CN116790582A - Thermodynamic guidance-based DNA nano structure for cancer diagnosis and treatment integration - Google Patents

Abstract

The application provides a DNA nano structure for cancer diagnosis and treatment integration based on thermodynamic guidance, which consists of A, B, C, D and H five oligonucleotide chains, wherein the DNA sequences of the oligonucleotide chain A and the oligonucleotide chain are respectively shown as SEQ ID No.1-No. 5. The DNA nanostructure has the combined effects of diagnostic imaging, chemotherapy and gene therapy. The reagent has the capability of recognizing multi-drug resistant 1 (MDR 1) mRNA, and the detection limit of MDR1 mRMA is 0.05 mu M; thermodynamic data indicate that the doxorubicin and daunorubicin have high drug loading rates; the DNA nano structure has good gene treatment effect and enhances the chemotherapy effect on drug-resistant cells.

Description

Thermodynamic guidance-based DNA nano structure for cancer diagnosis and treatment integration

Technical Field

The application belongs to the field of medicines, and particularly relates to a DNA nanostructure for cancer diagnosis and treatment integration based on thermodynamic guidance.

Background

Cancer is one of the major diseases threatening human health. Chemotherapy is currently considered one of the important means of treating cancer. Among various chemotherapeutic drugs, anthracyclines are a class of highly potent anticancer natural products that are widely used in the clinic. Doxorubicin (DOX, 14-hydroxydurobicin, fig. 1A) and daunorubicin (DAU, fig. 1B) are representative anthracyclines. They can lead to cancer cell death by inhibiting DNA replication and transcription. DOX is similar in structure to DAU except that one hydrogen atom on a side chain carbon atom is replaced with a hydroxyl group. However, the efficacy of anthracyclines is severely limited by resistance, resulting in chemotherapy failure. P-glycoprotein (P-gp) is an expression product of a multi-drug resistance 1 (MDR 1) gene, has ATP-dependent transmembrane transport activity, and can transport drugs out of cells, resulting in drug resistance in the cells. Gene therapy can inhibit the expression of drug resistance related proteins through gene silencing, thereby effectively overcoming drug resistance. Gene therapy generally uses nucleic acid drugs such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs). Compared to siRNAs, ASOs have the advantage of acting directly on drug-resistance related mRNAs and inhibiting their translation process without forming RNA-induced silencing complexes. However, the efficacy of gene therapy is difficult to assess due to the difficulty in determining the location of drug-resistance related mRNAs in living cells. Thus, accurate diagnosis of drug-resistance related mRNAs is essential for improving the efficacy of cancer gene therapy. The fluorescent nano-carrier containing the ASOs marked by the fluorescent switch can emit a fluorescent signal for detecting the positions of the drug-resistance related mRNAs in cells, and the fluorescent intensity is related to the gene silencing curative effect, so that in-situ imaging and the inhibition of the expression of the drug-resistance related mRNAs can be realized simultaneously.

Chemotherapeutic agents have the disadvantages of low solubility, systemic toxicity and nonspecific distribution. In addition, poor intracellular uptake of ASOs is a major obstacle leading to their use as therapeutic agents. The nano carrier can not only overcome the defects of chemotherapeutics and improve the chemotherapeutics effect, but also improve the uptake of cells to ASOs. DNA nanostructures have attracted considerable attention from researchers due to their well-defined size, good biocompatibility and high drug loading capacity. DNA tetrahedra are one of the DNA nanostructures of great interest because of their rigid structure, structural stability, and ease of synthesis.

A diagnostic integration strategy that loads chemotherapeutic drugs into ASOs-linked nanocarriers would provide better therapeutic results. However, to date, only ASOs-modified gold nanoparticles have been used for combination therapy and in situ imaging of drug resistance-associated mRNAs. There is no report of simultaneous loading of chemotherapeutics and ASOs into DNA tetrahedra. Furthermore, in the DNA nanostructure+drug complex, drug loading, release and cytotoxicity are closely related to the interaction between the DNA nanostructure and the drug. The interaction between the DNA nano structure and the medicine is researched based on a thermodynamic method, so that theoretical guidance can be provided for loading, releasing and curative effect of the medicine.

Disclosure of Invention

The present application aims to overcome the above-mentioned shortcomings of the prior art and to provide a DNA nanostructure having a combination of diagnostic imaging, chemotherapy and gene therapy.

A DNA nano-structure for cancer diagnosis and treatment integration based on thermodynamic guidance consists of A, B, C, D and H five oligonucleotide chains, wherein the DNA sequences of the oligonucleotide chain A and the oligonucleotide chain are respectively shown in SEQ ID No.1-No. 5.

