CN109646450B

CN109646450B - Application of DNA tetrahedron in preparation of medicine for treating corneal injury

Info

Publication number: CN109646450B
Application number: CN201910106315.7A
Authority: CN
Inventors: 林云锋; 刘楠馨; 张晓琳
Original assignee: Chengdu Tengda Tree Nano Biotechnology Co ltd
Current assignee: Chengdu jingrunze Gene Technology Co.,Ltd.
Priority date: 2019-01-31
Filing date: 2019-01-31
Publication date: 2021-03-12
Anticipated expiration: 2039-01-31
Also published as: CN109646450A

Abstract

In order to solve the problem that the existing cornea injury is lack of specific drugs, the invention provides application of a DNA tetrahedron in preparing a drug for treating cornea injury, wherein the DNA tetrahedron is a tetrahedron nanostructure formed by four DNA single-strands through base complementary pairing. The invention can effectively promote tissue regeneration, realize the healing of injured cornea, has no toxic or side effect and has simple preparation process.

Description

Application of DNA tetrahedron in preparation of medicine for treating corneal injury

Technical Field

The invention relates to the field of ophthalmic medicines, in particular to application of a DNA tetrahedron in preparing a medicine for treating corneal injury.

Background

Corneal injury is one of the clinically common and intractable ocular trauma, mainly chemical burns, and the most common of chemical burns is alkali burns. Aseptic corneal ulcer, perforation, eyeball adhesion, secondary glaucoma and the like which are generated after corneal injury are more common causes of ocular disability.

Clinical treatment modes of corneal injury comprise timely and thorough washing of conjunctival sac, sub-conjunctival injection of vitamin C, atropine mydriasis, local and systemic antibiosis and antiphlogosis, nourishing of cornea, amnion transplantation, autohemotherapy, covering of affected eyes and the like, but the medicines can only slightly relieve the state of an illness and have poor treatment effect.

The research on the use of corticosteroid hormone for treating corneal injury shows that the corticosteroid hormone has anti-inflammatory and immunosuppressive effects and has certain therapeutic effect. Corticosteroid hormones, however, inhibit tissue regeneration and have other potential side effects, and the therapeutic effect as a whole is still insufficient.

Disclosure of Invention

The invention aims to provide a novel medicine for treating corneal injury.

The invention firstly provides the application of a DNA tetrahedron in the preparation of a medicine for treating corneal injury, wherein the DNA tetrahedron is a tetrahedron nano structure formed by four DNA single chains through base complementary pairing; preferably, the injury is an alkali burn.

Further, the DNA tetrahedron is prepared by denaturation of the four DNA single strands at 90-98 ℃ for 10-15 min and annealing at 2-8 ℃ for 20-30 min.

Further, the DNA tetrahedron is prepared by the four DNA single strands through denaturation at 95 ℃ for 10min and annealing at 4 ℃ for 20 min.

Further, the edge length of the DNA tetrahedron is 10-100 bp.

Furthermore, the sequences of the four single strands forming the DNA tetrahedron are shown in SEQ ID NO. 1-4.

Further, the medicament is an eye drop.

Further, the using concentration of the medicine is 100-500 nM.

Further, the drug was used at a concentration of 250 nM.

The invention also provides a medicine for treating corneal injury, which is prepared by taking the DNA tetrahedron as an active ingredient and adding pharmaceutically acceptable auxiliary materials; preferably, the injury is an alkali burn.

Further, the medicament is an eye drop.

The inventor provides a drug which is different from the traditional drug pharmacology through the research on the corneal damage and the DNA tetrahedron. The traditional medicine mainly depends on inflammation diminishing, so that the cornea is recovered automatically, and the effect is poor; the invention has the function of promoting the proliferation and migration of corneal epithelial cells, and has better effect of treating corneal injury.

The invention has the following beneficial effects:

1) the DNA tetrahedron is safe and nontoxic;

2) the DNA tetrahedron of the invention can promote the proliferation and migration of corneal epithelial cells;

3) the DNA tetrahedron can promote tissue regeneration, and further promote corneal epithelium healing;

4) the DNA tetrahedron synthesis method is simple.

Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.

The foregoing aspects of the present invention are explained in further detail below with reference to specific embodiments. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.

Drawings

FIG. 1: schematic representation of DNA tetrahedral synthesis.

FIG. 2: DNA tetrahedral transmission electron microscopy.

FIG. 3: and detecting a particle size distribution diagram by DNA tetrahedral dynamic light scattering.

FIG. 4: three migration tests; a, cell scratch test; b, transwell test crystal violet staining result; c, cell scratch test 24h quantitative statistical result, D, transwell test statistical result, E, RTCA test result.

FIG. 5: three proliferation tests; a, CCK-8 test results; b, RTCA test results; c, BrdU immunofluorescence staining test results.

