CN116139285B

CN116139285B - Biological switchable miR nano-composite based on DNA tetrahedral framework nucleic acid and preparation method and application thereof

Info

Publication number: CN116139285B
Application number: CN202210794952.XA
Authority: CN
Inventors: 林云锋; 李松航; 田陶然; 蔡潇潇
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2022-07-07
Filing date: 2022-07-07
Publication date: 2024-05-31
Anticipated expiration: 2042-07-07
Also published as: CN116139285A

Abstract

The invention provides a biological switchable miR nano-composite based on DNA tetrahedron framework nucleic acid, a preparation method and application thereof, and belongs to the field of nucleic acid molecule medicines. The nano-composite is tetrahedral framework nucleic acid obtained by base complementary pairing of 3 oligonucleotide chains and 3 micro RNAs with enzyme cleavage sites connected to the tail ends; each oligonucleotide strand consists of 2 DNA strands and 2 RNA strands in the order of RNA ₁-DNA₁-RNA₂-DNA₂; the DNA ₁ strands of the 3 oligonucleotide strands form 3 sides of the tetrahedral framework nucleic acid by base complementary pairing; the 3 microRNAs with enzyme cutting sites connected with the tail ends are respectively in base complementation pairing with the RNA ₁ strand, the RNA ₂ strand and the DNA ₂ strand of the oligonucleotides to form the other 3 sides of the tetrahedral framework nucleic acid, each microRNA is respectively in base complementation pairing with the RNA ₂ strand of one oligonucleotide strand and the RNA ₁ strand of the other adjacent oligonucleotide strand, and the enzyme cutting sites connected with the tail ends of the microRNAs are in base complementation pairing with the DNA ₂ strand. The invention constructs an ideal carrier for carrying miR.

Description

Biological switchable miR nano-composite based on DNA tetrahedral framework nucleic acid and preparation method and application thereof

Technical Field

The invention belongs to the field of nucleic acid molecule medicines, and particularly relates to a biological switchable miR nano-composite based on DNA tetrahedral framework nucleic acid, and a preparation method and application thereof.

Background

MicroRNA (miR) plays a very important role in the field of nucleic acid medicines because of regulating almost all life processes of human bodies, and in recent years, miR treatment becomes a hot new star for research in the miR field. Briefly, miR therapies are either miR replacement therapies through the use of miR mimics or miR inhibitors inhibit the function of miR. However, miR mimics or miR inhibitors have very short half-lives in the blood, mainly due to their inherent instability, degraded by large amounts of ribonucleases in the blood stream, which also greatly limits the application of miR therapies. As such, more effort has also been expended in the development of miR delivery systems, developing diverse delivery systems such as viral vectors, liposomes, dendrimers, and the like. However, it is clear that the potential biosafety, high toxicity and low transfection efficiency of the delivery system described above also allows the vast majority of miR therapies to remain in preclinical stages. Slack et al also propose that the design of miR delivery vehicles is a major challenge for miR therapy, and that high stability, good biocompatibility and excellent delivery efficiency are required as optimal candidates for miR delivery vehicles. In the field of drug delivery, nucleic acid-based nanomaterials are preferred over other carriers because of their ability to utilize the base pairing rules waston-crick for very creative control of geometry and size. Among the most important members, self-assembled Framework Nucleic Acid (FNA) has multiple advantages.

Notably, in contrast to linear nucleic acids, there are some FNAs that can successfully cross cell membrane barriers through unique interactions with cells, which is certainly critical for drug delivery. Due to the structural characteristics and advantages, the application of FNA in the fields of drug delivery, tissue regeneration, targeted therapy and the like is greatly promoted. Studies have shown that tetrahedra of 21 base side length have a greater ability to penetrate cell membranes, while such FNAs with tetrahedra as three-dimensional structures minimize electrostatic repulsion primarily through "corner attack" patterns, ultimately being internalized by cells through the cellular protein-mediated pathway, which also demonstrates the structural dependence of tFNA in cell penetration.

Although delivery of mirnas and miR treatment are currently achieved using modifications of nucleic acid drugs at the tFNA apex or pendant arms, these approaches not only destroy the structural feature of tetrahedra, but also increase the size of the nanostructures, which would prevent tFNA from achieving optimal cell entry efficiency and tissue penetration. An ideal miR delivery system would need to ensure stable and intact delivery of the miR, however the above-described delivery modes expose the miR outside the carrier, ultimately giving more uncertainty in the delivery of the miR. How to find a new way of miRNA delivery to overcome the above problems requires further research.

Disclosure of Invention

The invention aims to provide a biological switchable miR nano-composite based on DNA tetrahedron framework nucleic acid, and a preparation method and application thereof.

