CN114306625B - Multifunctional nano-drug carrier integrating diagnosis and treatment and preparation method and application thereof - Google Patents
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Abstract
The invention discloses a multifunctional nano-drug carrier integrating diagnosis and treatment, a preparation method and application thereof. The invention discloses a multifunctional nano-drug carrier integrating diagnosis and treatment, which consists of the following structures: octahedral DNA origami, microRNA detectors, gene therapy drugs (siRNA) and chemotherapeutic drugs (doxorubicin Dox). According to the invention, three-dimensional DNA paper folding is selected as a carrier, a plurality of sites are designed on the bundle of paper folding to be connected with medicines or detection chains, wherein 48 sites are designed in a chamber to be connected with siRNA, so that the siRNA has a better protection function, 24 sites are designed outside the chamber to be connected with detectors, and the siRNA is fully contacted with target microRNA in cytoplasm; two medicines are loaded, and a detector is carried at the same time, so that the cancer diagnosis and treatment functions are integrated.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a multifunctional nano-drug carrier integrating diagnosis and treatment, a preparation method and application thereof.
Background
DNA folding has many advantages such as stable chemical properties; has good biocompatibility, programmability and addressability; the rigidity is strong; the size is controllable; is resistant to a variety of nucleases; the modifier is strong; cells and the like can be accessed without using a transfection reagent, and therefore, have been widely used in research in various fields, particularly in the field of drug delivery in recent years. There have been many related efforts in this regard. The Ding Baoquan group has achieved a lot of results, firstly, they adopt two-dimensional triangular piece-shaped DNA paper folding as a drug carrier, the anticancer drug doxorubicin Dox is loaded by inserting into guanine G-cytosine C base pairs, the carrier can effectively enter cells and exert drug effect (article: J.am. Chem. Soc.2012, 134 (32), 13396-13403), and then they develop a series of complex drug carriers by utilizing the strong addressability and the modifiable property of the DNA paper folding, such as an acid response paper folding carrier carrying tumor vaccine (article: nat. Mater.2021, 20, 421-430.) and a paper folding carrier simultaneously loading gene therapy drug small interfering RNAsiRNA and chemotherapeutic drug doxorubicin Dox and responding to glutathione GSH release (article: angew. Chem. Int. Ed.2020,59,1-6.).
DNA hybridization has strong specificity, can be used for detecting specific markers in cancer cells, such as microRNA, and can amplify relatively weaker detection signals by several orders of magnitude by skillfully designing toe ends toehold of reaction chains. For example, combining strand displacement techniques with fluorescence resonance energy transfer FRET effects, the concentration of target strands can be characterized by fluorescence intensity (article: adv. Function. Mater.2018, 1800490).
Disclosure of Invention
In order to construct the nano particle integrating cancer diagnosis and treatment, the invention utilizes octahedral DNA paper folding, the frame chamber is large, has high rigidity, can load gene therapy medicine Bcl2 small interference RNABcl2siRNA and chemotherapy medicine doxorubicin Dox at the same time, and has good protection function; in addition, a unique three-dimensional carrier integrating cancer diagnosis and treatment is built by combining a strand displacement amplification technology and FRET effect detection of low-concentration cancer cell markers, so that a specific strong signal can be generated for the low-concentration markers in cancer cells, and apoptosis guidance can be carried out on the cancer cells through two means of chemotherapy and gene therapy.
In order to achieve the above object, the present invention provides the following technical solutions: the invention discloses a multifunctional nano-drug carrier integrating diagnosis and treatment, which consists of the following structures: the kit comprises an octahedral three-dimensional DNA paper folding frame, a microRNA detector connected with a connecting chain extending outwards on a bundle at the edge of the frame, a gene therapeutic drug Bcl2siRNA connected with a connecting chain extending inwards on the bundle and a chemotherapeutic drug doxorubicin Dox embedded in the bundle. The microRNA detector is formed by hybridizing two DNA chains of a modified Cy5 fluorescent group and a BHQ2 quenching group, and the siRNA is formed by hybridizing two RNA chains of a sense chain and an anti-sense chain.
