A DNA nanoprobe for triple-negative breast cancer imaging and treatment, as well as its preparation method and application

Abstract

The present invention discloses a DNA nanoprobe for imaging and treating triple-negative breast cancer, as well as its preparation method and application. The invention relates to the technical field of DNA nanoprobes. The DNA nanoprobe comprises a DNA double helix structure and doxorubicin loaded by embedding the DNA double helix structure. The DNA double helix structure is self-assembled by a T strand, a hairpin strand 1, and a hairpin strand 2 through a hybridization chain reaction. The T strand is an AS1411-functionalized T strand. The sequence of the hairpin strand 1 contains a PARP1 antisense sequence and is modified with a Cy5 fluorescent group. The hairpin strand 2 is modified with a PD-L1 siRNA and a BHQ2 fluorescence quencher. The present invention is expected to achieve integrated diagnosis and treatment of triple-negative breast cancer through AS1411 aptamer targeting, PARP1-triggered "off-on" fluorescence imaging, and a chemotherapy-gene-immunotherapy combined therapy strategy.

Description

Technical Field

[0001] The present invention relates to the technical field of DNA nanoprobes, and in particular to a DNA nanoprobe for imaging and treating triple-negative breast cancer, and a preparation method and application thereof. Background Art

[0002] Triple-negative breast cancer (TNBC) is a subtype of breast cancer that lacks expression of estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2), accounting for approximately 10%-15% of all breast cancers. Due to the lack of a clear therapeutic target, traditional chemotherapy is the primary treatment, but it suffers from limited efficacy and significant toxic side effects. In recent years, nanoprobe-based integrated tumor diagnosis and treatment technologies have become a research hotspot. By integrating imaging and therapeutic capabilities, they can achieve precise diagnosis and efficient treatment of tumors, providing a new strategy for the clinical management of triple-negative breast cancer.

[0003] Currently, DNA nanoprobe technology for triple-negative breast cancer has the following main limitations:

[0004] (1) Insufficient imaging sensitivity and specificity: Traditional nano-imaging platforms mostly use an “always on” mode, where the probe continuously emits fluorescence in the blood circulation or non-target tissues, resulting in high background noise and limited detection sensitivity. For example, existing DNA-based fluorescent probes are difficult to distinguish between tumor microenvironment and normal tissues because they do not have an “activation-off” response mechanism, which limits their application in in vivo imaging.

[0005] (2) Separation of diagnostic and therapeutic functions: Existing DNA nanoprobes mostly focus on single tumor diagnosis (such as imaging) or treatment (such as chemotherapy, gene therapy), and are unable to achieve the synergistic effect of imaging and multimodal treatment through the same carrier. For example, some probes only load chemotherapy drugs or only integrate gene therapy components, but lack technical solutions that combine imaging functions with multimodal treatments such as chemotherapy, gene therapy, and immunotherapy, making it difficult to meet the clinical needs of "precise diagnosis-efficient treatment".

[0006] (3) Complex preparation process and component interference: When constructing multifunctional composite probes, conventional nanomaterials usually require complex chemical modification or multi-step assembly processes to integrate targeting ligands, imaging molecules, and therapeutic components, resulting in low preparation efficiency and easy interference between components (such as quenching of fluorescent groups and inactivation of therapeutic molecules). For example, some liposome- or polymer-based nanoprobes, when loaded with multiple therapeutic drugs, often release drugs prematurely due to unstable carrier structures, increasing the risk of off-target toxicity.

[0007] (4) Off-target toxicity risk: Traditional nanoprobes lack tumor-specific targeting capabilities and are easily taken up by normal tissues during circulation in the body, resulting in the release of therapeutic components (such as chemotherapy drugs and small interfering RNA) at non-target sites, causing systemic toxicity. For example, free doxorubicin, a commonly used chemotherapy drug for triple-negative breast cancer, has anti-tumor activity but has serious side effects such as cardiotoxicity, which limits its clinical application dose.

[0008] In summary, developing a DNA nanoprobe that combines high-sensitivity imaging, multimodal therapy, efficient assembly and low off-target toxicity is the key to solving the problem of precise diagnosis and treatment of triple-negative breast cancer. Summary of the Invention

[0009] The present invention discloses a DNA nanoprobe for the imaging and treatment of triple-negative breast cancer. To address the above-mentioned technical deficiencies, an activated DNA nanoprobe based on hybridization chain reaction self-assembly is proposed. Through AS1411 aptamer targeting, PARP1-triggered "off-on" fluorescence imaging and chemotherapy-gene-immunotherapy combined therapy strategy, it is expected to achieve integrated diagnosis and treatment of triple-negative breast cancer, thereby solving the above-mentioned difficulties existing in the existing DNA nanoprobe technology for triple-negative breast cancer.

[0010] The technical solution adopted in the present invention is as follows:

[0011] A DNA nanoprobe for imaging and treating triple-negative breast cancer, characterized in that the DNA nanoprobe comprises a DNA double helix structure and doxorubicin loaded by being embedded in the DNA double helix structure, the DNA double helix structure being self-assembled by a T strand, a hairpin strand 1, and a hairpin strand 2 through a hybridization chain reaction, the T strand being an AS1411-functionalized T strand; the sequence of the hairpin strand 1 contains a PARP1 antisense sequence, and the hairpin strand 1 is modified with a Cy5 fluorescent group; the hairpin strand 2 is modified with a PD-L1 siRNA and a BHQ2 fluorescence quencher.

[0012] Furthermore, the PARP1 antisense sequence is shown in SEQ ID NO: 1. SEQ ID NO: 1: CTG CAC CATGAT GGC CAT CCG GAG CGA GTC.

