CN108187062B

CN108187062B - Construction method of nucleic acid-based nano-drug carrier

Info

Publication number: CN108187062B
Application number: CN201711304393.5A
Authority: CN
Inventors: 吕浩源; 郭家义; 纪小婷; 丁彩凤
Original assignee: Qingdao University of Science and Technology
Current assignee: Qingdao University of Science and Technology
Priority date: 2017-12-11
Filing date: 2017-12-11
Publication date: 2020-10-02
Anticipated expiration: 2037-12-11
Also published as: CN108187062A

Abstract

The invention discloses a nucleic acid-based nano drug carrier, which is a columnar rigid structure formed by 6 groups of nucleic acids GJ1, GJ2, GJ3, GJ4, GJ5 and GJ6 through complementary pairing of bases, wherein the middle part of the columnar rigid structure is a through columnar hole with the diameter of 1.8-2nm, two ends of the carrier are blocked by locked nucleic acid single chains lock1 and lock3, one part of the blocking sequence lock1 at one end is a complementary sequence of target aptamer mRNA, the whole length of the carrier measured through dynamic light scattering is 40 +/-4.3 nm, when the target aptamer mRNA exists in a cell, an entropy substitution process occurs because the number of the pairing bases of the target mRNA and lock1 is far larger than the number of the combination of the lock1 and a basic structure port of the carrier, so that one end of the carrier is opened, and drugs in the carrier are released. The invention utilizes the nucleic acid existing in the organism as the raw material, regulates and controls the pairing condition among the basic groups through manual design, leads the nucleic acid to carry out three-dimensional folding to form a paper folding structure with controllable appearance, and is used as a basic carrier for loading the medicine.

Description

Construction method of nucleic acid-based nano-drug carrier

Technical Field

The invention relates to a construction method of a nucleic acid-based nano-drug carrier.

Background

The nano carrier is used as an important nano material, has a wide application prospect in the biomedical field due to excellent penetration and carrying performance and controllable drug release capacity, and one of the most important directions is the development of an anti-tumor targeted sustained-release drug.

The design of a drug delivery system aims at solving the problem of drug targeting, many chemical drugs achieve good treatment effects in vitro, but the effects are not obvious in human body, because the drugs lack targeting and stability in the human body environment, special drug delivery technologies need to be developed to overcome the problems of low solubility, instability in the organism, low bioavailability, potential biological toxicity to normal tissues and the like, the good drug delivery system is the key point for solving the problems, and the core is the design of a drug carrier with targeting.

At present, the core problems in the research field of nano-drug carriers mainly focus on the following three aspects:

1. how to improve the bioavailability of the drug. About 40% of the drugs developed by human beings are insoluble in water, while the circulatory system in the animal body is mainly based on the water phase, and the water-insoluble drugs are difficult to uniformly diffuse in the water-insoluble drugs, so that the bioavailability is reduced. Taking the antitumor drug paclitaxel as an example, the current common method mainly comprises the steps of wrapping the paclitaxel by using liposome, and controlling the particle size of the paclitaxel to form emulsion in vivo so as to ensure that the paclitaxel has better fluidity and dispersibility when being transported along with blood.

However, the method still has obvious problems in practical application, although the liposome is used for wrapping the drug, the affinity of the liposome and a cell phospholipid bilayer can be utilized to promote the endocytosis of the cell and improve the bioavailability of the drug, but the targeted delivery cannot be realized, so the biotoxicity of the biological tissue in a non-target area is obviously increased.

2. How to maintain stable release of the loaded drug. In clinical treatment, it is necessary to ensure that the drug can maintain sufficient concentration at a lesion site within a certain period of time, and at present, polymers such as polyethylene glycol (PEG), Polyethyleneimine (PEI) are commonly used to form a polymer protective layer by covalent bonding or self-assembly with drug molecules, so as to encapsulate the drug, prevent the drug from being rapidly metabolized or cleared in vivo, avoid phagocytosis by the reticuloendothelial system, and release the drug continuously and stably, thereby achieving the purpose of long-term blood circulation.