The invention relates to a preparation method of a multifunctional nano-drug carrier integrating diagnosis and treatment, which comprises the following steps:
(1) Octahedral DNA paper folding synthesis: m13mp18 scaffold DNA long strands and 120 DNA strand strands were mixed in Tris-acetate buffer (1 XTAE), 12.5mM magnesium acetate (MgAc) 2 ) In the solution, the mixed solution is put into pcr instrument, slowly cooling from 95 ℃ to 20 ℃ in one day to synthesize octahedral three-dimensional DNA folded paper with the concentration of 10nM;
(2) Purification of octahedral DNA paper folding: adding 500 μl of octahedral paper into 100kDa ultrafiltration tube, centrifuging in a centrifuge at 3000rcf for 8min, and repeating 2-4 times of washing operation after centrifuging, namely adding 1 xTAE and 12.5mM MgAc 2 The solution was made up to the initial volume and centrifuged to remove excess staple and concentrate the octahedral DNA fold to 20nM;
(3) Dispersing the ultrafiltered octahedron: in order to ensure that the octahedron is dispersed more uniformly and avoid the agglomeration phenomenon in the subsequent process, placing the ultrafiltered octahedron on a metal mixing instrument and vibrating at 900-1000rpm and 26 ℃ for overnight;
(4) siRNA, detector load: the siRNA consists of two single chains of a sense chain and an anti-sense chain, a detector comprises two single chains of a BHQ chain and a Cy5 chain, the ultra-filtered 20nM octahedral paper folding chain is mixed with the sense chain, the anti-sense chain, the BHQ chain and the Cy5 chain, and the temperature is reduced from 50 ℃ to 20 ℃ at a constant speed in a pcr instrument at a speed of 2.4 ℃/h;
(5) Doxorubicin Dox loading: the octahedral folded paper loaded with siRNA and detector and doxorubicin Dox are mixed according to the mass ratio of 1:10000, and placed on a metal mixer to react overnight at 500rpm and 25 ℃.
Further, in step (1), the scafold DNA long strand: the strand chain is 1:10.
Further, in the step (1), the octahedral paper folding and sense chain, the anti-sense chain, the BHQ chain and the Cy5 chain are in a mass ratio of 1:1:1:1:1.
The invention relates to application of a multifunctional nano-drug carrier integrating diagnosis and treatment in drugs.
The beneficial effects are that: according to the invention, three-dimensional DNA paper folding is selected as a carrier, a plurality of sites are designed on the bundle of paper folding to be connected with medicines or detection chains, wherein 48 sites are designed in a chamber to be connected with siRNA, so that the siRNA has a better protection function, and 24 sites are designed outside the chamber to be connected with detection chains so as to be in full contact with target microRNA in cytoplasm; two medicines are loaded, and a detector is carried at the same time, so that the cancer diagnosis and treatment functions are integrated.
Drawings
Fig. 1 is a load, operating diagram of a detector of the present invention. The gel electrophoresis results in FIG. 1 (a) and the quenching results of the "no amplification" set of fluorescent signals in FIG. 1 (b) may illustrate successful detector loading; fig. 1 (b) illustrates that the material has a high-efficiency detection effect, and can amplify the detection signal by 3 orders of magnitude.
FIG. 2 is a schematic representation of gel electrophoresis of gene therapy drug (siRNA) of the present invention loaded on octahedral paper sheet. The band hysteresis of lane 2 of the graph illustrates the successful loading of the drug.
FIG. 3 is a graph showing the loading effect of the chemotherapeutic drug (doxorubicin Dox) of the present invention on octahedral paper folding.
FIG. 4 is a representation of transmission electron microscopy, dynamic light scattering of the material of the present invention.
FIG. 5 is a schematic diagram of the detector reaction of the present invention.
FIG. 6 is a schematic diagram of an octahedral load detector and a drug according to the present invention.
Fig. 7 is a material synthesis roadmap of the invention.
Detailed Description
For a better understanding of the present invention, the content of the present invention will be further elucidated with reference to the examples and drawings, but the content of the present invention is not limited to the following embodiments. The experimental methods used therein are conventional methods unless otherwise specified.
Example 1
The invention discloses a multifunctional nano-drug carrier integrating diagnosis and treatment, which consists of the following structures: the kit comprises an octahedral three-dimensional DNA paper folding frame, a microRNA detector connected with a connecting chain extending outwards on a bundle at the edge of the frame, a gene therapeutic drug Bcl2siRNA connected with a connecting chain extending inwards on the bundle and a chemotherapeutic drug doxorubicin Dox embedded in the bundle. The microRNA detector is formed by hybridizing two DNA chains of a modified Cy5 fluorescent group and a BHQ2 quenching group, and the siRNA is formed by hybridizing two RNA chains of a sense chain and an anti-sense chain.