[0013] Furthermore, the PD-L1 siRNA is shown in SEQ ID NO: 2. SEQ ID NO: 2: GAC CTG GCT GCACTA ATT GTC TAT T.

[0014] Furthermore, the T strand is shown in SEQ ID NO: 3. SEQ ID NO: 3: GGT GGT GGT GGT TGT GGTGGT GGT GG TTT GGT GGG GGC CAT CCG GAG.

[0015] Furthermore, the hairpin strand 1 is shown in SEQ ID NO: 4. SEQ ID NO: 4: CTG CAC CAT GAT GGCCAT CCG GAG CGA GTC CTC CGG ATG GCC CCC ACC.

[0016] Furthermore, the unmodified strand of the hairpin strand 2 is shown in SEQ ID NO: 5. SEQ ID NO: 5: GAC TCGCAC CGG ATG GCC GGT GGG GGC CAT CCG GAG AG AAT AGA CAA TTA GTG CAG CCA GGTCAT.

[0017] Furthermore, the assembly molar ratio of the T strand, hairpin strand 1 and hairpin strand 2 is 1:3:3.

[0018] Furthermore, the loading molar ratio of the doxorubicin to the DNA double helix structure is 1:8.

[0019] The method for preparing any of the above-mentioned DNA nanoprobes for triple-negative breast cancer imaging and treatment comprises the following steps:

[0020] (1) Prepare T strand, hairpin strand 1, and hairpin strand 2, and modify hairpin strand 1 with a Cy5 fluorescent group, and modify hairpin strand 2 with PD-L1 siRNA and a BHQ2 fluorescence quencher;

[0021] (2) Annealing the T strand, hairpin strand 1, and hairpin strand 2 obtained in step (1) at 95°C for 5-10 minutes respectively;

[0022] (3) Add the annealed T strand, hairpin strand 1, and hairpin strand 2 to a Tris-Mg buffer solution containing Tris and MgCl2 at pH 8.0 and react at 37°C for 4 hours;

[0023] (4) Add doxorubicin and incubate at room temperature for 30 minutes to obtain the DNA nanoprobe.

[0024] Use of any of the above-mentioned DNA nanoprobes for imaging and treating triple-negative breast cancer in the preparation of integrated reagents or drugs for the diagnosis and treatment of triple-negative breast cancer.

[0025] Compared with the prior art, the present invention has achieved the following beneficial effects:

[0026] 1. This invention constructs an "off-on" response mechanism through the design of Cy5 fluorescent group / BHQ2 quenching group. The DNA nanoprobe remains in a fluorescence quenching state when there is no target, and activates the near-infrared fluorescence signal only when specifically triggered by PARP1 to disassemble. The detection limit of PARP1 is 0.73 nM (R 2 =0.9535). Specificity verification experiments showed that the fluorescence response rate of the probe to non-target sequences (such as Bcl-2 mRNA and random chains) was less than 5%;

[0027] 2. This invention achieves efficient recognition and specific entry into MDA-MB-231 cells through AS1411 aptamer-mediated nucleolin targeting. The DNA probe enters the target cells and undergoes specific disassembly, triggering multimodal therapy: ① PARP1 silencing inhibits base excision repair; ② PD-L1 siRNA interference reverses immunosuppression; ③ doxorubicin-induced cytotoxicity;

[0028] 3. This invention utilizes DNA hybridization chain reaction (HCR) to prepare probes. By extending nucleic acid strands or utilizing sticky ends of complementary sequences for ligation, the trigger strand (T strand), hairpin strand 1, and hairpin strand 2 are functionally modified. This simple approach allows for the rapid and efficient integration of a targeting aptamer (AS1411) and therapeutic components (PARP1 antisense sequence, PD-L1 siRNA, and the chemotherapy drug doxorubicin) into a single system. Experimental results demonstrate that the functional components of the probe do not interfere with each other, effectively achieving tumor targeting, specific imaging, and multimodal therapeutic capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The figure shows the electrophoresis results of the assembly feasibility of three groups of DNA nanoprobes with different sequences. In the figure, lane 1: T-1+H1-1+H2-1=1:1:1, lane 2: T-1+H1-1+H2-1=1:2:2, lane 3: T-2+H1-2+H2-2=1:1:1, lane 4: T-2+H1-2+H2-2=1:2:2, lane 5: T-3+H1-3+H2-3=1:1:1, lane 6: T-3+H1-3+H2-3=1:2:2, lane 7: marker (500bp, 200bp and 100bp are indicator bands, showing bright bands).

[0030] Figure 2This is the electrophoresis result of the assembly efficiency of DNA nanoprobes with different molar feed ratios. In the figure, lane 1: T, lane 2: H1, lane 3: H2, lane 4: T+H1, lane 5: T+ H2, lane 6: H1+ H2, lane 7: T+H1+H2=1:1:1, lane 8: T+H1+ H2=1:2:2, lane 9: T+H1+ H2=1:3:3, lane 10: marker (500bp, 200bp and 100bp are indicator bands, showing bright bands).

[0031] Figure 3 This is the electrophoresis result of the assembly efficiency of H2'-2 chain and S chain. In the figure, lane 1: H2'-2, lane 2: S, lane 3: H2'-2∶S=1:1, lane 4: H2'-2:S=1:1.2, lane 5: H2' -2:S=1:1.4, lane 6: H2' -2:S=1:1.6, lane 7: marker (500bp, 200bp and 100bp are indicator bands, showing bright bands).

