CN116059356A - DNA nano-enzyme with self-oxygen production capability and preparation method and application thereof - Google Patents
DNA nano-enzyme with self-oxygen production capability and preparation method and application thereof Download PDFInfo
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- CN116059356A CN116059356A CN202210990962.0A CN202210990962A CN116059356A CN 116059356 A CN116059356 A CN 116059356A CN 202210990962 A CN202210990962 A CN 202210990962A CN 116059356 A CN116059356 A CN 116059356A
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- photosensitizer
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Abstract
The invention provides a DNA nano enzyme with self-oxygen generating capacity, a preparation method and application thereof, wherein the DNA nano enzyme comprises a DNA nano flower, heme coenzyme and a photosensitizer Ce6, wherein the DNA nano flower is formed by rolling circle amplification of a primer chain and a substrate chain, the DNA nano flower forms an AS1411G quadruplex space structure in a high-potassium Tris buffer solution, the heme coenzyme is embedded into the AS1411G quadruplex structure, and the photosensitizer Ce6 is stacked in the AS1411G quadruplex structure through pi-pi action to form the DNA nano enzyme with the self-oxygen generating capacity; the DNA nano enzyme is efficiently and stably loaded with heme coenzyme and photosensitizer Ce6, and is enriched in tumor tissues through EPR effect, wherein AS1411G quadruplex recognizes tumor cells with high nucleolin expression to realize targeting positioning; under the environment of high hydrogen peroxide in a tumor, heme coenzyme efficiently catalyzes and produces oxygen, and obviously relieves the condition of hypoxia in the microenvironment of the tumor; meanwhile, the photosensitizer can convert the generated oxygen into Reactive Oxygen Species (ROS) with tumor cytotoxicity, and further kill tumor cells.
Description
Technical Field
The invention relates to the field of nano materials and nano biological medicines, in particular to a DNA nano enzyme with self-oxygen generating capacity, a preparation method and application thereof.
Background
Cancer is a significant problem affecting human life health and social development. Chemotherapy is the primary means of treating cancer clinically, but the heterogeneity of tumor microenvironment and single chemotherapy cause low efficacy, serious adverse reactions, and development of tumor resistance. Because photodynamic therapy (photodynamic therapy, PDT) has the advantages of good controllability, low toxicity, minimal invasiveness and the like, adverse reactions and swelling drug resistance induced by high doses are avoided.
However, PDT presents some obstacles in practical applications. Due to abnormal proliferation of tumor cells, lack of vascular structures and insufficient blood flow of tumor tissues, the tumor microenvironment often shows the characteristics of hypoxia, subacidity, over-expressed hydrogen peroxide, cytokines and the like. Tumor hypoxia upregulates the expression level of tumor hypoxia inducible factor-1 alpha (HIF-1 alpha), resulting in tumor cell resistance to therapies such as chemotherapy, PDT, etc. The photosensitizers convert oxygen into cytotoxic Reactive Oxygen Species (ROS) for photodynamic therapy, thereby exacerbating tumor hypoxia and severely affecting ROS production efficiency, ultimately leading to low PDT efficacy and poor prognosis. In addition, ROS cause cellular organelle dysfunction and damage by binding to biological macromolecules such as DNA, lipids, etc., ultimately leading to tumor cell death. Because of the short half-life (< 200 ns) and the small diffusion range (20 nm) of ROS, ROS localized to organelles are more tumor killing than in the cell membrane or cytoplasm, thus ensuring efficient delivery of photosensitizers is an important condition for achieving photodynamic therapy.
To address tumor hypoxia, a variety of oxygenation strategies have been developed to increase the oxygen levels of tumor tissue. By delivering oxygen carrying materials such as perfluorocarbon, hemoglobin or erythrocyte membranes, the tumor hypoxia can be directly improved. However, these oxygen-delivering materials often suffer from the disadvantages of poor oxygen carrying capacity, poor stability, such as short half-life of hemoglobin, etc., resulting in low oxygen delivery efficiency of the formulation. And the self-oxygenation strategy responding to the tumor microenvironment can just solve the problems, and target delivery of the peroxyEnzyme and nanomaterial with catalytic activity such as hydrogenase, manganese dioxide nanoparticle, tablet and Prussian blue nanoparticle, and can decompose H overexpressed in tumor cells 2 O 2 Up-regulating intracellular oxygen level, so as to raise photodynamic therapeutic effect, and its therapeutic effect is obvious in tumor hypoxia treatment. However, the above formulations have application limitations: (1) Catalase is inactivated to varying degrees in vivo; (2) Uncontrollable composition and biosafety of the nanomaterial. Therefore, the safe and efficient delivery carrier is constructed, the tumor hypoxia problem is solved while the targeting delivery of the photosensitizer is realized, and the problem to be solved in the field of photodynamic therapy research is realized.