The invention provides a nano-composite based on DNA tetrahedral framework nucleic acid, which is tetrahedral framework nucleic acid obtained by base complementation pairing of 3 oligonucleotide chains and 3 micro RNA with enzyme cutting sites connected to the tail ends; each oligonucleotide chain consists of 2 DNA chains and 2 RNA chains according to the sequence of RNA ₁-DNA₁-RNA₂-DNA₂;

The DNA ₁ strands of the 3 oligonucleotide strands form 3 sides of the tetrahedral framework nucleic acid by base complementary pairing; the 3 micro RNAs with enzyme cutting sites connected to the tail ends are respectively in base complementation pairing with the RNA ₁ strand, the RNA ₂ strand and the DNA ₂ strand of the oligonucleotides to form the other 3 sides of the tetrahedral framework nucleic acid, wherein each micro RNA is respectively in base complementation pairing with the RNA ₂ strand of one oligonucleotide strand and the RNA ₁ strand of the other adjacent oligonucleotide strand, and the enzyme cutting sites connected to the tail ends of the micro RNAs are in base complementation pairing with the DNA ₂ strand of the oligonucleotide strand.

Further, the micro RNA is a micro RNA inhibitor or a micro RNA mimic.

Further, the 3 micro RNAs are 3 micro RNAs with the same sequence.

Further, the 3' -end of the micro RNA is connected with an enzyme cutting site.

Further, the sequence of the oligonucleotide chain is shown as SEQ ID NO.1-3, and the sequence of the micro RNA is shown as SEQ ID NO. 4;

Or the sequence of the oligonucleotide chain is shown as SEQ ID NO.5-7, and the sequence of the micro RNA is shown as SEQ ID NO. 8.

The invention also provides a preparation method of the nanocomposite, which comprises the following steps:

adding oligonucleotide chain and micro RNA into TM buffer solution, maintaining at 95deg.C for 10min, rapidly cooling to 4deg.C, and maintaining for more than 30 min.

Further, the molar ratio of each oligonucleotide chain to micro RNA is 1:3;

Preferably, the concentration of each oligonucleotide strand is 1000nM; the concentrations of micro RNA were 3000nM.

The invention also provides the application of the nano-composite in preparing small nucleic acid medicaments.

Further, the small nucleic acid drug is a drug taking a micro RNA inhibitor as an active ingredient or a micro RNA mimic as an active ingredient.

The invention also provides application of the nano-composite in preparing anti-aging medicaments;

preferably, the medicament is a medicament for promoting hair growth.

Compared with the prior art, the invention has the advantages that:

The invention provides a biological switchable micro RNA nano compound based on DNA tetrahedral framework nucleic acid, which takes the DNA tetrahedral framework nucleic acid as a nucleic acid skeleton, and externally surrounds micro RNA (miR), so that the miR and the DNA tetrahedral framework nucleic acid are fused, and the structural characteristics of the DNA tetrahedral framework nucleic acid are maintained while the small size is maintained. Compared with the existing miR delivery mode, the compound disclosed by the invention has tissue permeability and shows extremely excellent stability in a complex serum environment. In order to facilitate the carried miR to be more easily identified after entering cells, the invention designs a DNA-RNA hybrid switch, so that the complex is triggered by RNase H after entering cells and is converted from a three-dimensional structure to a two-dimensional structure, and the carried miR plays a role in drug effect. In general, the invention constructs an ideal carrier for carrying miR, which has excellent cell entry capacity and tissue permeability, high stability, good biological compatibility and excellent carrying efficiency, and the construction of the compound enables the carrier not to be a big or short plate for miR treatment, has good application prospect in miR treatment, and provides a new idea for delivering other nucleic acid analogues (LNA, PNA).

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is a schematic diagram of BiRDS prepared according to the present invention.

Fig. 2 is a BiRDS design and manufacture: a is BiRDS composed of two parts: one internal nucleic acid core and three external mirs; b is BiRDS structural design drawing and ventral domain and caudal domain position drawing; c shows BiRDS gradual construction for AGE, and the gradual enhancement of fluorescence of Cy5 provides successful carrying of miR in a tetrahedron structure; d is the design of BiRDS, biRDS-BHQ and mispairing-BHQ, close proximity of BHQ and Cy5 results in a BiRDS-BHQ with lower relative fluorescence intensity (< 0.001 × P compared to BiRDS; # P < 0.05;, # compared to incorrect pairing); e is BiRDS showing a small tetrahedral structure in AFM images, scale bar: 25nm; f is the transformation of the nanostructure from BiRDS to Dart after cleavage by RNase H; g shows a conformational change of BiRDS for AGE and miRs is not disrupted; h is the design of the foothold (abdominal domain) in BiRDS, and PAGE shows that a foothold-mediated chain shift between the miR of interest and Dart occurs.

FIG. 3 shows the stability and cell uptake results of BiRDS: a is BiRDS and tFNA-miR stability in 10% fbs for 8 hours (< P <0.001 compared to tFNA-miR); b is BiRDS and tFNA-miR stability in 0-10% fbs (< P <0.001 compared to tFNA-miR); c is confocal images of cells taking free miR, tFNA and BiRDS at different time points, scale bar: 50 μm; d is a statistical analysis of confocal images taken by cells (< 0.01, <0.001, < ns: meaningless) compared to miR; e is the flow cytometry result of cell uptake of free miR, tFNA and BiRDS.

