WO2022086137A1 - Thermo-responsive dna nanogel and pharmaceutical use thereof - Google Patents

Abstract

The present invention relates to a thermo-responsive DNA nanogel and a pharmaceutical use thereof. The nanogel of the present invention enables cancer-specific intracellular delivery and treatment by a targeting and/or therapeutic nucleic acid component, and can exhibit an effective anticancer effect when photothermal therapy is used, and thus can be used as a targeted carrier for combined cancer treatment.

Description

Temperature Sensitive DNA Nanogels and Pharmaceutical Uses Thereof

The present invention relates to temperature-sensitive DNA nanogels and pharmaceutical uses thereof.

DNA hydrogel, a biomaterial, is a material attracting attention in the biomedical field due to its high biocompatibility and biodegradability. In addition, DNA is combined with pure DNA or organic/inorganic materials, and research is being actively conducted to apply it as a drug carrier based on multifunctionality, self-assembly through molecular recognition ability, and control of size and shape at the nano level. In particular, stimuli-sensitive nanogels made of DNA nanostructures can undergo structural transformations such as dissolution, separation, division and reconstitution of DNA using nucleotide sequence-specific properties, thereby inducing the release of loaded drugs. These stimuli-sensitive DNA nanogels have various advantages, such as bypassing biological barriers, providing high drug bioavailability, and reducing side effects through controllable drug release. To date, various types of stimulus-sensitive nanogels such as reducing agent (GSH)-reactivity, heat sensitivity, and pH sensitivity have been reported, but development of a platform capable of more effectively delivering combination anticancer drugs is insufficient.

It is an object of the present invention to provide a temperature-sensitive DNA nanogel for targeted delivery of a complex therapeutic agent including a nucleic acid.

Another object of the present invention is to provide a pharmaceutical use of the temperature-sensitive DNA nanogel as a drug delivery system or composition for anticancer.

In order to achieve the above object, the present invention provides a branched DNA unit in which each arm has an adhesive end; a photosensitive unit comprising gold nanoparticles and single-stranded DNA having an adhesive end; And it provides a temperature-sensitive DNA nanogel formed by self-assembly of a linking unit having an adhesive end.

The present invention also the temperature-sensitive DNA nanogel; And it provides a drug delivery system comprising a pharmaceutically acceptable carrier.

The present invention also provides an anticancer composition comprising the temperature-sensitive DNA nanogel.

The present invention also provides a method of treating cancer, comprising administering to a subject in need thereof an effective amount of the temperature-sensitive DNA nanogel.

The nanogel of the present invention enables efficient intracellular delivery of a drug by a target component, and exhibits a complex anticancer effect on light induction.

1 shows a controlled-release schematic diagram of a drug according to the fabrication and structural transformation of the temperature-sensitive DNA nanogel of the present invention.

2 is a result of confirming the synthesis of the DNA nanostructure of the present invention, (A) the sequential synthesis result of BUs revealed through gel electrophoresis (lane 1: DNA ladder; lane 2: X1; lane 3: X1 + X2; lane 4: X1 + X2 + X3; and lane 5: BUs), (B) gel electrophoresis analysis of AU preparation (lane 1: free AuNP; lane 2: 5'-AU; and lane 3: 3'-AU) , (C) Shows the surface density results of oligonucleotides adsorbed to AuNPs quantified using dissociation-based fluorescence analysis. Error bars represent standard deviation of three measurements (n = 3).

3 is a result of the preparation and light sensitivity confirmation of a DNA nanogel composed of a branched unit (BUs) and a photosensitive unit (AUs) of the present invention, (A) three adhesive ends (sticky) binding to AUs using electrophoresis; ends) functionalized BUs production confirmation result, (B) nanogel synthesis confirmation according to the ratio of AUs and BUs using electrophoresis, and (C) AUs-BUs synthesis confirmation and light-responsive decomposition using TEM degradation) is the result of confirmation.

Figure 4 shows the functional unit characterization results of Dgels, (A) melting curves of BUs-LUs with adjusted length of adhesive ends and (B) circular dichroism of BUs-LUs with 15 bp adhesive ends at various temperatures. Method spectra, (C) flow cytometry analysis of SKOV3 and NIH/3T3 cells treated with DOX-loaded BUs-LU, (D) CLSM images of SKOV3 and NIH/3T3 cells treated with DOX-loaded BUs-LU (scale) bar: 10 μm).

Figure 5 shows the CD spectral results of BUs-LU assembled with adhesive ends of different lengths and incubated at 20, 60 and 90 °C for 15 min.

6 shows the results of flow cytometry analysis of Apt-FAM-treated NIH/3T3 and SKOV-3 for verification of nucleolin expression levels.

7 shows CLSM images (scale bar: 10 μm) of SKOV3 cells treated with DOX-loaded BUs-LU for various time periods.

8 shows the results of size distribution analysis of BUs-LUs having different ratios. Error bars represent standard deviation of three measurements (n = 3).

9 shows SEM images (scale bar: 500 nm) of BUs-LU synthesized at different ratios.

10 shows the results of sequential synthesis of BUs-LU revealed through gel electrophoresis (lane 1: DNA ladder; lane 2: BU; lane 3: LU; lane 4: BUs-LU).

Figure 11 shows the morphology and physicochemical properties of Dgels, (A) absorbance spectra of free AuNPs, BUs-LU and Dgels, (B) SEM images of BUs-LU and Dgels (scale bar: 200 nm), (C) Dgels The particle size and surface charge of , (D) TEM images of Dgels before and after light irradiation (scale bar: 50 nm) are shown.

12 is an image (top) and thermometer measurement (bottom) of a thermal observation device for temperature change of a Dgel solution after light irradiation, showing the temperature rise due to light.

13 shows the temperature-responsive DOX release results of (A) pH and (B) Dgels error bars. Profiles represent the standard deviation of three measurements (n = 3).

14 is a gel electrophoresis analysis result of Dgel incubated for 30 minutes at each temperature showing the thermal reaction decomposition result of Dgels (lane 1: 25 °C, lane 2: 30 °C, lane 3: 35 °C, lane 4: 40 °C) , lane 5: 45°C, lane 6: 50°C, lane 7: 55°C, lane 8: 60°C).