Although the encapsulation of the copolymer can promote the safe and stable transportation and slow release of the drug, the biological safety of the method is also potentially hidden due to the similarity of the biological hormones of the copolymer monomers and the uncertainty of the degradation degree and the degradation period in vivo.

3. How to achieve targeted drug delivery. The method is mainly characterized in that the surface of a nano carrier is modified by utilizing components with target recognition capability such as specific aptamers, antibodies/antigens and the like, the modified carrier can overcome various physiological barriers encountered in the in-vivo delivery process of the drug, the drug is delivered to a target site and is slowly released, and for bioactive molecules with short half-life period such as protein and the like, the carrier can overcome the in-vivo physicochemical barrier due to the isolation effect of the carrier, so that the drug is prevented from being degraded before reaching the target site.

The method is convenient and feasible, but due to uncertainty of intermolecular handshake recognition, a target-off phenomenon often occurs, so that adverse reactions such as local normal tissue necrosis and the like are caused by early release of loaded drugs, and meanwhile, the targeting method cannot cope with drug resistance variation of target cells, normal drug delivery cannot be performed if the target disappears, and the treatment requirements of late-stage tumor diffusion and low specific expression at the early stage of pathogenesis cannot be met better.

Disclosure of Invention

The invention aims to solve the technical problem of establishing a novel nano-drug carrier platform by using nucleic acid which is abundant in organisms and good in biocompatibility as a material and adopting a paper folding structure of DNA, and the novel nano-drug carrier platform has the advantages of good wrapping affinity of conventional liposome, slow release stability of a copolymer structure and targeting effect of aptamer recognition, and simultaneously solves the problems that the liposome lacks targeting property, and the biological safety of the copolymer and the aptamer recognition effect are single.

The technical problem solved by the invention is realized by adopting the following technical scheme: a nucleic acid-based nano-drug carrier construction method is based on the design principle of a DNA origami structure, and is based on the control of the pairing condition among complementary bases, a nucleic acid nano-carrier structure is designed, the main body of the nucleic acid nano-carrier structure is 6 groups of columnar rigid structures (formed by mutually spirally hybridizing sequences GJ1, GJ2, GJ3, GJ4, GJ5 and GJ6 respectively, the combination state of the 6 nucleic acid sequences is shown in figure 2), the middle of the nucleic acid nano-carrier structure is a penetrating columnar hole with the diameter of 1.8-2nm, two ends of the carrier are blocked by locking nucleic acid single-chains lock1 and lock3, one part of the blocking sequence lock1 at one end of the nucleic acid nano-drug carrier structure is a complementary sequence of target aptamer mRNA, and the overall length of the carrier measured by dynamic light scattering is 40 +/-4.3 nm. When target aptamer mRNA exists in cells, the number of pairing bases of the target mRNA and lock1 is far larger than the number of combination of lock1 and a port of a basic structure of the carrier, so that an entropy substitution process occurs, one end of the carrier is opened, and the drug in the carrier is released. Meanwhile, due to the acidic environment in the tumor cells, after one end of the carrier is opened, the rigid structure is damaged, the integral stability is reduced, the pH response is generated, the integral framework is scattered, the original structure is lost, so that the micromolecule anti-tumor drug embedded in the double-stranded nucleic acid structure can be slowly released, the effect of carrying drugs with different mechanisms together is achieved, and the loading efficiency of micromolecule nucleic acid interference drugs is improved. On the surface of the nano-carrier structure, because the nucleic acid sequence forming the structure is subjected to carboxylation modification, free carboxyl exists, and the RGD cyclopeptide is modified on the surface of the structure by utilizing the condensation effect of the free carboxyl and the free amino on the surface of the RGD cyclopeptide, so that the combination of the nano-carrier and target cells and the promotion of endocytosis are realized.