FIG. 6: a graph of the healing effect of rabbit eye corneal injury; a, general appearance of rabbit eyes after corneal injury; b, the score statistic result of clinical examination, and the corneal transparency score of 8 rabbits at 7 days; c, scoring statistics of clinical examination, corneal transparency scoring of 4 rabbits at 14 days; d, the score statistic result of clinical examination, and the corneal epithelial healing rate of 8 rabbits at 7 days; e, score statistics for clinical examination, corneal epithelial healing rate of 4 rabbits at 14 days.

Detailed Description

EXAMPLES preparation and characterization of DNA tetrahedrons

1. Method of producing a composite material

1.1 preparation of DNA Tetrahedron (TDN)

TDN is synthesized by self-assembly of four uniquely designed DNA single strands (S1, S2, S3, S4) through a rapid, simple and specific PCR procedure (95 ℃ for 10min, rapid cooling to 4 ℃ for 20min, and long-term storage at 4 ℃). The four single strands were added in equimolar proportions (1. mu.l stock solution at 100. mu.M concentration per single strand) to a solution containing 96. mu.l of TM buffer (10mM Tris-HCl, 50mM MgCl. RTM.)₂pH 8.0) was heated to 95 ℃ for 10min, and then rapidly cooled to 4 ℃ to synthesize TDN.

1.2 specific sequences of the four DNA single strands are as follows:

1.3 polyacrylamide gel electrophoresis, Dynamic Light Scattering (DLS), Atomic Force Microscopy (AFM), Transmission Electron Microscopy (TEM) and Charge determination characterization for TDN:

r DLS: the synthesized TDN was diluted to 250nM with double distilled water and then observed on a ZETAPals analyzer.

AFM: the atomic force microscope is used for characterizing the surface topography of the TDN nano-particles and is completed by a Shimadzu SPM-9700 atomic force microscope in a tapping scanning mode. TDN was diluted to 20nM with TM buffer solution, then 10. mu.l of this solution was dropped on fresh mica plates to dry for about 15min before observation.

③ TEM: the transmission electron microscope observes the microstructure of the TDN, and shows that the TDN nano material is small particles with the particle size of 10nm and uniform distribution.

Zeta potential: the potential of the single-stranded, TDN was measured using Zetasizer Nano ZS90 (Malverm Instruments Ltd, U.K.).

2. Results

The tetrahedral structure appears under the field of transmission electron microscopy, indicating that the DNA tetrahedron assembly is successful (FIG. 2). The dynamic light scattering results show that the particle size of the tetrahedra is about 10nm (FIG. 3). From the above results, the synthesis of TDN was successful.

Experimental example 1 cell migration experiment

Wound healing is a dynamic, strictly ordered biological process in which re-epithelialization plays a very important role. Re-epithelialization of the wound surface relies primarily on the migration of epithelial cells from the wound margin to the center of the wound surface. To investigate whether TDN contributes to migration of human corneal epithelial cells, the inventors conducted the following experiment.

1. Method of producing a composite material

Scratch test, transwell test and RTCA method were used to examine the effect of TDN on migration of human corneal epithelial cells.

And (3) scratch test: cells were plated at 1.5X 10⁵And inoculating the cells into a 12-well plate at a density of one hole per hole for culture, scratching two vertically crossed scratches by using a sterile gun tip after the cells are fully paved by 80-90%, washing away cell fragments, adding a cell culture medium with the TDN concentration of 125nM, 250nM and 375nM to obtain a test group, and adding a cell culture medium without the TDN to obtain a control group. And collecting images with the scratches closed at 0h, 12h and 24h, and performing qualitative and semi-quantitative analysis.

Transwell test: the transwell membrane pore size was chosen to be 8 μm. Seeding of the upper chamber of a 24-well transwell cell 5X 10⁴Each well of cells, the volume of the culture medium is 250 mul, after 24h of culture, the growth medium is replaced by DMEM medium containing 1% fetal bovine serum and 125nM and 250nM TDN, this group is the experimental group; the control group was replaced with DMEM medium containing 1% fetal bovine serum and no TDN. After 24 hours, migrated cells were washed 3 times with PBS, fixed with methanol for 20min, stained with crystal violet and plotted, and the number of migrated cells in the experimental and control groups was analyzed.

RTCA migration test: adding 165 ul of culture medium containing 1% fetal calf serum into the lower-layer pore plate of the RTCA, adding 30 ul of culture medium with the same components into the upper-layer pore plate, and placing the upper and lower plates in a cell culture box for standing for 1 hour after the upper and lower plates are assembled. 1 hour, add cell suspension and TDN solution to make cell seeding density 5X 10⁴Per well, TDN concentrations of 0nM, 125nM and 250 nM. The cell migration was measured every 15 minutes for a total of 24 hours by the RTCA instrument, and a migration curve was generated.

2. Results

In the cell scratch test, the number of cell migration in the test group was larger than that in the control group at 12h and 24h, and the cell migration was fastest in the group with the TDN concentration of 250nM (FIG. 4A, C).

In the Transwell assay, the picture of crystal violet staining after 24 hours showed that TDN was able to promote cell migration and that the promotion was strongest at 250nM (fig. 4B, D).

