Drug delivery nanosystem and method based on DNA origami and doxorubicin prodrug

Abstract

The present invention discloses a drug delivery nanosystem and method based on DNA origami and doxorubicin prodrug, belonging to the field of biomedical technology. In view of the abnormally high expression of NQO1 enzyme in many tumor cells, the present application synthesizes a target-activated anti-tumor prodrug based on doxorubicin as a model, and its pharmacological action can be activated by the NQO1 enzyme; at the same time, a rectangular DNA origami with good biocompatibility is used as a nanocarrier to efficiently carry the prodrug, thereby constructing an activatable precision drug delivery nanosystem. This prodrug-embedded rectangular DNA origami is conducive to reducing the toxic and side effects of anticancer drugs on normal human liver cells (L02 cells), and has a significant inhibitory effect on human breast cancer cells (MCF-7 cells) with high expression of NQO1 enzyme. This therapeutic nanosystem is expected to reduce the systemic toxicity of tumor drugs due to non-specificity, improve patients' compliance and quality of life, and provide an effective strategy for precise clinical intervention of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a drug delivery nanosystem and method based on DNA origami and doxorubicin prodrug construction, and more particularly to a DNA framework nucleic acid precision drug delivery system carrying an NQO1 enzyme-activated prodrug and a construction method thereof. Background Art

[0002] Traditional chemotherapy is one of the main means of treating cancer clinically. It is an anti-tumor treatment for patients through systemic administration. However, due to the non-specific distribution characteristics of small molecule drugs used for treatment, there are often bottlenecks such as rapid clearance and off-target effects during the administration process. Nanosystems for targeted tumor drug delivery promote tumor accumulation through passive or active targeting strategies, which can alleviate the off-target effects of drugs to a certain extent. At present, some nanodrugs (such as Abraxane and Doxil) have been approved for clinical use by the FDA. However, recent studies have emphasized that due to complex tumor biology and biological barriers (such as the reticuloendothelial system and renal metabolism), in mouse models, an average of only 0.7% of the administered nanoparticles can eventually reach solid tumors. Injected nanodrugs are easily cleared by the reticuloendothelial system and are mainly distributed in the liver and spleen, which are rich in macrophages. Therefore, there is a problem of low bioavailability of nanoformulations. This phenomenon of high enrichment in healthy organs and low accumulation in diseased organs leads to the interaction of nanodrugs with normal tissues, which may cause potential systemic toxicity problems.

[0003] The application of nanotechnology to develop drug delivery systems for targeted transport and controlled release of anti-tumor drugs is one of the keys to solving this problem. Different from traditional small molecule anticancer drugs, multifunctional nanocarriers accumulate in the lesions with the help of the high permeability and long retention effect of tumors, which can improve the pharmacokinetic behavior of drugs, increase the bioavailability of drugs, and reduce systemic toxicity. Among them, DNA framework nucleic acid technology is a novel and unique DNA self-assembly nanotechnology, which uses hundreds of DNA staple chains as short chains to fold a long chain of DNA (usually M13 phage genomic DNA with a length of more than 7,000 bases), and self-assembles into a well-defined two-dimensional or three-dimensional structure through gradient cooling. These numerous short chains are different from each other, which endows DNA origami with easy modification and spatial addressability, making it possible to become a small molecule anti-tumor drug carrier. In addition, studies have shown that DNA origami nanostructures have good biological stability and low immunogenicity, thus broadening their applications in biosensing, bioimaging and biomedicine. Some aromatic ring small molecules with planar or near-planar structures (such as doxorubicin, daunorubicin, etc.) can be embedded in adjacent base pairs of DNA double strands, making DNA origami an excellent carrier for diagnostic and therapeutic reagents, which can promote the enrichment of anti-tumor drugs in tumor tissues and reduce the systemic toxicity of anti-tumor drugs to animals. Chinese patent CN 104368004A once disclosed a nucleic acid nanostructure for carrying anti-tumor drugs. Experiments at the cell level and animal level have shown that it can not only ensure the anti-tumor activity of the anti-tumor drugs it carries, but also improve the targeting of anti-tumor drugs, enrich anti-tumor drugs in tumor tissues, and significantly reduce the systemic toxicity of anti-tumor drugs due to non-specificity.

[0004] However, improving the carrier alone cannot achieve the ideal therapeutic effect. On this basis, selecting highly specific tumor markers as targets and constructing an activatable nanotherapeutic system are also of great significance for improving the selectivity of drug action, avoiding off-target risks, and reducing the systemic toxicity of anti-tumor drugs caused by non-specificity.