[0032] Figure 4 The graph shows the Cy5 fluorescence signal intensity when the probe binds to different sequences;

[0033] Figure 5 The graph shows the fluorescence intensity of the probe in response to different concentrations of PARP1 in buffer;

[0034] Figure 6 is the linear relationship between fluorescence intensity and PARP1 concentration;

[0035] Figure 7 The Cy5 fluorescence intensity histogram is shown when the probe is co-incubated with different sequences in the buffer;

[0036] Figure 8 The fluorescence intensity curve of DOX when different concentrations of probes were added to 1 μM DOX solution;

[0037] Figure 9 Flow cytometry images of MDA-MB-231 cells after incubation with the probe for 15 min;

[0038] Figure 10 This is a laser confocal microscopic image of cells after incubation with the probe for 15 minutes;

[0039] Figure 11 Laser confocal microscopy images of cells after incubation with probes for different time periods;

[0040] Figure 12 Laser confocal microscopic imaging of Cy5 after cells were co-incubated with the probe for 4 h under different treatments;

[0041] Figure 13 Laser confocal microscopic images of doxorubicin and Cy5 after cells were incubated with probes for different time periods;

[0042] Figure 14 Laser confocal microscopic imaging of Cy5 and doxorubicin after cells were co-incubated with probes under different treatments;

[0043] Figure 15 This is a bar graph showing the killing effect of different concentrations of DNA nanoprobes on MDA-MB-231 cells analyzed by MTS assay;

[0044] Figure 16 This is a bar graph showing the killing effect of different DNA nanoprobes on MDA-MB-231 cells analyzed by MTS assay;

[0045] Figure 17 The bar graph shows the relative expression levels of PARP1 and PD-L1 in MDA-MB-231 cells analyzed by RT-qPCR;

[0046] Figure 18 Figure 1 shows the relative expression of PARP1 and PD-L1 in MDA-MB-231 cells analyzed by Western blot. (a) is a bar graph showing the relative expression of PARP1 and PD-L1 in MDA-MB-231 cells analyzed by Western blot; (b) is a grayscale analysis histogram showing the relative expression of PARP1 and PD-L1 proteins in (a).

[0047] The symbols of significant differences and the corresponding P values ​​used in each figure are as follows: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns means P ≥ 0.05. DETAILED DESCRIPTION

[0048] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are for illustrating the present invention, rather than for limiting the present invention.

[0049] Throughout this specification, unless otherwise specified, the terms used herein should be understood to have the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In the event of any conflict, the present specification shall take precedence.

[0050] Unless otherwise specified, various raw materials, reagents, instruments and equipment used in the present invention can be purchased from the market or prepared by existing methods.

[0051] Example 1

[0052] In this example, electrophoresis experiments were used to verify the feasibility of assembling each sequence group and to investigate the assembly efficiency of DNA nanoprobes. Three different groups of DNA nanoprobes were designed based on the concept of the present invention. The nucleic acid sequences of the relevant DNA or RNA fragments in the DNA nanoprobes are shown in Table 1. The relevant DNA or RNA fragments were prepared by Shanghai Sangon Bioengineering Technology Service Co., Ltd.

[0053] Table 1 Nucleic acid sequences of related DNA or RNA fragments

[0054]

[0055] In this example, only the feasibility of assembling each sequence group and the assembly efficiency of the DNA nanoprobe were discussed, so the Cy5 fluorescent group and the BHQ-2 quenching group were not modified, and doxorubicin was not loaded.

[0056] The assembly process is as follows: the T chain, H1 chain (hairpin chain 1), and H2 chain (hairpin chain 2) (H2 chain is assembled from the unmodified chain H2' + PD-L1siRNA sequence, completed by Shanghai Sangon Bioengineering Technology Service Co., Ltd.) are annealed separately (annealing conditions: annealing at 95°C for 5-10 minutes). After annealing, the T chain, H1 chain, and H2 chain are added to a buffer solution (Tris-Mg buffer containing 10 mMTris, 5 mM MgCl2, pH 8.0) at different molar ratios, and the DNA nanoprobe is obtained after reaction at 37°C for 4 hours.

[0057] Assembly feasibility and efficiency evaluation: 6 μL of DNA nanoprobe, 2 μL of 6× Loading Buffer, and 2 μL of 100× SYBR Gold dye were mixed and slowly added to the sample reservoir. Electrophoresis was continued at 120 V in 1× Tris-Glycine electrophoresis buffer until the lowest band of the marker was reached approximately 2 / 3 of the way into the electrophoresis well. The sample was removed and photographed.

[0058] First, the feasibility of assembling three different groups of DNA nanoprobes shown in Table 1 was explored. T chain, H1 chain, and H2 chain were assembled into DNA nanoprobes at a molar feed ratio of 1:1:1 and 1:2:2, respectively. The six groups of DNA nanoprobes were subjected to electrophoresis experiments with markers. The results are shown in Figure 1. Figure 1 As shown, obvious product was generated in lane 4, indicating that the assembly of the second group of probes was feasible.

[0059] Secondly, to further investigate the assembly efficiency of the second group of probes, we set up multiple sets of molar feed ratios to assemble the second group of T chains, H1 chains, and H2 chains (T-2, H1-2, H2-2) into DNA nanoprobes, and then performed electrophoresis experiments on the obtained DNA nanoprobes with markers. The results are shown in Figure 2. Figure 2 As shown, it was found that the T chain, H1 chain, and H2 chain were successfully assembled. Further comparison of lanes 7, 8, and 9 showed that obvious products were generated when the T chain, H1 chain, and H2 chain were assembled in a ratio of 1:3:3, indicating that the probe assembly efficiency was the highest at this time.