Disclosure of Invention
In order to solve the technical problems, the invention provides a DNA nano enzyme with self-oxygen generating capacity, and a preparation method and application thereof, and aims to construct a safe and efficient delivery carrier, efficiently combine a heme coenzyme and a photosensitizer into a nano material, realize in vivo stable targeting of tumor tissues, effectively release and cooperate at tumor cells, improve the micro-environmental hypoxia condition of tumors and improve the photodynamic treatment effect of tumors.
In order to achieve the above object, the present invention provides a DNA nanoenzyme with self-oxygen generating capability, comprising a DNA nanoflower, heme-co-enzyme and photosensitizer Ce6, wherein the DNA nanoflower is formed by rolling circle amplification of a primer strand and a substrate strand, and the primer strand is shown in SEQ ID No. 1:
GTGGTGGTGTTGGTGGTGGT；
the substrate chain is shown as SEQ ID NO. 2:
CCACCAACACCACCACCACCTTTGACACACTAGCGATACGCGTATCGCTATGGCATATCG TACGATATGCCAGTGTGTCTTTCCACCA；
the DNA nanoflower has an AS1411G quadruplex structure, the heme coenzyme is embedded into the AS1411G quadruplex structure, and the photosensitizer Ce6 is stacked in the AS1411G quadruplex structure through pi-pi action.
Preferably, the shape of the DNA nanoenzyme is spherical, and the particle size is 100-200 nm.
Based on a general inventive concept, the invention also provides a preparation method of the DNA nano-enzyme with the self-oxygen generating capacity, which comprises the following steps:
s1, preparing DNA nanoflower: mixing a substrate chain and a primer chain, carrying out complementary pairing to form circular DNA with a notch, then adding DNA ligase to react to form complete circular DNA, finally introducing DNA polymerase to carry out PCR amplification to form a plurality of DNA long chains with copy chains, and synthesizing DNA nanoflower by the DNA long chains through base pairing and a self-assembly mode;
s2, AS1411G quadruplex structure formation: dissolving the DNA nanoflower in Tris buffer solution containing high potassium ions, and incubating at room temperature to form the DNA nanoflower with AS1411G quadruplex space structure;
s3, assembling to form DNA nano enzyme: mixing the DNA nanoflower with the AS1411G quadruplex space structure prepared in the step S2 with heme coenzyme, incubating for 4-6 hours at room temperature, adding a photosensitizer Ce6, uniformly mixing by vortex, continuing incubating for 3-4 h at room temperature, centrifuging and collecting precipitate to obtain the DNA nano enzyme with self-oxygen production capability.
Preferably, in the step S1, the annealing temperature is 95 ℃ water bath for 10-30 min, and then the temperature is slowly reduced to 25 ℃, and the temperature reduction time is more than 2.5h.
Preferably, the DNA ligase in the step S1 is T4-DNA ligase, and the DNA polymerase is phi29 DNA polymerase.
Preferably, the Tris buffer solution in the step S2 is a mixed solution of Tris-HCl, naCl and KCl, and the pH value is 7-8.
Preferably, in the step S3, the mixing mole ratio of the DNA nanoflower to the heme coenzyme is 1:40-50; the mixing mole ratio of the DNA nanoflower to the photosensitizer Ce6 is 1:500-600.
Use of the DNA nanoenzyme with self-generating capacity according to any one of claims 1 to 2 or the DNA nanoenzyme with self-generating capacity prepared by the preparation method according to any one of claims 3 to 7 in the preparation of anti-tumor photodynamic therapy drugs.