The invention relates to a preparation method of a multifunctional nano-drug carrier integrating diagnosis and treatment, which comprises the following steps:
(1) Octahedral DNA paper folding synthesis: m13mp18 scaffold DNA long strands and 120 stand strands were mixed in 1 XTAE, 12.5mM MgAc 2 In the solution, the mixed solution is put into a pcr instrument, and slowly cooled from 95 ℃ to 20 ℃ in one day to synthesize octahedral three-dimensional DNA paper folding with the concentration of 10nM; the scaffold DNA long chain: the strand chain is 1:10.
(2) Purification of octahedral DNA paper folding: adding 500 μl of octahedral paper into 100kDa ultrafiltration tube, centrifuging in a centrifuge at 3000rcf for 8min, and repeating washing operation 3 times after centrifuging, namely adding 1×TAE and 12.5mM MgAc 2 The solution was made up to the initial volume and centrifuged to remove excess staple and concentrate the octahedral DNA fold to 20nM;
(3) Dispersing the ultrafiltered octahedron: in order to ensure that the octahedron is dispersed more uniformly and avoid the agglomeration phenomenon in the subsequent process, placing the ultrafiltered octahedron on a metal mixing instrument for reaction overnight at 900rpm and 26 ℃;
(4) siRNA, detector load: mixing the ultrafiltered octahedral paper folding 20nM with gene therapeutic drug siRNA (sense chain and anti-sense chain), detector (BHQ chain and Cy5 chain), cooling from 50deg.C to 20deg.C at a speed of 2.4deg.C/h in pcr instrument; the octahedral paper folding chain, the sense chain, the anti chain, the BHQ chain and the Cy5 chain are mixed according to the mass ratio of 1:1:1:1:1.
(5) Doxorubicin Dox loading: the octahedral folded paper loaded with siRNA and detector and doxorubicin Dox are mixed according to the proportion of 1:10000 and placed on a metal mixing instrument to react overnight at 500rpm and 25 ℃.
The invention relates to application of a multifunctional nano-drug carrier integrating diagnosis and treatment in drugs.
Example 2
Example 2 differs from example 1 in that: the invention relates to a preparation method of a multifunctional nano-drug carrier integrating diagnosis and treatment, which comprises the following steps:
in step (2), octahedraDNA paper folding purification: adding 500 μl of octahedral paper into 100kDa ultrafiltration tube, centrifuging in a centrifuge at 3000rcf for 8min, and repeating 2 times of washing operations after centrifuging, namely adding 1×TAE and 12.5mM MgAc 2 The solution was made up to the initial volume and centrifuged to remove excess staple and concentrate the octahedral DNA fold to 20nM;
in step (3), the ultrafiltered octahedron is dispersed: in order to disperse the octahedron more uniformly and avoid the subsequent agglomeration phenomenon, the ultrafiltered octahedron is placed on a metal mixing instrument and is vibrated at 1000rpm and 26 ℃ overnight.
Example 3
Example 3 differs from example 1 in that: the invention relates to a preparation method of a multifunctional nano-drug carrier integrating diagnosis and treatment, which comprises the following steps:
in the step (2), octahedral DNA paper folding purification: adding 500 μl of octahedral paper into 100kDa ultrafiltration tube, centrifuging in a centrifuge at 3000rcf for 8min, and repeating washing operation 4 times after centrifuging, namely adding 1×TAE and 12.5mM MgAc 2 The solution was made up to the initial volume and centrifuged to remove excess staple and concentrate the octahedral DNA fold to 20nM;
in step (3), the ultrafiltered octahedron is dispersed: in order to ensure that the octahedron is dispersed more uniformly and avoid the subsequent agglomeration phenomenon, the ultrafiltered octahedron is placed on a metal mixing instrument and is oscillated overnight at the temperature of 26 ℃ at 950rpm, so as to prepare the multifunctional nano-drug carrier integrating diagnosis and treatment.
Detector, drug loading, running test of the invention:
tool device: centrifuge, pcr instrument and metal mixing instrument
Test example 1
The multifunctional nano-drug carrier integrating diagnosis and treatment and prepared in the embodiment 1 is adopted, and the feasibility of a diagnosis part is verified.