Fig. 4 shows the transdermal drug delivery results of BiRDS: a is fluorescence signal intensity after 1 day of local administration of miR, tFNA and BiRDS; b is fluorescence imaging of tissue sections 1 day after topical administration of miR, tFNA and BiRDS, scale bar: 100 μm; c is the fluorescence signal intensity after the miR, tFNA and BiRDS are locally administrated for 7 days; d is fluorescence imaging of tissue sections 7 days after topical administration of miR, tFNA and BiRDS, scale bar: 100 μm; * Compared to miR, P <0.01, P <0.001, ns: nonsensical.

FIG. 5 shows the results of BiRDS modulation of target proteins in HFSC: a is a schematic representation of BiRDS of the overall process from internalization into inhibition of a target protein; b is fluorescence imaging display BiRDS is mainly absorbed by HFSCs, scale: 50 μm; c and D are immunofluorescence assays of CLOCK and cytokeratin 14 expression after treatment with BiRDS, scale bar, respectively: 50 μm; e and F are statistical analyses of the immunofluorescence results of CLOCK and K14, respectively (. Times. P <0.05 compared to ctrl, # P <0.05 compared to tFNA, # P <0.05; ns: nonsensical).

Fig. 6 is a graph showing the results of BiRDS treatment of skin aging by TDD: a is a schematic of an anti-aging treatment schedule; b is a photograph of the back skin at various time points after WAX 1; c is at the end of WAX 1, with different degrees of baldness occurring in ctrl and tFNA groups, while the BiRDS groups had thicker hair; d is a back skin photograph at various time points after WAX 2; e is hair coverage quantification (< 0.001,/P compared to ctrl) at different time points in WAX 2; f is BiRDS accumulation at the ends of WAX 1 and WAX 2, scale bar: 100 μm; g is the quantification of the bald areas at the ends of WAX 1 and WAX 2 (< 0.05, <0.01, < P, <0.05, # compared to tFNA, # P, <0.05, # ns: nonsensical).

Fig. 7 is a histological level evaluation result of anti-aging treatment effect: a is an H & E stained image of the ends of WAX 1 and WAX 2; group BiRDS shows large and complete HF, scale bar: 200 μm. B is statistical analysis of the size of HF; c is the Masson stained image of the ends of WAX 1 and WAX 2, biRDS groups of epidermis layers thickened, scale bar: 200 μm; d is statistical analysis of hair follicles per millimeter; the size of sebaceous glands of group E BiRDS was increased in oil red O, scale bar: 50 μm; f is the statistical analysis of SG size; g is the key protein (CLOCK) highly expressed in the dermis of skin tissue treated with BiRDS in immunohistochemical staining, scale bar: 200 μm; h is the expression level of CLOCK (< 0.01 for P, <0.001 for P, <0.05 for # P, # P <0.01 for # P, # P <0.001 for ctrl, & P <0.05 for ns: nonsensical for tFNA).

Detailed Description

The materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

Example 1 preparation of micro RNA nanocomposites based on DNA tetrahedral framework nucleic acid

The synthesis method and specific sequence of the micro RNA (miR) nano-composite (BiRDS) based on DNA tetrahedral framework nucleic acid are as follows:

All oligonucleotide strand sequences (S1, S2 and S3 single strands, and miR-31 inhibitors) were made by Sangon (Sangon Biotechnology, shanghai). All the oligonucleotide strands were dissolved in TM buffer (50 mM MgCl ₂.6H₂ O,10mM Tris-HCl, pH 8.0) to give 1000nM final concentration of S1, S2 and S3 single strands and 3000nM final concentration of miR-31 inhibitor, thoroughly mixed and heated to 95℃for denaturation for 10 min, followed by maximum speed cooling to 4℃for 30min, all the assembly steps were carried out in a PCR thermal cycler, and BiRDS was finally prepared. The sequence of the entire oligonucleotide strand is shown in Table 1:

TABLE 1 oligonucleotide chain sequences of the invention

Note that: in the table, capital letters represent DNA monomers, lowercase letters represent RNA monomers, and underlined letters represent nucleotides to be methoxyethyl (2 '-OMe) modified (2' -OMe modification is a currently accepted modification method of miR inhibitors in order to enhance affinity for the target).

The structural schematic diagram of the BiRDS prepared is shown in figure 1: the central skeleton is connected by the base complementary pairing of the DNA parts of the three oligonucleotide chains S1, S2 and S3, and the three sides are connected by the base complementary pairing of the RNA parts of the three oligonucleotide chains S1, S2 and S3 and the miR-31 inhibitor, so that the miR-31 inhibitor surrounds the central skeleton.

Example 2 preparation of micro RNA nanocomposites based on DNA tetrahedral framework nucleic acid

By replacing the S1, S2 and S3 single strands, and the micro RNA with the sequences shown in Table 2, a complex carrying other micro RNA can be prepared by the method described in example 1.

TABLE 2 oligonucleotide chain sequences of the invention

Note that: in the tables, capital letters represent DNA monomers and lowercase letters represent RNA monomers.

Preparation of comparative examples 1, tFNA and tFNA-miR

1. TFNA preparation: a DNA tetrahedral framework nucleic acid (tFNA) was prepared according to the method described in the patent application 202110144200.4.

