prepared, incubated at 37℃ for 30 min and then subjected to agarose gel electrophoresis. The changes in the bands were observed to verify the molecular subtraction effect.

[0085] The results are as follows Figure 4 As shown, a is a schematic diagram of the chain displacement reaction between S1 / S and miR-182 / miR-30a; b is an agarose gel electrophoresis diagram of different reaction systems; where lane 1: miR-182; lane 2: miR-30a; lane 3: S1 / S2; lane 4: S1 / S2 + miR-30a; lanes 5-6: S1 / S2 + different concentrations of miR-182; lane 7: S1 / S2 + equal amounts of miR-182 and miR-30a; lane 8: S1 / S2 + miR-30a + excess miR-182. Figure 4 Lane 5-6 illustrates the mechanism by which miR-182 and miR-30a bind to, compete with, and achieve a "subtractive" reaction with the double-stranded probes S1 / S2. Lane 5-6: Upon addition of miR-182, S1 is replaced, and miR-30a is consumed. Figure 4In lane 7 of the DNA molecule b, after adding equal amounts of miR-182 and miR-30a, they were consumed together, and the S1 / S2 bands showed significant migration changes. In lane 8, when miR-182 was in excess, there was still residual free miR-182. The results show that DNA molecule subtraction can achieve equal consumption of the two miRNAs.

[0086] Fluorescence detection: A reaction system containing H1-TAMRA, H2-FAM, and different concentrations of miR-182 was prepared. After incubation at 37℃ for 1 h, the fluorescence intensity at 520 nm (FAM) and 560 nm (TAMRA) was detected using a fluorescence spectrometer under excitation light at 480 nm. The F-values ​​were calculated. Cy5 / F TAMRA The ratio was used to verify the concentration dependence and specificity of the HCR reaction.

[0087] Specificity verification: To verify the specificity of the probe for miRNA, H1-TAMRA and H2-FAM (200 nM each) were renatured in TE buffer, and then miR-182, miR-21, miR-30a, miR-101, or miR-143 (100 nM each) were added, and the mixture was incubated at 37°C for 2 h. The fluorescence intensities of FAM (520 nm) and TAMRA (580 nm) were recorded and the ratio (F_{TAMRA}} / F_{FAM}} was calculated.

[0088] Fluorescence emission spectroscopy detection of different reaction systems: The experimental groups were set up as follows: Mix1: H1; Mix2: H2; Mix3: H1 + H2; Mix4: H1 + H2 + miR-182 (50 nM) + miR-30a (70 nM); Mix5: H1 + H2 + miR-182 (150 nM) + miR-30a (30 nM); Mix6: S1 + S2 + H1 + H2 + miR-182 (50 nM) + miR-30a (70 nM); Mix7: S1 + S2 + H1 + H2 + miR-182 (150 nM) + miR-30a (30 nM). The concentrations of S1 and S2 were both 200 nM, and the concentrations of H1 and H2 were both 200 nM. After incubation at 37℃ for 2 h, each system was excited at 488 nm, and the fluorescence intensity at 520 nm and 580 nm was recorded. The ratio of (F_{\text{TAMRA}} / F_{\text{FAM}}) was calculated, and the result was the average of three independent experiments.

[0089] The results are as follows Figure 5 As shown. In this image, a is a gel electrophoresis image of HCR reactions triggered by different concentrations of miR-182; b is the F... Cy5 / F FAMLinear relationship between the ratio and miR-182 concentration; c represents the specificity verification of HCR for different miRNAs (miR-21, miR-30a, miR-101, miR-143); d represents the fluorescence emission spectra of different reaction systems; e represents the F-values ​​of different systems. Cy5 / F FAM Ratio comparison. Gel electrophoresis results showed that the higher the miR-182 concentration, the more HCR products were produced, confirming its triggering ability. Fluorescence spectroscopy results showed that the FRET effect between TAMRA / FAM was enhanced with increasing miR-182 concentration. Specificity experiments showed that the addition of non-target miRNAs such as miR-21, miR-30a, miR-101, and miR-143 did not significantly enhance the signal; only miR-182 significantly triggered fluorescence enhancement, indicating that the system has good sequence specificity and good reaction specificity. Fluorescence emission spectra of different systems showed that compared with conventional HCR and SHCR with the introduction of S1 / S2, the SHCR signal differed significantly when the miR-182:miR-30a concentration ratio was different (150:30 vs 50:70). The F... Cy5 / F FAM The ratio comparison results show that the signal difference of conventional HCR (Mix5-Mix4) is about 2.7 times; while the signal difference of SHCR of the present invention (Mix7-Mix6) reaches 15.4 times; indicating that the molecular subtraction of the present invention significantly improves the detection sensitivity and contrast.