Diluting 30 μl of the reaction product to 50-60 μl, performing fluorescence intensity measurement, diluting 20 μl to 40-50 μl, and performing gel electrophoresis characterization to test the load; and adding the target object and the fuel chain in vitro to measure the signal amplification factor. The gel electrophoresis results in FIG. 1 (a) and the quenching results of the "no amplification" set of fluorescent signals in FIG. 1 (b) may illustrate successful detector loading; fig. 1 (b) illustrates that the material has a high-efficiency detection effect, and can amplify the detection signal by 3 orders of magnitude.
Test example 2
siRNA load test: the loading was checked by gel electrophoresis characterization by diluting 20. Mu.l of the reaction product to 40-50. Mu.l. The band hysteresis of lane 2 in fig. 2 illustrates the successful loading of the drug.
Test example 3
Doxorubicin Dox load curve determination: taking 100-200 mu l of reaction products at different time points of the loading reaction, centrifuging at 11000-12000g for 12min, taking supernatant fluid, and measuring ultraviolet absorption value to obtain a loading curve of doxorubicin Dox. Fig. 3 is a graph of the time load of Dox measured, showing that the Dox load rate reaches 42.9%.
Test example 4
And carrying out transmission electron microscope visual characterization and dynamic light scattering characterization on the materials loaded with different parts. The transmission electron microscopy results in FIG. 4 (a) show that there is no change in three-dimensional morphology after the carrier is loaded with the detector or drug; the dynamic light scattering result in FIG. 4 (b) shows that the carrier particle size is about 80 nm.
Fig. 5 is a schematic diagram of detection by a detector, and a target microRNA and the detector undergo a strand displacement reaction to displace Cy5 strands, and similarly, a later-added fuel strand displaces microRNA, so that microRNA can continuously and circularly react to displace Cy5 strands, thereby achieving a signal amplification effect.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (2)
1. A multifunctional nano-drug carrier integrating diagnosis and treatment is characterized in that: the multifunctional nano-drug carrier consists of the following structures: the kit comprises an octahedral three-dimensional DNA paper folding frame, a microRNA detector connected with a connecting chain extending outwards on a bundle at the edge of the frame, a gene therapeutic drug Bcl2siRNA connected with a connecting chain extending inwards on the bundle and a chemotherapeutic drug doxorubicin Dox embedded in the bundle; wherein the microRNA detector is formed by hybridizing two DNA chains of a modified Cy5 fluorescent group and a BHQ2 quenching group, and the siRNA is formed by hybridizing two RNA chains of a sense chain and an anti-sense chain;
the preparation method of the multifunctional nano-drug carrier integrating diagnosis and treatment comprises the following steps:
(1) Octahedral DNA paper folding synthesis: m13mp18 scaffold DNA long chain and 120 DNA strand chains are mixed in Tris-acetic acid buffer solution 1 xTAE and 12.5mM magnesium acetate MgAc 2 solution, the mixed solution is put into a pcr instrument, and slowly cooled from 95 ℃ to 20 ℃ in one day to synthesize octahedral three-dimensional DNA origami with the concentration of 10nM; the scaffold DNA long chain: the strand chain is 1:10; the octahedral paper folding chain, the sense chain, the anti chain, the BHQ chain and the Cy5 chain are mixed according to the mass ratio of 1:1:1:1:1;
(2) Purification of octahedral DNA paper folding: adding 500 μl of octahedral folded paper into a 100kDa ultrafiltration tube, centrifuging in a centrifuge at 3000rcf for 8min, repeating 2-4 times of washing operation after centrifuging, adding 1×TAE and 12.5mM MgAc 2 solution to the initial volume, centrifuging, removing excessive staple, and concentrating the octahedral DNA folded paper to 20nM;
(3) Dispersing the ultrafiltered octahedron: in order to ensure that the octahedron is dispersed more uniformly and avoid the agglomeration phenomenon in the subsequent process, placing the ultrafiltered octahedron on a metal mixing instrument and vibrating at 900-1000rpm and 26 ℃ for overnight;
(4) siRNA, detector load: the siRNA consists of two single chains of a sense chain and an anti-sense chain, a detector comprises two single chains of a BHQ chain and a Cy5 chain, the ultra-filtered 20nM octahedral paper folding chain is mixed with the sense chain, the anti-sense chain, the BHQ chain and the Cy5 chain, and the temperature is reduced from 50 ℃ to 20 ℃ at a constant speed in a pcr instrument at a speed of 2.4 ℃/h;
(5) Doxorubicin Dox loading: the octahedral folded paper loaded with siRNA and detector and doxorubicin Dox are mixed according to the mass ratio of 1:10000, and placed on a metal mixer to react overnight at 500rpm and 25 ℃.
