CN108498451B - DNA nano-drug carrier with high drug loading effect, preparation method and application - Google Patents

DNA nano-drug carrier with high drug loading effect, preparation method and application Download PDF

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CN108498451B
CN108498451B CN201810630449.4A CN201810630449A CN108498451B CN 108498451 B CN108498451 B CN 108498451B CN 201810630449 A CN201810630449 A CN 201810630449A CN 108498451 B CN108498451 B CN 108498451B
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dna
seq
loading effect
drug
drug carrier
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CN108498451A (en
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潘林强
徐飞
石晓龙
姜素霞
王延峰
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Huazhong University of Science and Technology
Zhengzhou University of Light Industry
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Zhengzhou University of Light Industry
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    • A—HUMAN NECESSITIES
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Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a DNA nano-drug carrier with a high drug loading effect, a preparation method and application thereof, wherein the DNA nano-drug carrier is prepared from two DNA chains shown in SEQ ID NO.1 and SEQ ID NO.2, or two DNA chains shown in SEQ ID NO.3 and SEQ ID NO.4, or two DNA chains shown in SEQ ID NO.5 and SEQ ID NO.6, and can be applied to carrying anti-tumor drugs. The method for synthesizing the DNA nano-drug carrier has the advantages of simple steps, low cost, uniform shape and size, and can effectively improve the drug-loading effect.