The invention has the beneficial effects that:

1. the method takes nucleic acid as a raw material, utilizes a DNA paper folding technology, creates a new active transport carrier, provides a new carrier mode and thought for drug targeted transport, and is different from the prior liposome loading technology which lacks targeting and the biological toxicity of carrier microspheres in a high polymer sustained release microsphere technology. The biological safety is enhanced while the bioavailability is improved.

2. Nucleic acid existing in organisms is used as a raw material, and the pairing condition among bases is regulated and controlled through artificial design, so that the nucleic acid is subjected to three-dimensional folding to form a morphology-controllable paper folding structure which is used as a basic carrier for loading drugs.

3. By using the specific mRNA aptamer in the cell as a target instead of the traditional recognition of the cell surface aptamer, the problems of off-target, cell drug resistance variation, reduction of drug error release caused by recognition error, local tissue necrosis and the like are solved.

4. The high affinity of the nano-carrier and cells is realized through the modification of the surface biological affinity cyclopeptide, the cell utilization rate is improved, and the particle size of a finished product is easier to control relative to liposome, so that the drug transportation in a living body is facilitated.

5. The size of the carrier is accurately controlled, so that the medicine can enter organs and tissues through gaps on capillaries, the treatment effect is improved at a target part, and the toxic and side effects of other tissues or organs in a body are reduced.

6. Compared with the traditional capsule loading, the scheme realizes the double loading effects of content loading and structure insertion loading by utilizing high-concentration permeation and a nucleic acid insertion mechanism of small-molecule anticancer drugs, and effectively improves the drug loading efficiency.

7. The carrier structure is internally provided with uniform and consistent pore channels, so that the mixed loading of various medicines which accord with the pore size can be realized, and a microporous structure with different polydispersion sizes in the traditional medicine carrier is not adopted, so that the carrier structure is more suitable for the application of the cocktail therapy in the current clinical treatment.

8. The 6-strand double-helix rigid structure can effectively reduce the degradation effect on a carrier in a living body, has the pH response capability, and after entering the inside of a subacid tumor, one end of the double-helix rigid structure is opened on the premise of existence of target mRNA so as to break down a skeleton and slowly release the micromolecular drug embedded in the double-helix rigid structure.

Drawings

FIG. 1 is a general structure diagram of a nano-carrier

FIG. 2 is a schematic diagram showing the binding of single-stranded nucleic acids of nanocarriers to each other

FIG. 3 is a verification chart of the transport effect of the drug-loaded carrier

FIG. 4 is an ultraviolet characterization diagram of RGD modification on the surface of a drug-carrying carrier

FIG. 5 is a fluorescence characterization chart of the synergistic release of two drugs

Fig. 6 is a representation of dynamic light scattering of drug-loaded carriers.

Detailed Description

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further explained below by combining the specific drawings.

Example (b): the drug-loaded fluorescent paclitaxel and adriamycin are used;

construction of (A) basic vector Structure

1. Respectively taking 10 mu L of 7 nucleic acid single chains (GJ1, GJ2, GJ3, GJ4, GJ5, GJ6 and LOCK3) with the concentration of 10 mu Mol/L into a small centrifuge tube, uniformly mixing by using a mixing instrument, placing in a water bath at 95 ℃ for heating for 4 minutes, then taking out, slowly cooling to room temperature to obtain a basic carrier structure, and storing in an environment at 4 ℃ for later use.

(II) modification of RGD cyclic polypeptide on structure surface

1. 50 mu L of amino modified nucleic acid single-chain (MB) with the concentration of 10 mu mol/L is taken to be arranged in a centrifuge tube, then 100 mu L of NHS solution with the concentration of 0.2mol/L is added, and the mixture is reacted for 30 minutes at room temperature and then is mixed uniformly by a mixer for standby.

2. And (3) putting 10 mu L of magnetic beads with surface carboxyl modified into a centrifuge tube, adding 100 mu L of EDC solution with the concentration of 0.8mol/L, reacting for 30 minutes at room temperature, and uniformly mixing by using a mixing machine for later use.