Compared with the prior art, the invention has the following beneficial effects:
1. the DNA nano enzyme provided by the invention is used for efficiently and stably loading heme coenzyme and photosensitizer Ce6, and is enriched in tumor tissues through the EPR effect, wherein AS1411G quadruplex is used for identifying tumor cells with high nucleolin expression so AS to realize targeted positioning; under the environment of high hydrogen peroxide in a tumor, heme coenzyme efficiently catalyzes and produces oxygen, and obviously relieves the condition of hypoxia in the microenvironment of the tumor; meanwhile, the photosensitizer can convert generated oxygen into Reactive Oxygen Species (ROS) with tumor cytotoxicity, so that tumor cells are further killed, and heme coenzyme and the photosensitizer act synergistically to obviously enhance the photodynamic tumor treatment effect;
2. the key structure of the DNA nanoflower in the DNA nano enzyme is AS 1411G-quadruplex, the G-quadruplex is formed by the hydrogen bond connection between four adjacent guanine bases through an oligonucleotide chain rich in guanine (G), wherein heme coenzyme can be embedded into an aromatic plane to form the DNase with catalase activity, and hydrogen peroxide rich in tumor tissues is catalyzed to generate oxygen; meanwhile, the photosensitizer Ce6 can be stacked on an aromatic plane through pi-pi action, so that heme coenzyme and the photosensitizer Ce6 are stably loaded in a DNA nanoflower structure to form DNA nanoenzyme with good stability, and the AS 1411G-quadruplex is utilized for high-specificity recognition of tumors to realize tumor targeted delivery;
3. the DNA nano-enzyme has good enzyme degradation resistance, can realize stable delivery in vivo and target tumor cells, has no obvious side effects on heart, liver, spleen, lung, kidney and the like, and has good biological safety;
4. the preparation method of the DNA nano-enzyme is simple and controllable, and spherical nano-particles with the particle size of 100-200 nm are formed, so that the DNA nano-enzyme has a good application prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of the construction of DNA nanoenzyme of the invention;
FIG. 2 is a schematic diagram of rolling circle amplification in DNA nanoflower synthesis in example 1 of the present invention;
FIG. 3 is a graph of a nanoflower scanning electron microscope formed at different times in the rolling circle reaction in example 1 of the present invention;
FIG. 4 is a representation of the insertion of heme-CoA into DNA nanoflower and entrapment of photosensitizer in accordance with example 1 of the present invention, wherein FIG. 4A is a dynamic light flash image; FIG. 4B is a fluorescent image after photosensitizers are encapsulated; FIG. 4C is a transmission electron microscope image; FIG. 4D is an electron microscope elemental analysis map;
FIG. 5 is a graph showing the catalytic oxygen production and the enhancement of photodynamic effects of photosensitizers by DNA nanoenzymes in example 2 of the present invention, wherein FIG. 5A is a graph showing H under the catalysis of DNA nanoenzymes 2 O 2 Is a graph of consumption conditions; FIG. 5B is a DNA nanoenzyme catalyzed O 2 Is described herein; FIG. 5C is a graph of the free radical generation of photodynamic action catalyzed by DNA nanoenzyme (detected by SOSG kit); FIG. 5D is a diagram of the mechanism of the DNA nanoenzyme to catalyze the production of oxygen to enhance the photodynamic effect; FIG. 5E is a graph demonstrating the oxygen generation performance of a nanoenzyme; FIG. 5F is the protective properties of the nano-enzyme against DNase activity;
FIG. 6 is a graph showing transcellular transport, nuclear targeting, and tumor cell selectivity of the DNA nanoenzyme of example 3 of the present invention, wherein FIG. 6A is a graph showing time-dependent cellular uptake, and from the co-localization results, the nanoenzyme is mainly accumulated in the nucleus, FIG. 6B is a graph showing the result of DNA nanoenzyme uptake by tumor cells, and FIG. 6C is a graph showing the quantitative result of cellular uptake;
FIG. 7 is a graph showing the killing of tumor cells by DNase according to example 3 of the present invention, wherein FIG. 7A is a graph showing the cytotoxicity results of MTT assay characterization; FIG. 7B is a graph showing cytotoxicity results from staining live dead cells; FIG. 7C is a graph showing the results of fluorescent staining for the characterization of intracellular free radical generation;
FIG. 8 is a graph showing the targeted enrichment of DNase in tumor tissue in example 4, wherein free photosensitizer is used as a control to characterize the enrichment of nanoparticles in tumor tissue, and FIG. 8A is an in vitro imaging result and FIG. 8B is a fluorescence quantification result;
FIG. 9 is a graph showing in vivo antitumor activity characterization of DNase according to example 4 of the present invention, wherein FIG. 9A is a graph showing tumor volume monitoring dynamically during treatment, FIG. 9B is a graph showing tumor volume after treatment, and FIG. 9C is a quantitative result of tumor body weight;
FIG. 10 is a graph showing the evaluation of biosafety of DNase in example 4 of the present invention.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved more apparent, the following detailed description will be given with reference to the accompanying drawings and specific embodiments.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated; the reagents used in the examples were all commercially available unless otherwise specified.