15 shows CLSM images (scale bars: 10 μm) of SKOV3 cells treated with Dgel with or without AS1411 aptamer.

Figure 16 shows the potential application of Dgels in cancer treatment, (A) CLSM images of SKOV3 and NIH/3T3 cells treated with Dgel (scale bar: 10 μm), (B) light irradiation analyzed using MTT assay. Cell viability results in the absence and presence of Dgel. Data are presented as mean±SD values (n = 3).

17 shows CLSM images (scale bar: 10 μm) for AuNP removal analysis over time in SKOV3 cells.

18 shows the relative cell viability of SKOV3 cells treated with Bcl-2 AS-ODN-loaded Dgel after 2 minutes of light irradiation analyzed by MTT analysis. Error bars represent standard deviation of three measurements (n = 3).

Hereinafter, the configuration of the present invention will be described in detail.

The present invention relates to a branched DNA unit, each arm having an adhesive end; a photosensitive unit comprising gold nanoparticles and single-stranded DNA having an adhesive end; and a temperature-sensitive DNA nanogel formed by self-assembly of a linking unit having an adhesive end.

The DNA nanogel of the present invention is for targeted delivery and stimulus-responsive release of drugs, and includes a multifunctional branched DNA unit (also referred to as “Branched Unit; BU”) and a photosensitive unit (“Light-sensitive ssDNA-AuNP Unit; AU”). It is formed through self-assembly of DNA-based nanostructures of a linking unit (also referred to as “Linker Unit; LU”) and selectively delivers a drug to a target cell, and contains a photosensitive unit to increase the temperature when irradiated with light Through this, the units constituting the nanogel can be decomposed to release the loaded drug, and since the nanogel is DNA-based, various combinations of a nucleic acid therapeutic agent and a chemotherapeutic agent can be used as the drug.

The nanogel is designed to enable self-assembly through effective hybridization as each DNA unit has a sticky end group of a complementary sequence and to be decomposed when the temperature rises by light irradiation through photosensitive single-stranded DNA. , the connection unit is designed so that a nucleic acid drug that can be used as a therapeutic agent can bind, so that the nanogel can deliver various combinations of a nucleic acid drug and a chemotherapeutic agent. In addition, the nanogel has the characteristics of being able to control the physicochemical properties by controlling the length of the sequence of the adhesive end and the molar ratio of the nanostructure, and the release of various drug combinations through the stimulus response.

As used herein, the term “DNA unit” refers to a nanostructure composed of single-stranded or double-stranded DNA, which is assembled by sequence-specific interaction between complementary nucleic acids.

The term “temperature sensitivity” as used in the present invention means that the structure is switchable and/or its properties are changed according to a specific temperature, and in the present invention, the DNA nanogel is irradiated with light by the elevated temperature. It is characterized in that the nanogel is decomposed.

Adhesive ends of complementary sequences are bonded to the ends of single-stranded or double-stranded DNA units constituting the DNA unit, thereby enabling complementary hybridization between DNA units. The length of the adhesive end may consist of a nucleotide sequence of 5 to 30 bp, and when it is out of the above range, nanogel may not be formed or temperature sensitivity may be deteriorated. More specifically, adhesive ends of 10 to 20 bp in length may be used. More specifically, the sequence shown in Table 2 to be described below of about 15 bp may be used.

The branched DNA unit (BU) serves as the structural framework of the nanogel, and has an adhesive end that can connect between structures through self-assembly, a targeting function to target specific cells, and a low-molecular drug tower through binding to a DNA chain. It can be given multifunctionality such as talent, and can be prepared by hybridization of single-stranded DNA. The branched DNA is designed and synthesized so that each arm of the DNA molecule has complementary adhesive ends. In this case, the branched DNA can adjust the length of the cancer as needed.

The branched DNA may be X-type DNA, Y-type DNA or T-type DNA.

According to one embodiment of the present invention, each single-stranded DNA designated as X1, X2, X3 (including adhesive ends) and X4 (including adhesive ends) for one target is assembled to form an X-type branched form can form DNA. In this case, the single-stranded DNA may include an aptamer for targeting.

In addition, in the branched DNA, a nucleic acid component for targeting may be bound to the end of each arm of the DNA molecule, and the nucleic acid component may include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide (PNA). nucleic acid), LNA (morpholino and locked nucleic acid), GNA (glycol nucleic acid), oligonucleotide, plasmid DNA, antisense oligonucleotide, messenger RNA, micro RNA (microRNA), locked nucleic acid, DNA-based enzyme (DNAzyme), small interfering RNA (small interfering RNA), short hairpin RNA, RNA-based enzyme (RNAzyme) and nucleic acid aptamer ) can be used. More specifically, it may be a nucleic acid aptamer.

The photosensitive unit (AU) is composed of gold nanoparticles and single-stranded DNA bound thereto, and the single-stranded DNA is a polymerized adenosine (polyA) portion that binds to the gold nanoparticles and an adhesive end portion for self-assembly. Consists of. In addition, the photosensitive unit serves to increase the temperature in light sensitivity by using the characteristics of the gold nanoparticles. It enables the decomposition of the nanogel through the heat generated by such light irradiation, and single-stranded DNA consisting of an adhesive end and a 5'-polyA tail and a single-stranded DNA consisting of an adhesive end and a 3'-polyA tail. It can be formed through adsorption to gold nanoparticles (AuNPs). At this time, the polyA tail is strongly adsorbed to the gold surface, and this adsorption is similar to the binding affinity between Au-S. In addition, the DNA density on the surface of gold nanoparticles (AuNP) can be controlled by adjusting the length of the polyA tail.

The smaller the size of the gold nanoparticles, the less cytotoxicity, so to reduce cytotoxicity, a spherical shape having a diameter of 5 to 100 nm or a rod shape having a length of 5 to 70 nm may be used. More specifically, spherical gold nanoparticles having a diameter of about 5 to 13 nm may be used.

The linking unit (LU) is a single-stranded DNA or RNA, and has an adhesive end connecting the branched DNA unit and the photosensitive unit, and the middle portion of the structure may contain a nucleic acid drug. Specifically, 5'-LU and 3'-LU may be prepared for hybridization with 5'-AU and 3'-AU.