3. And (3) putting 100 mu L of the solution after the reaction in a centrifuge tube, placing the centrifuge tube in an environment of 37 ℃ of a shaking table, carrying out light-shielding oscillation reaction for 10h, taking out the centrifuge tube, carrying out magnetic separation and washing three times by using Tris-HCl buffer solution with the pH of 7.2, discarding supernatant, keeping magnetic beads, adding 100 mu L of LTris-HCl buffer solution, and uniformly mixing the mixture by using a mixing instrument for later use.

4. And (3) taking 50 mu L of the basic carrier structure obtained in the step (I) into a centrifuge tube, then adding 100 mu L of EDC solution with the concentration of 0.8mol/L, reacting for 30 minutes at room temperature, and then uniformly mixing by using a mixing machine for later use.

5. 50 mu L of RGD cyclopeptide solution with the concentration of 25 mu mol/L is taken to be put into a centrifuge tube, 100 mu L of NHS solution with the concentration of 0.2mol/L is added, and the mixture is reacted for 30 minutes at room temperature and then is mixed uniformly by a mixing instrument for standby.

6. Respectively putting 50 mu L of the solution after the reaction in a centrifuge tube, placing the centrifuge tube in an environment at 37 ℃ of a shaking table, carrying out a light-shielding oscillation reaction for 10h, taking out the centrifuge tube, uniformly mixing the solution by using a mixing instrument, adding 100 mu L of magnetic beads with nucleic acid chains bound on the surfaces, obtained in the previous step, reacting the mixture for 3 h in the shaking table at 37 ℃, taking out the mixture, using a Tris-HCl buffer solution with the pH of 7.2 in a water bath environment at 15 ℃, carrying out magnetic separation and washing for three times, discarding the supernatant, keeping the magnetic beads, adding 100 mu L of the Tris-HCl buffer solution, and uniformly mixing the mixture by using the mixing instrument for later use.

7. And heating the solution after the reaction in a water bath at 60 ℃ for 5 minutes, performing magnetic separation in a water bath at 50 ℃ for three times, and reserving the supernatant in a new centrifugal tube for later use to obtain the surface RGD cyclic polypeptide modified nano-carrier structure.

(III) drug Loading

1. And (3) putting 50 mu L of the nano carrier structure obtained in the step (II) and modified by the surface GD cyclic polypeptide into a centrifuge tube, sequentially adding 20 mu L of adriamycin solution with the concentration of 500 mu M and 20 mu L of taxol solution with the concentration of 500 mu M modified by the fluorescent group, carrying out vibration reaction in a shaking table at 37 ℃ in a dark place for 12 hours, and taking out for later use, thus completing the loading of the medicine.

(IV) end plugging and purification of vector

1. And (3) adding 50 mu L of the nucleic acid single strand (LOCK1) with the concentration of 10 mu mol/L and 30 mu L of the nucleic acid single strand (LOCK2) with the concentration of 10 mu mol/L into the solution obtained in the step (three) respectively, adding 50 mu L of the magnetic beads with the surface combined with the nucleic acid chains obtained in the step (two), placing the mixture into a shaking table, carrying out light-proof oscillation reaction at 37 ℃ for 3 hours, and then taking out the mixture.

2. Adding the solution obtained in the last step into a Tris-HCl buffer solution with the pH value of 7.2, performing magnetic separation and washing for three times in a water bath environment at 15 ℃, removing the supernatant, reserving a magnetic adsorption part, adding 100 mu of the LTris-HCl buffer solution, and uniformly mixing the solution with a mixing instrument for later use.

3. Heating the solution after the reaction in a water bath at 60 ℃ for 5 minutes, performing magnetic separation in a water bath at 50 ℃ for three times, and keeping the supernatant in a new centrifugal tube for later use to obtain the carrier which can complete the plugging of the carrier, so as to obtain the drug-loaded carrier separated and purified from the liquid medicine.