Description

DNA nano-drug carrier with high drug loading effect, preparation method and application
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a DNA nano-drug carrier with a high drug loading effect, a preparation method and application thereof.
Background
In recent years, DNA nanotechnology has become a popular field of research at home and abroad, and hundreds of articles about DNA nanotechnology have been published in international top-level journals such as nature and science. DNA is not only the code for life, but it is also a common element for the fabrication of nanoscale structures and devices. Through modern biotechnology, according to simple nucleic acid base pairing rules, DNA molecular chains can be artificially and directionally synthesized, which opens up a wide new place for the application of DNA and is not limited to the field of biological evolution in nature. By utilizing the structural DNA nanotechnology, the precise nanometer framework is realized through the excellent self-assembly and recognition capability of DNA molecules, the fast, sensitive and high-specificity nanometer chip can be manufactured, the DNA nanometer drug carrier which can more accurately position tumor cells and effectively improve drug delivery can be prepared, and the nanometer robot can be developed. Thus, DNA is often viewed in increasing research as a non-biological material rather than as a carrier of genetic information as in living cells.
The DNA nano-drug carrier is a promising tumor treatment tool, can transport various antitumor substances such as drug micromolecules, peptide fragments, proteins, accounting and the like, has more accurate positioning on tumor cells, has more active cell intake, can effectively improve drug delivery, and reduces side effects. At present, the construction of DNA nano-drug carriers is generally divided into two methods of DNA origamy and DNA module assembly. The DNA paper folding technology is that DNA is folded into various 2D and 3D structures like paper by utilizing the specific combination between the structure of DNA molecules and the molecules, but the nano-drug carrier constructed by the DNA paper folding technology has the advantages of uniform shape and size, high complexity, more short chains of the required DNA and high synthesis cost. The DNA module assembly decomposes a target structure into smaller structural units, and each unit is connected by using a viscous tail end which is designed in advance for each structural unit, so that a complete DNA nano-drug carrier is formed. Therefore, the application researches a method for preparing the DNA nano-drug carrier with uniform shape and size and improved drug loading efficiency.
Disclosure of Invention
The method for synthesizing the DNA nano-drug carrier has the advantages of simple steps, low cost, uniform shape and size, and can effectively improve the drug-loading effect.
The invention provides a DNA nano-drug carrier with high drug loading effect, which is prepared from two DNA chains shown in SEQ ID NO.1 and SEQ ID NO.2, or two DNA chains shown in SEQ ID NO.3 and SEQ ID NO.4, or two DNA chains shown in SEQ ID NO.5 and SEQ ID NO. 6.
The preparation method of the DNA nano-drug carrier with the high drug loading effect comprises the following steps:
s1, preparing the following pairs of DNA strands for use:
Staple 1:5’-aatcggaaatccgttcttatcaacgcaagcggacgagtgctg-3’，
Staple 2:5’-cgcttgcgttcagcactcgtcatttccgattgataagaacgg-3’；
alternatively, the length of the complete 3: 5'-gtttacttagggatggtacgaactcaacgcac-3',
Staple 4:5’-agttcgtagtgcgttgaagtaaacccatccct-3’；
alternatively, the length of the complete 5: 5'-aatcggaaatccgttcttatcaacgcaagcggacgagtgctg-3',
Staple 6:5’-gcttgcgttcagcactcgtcctttccgattgataagaacgga-3’；
preparing 1 XTEA buffer solution, wherein the 1 XTEA buffer solution contains 30mmol/L Mg2+20mmol/L Tris, 2mmol/L EDTA, pH 7.6 and ultrapure water as solvent;
s2, adding equal amount of substances such as two DNA strands of one pair in S1 into 1 × TEA buffer prepared in S1 for reaction, wherein the concentration of each DNA strand is 20 μmol/L, and the reaction conditions are as follows: cooling to room temperature at 95 deg.C at a cooling rate of 0.1 deg.C/min, and storing at 4 deg.C to obtain DNA nano-drug carrier with high drug-loading effect.
The application of the DNA nano-drug carrier with high drug-loading effect in carrying anti-tumor drugs.
Compared with the prior art, the invention has the beneficial effects that:
the DNA nano-drug carrier with high drug loading effect provided by the invention has three synthesis methods, is tubular, needs few DNA chains, only needs two single chains, improves the sequence reuse degree, has simple and easy operation of synthesis steps and low cost, has uniform shape and size, can effectively improve the drug loading effect, and has the prospect of large-scale application in follow-up research and clinic.
Drawings
FIG. 1 is a diagram showing the complementary pairing of regions corresponding to Stacke 1 and Stacke 2 in example 1 of the present invention;
FIG. 2 is a step-by-step microscopic view of a high drug loading effect DNA nano-drug carrier in example 1 of the present invention;
FIG. 3 is an atomic force microscope image of the DNA nano-drug carrier with high drug loading effect in example 1 of the present invention;
FIG. 4 is an atomic force microscope image of the DNA nano-drug carrier with high drug loading effect in example 2 of the present invention;
FIG. 5 is an atomic force microscope image of the DNA nano-drug carrier with high drug loading effect in example 3 of the present invention.
Detailed Description
An embodiment of the present invention will be described in detail with reference to fig. 1-5, but it should be understood that the scope of the present invention is not limited to the embodiment, and the reagents involved in the examples can be obtained through common channels.
Example 1
A DNA nano-drug carrier with high drug-loading effect is prepared from two DNA chains shown in SEQ ID NO.1 and SEQ ID NO.2, or two DNA chains shown in SEQ ID NO.3 and SEQ ID NO.4, or two DNA chains shown in SEQ ID NO.5 and SEQ ID NO. 6.
The preparation method of the DNA nano-drug carrier with the high drug loading effect comprises the following steps:
s1, preparing the following pairs of DNA strands (the DNA sequences involved in the invention are all synthesized by Shanghai Biotechnology Co., Ltd.) for use:
staple 1: 5'-aatcggaaatccgttcttatcaacgcaagcggacgagtgctg-3', shown in SEQ ID NO.1,
2, 5'-cgcttgcgttcagcactcgtcatttccgattgataagaacgg-3' as shown in SEQ ID NO. 2;
preparing 1 XTEA buffer solution, wherein the 1 XTEA buffer solution contains 30mmol/L Mg2+20mmol/L Tris, 2mmol/L EDTA, pH 7.6 and ultrapure water as solvent;
s2, adding the equal amount of substances such as two DNA strands in S1 into the 1 XTEA buffer prepared in S1 for reaction, wherein the concentration of the two DNA strands is 20 mu mol/L, and the reaction conditions are as follows: cooling to room temperature at 95 deg.C at a cooling rate of 0.1 deg.C/min, and storing at 4 deg.C to obtain DNA nano-drug carrier with high drug-loading effect. The DNA nano-drug carrier can be used for carrying anti-tumor drugs.
As shown in fig. 1-2, which are microscopic reaction process diagrams of the present invention, each single strand contains 42 nucleotides, both stage 1 (i.e., S1) and stage 2 (i.e., VS1) are in a horizontal "U" shape, stage 1 is divided into A, B, C, D four regions, each of which is composed of 10, 11, 10, and 11 nucleotides; the Staple 2 is divided into four regions of A ', B', C 'and D', and respectively consists of 10, 11, 10 and 11 nucleotides, and corresponding regions between two single-stranded modules are regularly combined through base complementary pairing to form a DNA double-helix structure.
In order to observe the structure of the DNA nano-drug carrier with high drug-loading effect, the atomic force microscope is characterized in that: dropping 3 μ L of the reaction product onto the surface of the mica which has just been cut, and keeping for 2 minutes for adsorption; mg in buffer2+Acting as glue ions to bind the negatively charged DNA to the negatively charged mica surface; two 20. mu.L aliquots of 1 XTAE buffer were then added to the mica and atomic force microscope tips, respectively, and the parameters were adjusted to obtain atomic force microscope images. FIG. 3 is an atomic force microscope scan of the tubular DNA nano-drug carrier synthesized in example 1 at 530nm scale length.
Example 2
Example 2 the only difference from example 1 is that the two DNA strands are replaced by:
staple 3: 5'-gtttacttagggatggtacgaactcaacgcac-3', shown in SEQ ID NO.3,
staple 4: 5'-agttcgtagtgcgttgaagtaaacccatccct-3', shown in SEQ ID NO. 4;
the remaining preparation steps and atomic force microscopy characterization were the same as in example 1. FIG. 4 is an atomic force microscope scanning image of the tubular DNA nano-drug carrier synthesized in example 2 at a length of 1.0 μm scale.
Example 3
Example 3 the only difference from example 1 is that the two DNA strands are replaced by:
staple 5: 5'-aatcggaaatccgttcttatcaacgcaagcggacgagtgctg-3', shown in SEQ ID NO.5,
standard 6: 5'-gcttgcgttcagcactcgtcctttccgattgataagaacgga-3' as shown in SEQ ID NO. 6;
the remaining preparation steps and atomic force microscopy characterization were the same as in example 1. FIG. 5 is an atomic force microscope scan of the tubular DNA nano-drug carrier synthesized in example 3.
It should be noted that the steps and methods adopted in the claims of the present invention are the same as those of the above-mentioned embodiments, and for the sake of avoiding redundancy, the present invention describes the preferred embodiments, but those skilled in the art can make other changes and modifications to these embodiments once they learn the basic inventive concept. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Sequence listing
<120> DNA nano-drug carrier with high drug loading effect, preparation method and application
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 42
<212> DNA
<213> Artificial sequence
<400> 1
aatcggaaat ccgttcttat caacgcaagc ggacgagtgc tg 42
<210> 2
<211> 42
<212> DNA
<213> Artificial sequence
<400> 2
cgcttgcgtt cagcactcgt catttccgat tgataagaac gg 42
<210> 3
<211> 32
<212> DNA
<213> Artificial sequence
<400> 3
gtttacttag ggatggtacg aactcaacgc ac 32
<210> 4
<211> 32
<212> DNA
<213> Artificial sequence
<400> 4
agttcgtagt gcgttgaagt aaacccatcc ct 32
<210> 5
<211> 42
<212> DNA
<213> Artificial sequence
<400> 5
aatcggaaat ccgttcttat caacgcaagc ggacgagtgc tg 42
<210> 6
<211> 42
<212> DNA
<213> Artificial sequence
<400> 6
gcttgcgttc agcactcgtc ctttccgatt gataagaacg ga 42