2. TFNA-miR preparation: (1) Firstly, preparing a DNA tetrahedron with 4 sticky end vertexes according to the method of the patent application No. 202111033236.1 in the example 1; (2) And replacing miRNA-2681 with miR-31inhibitor according to a method described in patent example 1 with the application number 202111033236.1, so as to prepare the stFNA-miR nanocomposite (tFNA-miR) carrying miR-31 inhibitor.

The beneficial effects of the present invention are demonstrated by specific test examples below.

Test example 1 physicochemical characterization of the miR nanocomposite BiRDS of the invention

1. Experimental method

And modifying Cy5 on miR-31inhibitor to obtain Cy5-cagcuaugccagcaucuugccucuua. Cy5 modified BiRDS was prepared as described in example 1. And the state of the miR-31inhibitor can be observed by using fluorescence in subsequent experiments.

Analysis was performed using agarose gel electrophoresis (agarose gel electrophoresis) on Cy5 modified BiRDS and the product tFNA-miR prepared in comparative example 1. The morphology of BiRDS prepared in example 1 was observed using an atomic force microscope and transmission electron microscope (CYPHER VRS, oxford Instruments, united Kingdom; libra200, zeiss, oberkochen, germany). The size and potential of BiRDS prepared in example 1 were measured using Zetasizer Nano ZS a 90 a.

Function verification of BiRDS biological switch: in order to determine that the biological switch is in a closed state after BiRDS is produced, the invention modifies Cy5 and BHQ-2 groups at the 5 'end and the 3' end of miR respectively. According to the design principle of BiRDS, if miR is fused in the tetrahedron frame, quenching of fluorescence occurs because the 5 'end of each miR is close to the 3' end of the adjacent miR, and finally the quenching result is analyzed by using Varioska LUX (Thermo Scientific, VL0L0TD 0). In this study, cleavage sites for RNase H were designed to effect BiRDS transformation from three-dimensional to two-dimensional. The structural changes were observed with AGE by mixing RNase H with RNase H Buffer (Sangon Biotechnology, shanghai) to different concentrations, mixing with BiRDS, and incubating for 1 hour at 37 ℃.

Feasibility of delivering miR inhibitor: to investigate whether a target miR can generate TMSD with Dart through toehold, dart (1. Mu.M) and an excessive target miR (5. Mu.M) are mixed in equal volumes and reacted for 1 hour at different temperatures (30 ℃,37 ℃ and 60 ℃). Finally, polyacrylamide gel electrophoresis is used for observing and analyzing the final product.

2. Experimental results

BiRDS prepared by the invention is divided into an internal nucleic acid framework and an external surrounding miR inhibitor, as shown in figures 2A and 2B: the internal nucleic acid skeleton consists of three nucleic acid chains containing DNA and RNA (from the 5' end, the fragment composition sequence is RNA1-DNA1-RNA2-DNA 2), the construction of the nucleic acid skeleton is realized by the three nucleic acid chains through the DNA1 part, and then the miR is combined with the RNA2 part through the RNA 1. It is particularly noted that in order to realize the bio-switchable delivery system (bioswitchable DELIVERY SYSTEM), the ribonucleotide of 4 bases is extended from the 3' -end of miR, and forms a DNA-RNA hybrid chain with the DNA2 part of the internal skeleton, and after entering cells, the RNA is degraded by RNase H widely existing in mammalian cells, so that the three-dimensional structure is converted into the two-dimensional structure. Thus, miR inhibitor is connected end to end and completely integrated into the framework nucleic acid, and a tetrahedron structure with 20 base pairs (bp) on each side is formed.

In order to more intuitively observe the successful construction of BiRDS, the invention modifies the 5' end of miR with Sulfo-Cyanine5 (Cy 5). A stepwise build-up of BiRDS can be observed. The gradual increase in Cy5 fluorescence further illustrates that mmiR inhibitor was successfully incorporated into the tetrahedral structure (fig. 2C). The invention also modifies a black hole quencher-2 (BHQ-2) (BiRDS-BHQ) at the 3 'end of the miR, and a miR mismatched at the 3' end (mispairing-BHQ). Fluorescence of BiRDS-BHQ was significantly quenched by successful frame closure, only 10% of BiRDS-Cy5, but the fluorescence signal of mispairing-BHQ was stronger than BiRDS-BHQ due to incomplete closure of the structure (fig. 2D).

To better define the morphology of BiRDS, an Atomic Force Microscope (AFM) was used to observe BiRDS. From FIG. 2E, the BiRDS tetrahedral structure is clearly captured and has dimensions much smaller than the manner in which the nucleic acid agent is suspended at the vertices of the tetrahedral structure, exhibiting dimensions similar to those of a 21 base tetrahedron, on the order of about 10 nm. Whereas BiRDS shows electronegativity due to the RNA involved in the construction of the nanostructure.