(V) transfecting the tissue cells by using the drug carrier;

1. and (3) putting 30 mu L of the nano drug-loaded carrier loaded with the drug into a centrifuge tube, adding 1ml of 1640 culture solution containing 20% of serum, uniformly mixing, adding into a cell culture dish for cell culture, and performing mixed culture for 3 hours in an incubator at 37 ℃ under the condition of 5% carbon dioxide.

(VI) releasing and detecting the drug;

1. taking out the culture dish cultured for 3 hours in the step (five), discarding the culture solution, adding fresh 1640 culture solution, repeatedly washing for three times, and adding 1ml culture solution containing 20% serum for standby.

2. The culture dish is moved to a laser confocal microscope to observe the fluorescence distribution condition in the tumor cells, emission lights with the wavelength of 525nm and the wavelength of 585nm are respectively collected by taking 495nm as an exciting light, the distribution of the fluorescence is consistent with the action mechanism of the anti-tumor drug, the orange-red fluorescence of the adriamycin is gathered in a cell nucleus area, and the green fluorescence of the fluorescent paclitaxel is gathered in a cytoplasm, so that the successful release of the fluorescent paclitaxel in the cells and the good anti-tumor performance of the drug are represented.

3. And (3) repeatedly washing the culture dish in the last step for 3 times by using PBS (phosphate buffer solution) with the pH value of 7.0, then crushing the cells in the culture dish by using an ultrasonic crushing instrument, centrifuging, taking supernatant, repeating for three times, taking 100 mu L of supernatant into a micro cuvette, scanning the fluorescence generation condition in the range of 500-700nm by using 495nm as an excitation wavelength in a fluorescence spectrophotometer, and observing two emission peaks of 525nm and 585nm, wherein the two emission peaks respectively correspond to the loaded drugs of the fluorescent paclitaxel and the doxorubicin, and further determining the successful release of the drugs in the cells.

As shown in figure 1, FITC-PTX and DOX are two antitumor drugs of fluorescent paclitaxel and adriamycin respectively, RGD is surface-modified cyclic peptide, 6 nucleic acid single chains are combined into a cylindrical framework structure, the specific combination mode is shown in figure 2, LOCK3 is a blocking nucleic acid single chain 1, and LOCK1 is a blocking nucleic acid single chain 2 with a target mRNA recognition sequence.

As shown in FIG. 2, 1, 2dock, 3, 4, 5dock and 6 in the figure correspond to GJ1, GJ2, GJ3, GJ4, GJ5 and GJ6 in the sequence table respectively, and the single strands form a single-helix structure with surrounding single strands in a manner of mutually pulling hands through complementary pairing between bases to form a main cylindrical hollow structure of the nano carrier.

Wherein the nucleic acid sequences of GJ1, GJ2, GJ3, GJ4, GJ5, GJ6, lock1, lock2, lock3 and MB are as follows:

as shown in fig. 3, where bright is a cell state diagram shown in a microscope bright field, PTX-FITC is an imaging image in a fluorescent paclitaxel channel, DOX is an imaging image in an doxorubicin channel, DAPI is a nuclear staining reagent for confirming the location of a cell nucleus, and Merge is a superimposed image of the above channels, it can be seen that since doxorubicin acts on the cell nucleus and the distribution thereof is consistent with DAPI, the superimposed image is pink, and fluorescent paclitaxel is distributed in cytoplasm and acts on tubulin, so the surrounding cytoplasm is green fluorescence.

As shown in fig. 4, three curves a, b, and c in the figure correspond to a carrier structure, a simple RGD cyclopeptide, and a carrier structure modified with the RGD cyclopeptide, respectively, and it is known from the figure that nucleic acid has characteristic ultraviolet absorption at 280nm, the RGD cyclopeptide has an obvious absorption peak at 215, and after the surface of the nucleic acid is modified with the RGD cyclopeptide, the ultraviolet spectrum of the nucleic acid has both absorption peaks and an overall blue shift.