[0005] Prodrugs refer to compounds that are inactive or less active in vitro after chemical structure modification, and release active drugs through enzymatic or non-enzymatic conversion in vivo to exert their efficacy. Designing prodrugs targeting the unique molecular defects of cancer cells (such as overexpression and mutation) is beneficial to improving pharmacokinetics while reducing off-target effects. However, a considerable number of prodrugs can be activated systemically by nonspecific stimuli (such as esterases, phosphatases, and low lysosomal pH), which may cause serious adverse reactions. For example, lysosomal acidification and intracellular redox environments exist in both cancer cells and healthy cells. This nonspecific stimulation can cause systemic drug efficacy recovery and induce systemic toxic side effects.

[0006] NAD(P)H quinone oxidoreductase 1 (NQO1 enzyme) is a cytoplasmic homodimeric flavoenzyme that catalyzes the two-electron reduction of quinones in the presence of NAD(P)H to protect cells from oxidative stress. It is abnormally highly expressed in many cancer cells. Therefore, the design of prodrugs targeting NQO1 is expected to achieve drug activation at the target site, which can significantly reduce off-target effects and improve the systemic toxicity of anti-tumor drugs caused by non-specificity. On this basis, if the designed prodrug targeting NQO1 can be effectively combined with nanocarriers, it will provide a new and effective strategy for precise clinical intervention of tumors. Summary of the invention

[0007] The purpose of the present invention is to improve the deficiencies in the prior art and provide a drug delivery nanosystem and method based on DNA origami and doxorubicin prodrug, which utilizes DNA framework nucleic acid materials to carry NQO1 enzyme-activated prodrugs for highly specific treatment of tumor cells.

[0008] The present invention is achieved by: a method for constructing a drug delivery nanosystem based on DNA origami and doxorubicin prodrug, comprising the following steps:

[0009] 1) Using doxorubicin as a model, synthesize a prodrug quinone propionic acid structure-doxorubicin conjugate molecule activated by tumor cell targets;

[0010] 2) Prepare custom-designed rectangular DNA origami;

[0011] 3) The rectangular DNA origami is mixed with the prodrug dispersed in ethanol, and the prodrug is embedded in the DNA double helix to achieve prodrug loading.

[0012] Furthermore, the rectangular DNA origami is a nucleic acid nanostructure formed by the self-assembly of the DNA scaffold chain and the DNA staple chain through the principle of base complementary pairing. During the preparation, the M13mp 18DNA scaffold chain and the DNA staple chain were mixed in 1×TAE-Mg 2+ The mixture was mixed in a buffer solution, programmed to anneal from 95°C to 25°C in a thermal cycler within 2 hours, ultrafiltered, and washed to complete the preparation.

[0013] Furthermore, the molar ratio of the M13mp 18 DNA scaffold chain to the DNA staple chain is 1:5-10, and the approximate dimensions of the length, width and height of the resulting rectangular DNA origami are 90×60×2 nm.

[0014] Furthermore, the quinone propionic acid structure-doxorubicin coupling molecule is generated by activating the quinone propionic acid ligand 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butyric acid using a carbodiimide condensation agent to form an active ester intermediate, which is then subjected to an amide condensation reaction with doxorubicin. The specific preparation process is as follows: under constant stirring, doxorubicin and 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butyric acid active ester are mixed and added to anhydrous DMF, DIPEA is added to the mixture, nitrogen is passed through, and the mixture is stirred and mixed at room temperature. After the reaction is completed, the prodrug is collected by rotary evaporation under reduced pressure and purification by column chromatography.

[0015] Furthermore, the molar ratio of doxorubicin to 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butyric acid active ester is 1:1-3.

[0016] Furthermore, the synthesized prodrug has the ability to embed into the double-stranded structure of DNA, and the prodrug loading is as high as 236 mg g –1 , the load efficiency reaches 94%.

[0017] Furthermore, the tumor cell target is the tumor-related NQO1 enzyme.

[0018] A drug delivery nanosystem is constructed according to the above-mentioned method for constructing a drug delivery nanosystem based on DNA origami and doxorubicin prodrug. The drug delivery nanosystem has acid-responsive prodrug release behavior. The prodrug is released under acidic pH stimulation in lysosomes and diffuses into the cytoplasm. The bulk drug is released after biotransformation by NQO1 enzyme in the cytoplasm and reaches the cell nucleus to exert its efficacy.