In the RTCA assay, migration of 24h cells was continuously measured, and the curves show that TDN stably promotes migration of corneal epithelial cells, and that TDN concentration of 250nM is the best effect (fig. 4E).

Experimental example 2 cell proliferation experiment

1. Method of producing a composite material

To examine the effect of TDN on the proliferative behavior of corneal epithelial cells, three experiments were performed.

CCK-8 test: the cells were cultured at 5X 10³The density of each well was plated in 96-well plates, and after 24h of growth medium, the plates were replaced with serum-free medium containing different concentrations (0nM, 125nM, 250nM, 375nM) of TDN, cck-8 absorptiometry assays were performed at 6h, 12h, 24h, 36h, respectively, according to the method used for cck-8 products, and statistical analysis was performed using SPSS.

BrdU cell proliferation assay: the corneal epithelial cells are expressed at 1 × 10⁴The cells/ml were inoculated in confocal dishes and after 24 cultures with growth medium, the medium was changed to medium containing 10 μm BrdU and different concentrations (0nM, 250nM) of TDN, after 24h the medium was aspirated, washed with PBS, acid-hydrolyzed and immunofluorescent-stained. Immunofluorescence pictures were analyzed to investigate proliferation.

RTCA cell proliferation assay: cells were plated at 4X 10³The density of each well was plated in 16-well plates and the growth medium was incubated overnight, the medium was changed the next day, with concentrations of TDN of 0nM, 125nM and 250 nM. After 65 hours of continuous detection, the proliferation rule of the corneal epithelial cells is analyzed according to the proliferation curve.

2. Results

The three proliferation tests all verified that TDN promotes proliferation of corneal epithelial cells, and the concentration of TDN is the optimal working concentration at 250nM (FIG. 5).

The results of experimental example 1 and experimental example 2 show that the DNA tetrahedron can promote the proliferation and migration of corneal epithelial cells and can promote the migration of corneal endothelial cells.

Experimental example 3 animal experiments

1. Method of producing a composite material

1.1 corneal Damage modeling

2.5-3 kg New Zealand white rabbits are used as experimental animals to manufacture an in-vivo alkali burn model, and the specific operations are as follows: the rabbit is subjected to muscle relaxation anesthesia, 10g/L of dicaine is dripped into eyes for surface anesthesia for 3 times, a filter paper sheet soaked with NaOH is attached to the center of a cornea by an ophthalmic forceps, the filter paper sheet is taken down after being closely contacted with the cornea for 20s, and the surface of the cornea and a conjunctival sac are immediately washed by physiological saline for 1 min. A discoid white lesion with a clear central border of the cornea is formed.

1.2 drug treatment

The model animals were 8 animals, the left eye was the control group, the right eye was the test group, and the following treatments were performed:

test groups: 250 nmRNA Tetrahedron (TDN) eye drops prepared by normal saline; control group: saline, one drop at a time, six times a day.

4 test rabbits were sacrificed 7d after dosing, and the remaining 4 test rabbits were sacrificed 14d after dosing. The speed of corneal epithelial healing and corneal light transmission were observed for the control and test groups.

2. Results

Cornea light transmission (fig. 6A, B, C) and corneal epithelium healing rate (fig. 6A, D, E) in clinical evaluation, it can be seen that the cornea treated with TDN eye drops heals faster than the control group, and the final healing effect is also superior to the control group.

Indicating that TDN can effectively promote the healing of injured cornea.

In conclusion, the DNA tetrahedron can effectively promote tissue regeneration and realize healing of injured cornea, and has no toxic or side effect and simple preparation process; the DNA tetrahedron has excellent industrialization prospect in the preparation of the cornea injury medicament.

SEQUENCE LISTING

<110> Sichuan university Hospital in western China

Application of <120> DNA tetrahedron in preparation of medicine for treating corneal injury

<130> CD007-701012531

<160> 4

<170> PatentIn version 3.5

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acggtattgg accctcgcat gactcaactg cctggtgata cgaggatggg catgctcttc 60

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Claims

Use of a DNA tetrahedron, the DNA tetrahedron being a tetrahedral nanostructure formed from four DNA single strands through base complementary pairing, in the manufacture of a medicament for treating corneal injury;

the sequences of the four single chains forming the DNA tetrahedron are shown in SEQ ID NO. 1-4.
2. Use according to claim 1, characterized in that: the injury is an alkali burn.
3. The use according to claim 1, wherein the DNA tetrahedron is prepared by denaturing the four DNA single strands at 90-98 ℃ for 10-15 min and annealing at 2-8 ℃ for 20-30 min.
4. The use according to claim 2, wherein the DNA tetrahedron is prepared from the four DNA singlestrands by denaturation at 95 ℃ for 10min and annealing at 4 ℃ for 20 min.
5. The use of claim 1, wherein the medicament is an eye drop.
6. The use according to claim 5, wherein the medicament is used at a concentration of 100 to 500 nM.
7. The use of claim 6, wherein the medicament is administered at a concentration of 250 nM.