In the following examples, details of the instruments and manufacturers used are shown in Table 1:
table 1: main instrument name and manufacturer
| Instrument name | Manufacturing factories |
| CP225 type electronic balance | Sidoris (Sartorius) Germany |
| BP224S type electronic balance | Sidoris (Sartorius) Germany |
| Vortex mixer | Shanghai Qinghai Shanghai analysis instruments works |
| Palm centrifugal machine | Sciloex Co., USA |
| TGL 20M table type high-speed refrigerated centrifuge | CHANGSHA YINGTAI INSTRUMENT Co.,Ltd. |
| Nano ZS90 particle size/potential analyzer | Malvern instruments Co., ltd |
| UV-2600 ultraviolet spectrophotometer | Shimadzu corporation of Japan |
| PHS 3C acidimeter | SHANGHAI INESA SCIENTIFIC INSTRUMENT Co.,Ltd. |
| Infinite M200PRO multifunctional enzyme labeling instrument | Austria TECAN Co |
| Tecnai G2F | |
| 20 type transmission electron microscope | FEI Co Ltd |
| MIN4-UVF water purifier | Hunan Colton Water Co.,Ltd. |
| SHA B water bath constant temperature oscillator | Changzhou Australia instruments Co Ltd |
| Forma Series type II CO 2 Incubator | Thermo Fisher Co., USA |
| IX70 type inverted fluorescence microscope | Olympus Japan Co Ltd |
| SW-CJ-2FD type vertical purification workbench | SUZHOU PURIFICATION EQUIPMENT Co.,Ltd. |
| IVIS Lumina III small animal living body imaging system | Perkinelmer Co., USA |
| Refrigerator at 4 DEG C | Medical refrigerator for Chinese sea |
| -20 ℃ refrigerator | Medical low-temperature preservation refrigerator for Chinese hals |
| Refrigerator at-80 DEG C | Medical low-temperature preservation refrigerator for Chinese hals |
In the following examples, the names of the main reagents used and the manufacturers are shown in Table 2:
table 2: main reagent name and manufacturer
| Reagent name | Production ofManufacturer' s |
| DNA (HPLC purification grade) | SANGON BIOTECH (SHANGHAI) Co.,Ltd. |
| Copper oxide nanoenzyme | Beijing De Kodak gold technology Co.Ltd |
| Nucleoside | Sigma Co., |
| 3,3', 5' -Tetramethylbenzidine (TMB) | Sigma Co., ltd |
| Anhydrous sodium acetate | Sinopharm Group Chemical Reagent Co., Ltd. |
| Glacial acetic acid | Sinopharm Group Chemical Reagent Co., Ltd. |
| 2-amino-2- (hydroxymethyl) -1, 3-propanediol (Tris) | Sinopharm Group Chemical Reagent Co., Ltd. |
| Sodium dihydrogen phosphate dihydrate | Sinopharm Group Chemical Reagent Co., Ltd. |
| Sodium chloride | SHANGHAI MACKLIN BIOCHEMICAL Co.,Ltd. |
| 30% hydrogen peroxide | Xilong Chemical Co., Ltd. |
Example 1
Preparation of DNA nanoflower (DF)
The basic flow chart is shown in fig. 2: the base at both ends of the substrate strand and the primer strand are complementarily paired by a one-step annealing method to form a circular DNA with a notch. Adding T4-DNA ligase to repair the notch on the substrate chain to form a complete circular DNA. Finally, phi29 DNase is introduced to identify a primer chain of the circular DNA, infinite replication is carried out, a DNA long chain with a plurality of copy chains is amplified, and the DNA long chain synthesizes the DNA nanoflower in a DNA base pairing and liquid crystal self-assembly mode. Wherein the primer strand has the sequence of GTGGTGGTGTTGGTGGTGGT, SEQ ID NO.1;
the sequence of the substrate strand is:
CCACCAACACCACCACCACCTTTGACACACTAGCGATACGCGTATCGCTATGGCATATCG TACGATATGCCAGTGTGTCTTTCCACCA，SEQ ID NO.2。
the specific operation is as follows: in 100. Mu. L T4-DNA ligase buffer (5 mM Tris-HCl,1mM MgCl) 2 Primer strand (final concentration 1.2. Mu.M) and substrate strand (final concentration 0.6. Mu.M) were added to 0.1mM ATP,1mM dithiothreitol, and the mixture was stirred and mixed uniformly, then heated in a 95℃water bath for 10min, transferred to a 250mL beaker containing 95℃hot water, and cooled slowly to 25℃for 3h. 2.5 mu L T4 ligase (10U/. Mu.L) was precisely aspirated and vortexed with the DNA annealing product described above and incubated at 25℃for 4h to close the nicks of the circular DNA.