The construction method of the DNA nano structure comprises the following steps:

s1, respectively placing A, B, C, D and H oligonucleotide chains in a container, centrifuging, and adding PBS to completely dissolve the mixture;

s2, four A, B, C, D oligonucleotide chains used for constructing TD are thoroughly mixed according to the ratio of 1:1:1:1, and are mixed in a thermostat at 95 ^o Annealing for 5 minutes under the condition of C, rapidly placing the annealed mixed solution in an ice-water bath, and cooling for 30 minutes to form a TD nano structure;

s3, H oligonucleotide chain at 90 ^o Annealing for 5 minutes under C, and naturally cooling to room temperature to form an ASOs hairpin structure;

s4, fully mixing the TD and the ASOs according to the ratio of 1:1, and placing the mixture on a microplates shaker (600 r/h, 1 hour) to form an ASOs-TD structure.

The DNA nano structure is applied to the preparation of anticancer drugs.

The beneficial effects are that: the DNA nanostructure has the combined effects of diagnostic imaging, chemotherapy and gene therapy. The reagent has the capability of recognizing multi-drug resistant 1 (MDR 1) mRNA, and the detection limit of MDR1 mRMA is 0.05 mu M; thermodynamic data indicate that the doxorubicin and daunorubicin have high drug loading rates; the DNA nano structure has good gene treatment effect and enhances the chemotherapy effect on drug-resistant cells.

Drawings

The structural formula of (A) DOX (B) DAU.

FIG. 2 construction of ASOs, TD and ASOs-TD.

FIG. 3 agarose gel electrophoresis characterization of ASOs-TD. From left to right: (1) 20 μ M A; (2) 20 μ M A +20 μ M B; (3) 10μ M A +10μ M B +10μ M C; (4) 10 μM TD; (5) 20 μ M H; (6) 5. Mu.M ASOs-TD; (7) Marker.

FIG. 4 fluorescence spectral response of ASOs-TD (0.25. Mu.M) to target MDR1mRNA (a-o: 0-5. Mu.M). Quenching fluorescence was restored after concentration-dependent addition of target mRNA (fig. 4 inset).

FIG. 5 Stern-Volmer plot of fluorescence quenching of (A) DOX (8. Mu.M) and (B) DAU (8. Mu.M) with or without ASOs-TD (0.12. Mu.M). K (K) ₄ [Fe(CN) ₆ ]The concentration of (2) is in the range of 0-0.1M.

FIG. 6 normalized fluorescence spectra of DOX (A)/DAU (B) in the presence of different concentrations of ASOs-TD. (C _DOX/DAU = 8 μM；a-m: 0-0.28 μМ)

FIG. 7 isothermal titration thermal profiles of DOX (A) and DAU (B) binding to ASOs-TD at 298.2K.

FIG. 8 binding enthalpy (. DELTA.) of ASOs-TD binding to DOX (A)/DAU (B)H ^o ) Graph of change with temperature.

FIG. 9 DSC of ASOs-TD (3. Mu.M), ASOs+DOX/DAU complex. (C _drug = 90 μM)

FIG. 10 fluorescence emission spectra of (A) DOX (30. Mu.M) and (B) DAU (30. Mu.M) with increasing ASOs-TD concentration. (a-d: 0-12.5. Mu. M)

FIG. 11 in vitro release profile of free or ASOs-TD loaded (A) DOX (30. Mu.M), (B) DAU (30. Mu.M) in PBS buffer (pH 7.4). DNase I concentration in drug-loaded ASOs-TD: 0, 10, 20, U/mL.

FIG. 12 in vitro inhibition of MCF-7/ADR (A, B) and MCF-7 (C, D) cells by free, TD or ASOs-TD loaded DOX (A, C), DAU (B, D).

FIG. 13 (A) CLSM images of MCF-7, MCF-7/ADR cells incubated with ASOs-TD, green fluorescence was generated by FAM. (B) CLSM images of MCF-7/ADR cells incubated with ASOs-TD+DOX for 6 hours.

Detailed Description

The application is further illustrated by the following specific examples, which are not intended to limit the application.

Example 1:

a DNA nano-structure for cancer diagnosis and treatment integration based on thermodynamic guidance, and synthesized ASOs-TD is composed of A, B, C, D and H five oligonucleotide chains. Each EP tube containing the corresponding oligonucleotide strand was centrifuged and then dissolved completely by adding an appropriate amount of 1 XPBS (pH 7.4). Four single strands used to construct TD were thoroughly mixed in a 1:1:1:1 ratio and annealed in a K30 dry thermostat (Hangzhou Quansheng instruments Co., ltd., china) (95 ^o C, 5 minutes). The annealed mixed solution was rapidly placed in an ice-water bath and cooled for 30 minutes to form the TD nanostructure. H chain is also at 90 ^o Annealing for 5 minutes at C, but naturally cooling to room temperature to form the ASOs hairpin structure. The TD and ASOs structures formed are shown in 4 ^o The mixture was left under C for 1 hour. TD and ASOs were mixed well in a 1:1 ratio and placed on a microplate shaker (600 r/h, 1 hour) to form an ASOs-TD structure. Fluorescently labeled ASOs-TD was also synthesized as described above, except that the H chain contained the phosphor carboxyfluorescein (FAM) and black hole quencher 1 (BHQ 1).