As a bio-switchable delivery system, the present invention also requires verification of the bio-switch of BiRDS. The invention uses RNase H enzyme to trigger switch in extracellular environment to simulate BiRDS into cell state. It is understood that RNase H can degrade RNA/DNA hybrid but does not affect either single-stranded or double-stranded RNA structure, which means that cleavage of RNase H does not affect miR inhibitor integrity within BiRDS structures. After RNase H hydrolysis, phosphodiester bonds among four ribonucleic acids at the tail of miR inhibitor are broken, and the whole nanostructure is opened outwards to change the conformation, so that a two-dimensional structure (Dart) similar to a return force standard is formed (figure 2F). As expected, after addition of 12.5U/ml RNase H, biRDS was changed in structure and as the enzyme activity unit increased, dart was not changed in other positions in Agarose Gel (AGE), which also means that no additional secondary structure was generated (FIG. 2G). Notably, cy5 fluorescence on miR remains in the same position as BiRDS, dart throughout, meaning that miR is not destroyed. After the miR is destroyed by RNase A, cy5 fluorescence does not appear at the same position as the nano structure, and the structural integrity of the miR is ensured by cutting by RNase H.

An ideal miR delivery system, besides being capable of delivering MIR MIMICS, also needs to be capable of delivering miR inhibitor to ensure the diversification of miR treatment. It is known that miR inhibitor is combined with a target miR through a single chain so as to inhibit the miR, so that toehold nt's toehold are arranged at the reverse folding position of each miR, and a strand displacement reaction between the target miR and Dart is ensured. After incubation of Dart with target miR, it was found that even at temperatures below human body temperature or cell culture temperature, target miR was still able to undergo strand displacement reaction with Dart, confirming BiRDS feasibility of carrying miR inhibitor.

Test example 2 evaluation of serum stability of miR nanocomposite BiRDS of the invention

1. Experimental method

And modifying Cy5 on miR-31inhibitor to obtain Cy5-cagcuaugccagcaucuugccucuua. Cy5 modified BiRDS and tFNA-miR were prepared as described in example 1 and comparative example 1, respectively.

BiRDS and tFNA-miR were mixed with 10% FBS (burning, new York, USA), respectively, and after incubation at 37℃for 0.5,1,2,4,8, 24 hours, the products were analyzed using AGE. To verify the stability of BiRDS and tFNA-miR in different serum concentrations, serum with the proportion of 0-10% is prepared, and after mixed incubation with nanomaterials at 37 ℃ for 1 hour, AGE results are observed.

2. Experimental results

The use of tFNA as a carrier for the transport of mirs has been previously reported, however the manner in which the miR is linked off the tFNA apex (tFNA-miR of comparative example 1) is such that the miR lacks sufficient protection upon and after entry into the cell. The present invention therefore seeks to investigate whether stability in serum environment is improved after fusion of miR inside tetrahedral structure. 37 ℃ was chosen to mimic human body temperature or cell culture environment, and BiRDS and tFNA-miR were incubated in mixture with 10% Fetal Bovine Serum (FBS), resulting in the observation that the fluorescence signal of Cy5 appeared at the same location as BiRDS within 8 hours, indicating that BiRDS structure was not destroyed and miR was still within BiRDS structure (fig. 3A). However, tFNA-mirs, after being in a complex serum environment, did not detect fluorescence signals of Cy5, although the position of the framework nucleic acid was still unchanged, indicating that the carried miR had been degraded. Similarly, the present invention also utilizes different proportions of serum to incubate both transport systems. It was observed that the Cy5 fluorescence signal intensity in BiRDS was not significantly changed over an incubation time of one hour, whereas almost no fluorescence signal was observed in tFNA-miR when the serum fraction reached above 4%, which further demonstrates the excellent stability of BiRDS (fig. 3B). BiRDS also means that the use of tetrahedral structures to achieve miR delivery does not require the lowering of serum proportions to ensure cargo integrity, which is certainly very advantageous in the fields of tissue engineering and regenerative medicine.

Test example 3 cell uptake Capacity of miR nanocomposites BiRDS of the invention

1. Experimental method

HeLa cells were purchased from Anwei-sci (Anweisci, shanghai) and cultured in incubator at 37℃and 5% CO ₂ concentration using MEM medium (Hyclone) and 10% FBS culture. Cells were passaged when cell densities reached above 80%. After various treatments with the drug (drug concentration of 250nM each), cells were fixed with paraformaldehyde and then treated with 0.5% Triton-100 for 10 min. After incubation staining with phalloidin and Dapi, respectively, cells were observed using confocal laser microscopy (a1rmp+, nikon, japan). Similarly, flow cytometry is also used to detect cellular uptake. After drug treatment, cells were digested with trypsin and washed repeatedly with PBS to avoid interference of the remaining drug with the results. Finally, the analysis was performed using a Flow cytometer (CytoFLEX, beckman Coulter). The medicines are respectively Cy5 modified BiRDS, tFNA and free miR-31inhibitor

2. Experimental results

In addition to ensuring BiRDS integrity, whether there is excellent transport capacity is also a key indicator for evaluation BiRDS. The invention selects Hela cells as model cells to observe the transfer efficiency of BiRDS. After treatment of cells with free miR, tFNA and BiRDS with fluorescent groups, it was observed that tFNA and BiRDS were already present in small amounts on the cell membrane after only 3 hours, that a large number of tFNA and BiRDS appeared on the cell membrane after 6 hours as compared to the cytoplasm, and finally tFNA and BiRDS showed similar strong fluorescent signals at 24 hours (fig. 3C). While accumulation of fluorescence signals occurred with time in the miR group, it eventually exhibited only a weak fluorescence signal (fig. 3D). The results of flow cytometry also showed the same trend as the above phenomenon, and therefore it was more believed that BiRDS could exhibit cell permeability as excellent as the small size tFNA, which was not important for the carrier (fig. 3E).