The nucleic acid drug is DNA (deoxyribonucleic acid), RNA (ribonucleic acid), PNA (peptide nucleic acid), LNA (morpholino and locked nucleic acid), GNA (glycol nucleic acid), oligonucleotide (oliogonucleotide), plasmid DNA (plasmid DNA) ), antisense oligonucleotide, messenger RNA, microRNA, locked nucleic acid, DNA-based enzyme, small interfering RNA, short hairpin It may have a form such as short hairpin RNA (RNA), an RNA-based enzyme (RNAzyme), or a nucleic acid aptamer. The nucleic acid may include a sequence encoding one or more proteins or a non-coding sequence.

Examples of the nucleic acid drug, polo-like kinase 1 (PLK1), apoptotic B-cell lymphoma 2 (Bcl-2), brain derived neurotrophic factor (BDNF), glial derived neurotrophic factor (GDNF), neurotrophic factor 3 (NT3), FGF (fibroblast growth factor), TGF (transforming growth factor), PDGF (platelet-derived transforming growth factor), MGF (milk growth factor), EGF (endothelial growth factor), ECDGF (endothelial cell-derived growth factors), NGF ( nerve growth factor), vascular endothelial growth factor (VEGF), 4-1BBR (4-1 BB receptor), TRAIL (TNF-related apoptosis inducing ligand), artemin (GFRalpha3-RET ligand), CXCL13 (B cell-attracting chemokine l ), B lymphocyte chemoattractant (BLC), B cell maturation protein (BCMA), bone-derived growth factor (BDF), megakaryocyte derived growth factor (MGDF), keratinocyte growth factor (KGF, thrombopoietin), platelet-derived growth factor (PGDF) ), coding for megakaryocyte derived growth factor (MGDF), keratinocyte growth factor (KGF), bone morphogenetic protein 2 (BMP2), BRAK, C-10, or Cardiotrophin 1 (CT1), etc. And/or a nucleic acid comprising a non-coding sequence may be introduced in various above-described forms applicable to the human body.

The DNA nanogel of the present invention may be loaded with a chemically and pharmaceutical active ingredient. The chemically active pharmaceutical ingredient may be an anticancer agent. Anticancer drugs are drugs that act on various metabolic pathways of cancer cells and exhibit cytotoxic or cytostatic effects on cancer cells. , anticancer antibiotics, hormones, or other drugs.

The anticancer agent is oxaliplatin, imatinib, docetaxel, pemetrexed, gefitinib, tegafur, capecitabine, erlotidib, doxyfluridine, paclitaxel, interferon alpha, gemcitabine, fludarabine, irinotecan, carboplatin, cisplatin, taxotere, doxorubicin, epirubicin, 5-fluorouracil, UFT, tamoxifen, goserelin, herceptin, anti-CD20 antibody, leuprolide (Lupron) or flutamide, etc. , but is not limited thereto.

The anticancer agent may be loaded onto the nanogel by incubation with the DNA nanogel.

According to one embodiment of the present invention, the DNA nanogel is synthesized by including the nucleic acid drug in the LU, and the DNA nanogel and the anticancer agent are incubated to simultaneously load the nucleic acid drug and the anticancer agent.

The DNA nanogel may first react with BU and LU to assemble a BU-LU complex. The molar ratio of BU and LU may be 2:1 to 1:4. Preferably, the molar ratio of BU and LU may be 1:2. Next, for preparing AU, the gold nanoparticle solution is reacted with a 5'-polyA or 3'-polyA DNA solution to prepare photosensitive AU. Finally, nanogels are prepared by mixing the stoichiometric contents of the BU-LU complex, 5'-AU and 3'-AU. The molar ratio of BU:LU:AU may be 1:2:0.25.

The nanogel shows spherical nanoparticles, and may have a diameter of approximately 50 nm to 300 nm. The size and shape of the DNA nanogel can be precisely controlled through the molar ratio between nanostructures and the length of the viscous end sequence.

The drug loaded on the nanogel is released in a pH- and temperature-dependent manner under conditions of pH 5.0 to 7.4 and 25 to 50 °C. That is, under the above conditions, a high concentration of H+ competes with the cationic drug inserted into the anionic DNA nanostructure to weaken the binding interaction, resulting in drug release. According to one embodiment of the present invention, the lower the pH, the higher the drug release, and the higher the temperature, the higher the drug release. In addition, the nucleic acid drug mounted on the linking unit can be released in a temperature-dependent manner by decomposition of the nanogel after light irradiation.

Accordingly, the present invention also provides the temperature-sensitive DNA nanogel; and

Provided is a drug delivery system comprising a pharmaceutically acceptable carrier.

The temperature-sensitive DNA nanogel of the present invention is manufactured by self-assembly of DNA nanostructures, so that a nucleic acid drug is included in the unit and/or a chemotherapeutic agent such as an anticancer agent is loaded, so that various combinations of a nucleic acid therapeutic agent and a chemotherapeutic agent are used to treat diseases It has the ability to deliver to target cells.

The present invention also relates to an anticancer composition comprising the temperature-sensitive DNA nanogel.

Since the DNA nanogel of the present invention contains gold nanoparticles, it can induce the death of cancer cells through heat generated during light irradiation, and thus can be used for photothermal therapy.

In addition, the DNA nanogel of the present invention can be loaded with a nucleic acid drug or a chemical anticancer agent, so that complex cancer treatment is possible through various combinations thereof.

The types of the nucleic acid drug and the anticancer agent are as described above.

The composition of the present invention may further include a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers that can be used in the drug delivery system or pharmaceutical composition of the present invention include carriers and vehicles commonly used in the pharmaceutical field, and specifically, ion exchange resins, alumina, aluminum stearate, lecithin, serum proteins (eg , human serum albumin); calcium hydride, sodium chloride and zinc salts), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic matrix, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene glycol or wool paper, etc. not limited

In addition, the composition of the present invention may further include a lubricant, a wetting agent, an emulsifier, a suspending agent, or a preservative in addition to the above components.

The composition of the present invention may be administered orally, rectal, transdermally, intravenously, intramuscularly, intraperitoneally, intramedullary, intrathecally or subcutaneously.

Formulations for oral administration may be, but are not limited to, tablets, pills, soft or hard capsules, granules, powders, solutions, or emulsions. Formulations for parenteral administration may be injections, drops, lotions, ointments, gels, creams, suspensions, emulsions, suppositories, patches, or sprays, but is not limited thereto.