TD and unlabeled ASOs-TD were characterized by agarose gel electrophoresis. Seven groups of samples, including A, A + B, A +b+c, TD, H, unlabeled ASOs-TD and markers, were injected into the gel channel from left to right. Electrophoresis experiments were performed on 2.5% agarose gel (100 v,90 min) in 1×tbe buffer. Imaging was performed using a 4600SF variable mode imager (Tanon, china).

As shown in Table 1, the constructed ASOs-TD consisted of five oligonucleotide strands, self-assembled according to Watson-Crick base pairing rules (FIG. 2). The H chain is used to recognize MDR1mRNA in multi-drug resistant cells. As shown in FIG. 3, ASOs-TD was characterized by agarose gel electrophoresis. The mobility of six groups of samples in the gel, except for the markers, can be used to assess successful synthesis of ASOs-TD. From lane 1 to lane 4, the mobility of the sample in the gel gradually slowed, indicating successful TD synthesis. Lane 6 corresponds to synthetic ASOs-TD, which are made up of H chains linked to TD. Because of the relatively high molecular weight, the synthesized ASOs-TD concentrated in the upper half of the gel with a distinct band. This indicates successful self-assembly of the ASOs-TD.

TABLE 1 DNA probe sequences

Experimental example: the following related assays for ASOs-TD

Fluorescence spectroscopy measurement:

all fluorescence spectroscopy measurements were performed on an F-7000 fluorescence spectrometer (Hitachi, japan) with a temperature controller. The measuring cell was a quartz cell of 0.5 cm ×0.5× 0.5 cm ×0.5 cm. Under experimental conditions, there was no background fluorescence and the absorbance at the excitation wavelength was less than 0.05, indicating that the effect of the internal filtration was negligible. All experimental data are averages of three measurements.

Ability to detect MDR1 mRNA:

the fluorophore FAM labeled on ASOs-TD was excited at 488 and nm and the recorded emission spectrum ranged from 510-650 nm. The width of the excitation and emission slits was 10 nm. Under the above spectral conditions, the effect of different concentrations of MDR1mRNA (0-5. Mu.M) on the fluorescence spectrum of ASOs-TD (0.25. Mu.M) at a given concentration was studied.

As shown in FIG. 4A, in the absence of MDR1mRNA, the fluorescent signal of FAM fluorophore in ASOs-TD was weak. This is because the H chain attached to the DNA tetrahedron is in the hairpin state, the FAM fluorophore and BHQ1 are close to each other, and the fluorescence of FAM is quenched. The fluorescence intensity of the FAM fluorophore increased with increasing MDR1mRNA target concentration in a linear relationship over a concentration range of 0-0.60. Mu.M (FIG. 4B). When the target MDR1mRNA is added, the hairpin structure of the H chain is opened, FAM and BHQ1 are far away from each other, and the FAM fluorophore exhibits concentration-dependent fluorescence enhancement. When the concentration of MDR1mRNA exceeds 0.60. Mu.M, the fluorescence intensity gradually reaches a plateau. The limit of detection of MDR1mRNA was calculated to be 0.05. Mu.M. This suggests that the constructed ASOs-TD can sensitively detect MDR1mRNA overexpressed in drug-resistant cancer cells.

Potassium ferrocyanide K ₄ [Fe(CN)] ₆ Quenching analysis:

the excitation wavelengths of DOX and DAU are 494 and 495 nm, respectively, and the emission wavelength ranges are 515-680 nm. The slit width was 10/10 nm. K was studied in the presence or absence of ASOs-TD ₄ [Fe(CN) ₆ ]Quenching of DOX and DAU, wherein DOX/DAU, ASOs-TD and K ₄ [Fe(CN) ₆ ]The concentrations of (2) are 8. Mu.M, 0.12. Mu.M and 0-0.1. 0.1M, respectively. With K ₄ [Fe(CN) ₆ ]And (3) measuring the increase of the concentration to obtain the fluorescence spectrum of the DOX/DAU and ASOs-TD+DOX/DAU binary system.