[0019] Beneficial effects:

[0020] (1) In view of the problem that most anticancer drugs or prodrugs have poor selectivity and large toxic side effects, this application starts from the differences in molecular biology between normal cells and tumor cells, selects the tumor-related biological enzyme NQO1 that is abnormally highly expressed in tumor cells as a stimulus, designs an activatable precursor, and utilizes the biological activation effect of tumor-related enzymes to accurately regulate the activity of the drug, improve the selectivity of the drug, and reduce the off-target effect of the drug;

[0021] (2) Compared with single-stranded or double-stranded DNA, rectangular DNA origami has enhanced physiological stability and can still maintain about 61% structural integrity after exposure to 10% fetal bovine serum for 24 hours, making it an excellent drug carrier for biomedical applications;

[0022] (3) Rectangular DNA origami with good biocompatibility is used as a nanocarrier, and a small amount of ethanol is used to dissolve the hydrophobic prodrug, which helps the prodrug to be more smoothly embedded in the DNA double helix, thereby achieving efficient loading of the prodrug. After the ethanol evaporates, the free prodrug settles due to its own hydrophobicity, and the DNA framework nucleic acid loaded with the prodrug is evenly dispersed in the solution. The free prodrug can be removed by low-speed centrifug, which provides a convenient new strategy for the loading of poorly soluble drugs.

[0023] (4) The DNA nanostructure loaded with prodrugs has an acid-responsive prodrug release behavior. Under the stimulation of acidic pH in lysosomes, the prodrug is released and diffuses into the cytoplasm. After biotransformation by NQO1 enzyme in the cytoplasm, the parent drug is released and finally reaches the cell nucleus to exert its efficacy. This drug efficacy mode helps to reduce the toxic side effects of anticancer drugs on normal human cells, and can help small molecule anticancer drugs directly reach the lesions and accumulate at the lesions, producing a more obvious inhibitory effect on cancer cells with high expression of NQO1 enzyme.

[0024] (5) Rectangular DNA origami has good biocompatibility and has no obvious toxic side effects on normal cells. Thanks to the efficient prodrug loading of DNA rectangular origami and the NQO1 enzyme responsiveness of the prodrug, the DNA nanostructure loaded with doxorubicin prodrug effectively alleviated the damage of doxorubicin to normal human liver cells L02, and showed a significant inhibitory effect on human breast cancer cells MCF-7 that overexpress NQO1, providing a new idea for the precision treatment of nanomedicines. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The assembly process of DNA rectangular origami loaded with doxorubicin prodrug and its mechanism of action at the cellular level;

[0026] Figure 2 This is a flow chart for synthesizing tumor-associated NQO1 enzyme-responsive prodrugs using doxorubicin as a model;

[0027] Figure 3 Panel a in the middle is the mass spectrum of the doxorubicin prodrug molecule, and panel b is the mass spectrum of the prodrug after activation by NQO1 enzyme;

[0028] Figure 4 Panel a in the middle is the agarose gel electrophoresis analysis of the rectangular DNA origami after exposure to 10% fetal bovine serum for different time periods, and panel b is the quantitative analysis of the corresponding residual amount of rectangular DNA origami;

[0029] Figure 5 Panel a in the middle is an atomic force microscope image of a rectangular DNA origami, and panel b is an atomic force microscope image of a rectangular DNA origami loaded with doxorubicin prodrug;

[0030] Fig. 6A is the loading curve of DNA origami on doxorubicin prodrug;

[0031] Figure 6B is the change in zeta potential of the DNA rectangular origami before and after loading with doxorubicin prodrug;

[0032] Figure 6C is the prodrug release behavior of prodrug-loaded rectangular DNA origami at different pH;

[0033] Figure 7 Panel a in the middle shows the effects of DNA origami, doxorubicin, and DNA origami loaded with prodrugs on the survival rate of L02 cells; Panel b shows the effects of doxorubicin and DNA origami loaded with prodrugs on the survival rate of MCF-7 cells. DETAILED DESCRIPTION

[0034] The preferred embodiments of the present invention are described in detail below so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0035] 1. Synthesis of prodrugs