Claims (3)

1. The DNA nano-drug carrier with the high drug loading effect is characterized by being prepared from two DNA chains shown in SEQ ID No.1 and SEQ ID No.2, or two DNA chains shown in SEQ ID No.3 and SEQ ID No.4, or two DNA chains shown in SEQ ID No.5 and SEQ ID No. 6.
2. The method for preparing the DNA nano-drug carrier with high drug loading effect according to claim 1, comprising the following steps:
s1, preparing the following pairs of DNA strands for use:
Staple 1:5’-aatcggaaatccgttcttatcaacgcaagcggacgagtgctg-3’，
Staple 2:5’-cgcttgcgttcagcactcgtcatttccgattgataagaacgg-3’；
alternatively, the length of the complete 3: 5'-gtttacttagggatggtacgaactcaacgcac-3',
Staple 4:5’-agttcgtagtgcgttgaagtaaacccatccct-3’；
alternatively, the length of the complete 5: 5'-aatcggaaatccgttcttatcaacgcaagcggacgagtgctg-3',
Staple 6:5’-gcttgcgttcagcactcgtcctttccgattgataagaacgga-3’；
preparing 1 XTEA buffer solution, wherein the 1 XTEA buffer solution contains 30mmol/L Mg2+20mmol/L Tris, 2mmol/L EDTA, pH 7.6 and ultrapure water as solvent;
s2, adding equal amount of substances such as two DNA strands of one pair in S1 into 1 × TEA buffer prepared in S1 for reaction, wherein the concentration of each DNA strand is 20 μmol/L, and the reaction conditions are as follows: cooling to room temperature at 95 deg.C at a cooling rate of 0.1 deg.C/min, and storing at 4 deg.C to obtain DNA nano-drug carrier with high drug-loading effect.
3. The use of the DNA nano-drug carrier with high drug loading effect of claim 1 in the preparation of anti-tumor drugs.
CN201810630449.4A 2018-06-19 2018-06-19 DNA nano-drug carrier with high drug loading effect, preparation method and application Active CN108498451B (en)

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CN110124047B (en) * 2019-04-25 2022-03-29 中国科学院上海应用物理研究所 Preparation method of DNA nano robot drug-loaded system and DNA nano robot drug-loaded system obtained by same
CN112451666B (en) * 2020-12-03 2021-07-27 中南大学 Nucleic acid-drug-loaded nano material for improving tumor permeability by VEGF response and preparation method and application thereof

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