In 100. Mu.L of RCA buffer (50 mM Tris-HCl,10mM (NH) 4 ) 2 SO 4 ，10mM MgCl 2 DNA cyclization product (final concentration 0.3. Mu.M), phi29 DNA polymerase (final concentration 1U/. Mu.L), dNTP (final concentration 2 mM) and BSA were added to 4mM dithiothreitol, mixed well and incubated in a water bath at 30℃for 3h. After the amplification reaction was completed, the reaction was stopped by incubating at 75℃for 10min and inactivating the phi29 polymerase at high temperature. The synthesis of nanoflower was monitored by scanning electron microscopy, and the results are shown in fig. 3, as can be seen from fig. 3: with the extension of the reaction time, the particle size of the nanoflower is larger and larger, but the particle number is largerThe less, the optimal reaction time is determined to be 3h. The DNA nanoflower was collected by centrifugation at 16000rpm for 15min, washed twice with ultrapure water to remove excess salt, sonicated for dispersion (40W, 12 s) and stored at-20℃for long periods.
Example 2
Preparation of photosensitizer-carrying DNA nanoenzyme (CH/DF)
The DNA nanoflower (DF) prepared in example 1 was mixed with an equal volume of 2 Xbuffer (20 mM Tris-HCl,40mM NaCl,40mMKCl,pH 7.6), incubated at room temperature for 1h to allow the AS411 domain sequence in the nanoflower structure to form a G-quadruplex space structure under high potassium ion conditions, and bound to heme-coenzyme (hemin) and photosensitizer Ce6 aromatic ligand by pi-pi action. Uniformly mixing DNA nanoflower and hemin (hereinafter referred to as H) in a molar ratio of 1:40, incubating for 4H at room temperature, centrifuging at 16000rpm for 10min, and performing ultrasonic redissolution on the precipitate by using an equal volume buffer solution to obtain H/DF; mixing H/DF and Ce6 (called C for short) in a molar ratio of 1:600 by vortex uniformly, incubating for 4 hours at room temperature, and centrifuging to collect precipitate to obtain the DNA nano enzyme (CH/DF) carrying the photosensitizer.
Experimental example 1
Characterization of photosensitizer-loaded DNA nanoenzymes (CH/DF)
Taking 10 μL DNA nano enzyme CH/DF sample, diluting with ultrapure water for 100 times, respectively measuring its hydration particle size and particle size distribution by dynamic light laser scattering (DLS) and Laser Doppler Velocimetry (LDV), and obtaining CH/DF with uniform particle size and average hydration particle size of about 300nm as shown in figure 4A
The entrapment of the photosensitizer Ce6 was monitored by a fluorescence spectrometer, and after Ce6 was loaded into the nanoflower, its fluorescence emission spectrum was significantly reduced, as shown in fig. 4B.
Scanning Electron Microscope (SEM) characterization: and taking out the newly prepared silicon wafer soaked in aqua regia overnight, cleaning with water and acetone, drying, dripping 10 μl of DNA nanoflower solution on the surface of the silicon wafer, and drying the sample in a 70 ℃ oven for 2 hours. Prior to SEM imaging, the sample was coated with gold to enhance its conductivity. As a result, as shown in fig. 4C, the nanoparticles exhibited a uniform spherical structure, and the particle diameter after drying was about 100nm:
transmission Electron Microscope (TEM) and energy spectroscopy (EDS) characterization: and (3) taking a proper amount of DNA nanoflower and CH/DF, performing ultrasonic dispersion, dripping the DNA nanoflower and the CH/DF on a copper mesh, and placing the copper mesh in a 70 ℃ oven for drying for 30min for TEM imaging and analyzing the composition of each element in the preparation through EDS. As a result, as shown in FIG. 4D, the nanoparticle has a uniform sphere shape, N/P elements in the structure are mainly attributed to DNA, and Fe elements are derived from encapsulation of heme.
From the above results, it is known that the DNA nanoflower successfully encapsulates heme coenzyme and photosensitizer Ce6, forming spherical nanoparticles with uniform particle size.