[0036] First, the broad-spectrum small molecule anticancer drug doxorubicin was used as a model to synthesize tumor-related NQO1 enzyme-responsive prodrugs. Figure 2 As shown, a carbodiimide condensation agent is used to activate a quinone propionic acid ligand to form an active ester intermediate, which then undergoes an amide condensation reaction with doxorubicin to generate a target compound. Specifically, the quinone propionic acid ligand 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-diene-1-yl)butyric acid was activated with reference to the public document "Enzyme-Instructed Activation of Pro-protein Therapeutics In Vivo" of DOI:10.1021 / jacs.9b08669 to obtain an active ester intermediate, and then, in 40 mL of anhydrous DMF, under constant stirring, adriamycin Dox (1.0 g) and the obtained 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-diene-1-yl)butyric acid active ester were mixed in a molar ratio of 1:1, and 2 equivalents of N,N-diisopropylethylamine (DIPEA) were added dropwise to the mixture, nitrogen was passed, and the mixture was stirred at room temperature. The reaction progress was monitored by thin layer chromatography, and after the reaction was completed, the mixture was evaporated under reduced pressure and purified by column chromatography to collect the generated prodrug.

[0037] From the mass spectrometry analysis ( Figure 3 In the middle a panel, the characteristic mass spectrometry peaks of the prodrug can be observed, indicating the successful synthesis of the doxorubicin prodrug. To further demonstrate the NQO1 enzyme responsiveness of the prodrug, 50 μg mL -1 The prodrug was added to the NQO1 enzyme (100 ng mL-1 ) and NADH (300mM) in 1×PBS buffer and incubated at 37°C overnight. Figure 3 As can be seen from the panel b), the prodrug has good NQO1 enzyme responsiveness. After exposure to NADH and NQO1 enzyme, the group containing the quinone propionic acid structure will be removed and the bulk drug will be recovered, verifying the NQO1 enzyme-mediated drug recovery effect.

[0038] 2. Construction of rectangular DNA origami loaded with prodrugs

[0039] Step 1: Self-assembly of rectangular DNA origami

[0040] The rectangular DNA origami chain was designed with reference to the open literature "Tuning the structural asymmetries of three-dimensional gold nanorod assemblies" with DOI:10.1039 / c5cc05295e, and slightly modified to remove the side DNA chain to avoid the stacking of adjacent DNA origami. In this example, the M13mp 18 DNA scaffold chain and the DNA staple chain with a molar ratio of 1:10 were added in 1×TAE-Mg 2+ The mixture was placed in a thermal cycler and programmed to anneal from 95°C to 25°C within 2 hours. The resulting mixed solution was ultrafiltered using an ultrafiltration tube with a molecular weight cutoff of 100 kDa, and then filtered with 1×TAE-Mg 2+ The purified product was then washed three times with 1% MgCl2 (containing 12.5 mM MgCl2) buffer to remove excess DNA strands. Finally, the purified product was collected and stored at 4°C until use.

[0041] from Figure 4 It can be seen that the rectangular DNA origami has good physiological stability. After being exposed to 10% fetal bovine serum for 24 hours, it can still maintain 61% of its structural integrity, making it an excellent drug carrier for biomedical applications.

[0042] Step 2: Prodrug loading

[0043] Take 3 volumes and disperse them in 1×TAE-Mg 2+Rectangular DNA origami (containing 16.5 mM MgCl2) and 1 volume of prodrug dispersed in ethanol are mixed in different molar ratios. In this step, the purpose of introducing a small amount of ethanol is to improve the dispersibility of the hydrophobic doxorubicin prodrug, thereby facilitating the loading of the prodrug. After the above mixed solution is gently shaken overnight, the centrifuge tube cover is opened to volatilize the residual ethanol. In this process, the unloaded prodrug itself is hydrophobic and precipitates, and the resulting rectangular DNA origami loaded with the prodrug is evenly dispersed in the supernatant. The unbound prodrug can be removed by centrifug (2,000 rpm, 10 minutes).

[0044] From the atomic force microscopy image ( Figure 5 ) It can be seen that the loading of the prodrug has almost no effect on the morphology of DNA origami.

[0045] Prodrugs derived from doxorubicin still retain the ability to intercalate into DNA duplexes. Fig. 6A It can be seen that the loading amount of DNA origami on prodrug increases with the increase of prodrug feed amount. Finally, the prodrug loading experimental conditions were optimized as follows: the mass ratio of prodrug to DNA origami was 250.0 mg / g. After prodrug loading, the zeta potential of DNA nanostructure changed from negative to positive ( Figure 6B ), demonstrating the efficient loading of the prodrug.