[0060] This example also discusses the assembly molar ratio of H2'-2 and PD-L1 siRNA sequences. Figure 3 As shown, it was found that the H2-2 chain can be successfully assembled when the H2'-2 and S chains of the DNA nanoprobe are assembled in a ratio of 1:1. By comparing lanes 3, 4, 5, and 6, it was found that when the proportion of the S chain was gradually increased, the change in the generation of the H2-2 chain was not obvious. Combined with the experimental results, it is considered that the best assembly ratio of the H2'-2 chain and the S chain is 1:1.

[0061] Example 2

[0062] This example investigates the response performance of DNA nanoprobes to targets and their loading capacity for doxorubicin. The DNA nanoprobes were modified with a Cy5 fluorescent group and a BHQ2 fluorescent quencher and loaded with doxorubicin. The nucleic acid sequences of the relevant DNA or RNA fragments involved in this example are shown in Table 2.

[0063] Table 2 Nucleic acid sequences of related DNA or RNA fragments

[0064]

[0065] In this example, experiments were conducted using the probe at the buffer level. The fluorescence intensity of Cy5 fluorescence (Cy5: Ex = 633 nm, Em = 650-750 nm) was measured using a fluorescence spectrophotometer to verify the feasibility of the probe-loaded PARP1 antisense sequence binding to PARP1 in PBS. The detection limit of the probe for PARP1 was calculated, and the specificity of the probe binding to PARP1 was examined. The doxorubicin fluorescence was measured using a fluorescence spectrophotometer to calculate the amount of doxorubicin loaded by the probe. The specific experimental process is as follows:

[0066] (1) Feasibility study of probe response target PARP1

[0067] A "Cy5 always-on" DNA nanoprobe (i.e., a DNA nanoprobe without a BHQ-2 quenching group) and a DNA nanoprobe (hereinafter referred to as the probe) were assembled separately. After the two probes were successfully assembled, the final concentration of the probe in the buffer was set to 50 nM. After PARP1 or PD-L1 target was added to the probe, the probe was incubated in PBS. The fluorescence value of Cy5 in each group was measured using a fluorescence spectrophotometer, and the response performance of the probe to the target PARP1 was examined in the buffer.

[0068] The Cy5 fluorescence signals in MDA-MB-231 cells of different groups were measured by fluorescence spectrophotometer. Figure 4 As shown, when the "Cy5 always-on" DNA nanoprobe, also known as the "probe-on" probe, was added alone to the buffer, the Cy5 fluorescence intensity was the strongest. However, when the DNA nanoprobe (with BHQ-2 modified on the H1 chain, also known as the "probe" probe) was added alone to the buffer, no obvious Cy5 fluorescence was detected. This indicates that compared to the "probe-on" probe, the Cy5 fluorescence of the "probe" probe was successfully quenched due to the BHQ-2 modification on the H1 chain during synthesis. The assembled DNA nanoprobe "probe" can be used to investigate the feasibility of probe response to the target PARP1. Further comparison of the Cy5 fluorescence intensity of each group revealed that the Cy5 fluorescence of the "probe+PARP1 target" and "probe+PARP1target+PD-L1 target" groups recovered, and the fluorescence intensities of the two groups were similar; the Cy5 fluorescence intensity of the "probe+PD-L1 target" group did not recover and was similar to that of the "probe" group. According to the above experimental results, it is shown that the probe can bind to the target PARP1 in the buffer, it is feasible for the probe to recognize the target PARP1 in the buffer, and the presence of the PD-L1 target does not affect the binding of the probe to the target PARP1.

[0069] (2) Determination of the detection limit of the probe response target PARP1

[0070] DNA nanoprobes were assembled. PARP1 mRNA at different concentrations was added to a buffer with a final probe concentration of 50 nM. Cy5 fluorescence activation was measured by spectrophotometry when the probe bound to different concentrations of PARP1 mRNA in the buffer. The limit of detection (LOD) of the probe for PARP1 mRNA was calculated (see formula (1)) to further evaluate the probe's ability to respond to the target. Three parallel experiments were performed, with three replicates for each concentration.

[0071] (1)

[0072] In formula (1), blank is the average fluorescence intensity of the blank buffer group measured by the fluorescence spectrophotometer, and a is the slope of the linear fitting equation of Cy5 fluorescence intensity and PARP1 concentration.

[0073] The fluorescence intensity of Cy5 in each group was measured by fluorescence spectrophotometer. Figure 5 As shown in Figure 2, when the concentration of PARP1 added gradually increased from 0 nM to 150 nM, the fluorescence intensity of Cy5 increased accordingly; Figure 6 As shown in Figure 2, when the concentration of PARP1 ranged from 0 nM to 10 nM, the fluorescence intensity of Cy5 was linearly proportional to the concentration of target PARP1 (R 2 = 0.9535). The detection limit was calculated to be 0.73 nM according to the LOD formula combined with the equation. The results showed that the DNA nanoprobe had a good response ability to PARP1.

[0074] (3) Investigation of the specificity of the probe response to the target PARP1

[0075] To verify the specificity of the PARP1 antisense sequence in the probe for binding to PARP1 after entry into cells and to exclude false positives caused by other highly expressed mRNAs in the cytoplasm, the probe's responsiveness to PARP1 and other highly expressed mRNAs (as shown in Table 2) was examined in buffer to verify the probe's specificity for PARP1. DNA nanoprobes were assembled, and the Cy5 fluorescence values ​​of each group were measured using a fluorescence spectrophotometer.

[0076] The Cy5 fluorescence intensity of each group was detected by fluorescence spectrophotometer. Figure 7 As shown, when the DNA nanoprobe was co-incubated with PARP1 in buffer, the Cy5 fluorescence intensity was the strongest, while when it was co-incubated with other sequences, no obvious Cy5 fluorescence was detected, indicating that the DNA nanoprobe did not bind to other highly expressed mRNAs in the cytoplasm and random DNA interferors with the same sequence length as PARP1 mRNA, proving that the probe can specifically respond to PARP1.