The composition of the present invention may contain additives such as diluents, excipients, lubricants, binders, disintegrants, buffers, dispersants, surfactants, colorants, flavorings or sweeteners, if necessary. The pharmaceutical composition according to one aspect of the present invention may be prepared by a conventional method in the art.

The active ingredient of the composition of the present invention will vary depending on the age, sex, weight, pathological condition and severity of the subject to be administered, the route of administration, or the judgment of the prescriber. Determination of the dosage based on these factors is within the level of the skilled artisan, and its daily dose is, for example, from 1 ng/kg/day to 10 mg/kg/day, specifically from 10 ng/kg/day to 1 mg/kg. / day, more specifically 0.1 μg/kg/day to 100 μg/kg/day, even more specifically 0.2 μg/kg/day to 20 μg/kg/day, but is not limited thereto. The composition of the present invention may be administered 1 to 3 times a day, but is not limited thereto.

The present invention also relates to a method of treating cancer comprising administering to a subject in need thereof an effective amount of said temperature-sensitive DNA nanogel.

The subject may be a human or non-human animal, for example, a non-human animal such as a cow, a monkey, a bird, a cat, a mouse, a rat, a hamster, a pig, a dog, a rabbit, a sheep, a horse.

In the treatment method of the present invention, the formulation and administration method of the composition are the same as described above.

Hereinafter, the present invention will be described in more detail through Examples according to the present invention, but the scope of the present invention is not limited by the Examples presented below.

<Example 1> Preparation of temperature-sensitive DNA nanogel

All DNA oligonucleotides used in the preparation of the temperature-sensitive DNA nanogels of the present invention were purchased from Integrated DNA Technologies (Coralville, Ionia, USA). Lyophilized DNA was resuspended in nuclease-free water and quantified prior to use in experiments. Doxorubicin (DOX) and 5 nm gold nanoparticles (AuNP) were purchased from Sigma Aldrich (St Louis, MO, USA). Magnesium chloride hexahydrate and sodium chloride were purchased from Deoksan Chemical (Ansan, Gyeonggi). Dulbecco's modified Eagle's medium (DMEM), phosphate buffered saline (PBS), fetal bovine serum (FBS) and penicillin-streptomycin solution (P/S) were purchased from Corning, Inc (Armonk, NY, USA). Bovine serum (BCS) and Hoechst-33342 were purchased from Thermo Fisher Scientific (Waltham, MA, USA).

(Synthesis of DNA nanogel)

For multifunctional branched DNA unit (MBU) assembly, 100 μl of four single-stranded DNAs (ssDNA, X1, X2, X3 and X4) at equal molar concentrations were mixed with MgCl ₂ (50 mM) and nuclease-free water. to obtain the final volume of The salt concentration and reaction temperature were optimized for MBU synthesis. using a thermocycler (Bio-Rad Laboratories) [SH Um et al. Nat Protoc 2006, 1, 995] as previously used assembly protocol (Roh et al, 2010, see non-patent literature [6]). The mixed DNA solution was denatured by heating to 95°C, annealed at 65°C, annealed at 60°C, and finally stored at 4°C for subsequent experiments.

For the assembly of BUs-LU, the synthesized BU and the calculated stoichiometric content of the DNA ligation unit (LU) were mixed in a tube to obtain a final volume of 100 μl. The LUs solution was heated at 60° C. for 2 min before addition to the solution. The mixed solution was then cooled to 25° C. over 3 hours. The synthesized BUs-LU was stored at 4°C for use in subsequent experiments.

DNA was adsorbed to AuNPs in a bis(p-sulfonatophenyl)phenylphosphine dihydrate dipotassium salt solution using a modified polyA sequence with adhesive ends [H Pei et al. J Am Chem Soc 2012, 134, 11876]. Adhesive ends were integrated into the 5'- or 3'-ends of the polyA sequence to synthesize 5'-polyA and 3'-polyA strands, respectively. To prepare ssDNA-AuNP units (AUs), AuNP solution (20 µL, 94 µM) was added to 5'-polyA or 3'-polyA DNA solution (480 µL, 2 µM) and without light exposure at 25 °C for 16 h. incubated. Then sodium chloride solution (10 μl, 5 M) was added to give a final concentration of 100 nM; Additional incubation was then followed for 48 hours. After incubation, sodium chloride solution was added back to the solution to a final concentration of 2M. The final solution was centrifuged at 16,000xg for 5 minutes and the pellet was washed twice with nuclease-free water to remove unreacted free DNA and AuNPs.

For the assembly of temperature-sensitive DNA-AuNP nanogels (Dgels), the calculated stoichiometric contents of MBU, 5'-AU, 3'-AU and LU synthesized in advance were mixed in a tube to obtain a final volume of 100 μl. The LU solution was heated at 60° C. for 2 minutes prior to addition to the mixture. The mixed solution was cooled to room temperature over 3 hours. The synthesized Dgels were stored at 4°C for subsequent experiments.

(characterization of nanostructures)

The polyA sequence adsorbed to the surface of AuNPs was quantified by dissociation-based fluorescence analysis using a multiplate reader (Victor X5, Perkin Elmer, Waltham, MA, USA). The polyA sequence adsorbed to AU was dissociated from AuNPs by adding KCN solution (1M) to obtain a final concentration of 20 mM. Then, SYBR Green I fluorescent dye for DNA was added to the solution to quantify the free polyA sequence released from the AU. Standard curves were obtained by plotting the fluorescence of free DNA in nuclease-free water at concentrations of 50, 25, 125, 625 and 313 nM.

(Analysis of morphology and structure of DNA nanogels)

High-resolution digital images of BUs-LU and Dgel were obtained for morphological characterization in a field emission scanning electron microscope (SEM; JEOL-7610F; JEOL, Tokyo, Japan). For SEM observation, a drop (10 μl) of the sample solution was placed on a silicon wafer and air-dried at room temperature. Then, the surface of the nanogel was irradiated at a voltage of 5 kV. High-resolution images of Dgels were obtained for morphological characterization at an accelerating voltage of 200 kV in a transmission electron microscope (TEM; JEM-F200; JEOL). For TEM observation, Dgel samples were diluted 10-fold and droplets (10 μl) of the diluted samples were air-dried at room temperature on a carbon/formbar coated TEM grid (200 mesh; Electron Microscopy Sciences, Hatfield, PA, USA).