[0046] 200 μL of DNA rectangular origami loaded with prodrug was added to two Slide-A-LyzerTM MINI dialysis devices with a molecular weight cutoff of 10 kDa. The dialysates were 1× PBS buffer (containing 12.5 mM MgCl2) at pH 7.4 and 5.0, respectively. At a predetermined time point, 100 μL of the solution was taken out from the dialysate and the same volume of buffer was added. The release rate of the prodrug was determined based on the characteristic absorbance of the prodrug at 502 nm. Figure 6C It can be seen that the prodrug-loaded DNA origami has acid-responsive prodrug release behavior, and acidic conditions accelerate the release of the prodrug. Given this behavior, it can be inferred that the acidic environment within the lysosome is conducive to the release of the prodrug.

[0047] Evaluation of therapeutic specificity of prodrug-loaded DNA rectangular origami

[0048] Human breast cancer cells MCF-7 with high expression of NQO1 and human normal liver cells L02 with low expression of NQO1 were selected and seeded in 96-well plates at a density of 5000 cells per well. After 24 hours of cell adhesion, they were incubated with DNA origami, doxorubicin, and DNA origami loaded with prodrug for 48 hours. Then, 10 μL of MTT (5.0 mg mL -1) and placed in a cell culture incubator. After 4 hours, the culture medium in each well was removed and 100 μL of DMSO was added to dissolve the formazan in the cells. The absorbance at 492 nm and 630 nm was measured with an ELISA reader, and the cell survival rate was determined by background correction.

[0049] from Figure 7 As can be seen from panel a, after 48 hours of co-culture with DNA origami and 1.5 nM prodrug-loaded DNA origami, the cells still maintained a high survival rate, indicating that the assembled DNA nanostructure has good biocompatibility. Figure 7 The two panels a and b show that this prodrug-embedded DNA rectangular origami is beneficial to reducing the toxic side effects of anticancer drugs on normal human liver cells (L02 cells), and exhibits a cell inhibitory effect comparable to that of doxorubicin in human breast cancer MCF-7 cells that highly express the NQO1 enzyme. This therapeutic nanosystem is beneficial to avoid the problem of indiscriminate harm in traditional chemotherapy, improve treatment specificity, and provide an effective strategy for precise clinical intervention of tumors.

[0050] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification and drawings, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. A method for constructing a drug delivery nanosystem based on DNA origami and doxorubicin prodrug, characterized in that: The steps include: 1) Using doxorubicin as a model, a tumor cell target activated prodrug quinone propionic acid structure-doxorubicin conjugate molecule was synthesized: the quinone propionic acid structure-doxorubicin conjugate molecule was generated by using a carbodiimide condensation agent to activate the quinone propionic acid ligand 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butyric acid to form an active ester intermediate, which was then reacted with doxorubicin to undergo an amide condensation reaction. The specific preparation process is as follows: under constant stirring, doxorubicin and 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butyric acid active ester were mixed and added to anhydrous DMF, DIPEA was added to the mixture, nitrogen was passed through, and the mixture was stirred and mixed at room temperature. After the reaction was completed, the prodrug was collected by vacuum rotary evaporation and column chromatography purification; 2) Preparation of customized rectangular DNA origami: Rectangular DNA origami is a nucleic acid nanostructure formed by the self-assembly of DNA scaffold chains and DNA staple chains through the principle of base complementary pairing. During preparation, the M13mp 18 DNA scaffold chain and DNA staple chain were mixed in 1×TAE-Mg 2+ The mixture was mixed in a buffer, programmed to anneal from 95°C to 25°C in a thermal cycler within 2 hours, ultrafiltered, and washed to complete the preparation; 3) The rectangular DNA origami is mixed with the prodrug dispersed in ethanol, and the prodrug is embedded in the DNA double helix to achieve prodrug loading.

2. The method for constructing a drug delivery nanosystem based on DNA origami and doxorubicin prodrug according to claim 1, characterized in that: The molar ratio of M13mp 18 DNA scaffold chain to DNA staple chain is 1:5-10.

3. The method for constructing a drug delivery nanosystem based on DNA origami and doxorubicin prodrug according to claim 1, characterized in that: The molar ratio of doxorubicin to 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohexa-1,4-dien-1-yl)butyric acid active ester is 1:1-3.

4. The method for constructing a drug delivery nanosystem based on DNA origami and doxorubicin prodrug according to claim 1, characterized in that: The tumor cell target is the tumor-associated NQO1 enzyme.

5. A drug delivery nanosystem, characterized in that: The drug delivery nanosystem is constructed according to the method for constructing a drug delivery nanosystem based on DNA origami and doxorubicin prodrug according to any one of claims 1 to 4.

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