2. The multifunctional nano-drug carrier integrating diagnosis and treatment into a whole, which is disclosed in claim 1, for preparing a drug.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017127033A1 (en) * | 2016-01-22 | 2017-07-27 | Yeditepe Universitesi | A preparation method for a dna origami based carrier system |
| CN107469088A (en) * | 2017-06-27 | 2017-12-15 | 郑州大学 | A kind of construction method of accurate identification targeted nano carrier based on DNA paper folding arts and its application |
| WO2018117333A1 (en) * | 2016-12-21 | 2018-06-28 | 한국생명공학연구원 | Bioprobe set capable of self-amplification of fluorescence signal for detecting microrna and use thereof |
| CN108273056A (en) * | 2018-02-01 | 2018-07-13 | 中国科学院长春应用化学研究所 | A kind of modified gold nano-material/nucleic acid probe nanometer system and preparation method thereof, application |
| WO2019109707A1 (en) * | 2017-12-07 | 2019-06-13 | Arizona Board Of Regents On Behalf Of Arizona State University | Dna nanorobot and methods of use thereof |
| WO2020107560A1 (en) * | 2018-11-28 | 2020-06-04 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for detecting micrornas based on rolling circle amplification and dna origami |
| CN111803511A (en) * | 2020-06-12 | 2020-10-23 | 南京邮电大学 | DNA tetrahedral nucleic acid frame type gastric cancer diagnosis and treatment integrated reagent and preparation method and application thereof |
-
2022
- 2022-01-06 CN CN202210008397.3A patent/CN114306625B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017127033A1 (en) * | 2016-01-22 | 2017-07-27 | Yeditepe Universitesi | A preparation method for a dna origami based carrier system |
| WO2018117333A1 (en) * | 2016-12-21 | 2018-06-28 | 한국생명공학연구원 | Bioprobe set capable of self-amplification of fluorescence signal for detecting microrna and use thereof |
| CN107469088A (en) * | 2017-06-27 | 2017-12-15 | 郑州大学 | A kind of construction method of accurate identification targeted nano carrier based on DNA paper folding arts and its application |
| WO2019109707A1 (en) * | 2017-12-07 | 2019-06-13 | Arizona Board Of Regents On Behalf Of Arizona State University | Dna nanorobot and methods of use thereof |
| CN108273056A (en) * | 2018-02-01 | 2018-07-13 | 中国科学院长春应用化学研究所 | A kind of modified gold nano-material/nucleic acid probe nanometer system and preparation method thereof, application |
| WO2020107560A1 (en) * | 2018-11-28 | 2020-06-04 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for detecting micrornas based on rolling circle amplification and dna origami |
| CN111803511A (en) * | 2020-06-12 | 2020-10-23 | 南京邮电大学 | DNA tetrahedral nucleic acid frame type gastric cancer diagnosis and treatment integrated reagent and preparation method and application thereof |
Non-Patent Citations (5)
| Title |
|---|
| Aptamer-Functionalized DNA Origami for Targeted Codelivery of Antisense Oligonucleotides and Doxorubicin to Enhance Therapy in Drug-Resistant Cancer Cells;Pan et al.;ACS Appl. Mater. Interfaces》;第12卷;第400页摘要 * |
| DNA Origami Frameworks Enabled Self-Protective siRNA Delivery for Dual Enhancement of Chemo-Photothermal Combination Therapy;Xu et al.;《Small》;第17卷;第2页左栏最后1段至右栏第1段，第11页左栏第3、6段以及右栏第1段 * |
| Efficient and Reliable MicroRNA Imaging in Living Cells via a FRETBased Localized Hairpin-DNA Cascade Amplifier;Liu et al.;《Anal. Chem.》;第91卷;第3675页摘要 * |
| Gold Nanorods Electrostatically Binding Nucleic Acid Probe for In Vivo MicroRNA Amplified Detection and Photoacoustic Imaging-Guided Photothermal Therapy;Yan et al.;《Adv. Funct. Mater.》;第1800490页 * |
| 金征宇等主编.《基因与纳米探针：医学分子成像理论与实践》.天津科学技术出版社,2017,第654页. * |
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