(Physicochemical properties of temperature-sensitive DNA nanogels)

The size distribution and zeta (ζ) potential of BUs-LU and Dgels were measured at 25°C using a particle size and ζ-potential analyzer (ELS-2000ZS; Otsuka Electronics Co, Osaka, Japan). For measurement, all nanogel samples were dispersed in nuclease-free water. Data were recorded in triplicate to obtain mean±SD values.

(Measurement of melting temperature of DNA nanogel)

The melting temperature (Tm) of BUs-LU was determined by fluorescence analysis using SYBR Green I dye. To determine the Tm value of the adhesive end between BU and LU, SYBR Green I dye (05 μl, 50x) was added to the BUs-LU solution (50 μl, 5 μM) and incubated for 1 hour at room temperature. After incubation, the fluorescence signal was measured from 10°C to 95°C in 0.5°C increments through a real-time PCR detection system (CFX96; Bio-Rad Laboratories Inc, Hercules, CA, USA). The Tm of BUs-LU was determined using the 50% decrease in fluorescence intensity in the graph. The SYBR Green I dye used in this experiment was inserted together with base pairs in the hybridized region of double-stranded DNA. A decrease in fluorescence intensity indicates separation of double-stranded DNA hybridized to ssDNA.

(Thermal Reactivity and Photothermal Characteristics of DNA Nanogels)

To characterize heat-induced conformational changes, BUs-LUs solutions (200 μL, 8 μM) with various adhesive end lengths were prepared using circular dichroism (J-815; JASCO, Mary's Court Easton, MD, USA) analyzed. Structural changes of BUs-LU incubated at 20, 60, and 90 °C for 10 minutes were observed. To investigate the photothermal properties of Dgels, an illumination lamp with a wavelength of 365 nm (Innocure 5000, Richchen, Gyeonggi-do, Korea), a thermal observation device (CompactXR; Seek Thermal Inc, CA, USA) and a thermometer were employed. The temperature of the Dgel solution was measured in real time using a thermal observation device and a thermometer after light irradiation for 0-4 minutes.

(Drug loading of DNA nanogels)

Dgel solution (0-400 nM) was incubated with DOX (5 μM; Sigma Aldrich) dispersed in PBS for 2 hours at room temperature to obtain a final volume of 100 μl. The solution was centrifuged at 16,000×g for 10 min to precipitate Dgel/DOX. Unbound free DOX in the supernatant was then quantified by measuring the mean fluorescence intensity at 595 nm (λex = 480 nm) using a multiplate reader (Victor X5). The amount of DOX loaded on the Dgel was calculated by subtracting the amount of DOX in the supernatant from the amount of added DOX. Nucleic acid therapeutics (Bcl-2 AS-ODN) were loaded onto the Dgel by incorporating the AS-ODN sequence into the LU.

Initial Dgel concentration = 400 nM

Initial DOX concentration = 5000 nM

DOX concentration of supernatant after centrifugation = 532 nM

(In vitro release of drug loaded on DNA nanogel)

The in vitro release of DOX from Dgel/DOX was analyzed under various pH conditions. To investigate the response of Dgel/DOX to pH, samples were dispersed in PBS (500 μl) at various pH values (5.0, 6.0 and 7.4) and incubated at 37°C. The Dgel/DOX solution was centrifuged at 16,000xg at regular time intervals for 5 minutes, and the supernatant was separated and transferred to the wells of a 96-well plate. Thereafter, the supernatant was replaced by adding a similar volume of fresh PBS each time. The amount of DOX in the supernatant was quantified by measuring the average fluorescence intensity at 595 nm (λex = 480 nm) using a multi-plate reader (Victor X5).

(cell culture)

SKOV3 ovarian cancer cells (Cell Biolabs Inc, San Diego, CA, USA) were cultured at 37° C. in a humidified atmosphere containing 5% CO ₂ in DMEM containing 10% FBS and 1% P/S. NIH/3T3 fibroblasts (Korean Cell Line Bank, Seoul, Korea) were cultured at 37° C. under a similar atmosphere in DMEM containing 10% BCS and 1% P/S. SKOV3 and NIH/3T3 cells used in this experiment were plated on T-flasks and cultured to 80% confluency before use.

(Intracellular delivery of DNA nanogels)

To investigate the selective intracellular delivery of DOX-loaded BUs-LU by flow cytometry, SKOV3 and NIH/3T3 cells (8Х10 ⁴ cells per well) were seeded in 6-well plates and incubated at 37°C for 24 hours. Then, the culture medium was removed and the cells were treated with DOX-labeled BU or BUs-LU (300 μl, 100 nM) in serum-free medium for 3 hours. Cells were then treated with diluted trypsin-EDTA (300 μl, 0.25×) for 2 min. Finally, cell solutions were collected in tubes for flow cytometry using LSRII (Becton Dickinson, Franklin Lakes, NJ, USA). SKOV3 and NIH/3T3 cells (8Х10 ⁴ cells per well) were seeded on cell culture slides and incubated at 37° C. for 24 h for measurement by confocal laser scanning microscopy. The culture medium was then removed and the cells were treated with DOX-loaded BU, BUs-LU or Dgel (300 μl, 100 nM) in serum-free medium for 2 hours. Thereafter, the cells were washed with PBS and fixed with 4% formaldehyde at room temperature for 15 minutes. Then, the cells were washed with PBS and the cell nuclei were stained with Hoechst-33342 at room temperature for 15 minutes. Finally, cells were washed twice with PBS, mounted and scanned (LSM 700; Carl Zeiss, Thornwood, NY, USA).