K as an anion quencher ₄ [Fe(CN) ₆ ]Fluorescence of many small molecules can be quenched. K (K) ₄ [Fe(CN) ₆ ]And the anionic phosphate backbone of the DNA, so that the DNA can protect the intercalating bound small molecules. However, DNA offers less protection to small molecules that are bound in a groove-like manner. To determine the binding pattern between DOX/DAU and ASOs-TD, the K-exposure of ASOs-TD to DOX/DAU was studied ₄ [Fe(CN) ₆ ]The effect of quenching and the quenching constant was calculated by the Stern-Volmer equationK _sv ：

(1)

[Q]Is K ₄ [Fe(CN) ₆ ]Is used for the concentration of (a),F ₀ andFthe fluorescence intensity of DOX/DAU in the absence or presence of ASOs-TD, respectively. Embedding typeOf inserted combined medicamentsK _sv The value should be significantly smaller than that of the free drugK _sv Values. FIG. 5 shows K with or without ASOs-TD ₄ [Fe(CN) ₆ ]Stern-Volmer plot of fluorescence quenching for DOX/DAU. Fluorescence of free DOX/DAU is easily affected by K ₄ [Fe(CN) ₆ ]Quenching. After being loaded on ASOs-TD, the DOX/DAU has greatly reduced fluorescence quenching degree, and the method corresponds toK _sv The values were reduced by 70.66% and 50.30%, respectively. This indicates that ASOs-TD protects DOX/DAU and that DOX/DAU is combined with ASO-TD by intercalation. DOX compared to DAUK _sv The decrease in value was more pronounced, indicating a stronger binding between DOX and ASOs-TD, resulting in a better protection of DOX by ASOs-TD.

Fluorescence measurement of binary complexes:

in the ASOs-TD+DOX/DAU binary complex, the concentration of the two drugs was 8. Mu.M. The concentration of ASOs-TD was 0, 0.02, 0.04, 0.06, 0.08, 0.10, 0.12, 0.14, 0.16, 0.18, 0.20, 0.24 and 0.28. Mu.M, respectively. After 30 min incubation at 298.2K, the fluorescence spectra of DOX/DAU in the presence of different concentrations of ASOs-TD were measured. The measurement parameters are as described in the above section.

FIG. 6 shows fluorescence spectra of DOX and DAU in the presence of ASOs-TD at different concentrations. As the concentration of ASOs-TD increased, the fluorescence intensity of DOX and DAU gradually decreased, indicating interaction between DOX/DAU and ASOs-TD. Fluorescence quenching of DOX and DAU is due to their intercalation into the duplex structure of ASOs-TD, resulting in partial shielding of its chromophore atoms. Fitting to obtain the number of combined sites of interaction between ASOs-TD and DOX/DAU according to Scatchard equationn) And binding constant [ ]K _a )：

(2)

C _f Is the concentration of free DOX/DAU,ris the concentration ratio of bound DOX/DAU to ASOs-TD. From the following componentsr/C _f For a pair ofrCalculated from the Scatchard graph (inset of FIG. 6)nAndK _a the values of (2) are shown in Table 2.

The number of binding sites for DOX and DAU in ASOs-TD was 68.55 and 59.96, respectively, indicating that ASOs-TD has high loading capacity for both drugs. DOX has more binding sites, which suggests that its molecular structure is more suitable for intercalation into ASOs-TD.K _a Values of 5 to 6 orders of magnitude indicate that DOX and DAU, and in particular DOX, bind strongly to ASOs-TD. The weaker binding of DAU may be due to the weakening of the H bond by methyl groups on the DAU side chains.

Isothermal Titration Calorimetry (ITC) measurement:

ITC measurement at MicroCal ITC ₂₀₀ Titration calorimeter (micro, GE Healthcare). Titration temperatures were 298.2, 302.2, 306.2 and 310.2K, respectively. Each titration experiment included 20 injections in which the DOX/DAU solution (600 μm) in the syringe was titrated into the ASOs-TD solution (0.5 μm) in the pool. Control experiments of DOX/DAU titration of PBS solution and PBS titration of ASOs-TD solution were performed to subtract the respective heat of dilution. The first drop (0.4 μl) was removed before data analysis was performed. Data analysis was performed using the Origin 7.0 software provided by ITC.