[0090] Example 2

[0091] Preparation of lipid nanoparticles (SHLNP / HLNP / BLPN)

[0092] Materials preparation: First, dissolve 4-(N,N-dimethylamino)butyrate (dilinoleyl) methyl ester, cholesterol, distearate phosphatidylcholine, and dimyristoylglycerol-polyethylene glycol in ethanol at a molar ratio of 50:37.5:7.5:5 to prepare solution #1; dissolve S1 / S2, H1, and H2 in 10mM citrate buffer (pH=4) at a molar ratio of 1:2:2 to prepare solution #2.

[0093] Preparation by ethanol injection method: 1 part of solution #1 is slowly injected into 10 parts of solution #2, and the mixture is continuously stirred at 37°C for 30 minutes to remove ethanol. Then, the mixture is concentrated by distillation under reduced pressure to obtain a solution of lipid nanoparticles containing delivery carriers S1 / S2, H1 and H2 of the desired concentration.

[0094] Characterization: Particle size and zeta potential were determined by dynamic light scattering (DLS), and morphology was observed by transmission electron microscopy (TEM).

[0095] The results are as follows Figure 6The figures in Figure ac show the particle size distribution of BLPN, HLNP, and SHLNP. Figure a shows the TEM image of SHLNP; and figure c shows the zeta potential of the three LNPs. The particle sizes of the three lipid nanoparticles (LNPs)—BLNP (blank), HLNP (containing H1 / H2), and SHLNP (containing S1 / S2 / H1 / H2)—are approximately 120-150 nm. The transmission electron microscopy (TEM) image of SHLNP shows its spherical morphology. The surface potential (zeta potential) of the three nanoparticles is approximately +20 mV, which is beneficial for cellular uptake.

[0096] Cell experiments

[0097] Cell culture: MCF-7 (human breast cancer cells) and MCF-10A (human normal breast epithelial cells) were cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2. The relative expression levels of miR-182 and miR-30a were detected: miR-182 in MCF-7 was about 2.9 times that of MCF-10A; miR-30a in MCF-7 was about half that of MCF-10A.

[0098] Transfection: Incubate cells with 500 nM (calculated as H1) of SHLNP, HLNP, and BLPN for 1 h, then replace with fresh medium and continue culturing for 24 h.

[0099] Imaging and Detection: Detection of intracellular F by flow cytometry Cy5 / F FAM The ratio is used to achieve imaging. The detection procedure on the flow cytometer is as follows: excitation is performed using only a 488nm laser. Fluorescence emission signals of FAM (donor) and Cy5 (acceptor) are collected separately, and the FRET ratio is calculated accordingly.

[0100] The results are as follows Figure 6 As shown in d, Figure 6 In this context, d represents the F cells of the two cell lines after treatment with HLNP and SHLNP. Cy5 / F FAM Ratio cell imaging fluorescence ratio results showed that the F of MCF-7 / MCF-10A in HLNPs was higher than that in HLNPs. Cy5 / F FAM The ratio was only 1.5 times; while SHLNP improved it to 3.5 times, resulting in a significant improvement in imaging contrast. The results show that the SHCR system can distinguish between cancer cells and normal cells.