(in vitro cytotoxicity assay)

The cytotoxicity of Dgels and Dgel/DOX was assessed using the MTT assay. Briefly, SKOV3 cells (6Х10 ³ cells per well) were seeded into each well of a 96-well plate and incubated at 37°C for 24 h. Cells were sequentially treated with Dgel or Dgel/DOX at different AS-ODN concentrations (400, 200, 100 and 50 nM) in serum-free medium for 3 hours upon light irradiation. Cells were then washed with PBS and further incubated for 48 hours in fresh medium containing 10% FBS. Then, MTT solution and DMSO were added according to the manufacturer's instructions. Finally, the absorbance of each well was measured at a wavelength of 490 nm using a multi-plate reader (Victor X5). To investigate the cytotoxicity of Dgels and Dgel/DOX under light irradiation, cells were irradiated for 3 hours after treatment with Dgels or Dgel/DOX at various concentrations (400, 200, 100 and 50 nM) in serum-free medium. Relative cell viability was calculated by comparing the cell viability of SKOV3 cells treated with Dgel loaded with Bcl-2 AS-ODN with the viability of cells treated with Dgel loaded with scrambled sequences.

(statistical analysis)

All experimental data were recorded in triplicate to express mean±SD values. Statistical analysis of the data was performed using Scheff's test of SPSS software package version 240 (Armonk, NY, USA) with one-way ANOVA. Results are statistically expressed as *p < 005, **p < 001 or ***p < 0005.

<Experimental Example 1> Synthesis of temperature-sensitive DNA nanogel

As shown in FIG. 1, smart DNA nanogels (Dgels) were synthesized for targeted co-delivery of cancer therapeutic agents and stimulatory-responsive release of loaded drugs.

Dgels were composed of three types of functionalized DNA nanostructures: a multifunctional branched DNA unit (BU), a photosensitive single-stranded DNA (ssDNA)-AuNP unit (AU), and an ssDNA linker unit (LU). . In particular, branched X-type DNA (X-DNA) nanostructures served as structural backbones for decoration with functional moieties. AuNPs adsorbed with oligonucleotides were adopted to enable temperature rise by light irradiation. The linking unit is designed to incorporate a therapeutic oligonucleotide and sequence-dependently programmed degradation. Morphological and physicochemical analyzes of self-assembled Dgels were performed to demonstrate successful synthesis and controllable thermal reactivity. Thereafter, intracellular delivery of Dgels for co-delivery of anticancer drugs to target cells, anticancer drug efficacy and cell viability were analyzed for potential therapeutic applications.

This Dgel has the following characteristics. 1) With regard to structural tunability, the nanogel precisely controlled physicochemical properties by adjusting the length of the sequence at the adhesive end and the molar ratio of the nanostructure. 2) Combination therapeutics including nucleic acid drugs were adopted for stimuli-responsive drug release through dual triggers for application in cancer treatment.

First, the synthesis of three functional nanostructures of Dgel was confirmed. BU was assembled into four ssDNA oligonucleotide sequences through complementary hybridization, and the ends of the X-shaped branches of BU were anisotropically functionalized with nucleic acid aptamers for cancer-specific targeting and adhesive ends for supramolecular assembly (Table 1). . The programmed BU assembly was confirmed through DNA band shift during gel electrophoresis (Fig. 2A). The BU band (lane 5) migrated slower than the ssDNA band (lane 2) and other partially hybridized units (lanes 3 and 4), demonstrating successful assembly of the designed complementary sequence in high yield. Second, AU was fabricated through sequence-specific adsorption of a polyA tail with an integrated adhesive end to AuNPs for light-induced temperature increase and degradation of Dgel. PolyA was strongly adsorbed on the Au surface, and the adsorption potential was similar to the Au–S binding affinity. In addition, the DNA density on the surface of AuNPs can be controlled simply by adjusting the length of the polyA tail. Since the smaller size AuNPs are less toxic, 5 nm AuNPs were adopted for AU assembly. 5'- and 3'-AU were prepared for hybridization to 5' and 3' LU adhesive ends, respectively. Successful AU synthesis was confirmed through gel electrophoresis (Fig. 2B). Compared to the bands of naked AuNPs (lanes 1), the bands of AU (lanes 2 and 3) showed slow migration due to the adsorption of polyA-containing ssDNA. As a result of surface density quantification, it was found that about 3.27 ± 0.15 and 3.60 ± 0.14 DNA strands were adsorbed to the 5'-AU and 3'-AU surfaces, respectively (FIG. 2C).

3A is an electrophoresis result of synthesizing BUs that directly binds to AUs to synthesize a DNA nanogel composed of AUs and BUs without a linking unit. BUs with one aptamer sequence and three adhesive ends capable of binding to AUs were synthesized using four single-stranded DNAs. When nanogels were synthesized using the corresponding BUs by complementary bonding with AUs, nanogels were gradually formed as the ratio of AUs to BUs increased, and was best formed at a ratio of 1.6:1 (FIG. 3B). When the temperature sensitivity of the formed nanogel was confirmed with a transmission electron microscope, it was confirmed that the formed nanogel was decomposed to a single particle level after light irradiation (FIG. 3C).

Finally, the LU constructed ssDNA containing adhesive ends and nucleic acid therapeutic regions. The lengths of the adhesive ends connecting different nanostructures to form a complete Dgel were designed to be 10, 15 and 20 bp for precise control of thermal reactivity (Table 2). These three types of nanostructures are assembled in a sequence-specific manner to form multifunctional DNA nanogels. BU and AU promoted aptamer-based cell targeting and light-induced temperature increase through photothermal effects. In addition, LU is connected between BU and AU with adhesive ends, facilitating controllable degradation of nanogels through temperature rise. After forming a supramolecular structure through self-assembly, the function of each nanostructure was confirmed.

First, the thermally reactive decomposition of the supramolecular structure was analyzed according to the length of the adhesive end of the LU. The melting curve of hybridized BUs-LU (ratio 1:2) was analyzed via real-time PCR (Fig. 4A). The melting temperatures (Tm) of hybridized BUs-LUs using nanostructures with adhesive end lengths of 10, 15, and 20 bp were 45.0 ± 0.3, 54.2 ± 1.1 and 58.2 ± 0.9 °C, respectively. Circular dichroism (CD) of BUs-LU with 15 bp adhesive ends showed that the ellipticity of the spectrum decreased with increasing temperature, showing a structural change in supramolecular assembly (Fig. 4B and Fig. 5). Consistent with the real-time PCR results, BUs-LUs with adhesive ends of 10 bp showed a faster decrease in ellipticity, whereas BUs-LUs with adhesive ends of 20 bp were relatively resistant to heat, resulting in ellipticity only at 90 °C. rate change was observed. In particular, at high temperatures, BU is expected to remain partially hybridized, preventing a decrease in ellipticity similar to that of the ssDNA control. These results indicate that supramolecular degradation can be precisely controlled by designing appropriate hybridization interactions between DNA sequences. Consequently, 15 bp adhesive ends were chosen for further application at physiological temperatures.