[0077] (4) Investigation of the probe's loading capacity for doxorubicin

[0078] DNA nanoprobes were assembled and various concentrations of the probes were added to a doxorubicin solution with a final concentration of 1 μM. After incubation with doxorubicin for 30 minutes, the fluorescence intensity of doxorubicin after the addition of the different concentrations of the nanoprobes was measured using an FL970 fluorescence spectrophotometer. The probe-to-doxorubicin ratio was calculated as the probe drug loading (doxorubicin: Ex = 488 nm, Em = 500-550 nm and 570-620 nm).

[0079] Doxorubicin itself has fluorescence, and its fluorescence is quenched after being embedded in the CG base pair of the DNA double strand. This experiment uses this special property of doxorubicin to calculate the drug loading capacity of the DNA nanoprobe by detecting the fluorescence intensity of doxorubicin. Figure 8 As shown in the figure, with the fluorescence at a doxorubicin concentration of 1 μM as the background, as the concentration of the added DNA nanoprobe gradually increased, the fluorescence intensity of doxorubicin gradually weakened until the concentration ratio of the DNA nanoprobe to doxorubicin reached 1:8, the fluorescence intensity of doxorubicin was quenched to the maximum extent. This was the maximum loading capacity of the DNA nanoprobe for doxorubicin, that is, probe: doxorubicin = 1:8.

[0080] Example 3

[0081] Based on the research in the examples, this example further investigates the DNA nanoprobe's ability to recognize cells, the feasibility and specificity of intracellular imaging, the ability to release doxorubicin intracellularly, and the cell-killing effect at the MDA-MB-231 cell level. The triple-negative breast cancer cell line used in this example is MDA-MB-231, purchased from iCell. The cell treatment process is as follows:

[0082] The specific experimental process is as follows:

[0083] (1) Investigation of the probe's ability to recognize cells

[0084] This experiment was divided into three groups: Group A: MDA-MB-231 cells; Group B: Cy5 always-on DNA nanoprobe (with the AS1411 aptamer in the T strand replaced with a random sequence) + MDA-MB-231 cells; Group C: Cy5 always-on DNA nanoprobe (with the AS1411 aptamer functionalized in the T strand) + MDA-MB-231 cells. Flow cytometry was used to measure the Cy5 fluorescence signal in each group. The Cy5 fluorescence signal in the blank control group (Group A) was adjusted to zero as background.

[0085] Trypsin-digested MDA-MB-231 cells (density approximately 2×10 5 Cells were resuspended in 200 μL of phosphate-buffered saline (PBS, 10 mM) and DNA nanoprobes were added to a final concentration of 50 nM. The cells were incubated at 37°C in the dark for 15 minutes and then centrifuged three times (2,000 rpm, 5 minutes each). Flow cytometry was used to detect the Cy5 marker signal using the FL6 fluorescence channel, with the detection threshold set to 1 × 10 cells / mL. 4 During the implementation of the research plan, all data collection was repeated at least 3 times in parallel.

[0086] like Figure 9 As shown, after the MDA-MB-231 cells in group B were co-incubated with the random chain functionalized "Cy5 always-on" DNA nanoprobe radprobe, the Cy5 fluorescence signal on the cells was no different from that in the blank control group, indicating that the probe failed to successfully identify the MDA-MB-231 cells; while after the MDA-MB-231 cells in group C were co-incubated with the AS1411 aptamer functionalized "Cy5 always-on" DNA nanoprobe aptprobe, obvious Cy5 fluorescence was detected on the cells. This result proves that the DNA nanoprobe can recognize MDA-MB-231 cells, and its recognition ability of the target cell MDA-MB-231 comes from the AS1411 aptamer sequence functionalized on the probe.

[0087] To further investigate the probe recognition ability of MDA-MB-231 cells, imaging was performed on MDA-MB-231 cells co-incubated with radprobe and aptprobe probes. MDA-MB-231 cells were seeded in 35 mm confocal microplate dishes. When the cell density reached approximately 70%, well-grown cells were selected and washed twice with prewarmed PBS. 200 μL of PBS (containing the DNA nanoprobe at a final concentration of 100 nM) was added to each dish. The cells were incubated in the dark at 37°C for 15 minutes and then washed three times with PBS. Hoechst 33342 was added for nuclear staining. The cells were incubated at 37°C for an additional 5 minutes and then washed twice with PBS. Fluorescence was observed using a laser confocal microscope (100× oil immersion lens). (Cy5: Ex = 640 nm, Em = 663-738 nm; Hoechst 33342: Ex = 405 nm, Ex = 425-475 nm). All imaging experiments were repeated three times in parallel. Figure 10 As shown in the figure, no obvious Cy5 fluorescence signal was detected on the MDA-MB-231 cells incubated with the radprobe probe, while obvious Cy5 fluorescence could be detected on the cells incubated with the aptprobe probe. This result once again proved that the DNA nanoprobe assembled in this experiment can recognize MDA-MB-231 cells through the AS1411 aptamer functionalized thereon.

[0088] (2) Investigation of the feasibility and specificity of probes for intracellular imaging

[0089] MDA-MB-231 cells were dispersed and cultured in 35 mm culture dishes. After the cells were completely attached to the wall, the culture medium was removed and the cells were washed three times with PBS. The feasibility and specificity of imaging the probe in MDA-MB-231 cells were investigated using laser confocal microscopy.