Experimental example 2
Oxygen production and stability detection of DNA nanoenzyme (CH/DF)
1. Catalase Activity assay
Based on G (th) theory, H 2 O 2 Combining ammonium molybdate to form stable and irreversible yellow complex, calculating H according to absorbance at 405 and nm in ultraviolet spectrogram 2 O 2 Concentration. The method for detecting the peroxidase activity of the DNA nanoflower by adopting the ammonium molybdate method comprises the following specific operations: suction 0.5mLH 2 O 2 (1 mM) and 0.1mL of CH/DF (0.5. Mu.M) were mixed uniformly in a 1.5mL EP tube, reacted at room temperature for 1min, and then 0.5mL of ammonium molybdate solution (32.4 mM) was added to terminate the reaction, and after standing for 10min, A405nm was detected using an ultraviolet spectrophotometer. The results are shown in FIG. 5A, which demonstrates that CH/DF can consume H in a test tube 2 O 2 。
2. Investigation of oxygen production capacity
In order to further study the self-oxygen generating performance of CH/DF, a portable dissolved oxygen meter is adopted to monitor the dissolved oxygen level of the system in real time. The main operation is as follows: draw 4mL CH/DF (0.5. Mu.M) in a 10mL beaker and add H 2 O 2 Stock was diluted to a final concentration of 1mM. The low-speed stirring is kept in the detection process, and the dynamic change of the oxygen in the photodynamic therapy of CH/DF is studied. The results are shown in FIG. 5B, which shows that CH/DF can rapidly catalyze H 2 O 2 Production of O 2 。
3. ROS production capability investigation
SOSG fluorescent probe is free from other reactive oxygen species such as hydroxyl radicals (OH) or superoxide anions (O) 2– ) Influence, can specifically bind to singlet oxygen (1O) 2 ) Thereby reducing background interference. Suction CH/DF (0.5. Mu.M), SOSG (2.5. Mu.M) and H 2 O 2 (1 mM) in a 96-well plate, the mixture was homogenized, and the buffer was added to a final volume of 100. Mu.L. The power used is 0.75W/cm 2 The plate was irradiated with the 660nm laser light for 1min, and the fluorescence intensity of SOSG was recorded every 10s (ex=504 nm, em=524 nm). The results are shown in FIG. 5C, where SOSG fluorescence is rapidly enhanced under light conditions, demonstrating CH/DF photodynamic activity.
4. Enzyme stability investigation
The experiment aims at researching the enzyme degradation resistance of DNA nanoflower and free G-quadruplex (CH/G4), and the specific operation is as follows: g-quadruplex was loaded with hemin, ce6, prepared CH/G4 according to the procedure of example 2, and then both CH/G4 and CH/DF formulations were incubated with 10% FBS for 12h, respectively, to simulate enzymatic degradation. The self-oxygen generating properties and ROS generating ability of the two formulations after enzyme treatment were examined according to the above methods. As shown in FIG. 5E and FIG. 5F, the activity of CH/G4 is obviously reduced after being treated by FBS, and the CH/DF still maintains the original activity, thus confirming the enzymatic degradation resistance of the DNA nanoflower.
From the above results of this experimental example, it was found that DNA nanoenzyme (CH/DF) can catalyze H with high efficiency 2 O 2 Production of O 2 At the same time, CH/DF can also catalyze and generate singlet oxygen 1O 2 Assisting photodynamic therapy (PDT), the basic principle of which is shown in fig. 4D; meanwhile, CH/DF can effectively resist enzyme degradation, has stable structure and can facilitate in vivo effective delivery.
Experimental example 3
1. Cell uptake assay: a549 or HEK-293 cells were seeded at a cell density of 2.5×104 cells per well in 24-well plates (0.5 mL cell suspension/well) and incubated in cell culture incubator for 24h. The waste culture medium is sucked off, CH/DF is added, and the incubation is carried out for 1, 2 or 4 hours respectively. The cells were incubated with 4% paraformaldehyde for 20min after three washes in PBS. After fixation, the nuclei were stained with DAPI (1. Mu.g/mL) and blotted. After incubation for 10min, the cells were washed twice with PBS, placed in a cell imager and the fluorescence distribution was observed. As shown in fig. 6A, the fluorescence of nanoflower in cells gradually increased with prolonged incubation time, demonstrating time-dependent cellular uptake; fig. 6B and 6C show that the fluorescence intensity of the nanoparticle in tumor cells is significantly higher than that of normal cells, confirming the tumor targeting selectivity of the formulation CH/DF.
2. Cytotoxicity experiment: a549 cells were grown at a density of 5×10 3 The cells/wells were seeded in 96-well plates and incubated in cell culture boxes for 24h. The old culture medium is sucked and abandoned, PBS is washed twice, and then the treatment is carried out by using DF, C/DF, CH/DF, C/DF+L and CH/DF+L in a grouping way, wherein the C/DF+L and the CH/DF+L are control groups using illumination; in addition, the sample solutions of different concentrations were selected for comparison in groups of treated cells. After 24h incubation the laser group was given 0.75W/cm 2 The culture was continued for 24 hours by laser irradiation for 1min. mu.L MTT (5 mg. Multidot.mL) was added to each well -1 ) The solution was incubated for 4h, the supernatant was aspirated off, 100. Mu.L of DMSO was added and the solution was placed in a constant temperature water bath shaker tank at 90rpm/min for 10min. The absorbance (a) value at 570nm wavelength per well was measured using a microplate reader, and the cell viability of each group was calculated from the viability (%) = (sample group a-zero group a)/(blank group a-zero group a). The results are shown in FIG. 7A: DF is not cytotoxic, indicating the biosafety of the material; after the photosensitizer Ce6 is encapsulated, the C/DF shows a certain phototoxicity; further entrapping heme, and enhancing toxicity; under the illumination condition, CH/DF shows the strongest cytotoxicity, and the synergy of the self-oxygen generating capacity and photodynamic therapy is proved.