To confirm the targeting potential and cancer-specific cellular uptake of the BUs-LU complex, BU was decorated with an AS1411 aptamer that selectively binds to cancer cells overexpressing nucleolins. Prior to cell delivery of BUs-LU, target site expression levels were confirmed by flow cytometry using FAM-labeled AS1411 aptamer (FIG. 6). SKOV3 cells showed significant changes in FAM fluorescence compared to NIH/3T3 cells, indicating nucleolin upregulation. Aptamer-mediated delivery of the BUs-LUs complex was analyzed using confocal laser scanning microscopy (CLSM) and flow cytometry after loading with doxorubicin (DOX), a representative anticancer agent inserted into the DNA duplex. Significantly increased red fluorescence intensity indicative of the presence of DOX was observed in SKOV3 ovarian cancer cells (nucleolin positive) but not in NIH/3T3 normal fibroblasts (nucleolin negative) as revealed by flow cytometry (Fig. 4C). CLSM further revealed that BUs-LUs complexes were more efficiently delivered to SKOV3 cells than BUs, indicating that the particle size of BUs-LUs complexes was more suitable for cell delivery (Fig. 4D). Conversely, BU and BUs-LU were hardly delivered to NIH/3T3 cells. The uptake behavior of BUs-LU was analyzed at various incubation periods and BUs-LU was successfully delivered after 1.5 h (Fig. 7). These results demonstrated that nanostructures and their supramolecular structures were selectively delivered to target cancer cells through the cellular uptake mechanism of AS1411 aptamer. The physicochemical properties of the synthesized Dgels were also analyzed. Prior to AU integration, BUs-LUs complexes were prepared to identify the optimal rate of supramolecular structure assembly. The hydrodynamic diameters obtained using the BUs:LUs molar ratios of 2:1, 1:1, 1:2 and 1:4 were 258.8 ± 57.5, 219.8 ± 59.5, 196.9 ± 33.4 and 287.9 ± 13.9 nm, respectively (Fig. 8). Scanning electron micrographs (SEM) showed well-dispersed and uniform spherical nanoparticles for all molar ratios of sizes 217.2 ± 44.8, 216.1 ± 65.7, 162.4 ± 35.9 and 252.8 ± 42.1 nm, consistent with the dynamic light scattering results (Fig. 9). . The BUs-LUs ratio of 1:2 was chosen for further biological applications as the relevant nanogel size adequately supports efficient intracellular delivery and extended in vivo blood circulation. In addition, gel electrophoresis analysis to confirm the synthesis of the BUs-LU complex showed that the band of BUs-LU moved slower than the bands of BU and LU and showed more smearing, indicating successful assembly (FIG. 10).

Then, Dgel was synthesized by adding AU to the BUs-LU complex at a fixed ratio (BU:LU:AU molar ratio 1:2:0.25). The Dgel absorption spectrum showed that the plasmon resonance peak shifted to a longer wavelength compared to that of AuNPs alone (Fig. 11A). SEM images showed monodisperse and spherical Dgel morphology (Fig. 11B). The average Dgel size was 206.2 ± 58.6 nm, which was similar to that of BUs-LUs synthesized with the same BUs:LUs ratio (Fig. 11C). The zeta (ζ) potential of Dgels (-17.9 ± 0.7 mV) was slightly higher than that of BUs-LU (-11.5 ± 0.2 mV) due to the addition of negatively charged ssDNA-AuNPs (AU). These results show that the physicochemical properties of Dgel did not change significantly when AU was added by successfully integrating AU into Dgel through sequence-assisted self-assembly.

Dgels are designed to be dissociated by a light trigger. When the Dgels were exposed to light, the AuNPs contained in the Dgels were heated through the photothermal effect. The generated heat was then transferred to adjacent DNA nanostructures. When the temperature reaches a specified threshold, the complementary linkage is disrupted and supramolecular assembly ceases. In order to confirm the temperature response by light irradiation, the photothermal properties of the Dgel were monitored using a thermal observation device and a thermometer (FIG. 12). After 2 minutes of light irradiation, the temperature of the Dgel solution increased by 12°C. The AuNP distribution of the Dgels was monitored via transmission electron microscopy (TEM) to confirm the light-induced degradation of the Dgels (Fig. 11D). In the TEM image of Dgels before light irradiation, AuNPs are adjacent to the range of about 100 nm. After 2 min of light irradiation, most of the AuNPs are well dispersed, indicating the degradation of the Dgel. These results show successful light-induced Dgel degradation, which is closely related to the release mechanism of the loaded drug.

The applicability of Dgel for complex drug loading and stimulus-response drug release was investigated by incorporating antisense oligonucleotides (AS-ODN) and chemical drugs (DOX). As a proof-of-concept, AS-ODN was selected, which targets an anti-Bcl-2 (apoptotic B-cell lymphoma 2) family protein that is frequently overexpressed in cancer cells. Bcl-2 protein is implicated in chemotherapy resistance, which is involved in the regulation of DOX and apoptosis. Bcl-2 AS-ODN was incorporated into the LU and DOX was inserted into the DNA duplex. The loading efficiency of AS-ODN was about 100%, as little LU was observed in gel electrophoresis. The loading efficiency of DOX was 89.4%. The amount of DOX in Dgels was quantified as 12.5 equivalents per AS-ODN in Dgels. Stimuli-responsive release of drug loaded in Dgels at different pH and temperature was confirmed by monitoring the amount of released DOX. The DOX release behavior of Dgel / DOX in the endosomal environment was evaluated under various pH conditions (5.0, 6.0 and 7.4) (FIG. 13A). Cumulative DOX release from Dgels increased in a pH-dependent manner: 32.3, 38.7 and 41.1% at pH 7.4, 6.0 and 5.0, respectively, within 4 h. High concentrations of H+ compete with cationic DOX incorporated into anionic DNA nanostructures, weakening binding interactions and leading to DOX release. The thermally responsive release of DOX was evaluated at different temperatures (25, 37 and 50° C.) ( FIG. 13B ). DOX was released more rapidly with increasing temperature, about 57% was released after 4 hours of incubation. In particular, although DOX was not completely released by pH and temperature triggers, the unreleased DOX was readily accessible through enzymatic degradation under physiological conditions. These results suggest that DOX can be released from cells by pH and temperature triggers. In addition, Bcl-2 was expected to be released in a temperature-dependent manner at 40 °C by degradation of the supramolecular structure, facilitating light-triggered release of oligonucleotides on demand (Fig. 14).