[0101] The expression levels of STING mRNA and IFN-β mRNA were detected using qPCR. MCF-10A and MCF-7 cells were cultured for 48 hours in medium containing 1 mL of HLNP or SHLNP solution, respectively. The amount of HLNP or SHLNP added was such that the final concentration of DNA probes (S1 / S2, H1, and H2) in the medium was 500 nM. Total RNA was then extracted from MCF-10A or MCF-7 cells using Beyozol reagent (Beyotime, China) and quantified using a NanoDrop micro-spectrophotometer (Thermo Scientific, USA). The expression levels of cGAS-STING and IFN-β mRNA were detected using quantitative real-time PCR (qRT-PCR) with β-actin as an internal control on a Bro-Rad T100 PCR instrument (Bio-Rad, USA). All primers used were specific primers, as shown in SEQ ID NO.7-SEQ ID NO.12. qRT-PCR was performed using a one-step real-time quantitative PCR kit (Beyotime, China). Total RNA was first extracted from MCF-7 and MCF-10A cells using Beyotime reagent (China). The reaction mixture (total volume 20 μL) was prepared directly according to the kit instructions. STING and IFN-β were used as target genes, and β-actin was used as an internal control. The reaction was performed on a quantitative PCR instrument: 95℃ pre-denaturation for 10 min; 95℃ for 15 s, 60℃ for 1 min, for a total of 40 cycles. Relative expression levels were calculated using the 2^(-ΔΔCt) method. All experiments were performed in triplicate, and results are expressed as mean ± standard deviation.

[0102] The expression level of IFN-β protein was detected using an ELISA kit. The procedure was as follows: MCF-7 and MCF-10A cells were cultured at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 μL in 24-well plates containing 500 μL of culture medium and cultured for approximately 24 hours until cell confluence reached approximately 80%. The medium was then replaced with Opti-MEM (Thermo Fisher Scientific, USA), and cells were transfected with either HLNP (100 nM as H1) or SHLNP (100 nM as H1). One hour after transfection, the medium was replaced with standard growth medium, and cells were cultured for another 18 hours. Finally, the expression levels of IFN-β protein in MCF-7 and MCF-10A cells were detected using a Human IFN-β ELISA Kit (Bioswamp, China).

[0103] Apoptosis rate was detected using the Annexin V-FITC / PI double staining kit (Beyotime, China). The procedure was as follows: MCF-10A and MCF-7 cells were added to culture medium containing 1 mL of HLNP (500 nM H1) or SHLNP (500 nM H1), respectively, and cultured for 48 hours. Subsequently, the cells were washed three times with 500 μL PBS and resuspended in 1×Annexin V binding buffer (Beyotime, China) to a concentration of 1×10⁻⁶. 6 Cells / mL. Add 5 μL Annexin V-FITC (20×) (Beyotime, China) and 5 μL pyridine iodide (PI) (Beyotime, China) to the cell suspension, mix gently, and incubate at room temperature (20–25°C) in the dark for 15 minutes. After incubation, wash the cells three times with pre-chilled 1× Annexin V binding buffer, and resuspend in 400 μL of pre-chilled 1× Annexin V binding buffer. Finally, analyze the samples using a flow cytometer (CytoFLEX, Beckman Coulter, USA).

[0104] The results are as follows Figure 7As shown in the figure, a represents the expression level of STING mRNA; b represents the expression level of IFN-β mRNA; c represents the expression level of IFN-β protein; d represents the apoptosis rate; and e represents the cell survival rate at different probe concentrations. The STING mRNA expression level results show that the expression level of STING mRNA in the MCF-10A+SHLNP group was significantly lower than that in the MCF-10A+HLNP group, while the change was not significant in the MCF-7 cell culture group. The trend of IFN-β mRNA expression level was similar to that of STING mRNA. The IFN-β protein expression level results show that the IFN-β protein expression level in the MCF-7+HLNP group (100 nM based on H1) was similar to that in the MCF-7+SHLNP group (100 nM based on H1); the IFN-β protein expression level in the MCF-10A+SHLNP group was significantly decreased. The apoptosis assay results showed that 27% of MCF-10A cells in the MCF-10A+HLNP group underwent apoptosis, while 18% in the MCF-10A+SHLNP group. The selectivity of the MCF-10A+SHLNP group for normal and cancer cells (selectivity defined as the ratio of drug-induced apoptosis rate in normal cells to drug-induced apoptosis rate in cancer cells) was increased, indicating that SHCR can reduce damage to normal cells and improve selectivity. Cell viability results at different probe concentrations showed that with increasing concentration, SHLNP maintained a strong killing effect on MCF-7 cells while enhancing its protective effect against MCF-10A cells (statistical significance: p<0.05, p<0.01, p<0.001).