3. Live/dead cell staining experiments: a commercially available Calcein-AM/PI kit is adopted for carrying out a live/dead cell staining experiment, and Calcein acetoxymethyl ester (Calcein-AM) is used as a fluorescent dye for marking living cells, so that the living cells can easily pass through living cell membranes. After cell entry, the Calcein-AM is hydrolyzed by intracellular esterase into a non-membrane permeation type fluorescent molecule Calcein, and the non-membrane permeation type fluorescent molecule Calcein stays in cells and emits green fluorescence. Propidium Iodide (PI) cannot enter living cells, and because of good cell membrane permeability of dead cells, PI is combined with DNA at cell nuclei after entering cells, and strong red fluorescence is emitted. The density of 5X 10 was obtained by the method described above 4 cells/mL of a549 cell suspension were seeded in 96-well plates at a density of 5 thousand cells per well and incubated in a cell incubator for 24h. And after the DF, the C/DF, the CH/DF, the C/DF+L and the CH/DF+L are added in groups for intervention for 24 hours, the laser group is additionally irradiated by a light source for 1min. Continuing to incubateAfter 24 hours, the old culture medium is removed, PBS is gently washed for 2-3 times, and the cells are prevented from being blown away. Precisely sucking 10 μl of Calcein-AM working solution into 10mLPBS, vortex mixing, adding 10 μl of PI working solution, and mixing thoroughly. 100 μl of staining solution was added to each well, and after incubation for 30min, the supernatant was aspirated off, and the green and red fluorescence distribution was observed with a fluorescence microscope. Results as shown in fig. 7B, living cells appeared green by staining, and apoptotic cells appeared red, and cytotoxicity results of each preparation were highly consistent with MTT experiments by staining.
4. Intracellular ROS level investigation: the present experiment uses the DCFH-DA kit to monitor intracellular ROS expression levels. The working principle is that DCFH-DA can enter the cell through free transmembrane, endogenous esterase is hydrolyzed to generate non-fluorescent DCFH, and the DCFH distributed in the cell is rapidly oxidized into DCF with strong fluorescence by intracellular ROS because the DCFH cannot pass through cell membranes, and the green fluorescence intensity is in direct proportion to the ROS level. A549 cells were plated at 5×10 per well 4 The density of each mL was inoculated into 24-well plates, and a control group, a DF group, a C/DF group, an H/DF group, a CH/DF group, a CD/DF+L group, and a CHD/DF+L group were set. After incubation of 10nM samples with cells for 4h, the cells were washed 3 times with pre-chilled PBS. DCFH-DA (10. Mu.M) was diluted with a medium without fetal bovine serum, added to the wells, incubated for 30min, and then irradiated with additional 660nm laser for 1min (0.75W/cm 2). Finally, observing the intracellular fluorescence distribution by using a cell imager. The results are shown in FIG. 7C, wherein the intracellular fluorescence of the C/DF and CH/DF groups is significantly enhanced after illumination, confirming the photodynamic effect of the photosensitizer. At the same time, CH/DF has a stronger intracellular fluorescence signal due to the photodynamic enhancement of the self-oxygenating activity of the nanoenzyme.
Example 4
(1) Molding tumor-bearing nude mice: nude mice are purchased and raised uniformly under the condition meeting the requirements of animal experiments by the university of south and middle school laboratory animal school. All animal care and treatments were approved by the local ethics committee and were conducted in accordance with guidelines of the chinese related animal care laws. The right front side of the nude mice is subcutaneously inoculated with a suspension prepared in advance and containing A549 cells, PBS and matrigel, the inoculation amount of each nude mouse is about 5x 106 cells, the nude mice after inoculation are normally fed, and the nude mice are used for the subsequent animal experiments after the tumor volume grows to a proper size.
(2) In vivo imaging: cy 5-labeled DNA nanoenzyme was injected into tumor-bearing nude mice via the tail vein. A control group was also set up and given to the equivalent Cy5 tail vein. At 1h and 24h time points after injection, mice in each group were subjected to live animal imaging. Fig. 8A shows the in vitro imaging results, and fig. 8B shows the fluorescence quantification results. Compared with free Ce6, CH/DF has stronger tumor fluorescence signal, and the tumor targeting enrichment capability of the nano preparation is proved.