Potential drug delivery applications were determined by validating the intracellular delivery, anticancer drug efficacy, and cell viability of Dgels. To analyze aptamer-mediated intracellular delivery and endosomal escape of Dgel to confirm proper function in target cells, SKOV3 cells were treated with Dgel and cultured for an additional 6 hours, and the internalized Dgel was subjected to differential interference contrast (DIC). visualized. Dgels with aptamer function were observed in SKOV3 cells, whereas Dgels without aptamer were hardly observed in SKOV3 cells (FIG. 15). In addition, Dgels did not deliver to NIH/3T3 cells, indicating selective delivery to cancer cells ( FIG. 16A ). In addition, pH-induced formation of AU clusters inside the cells was observed. PolyA sequence integrated into AU causes the formation of a pH-dependent AU cluster by forming a parallel duplex in the acidic environment of endosomes, which is expected to induce endosomal escape by gradual AU cluster growth. In particular, AU clustering was not observed immediately after treatment of SKOV3 cells with Dgel, and clusters were gradually formed after 3 hours of incubation, and mostly disappeared after 48 hours. However, clusters were hardly formed in NIH/3T3 cells or SKOV3 cells when Dgel was treated without an aptamer even after 6 hours of incubation ( FIG. 17 ).

To evaluate the therapeutic efficacy of the loaded drug, the viability of SKOV3 cells incubated with Dgel at different concentrations was analyzed. Cells treated with Dgel/Bcl-2 without DOX after light irradiation demonstrated increased therapeutic efficacy in a concentration-dependent manner. The relative cell viability was 44.9% at an effective Bcl-2 AS-ODN concentration of 1 μM, which was induced by Bcl-2 regulated apoptosis ( FIG. 18 ). The effect of simultaneous delivery of Bcl-2 AS-ODN and DOX using Dgels, ie, Dgel/Bcl-2/DOX, was subsequently investigated. Treatment of SKOV3 cells with Dgel loaded with DOX without Bcl-2 AS-ODN or light irradiation did not alter cell proliferation ( FIG. 16B ). In particular, Dgel/Bcl-2/DOX treatment significantly reduced the relative cell viability to 51.4% after light irradiation with 400 nM Bcl-2 AS-ODN, potentially indicating the efficacy of the combination treatment of the Dgel/Bcl-2/DOX system. has been proven. Through light-induced Dgel degradation, AS-ODN and DOX inside cells were successfully released, and co-drugs loaded on Dgel significantly reduced cell viability as the drug-free gel showed negligible toxicity to SKOV3 cells. . These results demonstrate the combined therapeutic efficacy of Dgel containing AS-ODN and DOX against target cancer cells.

In conclusion, the present invention provides an intelligent DNA-based nanogel formed from functionalized nanostructures. By integrating appropriate sequences and multiple functions, this platform can selectively deliver drugs to target cancer cells and precisely control the release of loaded drugs through temperature-induced degradation. The use of this DNA nanogel platform therefore offers a promising strategy for precision medicine and cancer treatment by employing diverse combinations of nucleic acid and chemotherapeutic agents.

The present invention can be applied to the field of drug delivery or anticancer.

Claims (13)

1. branched DNA units, each arm having an adhesive end;

   a photosensitive unit comprising gold nanoparticles and single-stranded DNA having an adhesive end; and

   A temperature-sensitive DNA nanogel formed by self-assembly of linking units with adhesive ends.
2. The method of claim 1,

   The adhesive end consists of a nucleotide sequence of 5 to 30 bp, temperature-sensitive DNA nanogel.
3. According to claim 1,

   wherein the branched DNA unit is selected from X-type DNA, Y-type DNA and T-type DNA.
4. According to claim 1,

   wherein the branched DNA unit further comprises a targeting nucleic acid component.
5. According to claim 1,

   The photosensitive unit is a temperature-sensitive DNA nanogel formed by adsorbing single-stranded DNA including an adhesive end and a polyA tail to gold nanoparticles.
6. According to claim 1,

   Gold nanoparticles are in the form of a spherical shape having a diameter of 5 to 100 nm or a rod having a length of 5 to 70 nm, a temperature-sensitive DNA nanogel.
7. According to claim 1,

   The temperature-sensitive DNA nanogel, wherein the molar ratio of the branched DNA unit and the linking unit is 2:1 to 1:4.
8. The method of claim 1,

   The linking unit further comprises a nucleic acid drug, temperature-sensitive DNA nanogel.
9. 9. The method of claim 8,

   Nucleic acid drugs are DNA (deoxyribonucleic acid), RNA (ribonucleic acid), PNA (peptide nucleic acid), LNA (morpholino and locked nucleic acid), GNA (glycol nucleic acid), oligonucleotides, plasmid DNA , antisense oligonucleotide, messenger RNA, microRNA, locked nucleic acid, DNA-based enzyme, small interfering RNA, short hairpin RNA (short hairpin RNA), RNA-based enzyme (RNAzyme), and at least one selected from the group consisting of nucleic acid aptamer (aptamer), temperature-sensitive DNA nanogel.
10. The method of claim 1,

    Temperature-sensitive DNA nanogel is a metabolic antagonist, plant alkaloid, topoisomerase inhibitor, alkylating agent, anticancer antibiotic and one or more anticancer agents selected from the group consisting of hormones, temperature-sensitive DNA nanogels that can be loaded.
11. The temperature-sensitive DNA nanogel of any one of claims 1 to 10; and

    A drug delivery system comprising a pharmaceutically acceptable carrier.
12. An anticancer composition comprising the temperature-sensitive DNA nanogel of any one of claims 1 to 10.
13. A method for treating cancer comprising administering to a subject in need thereof an effective amount of the temperature-sensitive DNA nanogel of any one of claims 1 to 10.

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