Test example 4 effect test verification of the invention BiRDS

1. Test method

Cy5 modified BiRDS, tFNA and free miR-31inhibitor (miR) were prepared.

(1) Skin penetration test

BiRDS, tFNA and miR are mixed with moisturizing cream (Aquaphor Healing Ointment (Eucerin)) thoroughly, and then uniformly coated on the back of C57/BL mice dehaired by beeswax. BiRDS, tFNA and miR are all administered at a dose of 100 mu L per day and a concentration of 1000nM. The mass ratio of BiRDS, tFNA and miR to the moisturizing cream is 1:1. The moisturizing cream only has the effect of keeping the back moist and has no other effect. The back was then covered with Tegaderm ^TM to ensure that the drug remained in the back area. In addition, mice need to be kept under dim light conditions to avoid accidental fluorescence quenching. After 1 day and 1 week of treatment, the mice were rubbed off residual drug on the back with emollient cotton to avoid false positive results. Finally, the fluorescence of the back of the mice was observed using a living imaging system (PERKINELMER IVIS luminea III). The skin of the back of the mice was removed and fixed in paraformaldehyde, and tissue sections were incubated with Cytokeratin (ET 1610-42,1:200, huaboo, china) and Dapi, respectively, and finally the penetration results were observed using a confocal laser microscope.

(2) Target validation in hair follicle stem cells

Mouse-derived Hair Follicle Stem Cells (HFSCs) were purchased from Anwei-sci (Anweisci, shanghai) and were screened for purity of 90% or more by flow cytometry (flurescence ACTIVATING CELL Sorter). HFSCs cultures were carried out using Keratinocyte Growth Medium (PriMed-AWCell-010, anwei-sci, china). After cells were seeded on Cell culture slides at a density of 20000 cells/well, the cells were stimulated with the addition of BiRDS, tFNA and miR, respectively, at a concentration of 250nM for BiRDS, tFNA and miR, 2mL of medium and drug mixture per well. After 48h of treatment with the drug, cells were fixed with paraformaldehyde. Then, the mixture was incubated with 0.5% Triton-100 for 10 minutes, and after rinsing, 5% goat serum was added thereto and incubated at 37℃for 1 to 1.5 hours. The CLOCK and Cytokeratin-14 antibodies to be detected (CLOCK: ET1704-82,1:200;Cytokeratin 14:ET1610-42,1:200; huaboo, china) were added and incubated overnight at 4 ℃. Following the following day of rinsing the cells, the cells were incubated for 1 hour with secondary antibody (ZSGB-BIO, china) and stained with phalloidin and DAPI. Finally, a confocal laser microscope is used for observing the cell sample.

(3) Construction of skin aging model

The animal experiments of the invention have been approved by the ethical committee of oral hospital research at university Hua Xi of Sichuan. C57/BL mice for P50 were purchased from GEMPHARMATECH Co., ltd and cultured in a suitable environment (18-23 ℃,40-60% humidity). In order not to cause death of mice by overdosing the radiation dose, the present invention wears a tin plate with rectangular holes (2 cm x3 cm) to the mice and only partially irradiates the upper back of the mice. Gamma irradiation was performed in RS2000 (Rad Source, atlanta, USA) and the radiation dose was severely limited to 10-Gy. After irradiation, mice were fed with drinking water containing neomycin.

(4) Wax dehairing and TDD

Construction of the skin aging model of "(3) above" constructed mice irradiated area was subjected to first dehairing (WAX 1) using WAX while also ensuring that no hair remained and additional wounds were created. After depilation (WAX 1), carrying out anti-aging treatment, uniformly coating BiRDS or tFNA mixed with moisturizing cream according to a mass ratio of 1:1 on a depilation area every 24 hours in the course of the anti-aging treatment, wherein the administration dose of BiRDS and tFNA is 100 mu L each time and the concentration is 1000nM, and then using Tegaderm ^TM to prevent the loss of medicines, wherein the WAX 1 is administered for 4 weeks. The mice were then exposed to a second dehairing (WAX 2) using WAX, and after dehairing, anti-aging treatment was performed, as above, for 4 weeks after WAX 2.

Photographs of the mouse dehairing area were taken on days 0, 5, 10, 15, 20 and 25 of WAX 1 and WAX 2 using CANON EOS7D (tokyo, japan). The bald areas and hair coverage areas were analyzed using Image J (V1.53). The mice were still treated for anti-aging with BiRDS with Cy5 in this experiment, and the present invention collected back skin for 1 month and two months of treatment for the same fluorescent staining as in skin penetration experiments.