Figure 7 shows the corresponding ITC curve at 298.2K. Performing a class of site fitting on the data to obtain the number of binding sites [ ]n) Binding constant [ ]K _a ) And enthalpy change of binding process (deltaH ^o ). The Gibbs free energy variation (delta) is then calculated according to the following formulaG ^o ) And entropy change (delta)S ^o )：

(3)

(4)

The thermodynamic parameters obtained are listed in table 2. Consistent with the fluorescence results, ASOs-TD had more binding sites for DOX and DAU and more binding sites for DOX, indicating the high drug loading capacity of ASOs-TD. Furthermore, the number of binding sites for both drugs decreases with increasing temperature.This may be related to structural changes of the ASOs-TD at different temperatures. The data in Table 2 also show that DOX and DAU, and in particular DOX, have strong binding to ASOs-TD, which is consistent with the results of fluorescence spectroscopy.K _a The value decreases with increasing temperature, indicating that lower temperatures favor the combination of DOX and DAU. We note that at 298.2K, it was obtained by ITC and fluorescence spectroscopynAndK _a the values are different due to the different sample concentrations and data processing models in the two methods.

TABLE 2 thermodynamic parameters of DOX/DAU binding to ASOs-TD by isothermal titration calorimetry and fluorescence spectroscopy

ΔH ^o And deltaG ^o The negative value of (C) indicates that DOX/DAU interaction with ASOs-TD is exothermic and spontaneous. -deltaH ^o >TΔSThe degree indicates that deltaHDEG vs deltaGThe contribution of the degree is greater and the binding process is driven mainly by enthalpy. Thus, DOX and DAU are mainly inserted in base pairs of ASOs-TD, which is similar to K ₄ [Fe(CN) ₆ ]Fluorescence quenching was consistent. Negative deltaHThe values of degree are mainly due to hydrogen bonds, van der Waals forces and electrostatic forces (vs. deltaHThe negative contribution of °) exceeds the hydrophobic effect (on deltaHPositive contribution of °). DeltaS ^o In most cases negative, mainly due to the reduced flexibility of the DNA helix and drug molecule after intercalation and conjugation. However, for the binding of DAU at 298.2K and 302.2K, the contribution of solvent molecule release exceeds the contribution of reduced flexibility, resulting in ΔS ^o Positive values of (2). Delta of ASOs+DOX and ASOs+DAU with increasing temperatureS ^o Value decreases, while the ASOs+DAU system is deltaS ^o The value drops even significantly from a positive value to a negative value. This is probably due to the disordered structure of the solvated layer increasing at high temperature, weakening the solvent molecular release vs. deltaS ^o Is a positive contribution of (c). Delta of ASOs+DOX at the same temperatureH ^o And deltaS ^o Value ratio ASOs+DAU corresponding valueMore negative, this is due to the stronger binding between DOX and ASOs-TD.

ΔH ^o Can be varied in a temperature-dependent manner by a significant thermodynamic parameter molar heat capacity (deltaC _p ^o ) To indicate that it is related to the nature of the intermolecular interactions during binding. We calculated the delta of DOX/DAU interaction with ASOs-TD by linear fitting (FIG. 8) according to the following formulaC _p ^o Values are listed in table 2.

(5)

ΔC _p ^o The negative value of (2) indicates that enthalpy change is inversely related to temperature, and that DOX/DAU binding to ASOs-TD results in hiding of nonpolar surface areas. Delta of interaction between DAU and ASOs-TDC _p ^o The value is negative, mainly due to the release of more solvent molecules around the DAU molecules versus deltaC _p ^o Is a negative contribution of (c).

Differential Scanning Calorimetry (DSC) measurements:

DSC measurements were performed on a VP-DSC calorimeter (microfcal, GE Healthcare). The concentrations of ASOs-TD and DOX/DAU were 3. Mu.M and 90. Mu.M, respectively. By scanning the sample and reference cells with buffer, a stable and repeatable baseline is obtained. After sufficient agitation and degassing, the sample solution (ASOs-TD or ASOs-TD+DOX/DAU) and PBS buffer were loaded into the sample cell and the reference cell, respectively. Sample at 90 °c.h ^-1 Is scanned from 15 ℃ to 100 ℃. Melting temperature [ ]T _m ) Corresponds to the maximum of the DSC peak. Data analysis was performed using Origin 7.0 software provided by VP-DSC.

FIG. 9 shows the thermal denaturation curve of ASOs-TD before and after DOX/DAU loading. As shown, ASOs-TD denatured with or without DOX/DAU, and produced a single endothermic peak. Corresponding to ASOs-TD, ASOs-TD+DAU and ASOs-TD+DOXT _m The values are (74.83.+ -. 0.45), (81.62.+ -. 0.21) and (82.73.+ -. 0.17), respectively ^o C. It has been reported that intercalation binding of small molecules can be increasedThermal stability of double helix DNA, leading toT _m Value increase of 5-8 ^o C. This suggests that DOX/DAU is combined with ASOs-TD intercalation, and that DOX/DAU loading increases the thermal stability of ASOs-TD. This is consistent with the results of the ITC analysis.