[0105] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art can make other variations or modifications based on the above description. It is impossible to exhaustively list all possible implementations here. All obvious variations or modifications derived from the technical solutions of this invention are still within the scope of protection of this invention.

Claims

1. A cancer cell classification imaging system, characterized by, The application relates to a cancer cell classification imaging system and a liposome thereof. The system comprises: a double-stranded DNA structure containing S1 and S2 single strands; the S2 single strand contains a specific hybridization domain t1 and a specific hybridization domain t2; wherein the specific hybridization domain t1 can be complementary to a target miRNA, and the specific hybridization domain t2 can be complementary to a non-target miRNA; the expression level A of the target miRNA in cancer cells is higher than the expression level B of the target miRNA in normal cells; the expression level C of the non-target miRNA in cancer cells is lower than the expression level D of the non-target miRNA in normal cells; A is greater than C; a hybridization chain reaction system of two single-stranded hairpin DNA chains H1 and H2; wherein H1 and H2 are partially complementary; the target miRNA opens the hairpin structure of H1 through base complementary pairing, the unpaired part of H1 opens the hairpin structure of H2, and the hybridization chain reaction of the H1 chain and the H2 chain is triggered to form a long-chain double-stranded DNA. The cancer cells are breast cancer cells or non-small cell lung cancer cells. The cancer cells are breast cancer cells MCF-7, non-small cell lung cancer cells A549, non-small cell lung cancer cells H1299, non-small cell lung cancer cells H358 or non-small cell lung cancer cells PC9.

2. The cancer cell classification imaging system of claim 1, wherein, The target miRNA is miR-182, and the non-target miRNA is miR-30a.

3. The cancer cell classification imaging system of claim 1, wherein, The base length of the specific hybridization domain t1 is 6-10; and the base length of the specific hybridization domain t2 is 6-10.

4. The cancer cell classification imaging system of claim 1, wherein, H1 comprises a stem part and a loop part, the stem part is composed of 16 pairs of complementary base pairs and 5' end n-14 overhanging base sequences, the loop part is composed of 6 bases, and the total number of bases is n+38-14, wherein n is the base number of the target miRNA; H2 comprises a stem part and a loop part, the neck part is composed of 15 pairs of complementary base pairs and 3' end n-14 overhanging base sequences, the loop part is composed of 8 bases, and the total number of bases is n+38-14, wherein n is the base number of the target miRNA; the 3' end of H1 is marked with a first fluorescent group, the loop part of H2 close to the 3' end position is marked with a second fluorescent group, and the first fluorescent group and the second fluorescent group can produce a Forster resonance energy transfer.

5. The cancer cell classification imaging system of claim 1, wherein, The liposome comprises the cancer cell classification imaging system according to any one of claims 1-6.

6. The cancer cell classification imaging system of claim 1, wherein, The liposome further comprises: 50-60 parts of 4-(N,N-dimethylamino) butyric acid (dilinoleyl) methyl ester, 30-45 parts of cholesterol, 5-15 parts of distearoyl phosphatidylcholine, and 3-8 parts of dimyristoyl glycerol-polyethylene glycol.

7. A liposome, characterized by, 9. The cancer cell classification imaging system according to any one of claims 1-6 or the liposome according to claim 7 or 8 is applied to identification of cancer cells.

8. The liposome of claim 7, wherein, 10. The cancer cell classification imaging system according to any one of claims 1-6 or the liposome according to claim 7 or 8 is applied to preparation of a reagent for treating cancer cells. ​ ​

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