As shown in FIG. 5, a in the graph is a blank group, b is an experimental group, and it can be seen from the graph that the experimental group has relatively strong characteristic emission peaks near 525nm and near 585nm, which are consistent with the emission wavelengths of fluorescent paclitaxel and doxorubicin, and the drug release effect is proved to be good.

Fig. 6 shows that a is a dynamic light scattering characterization diagram of a basic structure of a nano-carrier, and B is a dynamic light scattering characterization diagram of a carrier with RGD cyclopeptide modified on the surface, as shown in the diagram, before the RGD cyclopeptide is modified, the particle size distribution of the carrier is about 20nm, and after the RGD cyclopeptide is modified, the particle size distribution of the carrier is about 40nm, which meets the design expectation of a system, and the particle size can meet the requirement of diffusion transportation in a living body.

While there have been shown and described what are at present considered the fundamental principles and essential features of the invention and its advantages, it will be understood by those skilled in the art that the invention is not limited by the embodiments described above, which are merely illustrative of the principles of the invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the invention as defined by the appended claims and their equivalents.

Sequence listing

<110> Qingdao university of science and technology

<120> construction method of nucleic acid-based nano-drug carrier

<160>10

<170>SIPOSequenceListing 1.0

<210>1

<211>50

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

agcgaacgtg gattttgtcc gacatcggca agctcccttt ttcgactatt 50

<210>2

<211>86

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

ccgatgtcgg acttcgtgcg cttttagtca cgttacacga tcttcgcctg ctgggttcga 60

gggtcttttc atcatttggg agcttg 86

<210>3

<211>50

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

cgaagatcgt gtttttccac agttgattgc ccttcacttt tcccagcagg 50

<210>4

<211>50

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

aatcaactgt ggtttttctc actggtgatt agaatgcttt tgtgaagggc 50

<210>5

<211>86

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

tcaccagtga gattgcacgg cttttcgacg gatttgtcgt accaggtgca tggatttacc 60

gagtttttta tcatcttgca ttctaa 86

<210>6

<211>50

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

cctggtacga catttttcca cgttcgctaa tagtcgattt tatccatgca 50

<210>7

<211>77

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

caccgatcgg tgagctcggt tgtcctggat gatgatgttt tcaaccctcg gttgtcctga 60

tgatgatgtt ttcaacc 77

<210>8

<211>14

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

ctcaccgatc ggtg 14

<210>9

<211>126

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

gggagacaag aataaacgct caagcagttg atcctttgga taccctggtt cgacaggagg 60

ctcacaacag gcgaaaatca gtgctttttt ttcgtgccga aaagctgcct ttttttttgc 120

acgcgc 126

<210>10

<211>11

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

ctcccttttt t 11

Claims

1. A nano-drug carrier based on nucleic acid is characterized in that: the drug carrier is a columnar rigid structure formed by 6 groups of nucleic acids GJ1, GJ2, GJ3, GJ4, GJ5 and GJ6 through complementary pairing of bases, the middle part of the drug carrier is a through columnar hole with the diameter of 1.8-2nm, two ends of the carrier are blocked by locking nucleic acid single chains lock1 and lock3, part of a blocking sequence lock1 at one end of the drug carrier is a complementary sequence of target aptamer mRNA, the overall length of the carrier measured through dynamic light scattering is 40 +/-4.3 nm, when the target aptamer mRNA exists in a cell, an entropy substitution process is caused to occur due to the fact that the number of the paired bases of the target mRNA and lock1 is far larger than the number of combination of the lock1 and a basic structure port of the carrier, one end of the carrier is opened, and drugs in the carrier are released; wherein, the sequences of GJ1, GJ2, GJ3, GJ4, GJ5 and GJ6 are respectively shown as SEQ ID NO. 1, 2, 3, 4, 5 and 6, the sequence of lock1 is shown as SEQ ID NO. 7, and the sequence of lock3 is shown as SEQ ID NO. 9.