[0090] Feasibility study: Add 200 μL of PBS containing 50 nM DNA nanoprobes to MDA-MB-231 cells and incubate them in the dark at 37°C for 1, 2, 3, 4, and 5 hours. After the cells and probes are incubated, laser confocal microscopy is performed to observe the changes in Cy5 fluorescence. After selecting the optimal imaging time, the specificity of the probe imaging in the cells is investigated. Figure 11 As shown in the figure, as the DNA nanoprobe was incubated with MDA-MB-231 cells in the dark for an increasing time, the intracellular Cy5 fluorescence gradually recovered. These results indicate that the DNA nanoprobe prepared in this study can respond to the target PARP1 in MDA-MB-231 cells, enabling intracellular imaging of the DNA nanoprobe. Furthermore, by comparing the fluorescence intensity of Cy5 in cells after different incubation times in the dark, it was found that Cy5 fluorescence reached its peak after 4 and 5 hours of incubation with the DNA nanoprobe. Based on these experimental results, this study selected 4 hours as the optimal imaging time for subsequent experiments.

[0091] Specificity assessment: DNA nanoprobes were incubated with differently treated MDA-MB-231 cells at 37°C in the dark for 4 hours, followed by laser confocal microscopy imaging. The cell treatments and corresponding groups are as follows: (A) MDA-MB-231 cells treated with lipofectamine-3000; (B) MDA-MB-231 cells treated with lipofectamine-3000 and encapsulated with a PARP1 antisense sequence (hereafter referred to as anti-PARP1); (C) MDA-MB-231 cells treated with lipofectamine-3000 and encapsulated with PD-L1 siRNA; and (D) MDA-MB-231 cells treated with lipofectamine-3000 and encapsulated with both anti-PARP1 and PD-L1 siRNA.

[0092] After incubation, MDA-MB-231 cells were washed with PBS to remove unbound probes. PBS was then added, and fluorescence was acquired using a laser confocal imaging system with a 100× oil-immersion objective: Cy5: Ex = 640 nm, Em = 663-738 nm. All imaging experiments were repeated three times.

[0093] like Figure 12As shown in the figure, both MDA-MB-231 cells transfected with lipofectamine-3000 and MDA-MB-231 cells transfected with PD-L1 siRNA by lipofectamine-3000 exhibited obvious Cy5 fluorescence; however, in the other two groups, namely, MDA-MB-231 cells transfected with PARP1 antisense sequence by lipofectamine-3000 and MDA-MB-231 cells transfected with PARP1 antisense sequence and PD-L1 siRNA by lipofectamine-3000, almost no Cy5 fluorescence was observed. MDA-MB-231 cells were transfected with lipofectamine-3000 containing PARP1 antisense sequences (anti-PARP1) and PD-L1 siRNA. RT-qPCR was used to detect the expression of PARP1 and PD-L1 nucleic acids in MDA-MB-231 cells under different treatments. The test results are shown in Table 3. The above treatments can downregulate the expression of PARP1 and PD-L1 nucleic acids in cells, thereby affecting the binding of anti-PARP1 and PD-L1 siRNA loaded on the probe to the corresponding targets in the cells. These results indicate that the DNA nanoprobe specifically recognizes and binds to PARP1 in MDA-MB-231 cells through the PARP1 antisense sequence loaded on it, activating and restoring the Cy5 fluorescence in the cells, thereby achieving specific imaging of the DNA nanoprobe in MDA-MB-231 cells.

[0094] Table 3 Relative expression of PARP1 and PD-L1 nucleic acids in cells detected by RT-qPCR

[0095]

[0096] Note: In this table, * indicates significant difference P value < 0.05, ** indicates significant difference P value < 0.01.

[0097] (3) Investigation of the probe's ability to release doxorubicin in cells

[0098] MDA-MB-231 cells were seeded into confocal microscopy-specific culture dishes (35 mm size) and cultured until the cell density reached about 70%. The cell status was observed under a microscope, and culture dishes with good growth status were selected.

[0099] Drug release feasibility study: DNA nanoprobes (final concentration of 200 nM) were added to the well-growing culture dish of the above-mentioned MDA-MB-231 cells. The probes were incubated with the cells at 37°C for 1, 2, 3, and 4 hours, respectively. The cells were then washed with PBS buffer and observed using a laser confocal microscope to analyze the fluorescence signals of doxorubicin and Cy5. Figure 13As shown in the figure, within 1-4 h, the fluorescence of doxorubicin and Cy5 showed a gradually increasing trend with the increase of incubation time, and the fluorescence signal intensity of the two reached the highest at 4 h. The experimental results showed that the doxorubicin loaded by the DNA nanoprobe was successfully released in the MDA-MB-231 cells.

[0100] Drug release specificity study: MDA-MB-231 cells treated with different methods were incubated with DNA nanoprobes (37°C, 4 h). After incubation, the cells were washed and the fluorescence signals of Cy5 and doxorubicin were collected and observed using a laser confocal microscope (100× oil immersion lens). DOX: Ex = 488 nm, Em = 570-620 nm; Cy5: Ex = 640 nm, Em = 663-738 nm; Hoechst 33342: Ex = 405 nm, Em = 425-475 nm. Figure 14 As shown in the figure, in MDA-MB-231 cells treated with lipofectamine-3000 and MDA-MB-231 cells transfected with PD-L1 siRNA by lipofectamine-3000, there were obvious Cy5 and doxorubicin fluorescence; while in MDA-MB-231 cells transfected with PARP1 antisense sequence by lipofectamine-3000 and transfected with PARP1 antisense sequence and PD-L1 siRNA by lipofectamine-3000, there was almost no Cy5 and doxorubicin fluorescence. The above results indicate that, after the PARP1 antisense sequence is transfected into MDA-MB-231 cells via lipofectamine 3000, it binds to PARP1 and silences the expression of PARP1 in the cells, preventing the DNA nanoprobe from binding to PARP1 in the cells and causing further disassembly. Consequently, the doxorubicin loaded on the probe's CG base pair is not successfully released. However, when the DNA nanoprobe successfully recognizes and binds to PARP1 in MDA-MB-231 cells via the PARP1 antisense sequence loaded on it, the probe further disassembles, ultimately releasing doxorubicin in the cells. In summary, the release of doxorubicin in MDA-MB-231 cells is a specific result of probe disassembly caused by the binding of the DNA nanoprobe to intracellular PARP1.