(5) Histological analysis

The skin samples of the first treatment phase (WAX 1) and the second treatment phase (WAX 2) were fixed, followed by paraffin embedding. Sections were taken along the long axis of the mouse body and samples were H & E stained and masson stained. The main organs of the mice were also dissected and subjected to H & E staining. Soaking the slice in oil red O dye liquor, then soaking the slice in hematoxylin, and finally finishing the oil red O dyeing of the sample. To observe protein expression at the histological level, the present invention placed Anti-CLOCK antibody (CLOCK: ET1704-82,1:50, huaboo, china) into the sample for incubation overnight after the sample was sealed with hydrogen peroxide. The following day the staining was done with secondary anti-incubation antibody for 1 hour, followed by diaminobenzodine and hematoxylin. All of the above sections were observed after scanning with VS200 (Olympus).

2. Experimental results

(1) BiRDS have perfect tissue penetration to achieve transdermal drug delivery

Transdermal Drug Delivery (TDD) is a hot topic due to its non-invasive mode of administration and extremely high patient compliance. The study required a search for whether BiRDS had tissue permeability, thereby making the route of administration of BiRDS more likely. After dehairing the backs of the mice using beeswax, three drugs (BiRDS, tFNA and miR) were thoroughly mixed with moisturizing cream (moisturizer, aquaphor Healing Ointment (Eucerin)) respectively, and applied to three areas of the backs of the mice, and the administration areas were protected using Tegaderm ^TM, and then the permeation of the drugs was observed over 24 hours using a living body imaging system (PERKINELMER IVIS luminea III), and the results are shown in fig. 4A and 4B: after 24 hours of drug permeation, no significant permeation of miR occurred, whereas BiRDS exhibited a strong fluorescent signal similar to tFNA. Histological levels observe that miR does not accumulate within the cortex, interestingly, both tFNA and BiRDS accumulate predominantly in hair follicles and sweat glands. Experimental results show that BiRDS prepared by the invention has excellent percutaneous permeability.

To further observe the drug accumulation and tissue penetration results after long term administration, the present invention treated back dehaired mice with three drugs (BiRDS, tFNA, and miR) for one week, respectively, and observed using a living imaging system after wiping off the drug on the back surface. miR still did not accumulate after up to one week of dosing, while tFNA and BiRDS both produced stronger fluorescent signals (shown in figures 4C and 4D). Although fluorescence signal of miR can be observed at histological level, it is only present in the epidermis and does not penetrate like deeper tissue interiors. BiRDS exhibit a strong fluorescent signal accumulation and it is evident that there is more accumulation in the skin appendages. The excellent tissue permeability of BiRDS means that the preparation can be applied through TDD, and provides an attractive application path for microRNA treatment.

(2) BiRDS treatment of skin aging by transdermal route of administration

Since ancient times, humans have never stopped and fight against aging. As a hallmark of aging, slow hair growth and thin hair density have been a major problem for humans. In a recent study, zhang et al found that miR-31, which is highly expressed in Hair Follicle Stem Cells (HFSCs), was closely related to baldness and Hair Follicle (HF) miniaturization, and miR-31 was targeted directly to the circadian gene CLOCK. The importance of CLOCK is self-evident, for example, in that chronic circadian rhythm disorders inhibit the expression of CLOCK, leading to the occurrence of skin aging symptoms such as hair loss, baldness, etc. Although miR-31 was found to be a key element in HFSCs early skin aging, it is limited by the limitations of miR delivery in the prior art, and this key element still cannot be utilized to solve the problem.

The invention carries miR-31inhibitor into cells through BiRDS, and changes BiRDS into a two-dimensional conformation through enzymolysis of RNase H in the cells, so that the miR-31inhibitor is beneficial to participating in subsequent reactions (figure 5A). After BiRDS treatment HFSCs, significant upregulation of CLOCK protein expression was observed, which also indicated successful inhibition of miR-31 by miR-31 inhibitor. In addition, up-regulation of Cytokeratin 14 was observed. Since Cy5 modification is carried out at the 3' -end of miR inhibitor during the design BiRDS, the invention can clearly see that BiRDS is largely ingested by HFSCs, and the invention also demonstrates the strong application potential of BiRDS in the field of stem cell therapy (FIGS. 5B-5F). Ctrl in fig. 5 is HFSCs untreated.

To further explore the efficacy of BiRDS in vivo while fully exploiting the advantages of BiRDS, the present invention selects the currently highly compliant and attractive TDD as a route of administration for anti-aging treatment of skin aging mice (fig. 6A). Physiological skin aging is a lengthy and complex process, so the present invention selects the local radiation (LIR) mode to construct a model of skin aging. After first dehairing (WAX 1) of LIR treated P50 mice, anti-aging treatment was performed for one month using the drug application in the manner described previously, followed by dehairing again (WAX 2) to induce repeated hair regeneration.

After the mice were subjected to WAX1, although the control group (ctrl, treated with moisturizing cream and physiological saline alone after dehairing by irradiation), tFNA and BiRDS were more similar in terms of hair growth rate, the BiRDS group had deeper hair color than ctrl group and tFNA group and had higher hair density (fig. 6B). In addition, both ctrl and tFNA groups showed different degrees of baldness, while BiRDS group had thicker and thicker hair (fig. 6C). After the mice were subjected to WAX2, the blanc group (without irradiation, after depilation, with moisturizing cream and physiological saline alone) and BiRDS groups observed hair growth on days or so, tFNA groups observed hair growth on days or so, while ctrl group and WAX1 performed the same and no significant hair growth was observed until days or so. The hair regrown in the final ctrl and tFNA groups also appeared bald, while the BiRDS group showed little appearance (fig. 6D). By analysis of the hair coverage area, the present invention obtains a hair growth rate profile. As can be seen from fig. 6E, the hair growth rate of group BiRDS was significantly improved during the WAX2 phase, although the growth rate of fully healthy mice was still not achieved.