In addition, another important parameter, enthalpy (delta) can be obtained from DSC measurementsH _cal ) Delta of ASOs-TD, ASOs-TD+DAU and ASOs-TD+DOXH _cal The values were (2.51.+ -. 0.49). Times.10, respectively ⁵ 、 (3.18 ± 0.27) × 10 ⁵ And (3.97.+ -. 0.12). Times.10 ⁵ cal·mol ^-1 . Clearly, intercalation of the DAU or DOX results in more heat being required for the ASOs-TD to melt. Delta for the ASOs-TD+DOX ComplexH _cal The values were higher, mainly due to the higher binding affinity of DOX to ASOs-TD.

Drug loaded in vitro release studies:

in vitro release studies before and after DOX/DAU loading were performed at 37 oC in a XT5508-R05C shaker (carbon in snow constant temperature technology Co., ltd., hangzhou, china). The effect of varying concentrations of nuclease (DNase I,10 and 20U/mL) on DOX/DAU release loaded by ASOs-TD was investigated. A mixture of DOX/DAU (30. Mu.M) and ASOs-TD (12. Mu.M) was incubated at room temperature for 12 hours to form an ASOs-TD+DOX/DAU binary complex. The test samples (DOX/DAU, ASOs-TD+DOX/DAU or ASOs-TD+DOX/DAU+DNase I, 500. Mu.L) were placed in Mini-Pur-A-Lyzer tubes (Sigma Aldrich) with a molecular weight of 3.5 kDa and immersed in an outer tube containing 15 mL PBS (0.1M, pH 7.4). Release medium of 1 mL was collected at different time points (0.25, 0.5, 1, 2, 4, 6, 9, 12, 24, 36, 48, 60, 72, 84, 96 hours) and PBS of 1 mL was added. Fluorescence of the release medium was recorded and the corresponding release amounts of DOX/DAU at different time points were calculated.

The efficient release of DOX/DAU loaded by DNA nanostructures after reaching tumor cells is a precondition for achieving therapeutic effects. According to the calculatedK _a Andnthe value, the percentage of binding (bound drug concentration) of DOX or DAU added was obtained by the following formulaC _b Total drug concentrationC _t )：

(6)

(7)

Where Θ is the ratio of occupied sites to total sites,M _t andC _f the total concentration of ASOs-TD and the free concentration of DOX/DAU, respectively. From the ITC results of 310.2 and K, the binding rates of DOX (30. Mu.M) and DAU (30. Mu.M) to ASO-TD (12. Mu.M) were calculated to be 99.00% and 97.93%, respectively, indicating that the added drug was almost completely bound to ASOs-TD. To further confirm the reliability of the above binding rates, we measured the fluorescence intensity of DOX/DAU (30. Mu.M) with ASOs-TD at different concentrations. As shown in FIG. 10, the fluorescence intensity of DOX/DAU was almost completely quenched, further demonstrating that they were completely bound to ASOs-TD.

Fig. 11 shows the release profile of all samples in PBS buffer at pH 7.4. Free DOX and DAU are released rapidly. In contrast, DOX and DAU release rates were significantly slower after ASOs-TD loading, with the release percentages over 96 hours being 34.23% and 42.40%, respectively. This suggests that the ASOs-TD+DOX/DAU complexes are stable in PBS, which is associated with their strong binding to ASOs-TD. In the presence of 10U/mL DNase I, DOX and DAU released 82.71% and 94.80% within 96 h, respectively. When the DNase I concentration was increased to 20U/mL, DOX and DAU were almost completely released within 72 h. In the presence of DNase I, accelerated release of DOX and DAU is due to degradation of DNA strands by DNase I. Thus, sensitivity of ASOs-TD to nucleases can provide theoretical support for the regulation of drug release by intracellular nucleases.

Furthermore, the release mechanisms of DOX and DAU in ASOs-TD were studied using three different release models:

zero order release model (8)

Primary release model (9)

Higuchi release model (10)

Wherein the method comprises the steps oftFor the release time (h),K ₀ ，K ₁ andK _H the release rate constants of the three models are respectively.M _∞ AndM _t is the original amount of DOX/DAU in the tube and their timetIs released. Table 3 shows the release rate constants for DOX and DAU release from ASOs-TD in different release models.R ² The values indicate that the release of DOX and DAU followed a first order release model, which is characterized by a concentration dependent release. The release rate constant of DAU is greater than DOX, as opposed to their binding affinity for ASOs-TD. This means that the stronger the binding of DOX to ASOs-TD, the slower the release rate. Furthermore, the release rate constants of DOX and DAU increase with increasing DNase I concentration, so it can be inferred that DNase I in cancer cells is beneficial for drug release.