[0101] (4) Investigation of the killing effect of the probe on cells

[0102] MTS experiment: MDA-MB-231 cells were seeded in 96-well plates. When the cell density reached about 40%, the culture medium was removed and 100 μL of probe was added to each well (4 replicate wells / group). After culturing for 48 h, the supernatant was removed and MTS detection solution (10 μL / well) was added. After incubation at 37 °C for 2 h, the cells were taken out and gently mixed on a shaker before reading. The absorbance of different wells at a wavelength of 490 nm was measured using an enzyme-linked microplate reader, and the cell survival rate of each well was calculated according to formula (2).

[0103] (2)

[0104] Among them, OD treated ,OD blank ,OD control Represent the UV absorption values ​​of the experimental group, blank control group, and control group, respectively.

[0105] The killing effect of different concentrations of DNA nanoprobes on MDA-MB-231 cells was detected by MTS assay, and the optimal concentration was selected and grouped. Figure 15 As shown in the figure, compared with the control group, with the gradual increase of probe concentration, the survival rate of MDA-MB-231 cells gradually decreased, indicating that the killing effect of the probe was related to the concentration of the probe (P < 0.0001). When the probe concentration reached 625nM (at this time, the doxorubicin concentration was 5 μM), the cell survival rate was 29.88%. As the probe concentration continued to increase to 937.5nM and 1250nM, the cell survival rates of the two decreased, but there was no significant difference compared with the probe concentration of 625nM (P > 0.05). Therefore, this study selected the probe concentration of 625nM as the optimal concentration for subsequent experiments.

[0106] To further compare the differences in the killing effects of single treatment mode and combined treatment mode on MDA-MB-231 cells, this study continued to use the MTS assay to examine the survival rate of MDA-MB-231 cells in each group after different treatments. The groups are as follows:

[0107] Group A: MDA-MB-231 cells

[0108] Group B: free doxorubicin + MDA-MB-231 cells

[0109] Group C: MDA-MB-231 cells carrying PARP1 antisense sequence probe

[0110] Group D: PD-L1 siRNA probe + MDA-MB-231 cells

[0111] Group E: PARP1 antisense sequence@PD-L1 siRNA probe + MDA-MB-231 cells

[0112] Group F: Doxorubicin@PARP1 antisense sequence probe + MDA-MB-231 cells

[0113] Group G: Doxorubicin@PD-L1 siRNA probe + MDA-MB-231 cells

[0114] Group H: Doxorubicin@PARP1 antisense sequence@PD-L1 siRNA probe + MDA-MB-231 cells

[0115] The experimental results are as follows Figure 16 As shown, both free doxorubicin and DNA nanoprobes loaded with different functional components can kill MDA-MB-231 cells. By comparing the killing effects of various groups of DNA nanoprobes on MDA-MB-231 cells, it was found that the following three groups of DNA nanoprobes treated with chemotherapy combined with gene therapy strategies (i.e., probes loaded with doxorubicin@PARP1 antisense sequence, doxorubicin@PD-L1 siRNA, and doxorubicin@PARP1 antisense sequence@PD-L1 siRNA) had stronger killing effects on cells and lower cell survival rates, at 59.95%, 61.77%, and 37.71%, respectively. There was no significant difference in the killing ability of the two probes containing doxorubicin@PARP1 antisense sequence and doxorubicin@PD-L1 siRNA against MDA-MB-231 cells (P>0.05). However, the killing ability of the probe containing doxorubicin@PARP1 antisense sequence@PD-L1 siRNA against MDA-MB-231 cells was significantly higher than that of the probes containing doxorubicin@PARP1 antisense sequence and doxorubicin@PD-L1 siRNA (P<0.0001). In summary, the DNA nanoprobes prepared in this study have a strong killing ability against MDA-MB-231 cells, and the combination of chemotherapy and gene therapy can enhance the killing effect of DNA nanoprobes on MDA-MB-231 cells.

[0116] (5) Investigation of the effects of probes on gene interference and gene silencing in MDA-MB-231 cells

[0117] RT-qPCR analysis: This study used RT-qPCR to analyze the expression levels of PARP1 and PD-L1 in each group of cells to evaluate the effect of DNA nanoprobes on gene interference and gene silencing in MDA-MB-231 cells. MDA-MB-231 cells were divided into groups and treated differently, and the groups were as follows:

[0118] Group A: control group

[0119] Group B: PARP1 antisense sequence probe

[0120] Group C: PD-L1 siRNA probe

[0121] Group D: PARP1 antisense sequence@PD-L1 siRNA probe

[0122] Group E: lipofectamine-3000 + PARP1 antisense sequence

[0123] Group F: lipofectamine-3000 + PD-L1 siRNA

[0124] Group G: lipofectamine-3000 + PARP1 antisense sequence + PD-L1 siRNA

[0125] The experimental results are as follows Figure 17 As shown, compared with the control group A, the relative expression of PARP1 in groups B and E was downregulated (P < 0.05), the relative expression of PD-L1 in groups C and F was downregulated (P < 0.05), while the relative expressions of PARP1 and PD-L1 in groups D and G were both downregulated (P < 0.01). The experimental results showed that the use of DNA nanoprobes to load the antisense sequence of PARP1 and the transfection of the antisense sequence of PARP1 into MDA-MB-231 cells through lipofectamine-3000 could both downregulate the expression of PARP1 in cells; the use of DNA nanoprobes to load PD-L1 siRNA and the transfection of PD-L1 siRNA into MDA-MB-231 cells through lipofectamine-3000 could both downregulate the expression of PD-L1 in cells; when the DNA nanoprobes were used to load the antisense sequence of PARP1 and PD-L1 siRNA together or the PARP1 antisense sequence and PD-L1 siRNA were transfected into cells through lipofectamine-3000, the expression of PARP1 and PD-L1 in MDA-MB-231 cells was downregulated. In summary, the DNA nanoprobes loaded with PARP1 antisense sequence and PD-L1 siRNA prepared in this study can effectively downregulate the relative expression levels of PARP1 and PD-L1 nucleic acids in MDA-MB-231 cells, achieving gene interference and silencing of MDA-MB-231 cells.

[0126] Western blot analysis: This study used Western blot analysis to analyze the protein expression levels of PARP1 and PD-L1 in MDA-MB-231 cells under different treatments to evaluate the effect of DNA nanoprobes on gene interference and gene silencing in MDA-MB-231 cells. The experimental groups were the same as those for RT-qPCR analysis. The experimental results are shown in Figure 2. Figure 18As shown, compared with the control group A, the expression of PARP1 protein in MDA-MB-231 cells in groups B and E was decreased, the expression of PD-L1 protein in groups C and F was decreased, while the expression of PARP1 and PD-L1 proteins in groups D and G were both downregulated. According to the experimental results, this study demonstrated that the use of DNA nanoprobes to load PARP1 antisense sequences and the transfection of PARP1 antisense sequences into MDA-MB-231 cells via lipofectamine-3000 can both downregulate the expression of PARP1 in cells; the use of DNA nanoprobes to load PD-L1 siRNA and the transfection of PD-L1 siRNA into MDA-MB-231 cells via lipofectamine-3000 can downregulate the expression of PD-L1 in cells; in addition, when DNA nanoprobes were used to load PARP1 antisense sequences and PD-L1 siRNA at the same time or when lipofectamine-3000 was used to transfect PARP1 antisense sequences and PD-L1 siRNA into cells, the expression of both PARP1 and PD-L1 in MDA-MB-231 cells was downregulated. In summary, the DNA nanoprobes loaded with PARP1 antisense sequence and PD-L1 siRNA prepared in this study can effectively downregulate the relative protein expression of PARP1 and PD-L1 in MDA-MB-231 cells, achieving gene interference and silencing functions in MDA-MB-231 cells.

[0127] Finally, it should be noted that the terms "comprises," "comprising," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus that includes a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus. Although preferred embodiments of the present invention have been described, additional changes and modifications may be made to these embodiments by those skilled in the art once the basic inventive concepts are known. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the invention.

[0128] The above-described embodiments merely represent specific implementation methods of the present application. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of protection of the present application. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the technical concept of the present application, and all such variations and improvements fall within the scope of protection of the present application.

Claims

1. A DNA nanoprobe for imaging and treatment of triple-negative breast cancer, characterized in that: The DNA nanoprobe includes a DNA double helix structure and doxorubicin loaded by being embedded in the DNA double helix structure. The DNA double helix structure is self-assembled by a T chain, a hairpin chain 1, and a hairpin chain 2 through a hybridization chain reaction. The T chain is an AS1411-functionalized T chain, as shown in SEQ ID NO: 3; the sequence of the hairpin chain 1 contains a PARP1 antisense sequence, and the hairpin chain 1 is modified with a Cy5 fluorescent group, as shown in SEQ ID NO: 4; the hairpin chain 2 is modified with a PD-L1 siRNA and a BHQ2 fluorescence quencher, and the unmodified chain of the hairpin chain 2 is shown in SEQ ID NO:

5.

2. The DNA nanoprobe for triple-negative breast cancer imaging and treatment according to claim 1, wherein: The PARP1 antisense sequence is shown in SEQ ID NO:

1.

3. The DNA nanoprobe for triple-negative breast cancer imaging and treatment according to claim 1, wherein: The PD-L1 siRNA is shown in SEQ ID NO:

2.

4. The DNA nanoprobe for triple-negative breast cancer imaging and treatment according to claim 1, wherein: The assembly molar ratio of the T chain, hairpin chain 1 and hairpin chain 2 is 1:3:

3.

5. The DNA nanoprobe for triple-negative breast cancer imaging and treatment according to claim 1, wherein: The loading molar ratio of the doxorubicin to the DNA double helix structure is 1:

8.

6. The method for preparing a DNA nanoprobe for imaging and treating triple-negative breast cancer according to any one of claims 1 to 5, wherein: The steps include: (1) Prepare T strand, hairpin strand 1, and hairpin strand 2, and modify hairpin strand 1 with a Cy5 fluorescent group, and modify hairpin strand 2 with PD-L1 siRNA and a BHQ2 fluorescence quencher; (2) Annealing the T strand, hairpin strand 1, and hairpin strand 2 obtained in step (1) at 95°C for 5-10 minutes respectively; (3) Add the annealed T strand, hairpin strand 1, and hairpin strand 2 to a Tris-Mg buffer solution containing Tris and MgCl2 at pH 8.0 and react at 37°C for 4 hours; (4) Add doxorubicin and incubate at room temperature for 30 minutes to obtain the DNA nanoprobe.

7. Use of the DNA nanoprobe for imaging and treating triple-negative breast cancer according to any one of claims 1 to 5 in the preparation of an integrated reagent or drug for the diagnosis and treatment of triple-negative breast cancer.

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