(3) In vivo anti-tumor experiments: establishing a tumor nude mouse model of subcutaneous lotus A549 allograft according to the method until the tumor grows to about 60mm 3 At this time, different treatments were given, and then labeling, weighing and tumor diameter measurement were performed on each group of tumor-bearing nude mice, and then tail vein administration was performed on each tumor-bearing nude mice according to the set group. While weighing and tumor diameter determination were performed on alternate days during the treatment period. After day 16 of treatment, tumor-bearing nude mice were euthanized and tumors were removed and weighed. Fig. 9A is a graph of tumor volume monitoring dynamically during treatment, fig. 9B is a graph of tumor volume after treatment, and fig. 9C is a quantitative result of tumor body weight. The results show that the antitumor efficacy of each group preparation is ordered as CH/DF+L>C/DF+L>CH/DF>C/DF is about DF, wherein CH/DF has the strongest anti-tumor capability under the illumination condition, and the tumor treatment potential of the nano-enzyme is further proved. After treatment, the main organs of each group of mice were taken and pathological observation was performed, and the results are shown in fig. 10: after treatment, all organs of the mice are not obviously changed, and the biological safety of the DNA nano enzyme preparation is proved.
The above embodiments are only preferred embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to apply equivalents and modifications according to the technical solution and the concept of the present invention within the scope of the present invention.
Claims (8)
1. The DNA nano enzyme with the self-oxygen generating capacity is characterized by comprising a DNA nano flower, heme coenzyme and a photosensitizer Ce6, wherein the DNA nano flower is formed by rolling circle amplification of a primer chain and a substrate chain, and the primer chain is shown as SEQ ID NO. 1:
GTGGTGGTGTTGGTGGTGGT；
the substrate chain is shown as SEQ ID NO. 2:
CCACCAACACCACCACCACCTTTGACACACTAGCGATACGCGTATCGCTATGGCATATCGTACGATATGCCAGTGTGTCTTTCCACCA；
the DNA nanoflower has an AS1411G quadruplex structure, the heme coenzyme is embedded into the AS1411G quadruplex structure, and the photosensitizer Ce6 is stacked in the AS1411G quadruplex structure through pi-pi action.
2. The DNA nanoenzyme with self-oxygen generating capacity according to claim 1, wherein the DNA nanoenzyme has a spherical shape and a particle diameter of 100 to 200nm.
3. A method for preparing the DNA nanoenzyme with self-oxygen generating capacity according to any one of claims 1 to 2, comprising the steps of:
s1, preparing DNA nanoflower: mixing a substrate chain and a primer chain, carrying out complementary pairing to form circular DNA with a notch, then adding DNA ligase to react to form complete circular DNA, finally introducing DNA polymerase to carry out PCR amplification to form a plurality of DNA long chains with copy chains, and synthesizing DNA nanoflower by the DNA long chains through base pairing and a self-assembly mode;
s2, AS1411G quadruplex structure formation: dissolving the DNA nanoflower in Tris buffer solution containing high potassium ions, and incubating at room temperature to form the DNA nanoflower with AS1411G quadruplex space structure;
s3, assembling to form DNA nano enzyme: mixing the DNA nanoflower with the AS1411G quadruplex space structure prepared in the step S2 with heme coenzyme, incubating for 4-6 hours at room temperature, adding a photosensitizer Ce6, uniformly mixing by vortex, continuing to incubate for 3-4 hours at room temperature, centrifuging and collecting precipitate to obtain the DNA nano enzyme with self-oxygen production capability.
4. The method according to claim 3, wherein the annealing temperature in the step S1 is 95 ℃ water bath for 10-30 min, and then slowly cooling to 25 ℃, and the cooling time is more than 2.5h.
5. The method according to claim 3, wherein the DNA ligase in the step S1 is T4-DNA ligase and the DNA polymerase is phi29 DNA polymerase.
6. The method according to claim 3, wherein the Tris buffer in step S2 is a mixture of Tris-HCl, naCl and KCl, and the pH is 7-8.
7. The method according to claim 3, wherein the mixing molar ratio of the DNA nanoflower to the heme-coenzyme in the step S3 is 1:40-50; the mixing mole ratio of the DNA nanoflower to the photosensitizer Ce6 is 1:500-600.
8. Use of the DNA nanoenzyme with self-generating capacity according to any one of claims 1 to 2 or the DNA nanoenzyme with self-generating capacity prepared by the preparation method according to any one of claims 3 to 7 in the preparation of anti-tumor photodynamic therapy drugs.
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