TABLE 3 release rate constants of DOX/DAU in ASOs-TD from different release models at pH 7.4

In vitro cytotoxicity investigation of DOX/DAU-loaded:

MCF-7 and MCF-7/ADR cells expressing small or large amounts of MDR1mRNA, respectively, were cultured in HERAcell 150i cell incubator (Thermo, USA) at 37 ^o C，CO ₂ The content is 5%. The MTT method was used to detect cytotoxicity of DOX/DAU to both cells before and after TD/ASOs-TD loading. Cells were first seeded into 96-well plates at a density of 3000 cells/well and at 37 ^o Culture 24 h under C. The culture medium was replaced with the prepared samples (DOX/DAU and TD/ASOs-TD+DOX/DAU). For MCF-7 cells, the DOX/DAU concentrations in the prepared samples were 0.2, 0.5, 1.0, 1.5, 2.0 and 3.0. Mu.M, respectively. For MCF-7/ADR cells, the corresponding concentrations of DOX/DAU were respectively2.4, 8, 10, 12 and 15. Mu.M. The concentration ratio of DOX/DAU to TD/ASOs-TD was 2.5:1. after 48 hours of incubation, 20. Mu.L of MTT (5 mg/mL) was added to each well and incubated for 4 hours. The liquid was emptied, 150. Mu.L of DMSO was added to each well and shaken for 5 minutes. The cytostatic ratio was obtained from the absorbance values at 570 nm of the control group cells (a) and the treatment group cells (B) using the following formula:

(11)

intracellular imaging of ASOs-TD and intracellular distribution of DOX:

diagnostic imaging of intracellular MDR1mRNA by ASOs-TD and intracellular distribution of ASOs-TD loaded DOX. MCF-7 and MCF-7/ADR cells were seeded in 15 mm confocal dishes, incubated for 24 hours, and then the medium was decanted. For intracellular imaging of ASOs-TD, further incubation was performed for 4 hours after addition of ASOs-TD of 100 nM to MCF-7 and MCF-7/ADR cells. For intracellular distribution of DOX, MCF-7/ADR cells were further incubated for 6 hours after addition of ASOs-TD+DOX. DOX and ASOs-TD concentrations were 30. Mu.M and 12. Mu.M, respectively. After incubation, the liquid in the dish was poured out. Cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. Confocal fluorescence images were recorded on an LSM-880 confocal laser scanning microscope (CLSM, zeiss, germany). The green fluorescence of FAM was collected under 488 nm laser excitation. DOX and nuclei stained with Hoechst 33258 were collected in red and blue channels, respectively, under laser excitation conditions 488 nm and 405 nm.

The inhibition of MCF-7 and MCF-7/ADR cells by the different samples is shown in FIG. 12, half maximal inhibitory concentration (IC ₅₀ ) The values of (2) are listed in Table 4. IC of DOX and DAU to MCF-7/ADR cells ₅₀ The values were 8.52. Mu.M and 10.39. Mu.M, respectively. By IC ₅₀ The values may infer that DOX inhibits both cell types more than DAU. IC of DOX and DAU to MCF-7/ADR cells ₅₀ The higher values may be due to drug transport from drug-resistant cells to drug-transport machinery. After loading in TD or ASOs-TD, both DOX and DAU have enhanced cytotoxicity to cancer cells. This is possible because of loadingDOX/DAU in DNA nanostructures is more easily endocytosed into cells. The cytotoxicity of TD and ASOs-TD loaded DOX/DAU on MCF-7 cells was identical. However, ASOs-TD-loaded DOX/DAU was more cytotoxic to MCF-7/ADR cells than TD-loaded identical drugs, confirming the effectiveness of gene therapy. MDR1mRNA expression is inhibited by ASOs, and the sensitivity of drug-resistant cells to chemotherapeutic drugs is improved. This indicates that DOX/DAU-loaded ASOs-TD system can effectively improve the drug resistance of cancer cells.

TABLE 4 determination of half-inhibitory concentration values of DOX/DAU, TD+DOX/DAU ASOs-TD+DOX/DAU in MCF-7 and MCF-7/ADR cells by MTT method

Intracellular imaging of ASOs-TD and intracellular distribution of DOX

The ability of ASOs-TD to detect MDR1mRNA in MCF-7 and MCF-7/ADR cells was studied using confocal fluorescence microscopy (CLSM). As shown in FIG. 13A, bright green fluorescence was seen in MCF-7/ADR cells after treatment with ASOs-TD, but little fluorescence was detected in MCF-7 cells. This is due to the overexpression and overexpression of MDR1mRNA in MCF-7/ADR and MCF-7 cells, respectively. When the ASOs recognize MDR1mRNA, its hairpin structure opens. The FAM and BHQ1 on the H chain are far away, and the FAM fluorophore emits a fluorescent signal. This suggests that labeled ASOs-TD has good MDR1mRNA imaging ability and that fluorescent signal is closely related to mRNA expression levels. ASOs-TD is a good in situ imaging nanostructure that can distinguish drug-resistant cells from non-drug-resistant cells.

The distribution of DOX in MCF-7/ADR cells was also studied. As shown in FIG. 13B, after staining with Hoechst 33258, the nuclei exhibited blue fluorescence, and the red fluorescence of DOX was mainly distributed in the nuclei. Rubrene in the cytoplasm is due to the release of DOX loaded in the asas hairpin structure. This is because after recognizing MDR1mRNA in cells, the hairpin structure of ASOs is opened and DOX is released. After ASOs-TD enters the nucleus, a large amount of DOX is released, thereby effectively killing MCF-7/ADR resistant cells. This means that the constructed ASOs-TD is an effective diagnosis and treatment nano-carrier integrating diagnostic imaging, gene therapy and chemotherapy.

In summary, the present application provides an ASOs-TD nanostructure for loading of chemotherapeutic drugs DOX/DAU, and diagnostic imaging and gene therapy based on MDR1 mRNA. Fluorescence detection shows that ASOs-TD has good detection capability on MDR1 mRNA. K (K) ₄ [Fe(CN) ₆ ]The results of fluorescence quenching assays, ITC and DSC experiments indicate intercalation binding of DOX/DAU to ASOs-TD. The number of binding sites, binding constants, enthalpy change and entropy change of the binding process were determined using fluorescence spectroscopy and ITC.nAndK _a the values indicate that ASOs-TD has high loading capacity and strong binding affinity for both drugs. From deltaH ^o And deltaS ^o Is the combination process is enthalpy driven. DOX has more binding sites and stronger binding affinity, indicating that its molecular structure is more suitable for intercalation into ASOs-TD. In vitro release experiments showed that the release of DOX and DAU followed a first order release model. The strong binding strength of DOX to ASOs-TD results in a slow release rate. The presence of DNase I accelerates the release of DOX and DAU. The loading of TD or ASOs-TD enhanced the cytotoxic effects of DOX/DAU on MCF-7 and MCF-7/ADR cells. The cytotoxicity of DOX/DAU loaded by ASOs-TD on MCF-7/ADR cells is stronger than that of TD, and the gene therapy effect of ASOs-TD is proved. Intracellular imaging experiments show that ASOs-TD has good imaging effect on MDR1 mRNA. Cellular uptake showed that ASOs-TD loaded DOX was distributed predominantly in the nuclei of MCF-7/ADR cells. Experimental results show that the constructed ASOs-TD has good diagnostic imaging and gene therapy effects on MDR1mRNA, and further improves the chemotherapeutic effect of DOX/DAU loaded by the ASOs-TD. In conclusion, the binding mechanism of DOX/DAU and ASO-TD is related to the therapeutic effect of the drug, which provides a theoretical basis for the combination of DNA nanostructure-based diagnostic imaging, gene therapy and chemotherapy.

The above examples are provided for further illustration of the present application and are not intended to limit the scope of the patent claims, but are intended to be included within the scope of the patent claims.

Thank to national natural science foundation (22073039), shandong province natural science foundation (ZR 2020MB 052), subsidized the project.

Claims (3)

1. The DNA nano structure for cancer diagnosis and treatment integration based on thermodynamic guidance is characterized by comprising A, B, C, D and H five oligonucleotide chains, wherein the DNA sequences of the oligonucleotide chain A and the oligonucleotide chain are respectively shown as SEQ ID No.1-No. 5.

2. The method for constructing a DNA nanostructure according to claim 1, comprising the steps of:

s1, respectively placing A, B, C, D and H oligonucleotide chains in a container, centrifuging, and adding PBS to completely dissolve the mixture;

s2, four A, B, C, D oligonucleotide chains used for constructing TD are thoroughly mixed according to the ratio of 1:1:1:1, and are mixed in a thermostat at 95 ^o Annealing for 5 minutes under the condition of C, rapidly placing the annealed mixed solution in an ice-water bath, and cooling for 30 minutes to form a TD nano structure;

s3, H oligonucleotide chain at 90 ^o Annealing for 5 minutes under C, and naturally cooling to room temperature to form an ASOs hairpin structure;

s4, fully mixing the TD and the ASOs according to the ratio of 1:1, and placing the mixture on a microplates shaker (600 r/h, 1 hour) to form an ASOs-TD structure.

3. The use of the DNA nanostructure obtained according to claim 1 for the preparation of anticancer drugs.