To clarify the accumulation of drug during anti-aging treatment, the present invention observed fluorescence signals from the dorsal skin of mice treated with BiRDS for 1 month and two months. After one month of treatment, cy5 fluorescent signals accumulated to some extent within the hair follicle and dermis, and BiRDS was observed to be a major penetration through the skin appendages; after two months of treatment, a large number of fluorescence signals on BiRDS were observed at the epidermis layer and dermis layer, and the entire dermis layer exhibited uniform and intense fluorescence signals (fig. 6F). It can also be seen from figure 6G that BiRDS bald rates were significantly lower than Ctrl and tFNA, both for 1 month and 2 months of treatment.

Looking at the hair follicle and its surroundings, after BiRDS treatment, the entire follicle became larger and full, while the hair follicle of ctrl group remained in a contracted state, which was also responsible for the increased final bald area (fig. 7A, 7B and 7D). The BiRDS set of epidermis layers were seen to thicken in masson stain and the junction of the epidermis and dermis was no longer flat (fig. 7C). In addition, the present invention also observed a slight increase in sebaceous gland size in group BiRDS, becoming more filled (fig. 7E, 7F). Finally, the invention still detects the key target protein CLOCK at the histological level, and can find that the CLOCK is highly expressed in dermis layer in skin tissue treated by BiRDS. This suggests BiRDS that regulation of the target can be achieved by TDD (fig. 7G, 7H).

BiRDS maintains excellent biological safety in terms of safety. No significant skin inflammation occurred, either on the skin applied BiRDS days, or even one or two months, which was also consistent with H & E staining. These manifestations show excellent biocompatibility BiRDS, which lays a solid foundation for its application in numerous fields.

In summary, the invention provides a biological switchable micro RNA nano complex based on DNA tetrahedral framework nucleic acid, which takes the DNA tetrahedral framework nucleic acid as a nucleic acid skeleton, and surrounds micro RNA (miR) outside, so that the miR and the DNA tetrahedral framework nucleic acid are fused, and the structural characteristics of the DNA tetrahedral framework nucleic acid are maintained while the small size is maintained. Compared with the existing miR delivery mode, the compound disclosed by the invention has tissue permeability and shows extremely excellent stability in a complex serum environment. In order to facilitate the carried miR to be more easily identified after entering cells, the invention designs a DNA-RNA hybrid switch, so that the complex is triggered by RNase H after entering cells and is converted from a three-dimensional structure to a two-dimensional structure, and the carried miR plays a role in drug effect. In general, the invention constructs an ideal carrier for carrying miR, which has excellent cell entry capacity and tissue permeability, high stability, good biological compatibility and excellent carrying efficiency, and the construction of the compound enables the carrier not to be a big or short plate for miR treatment, has good application prospect in miR treatment, and provides a new idea for delivering other nucleic acid analogues (LNA, PNA).

Claims

1. A nanocomposite based on DNA tetrahedral framework nucleic acid, characterized in that: the tetrahedral framework nucleic acid is obtained by base complementary pairing of 3 oligonucleotide chains and 3 micro RNA with enzyme cutting sites connected to the tail ends; each oligonucleotide chain consists of 2 DNA chains and 2 RNA chains according to the sequence of RNA ₁-DNA₁-RNA₂-DNA₂;

2. The nanocomposite according to claim 1, characterized in that: the micro RNA is a micro RNA inhibitor or a micro RNA mimic.

3. The nanocomposite according to claim 1, characterized in that: the 3 micro RNAs are 3 micro RNAs with the same sequence.

4. The nanocomposite according to claim 1, characterized in that: and the 3' -end of the micro RNA is connected with an enzyme cutting site.

5. The nanocomposite according to any one of claims 1 to 4, wherein: the sequence of the oligonucleotide chain is shown as SEQ ID NO.1-3, and the sequence of the micro RNA is shown as SEQ ID NO. 4;

6. The method for preparing a nanocomposite according to any one of claims 1 to 5, characterized in that: it comprises the following steps:

adding oligonucleotide chain and micro RNA into TM buffer solution, maintaining at 95deg.C for 10min, rapidly cooling to 4deg.C, and maintaining for 30min or more.

7. The method of manufacturing according to claim 6, wherein: the molar ratio of each oligonucleotide chain to micro RNA is 1:3.

8. The method of manufacturing according to claim 7, wherein: the concentration of each oligonucleotide strand is 1000nM; the concentrations of micro RNA were 3000nM.

9. Use of the nanocomposite of any one of claims 1-5 in the preparation of small nucleic acid pharmaceuticals.

10. Use according to claim 9, characterized in that: the small nucleic acid drug is a drug taking a micro RNA inhibitor as an active ingredient or a micro RNA mimic as an active ingredient.

11. Use of the nanocomposite of claim 5 in the manufacture of an anti-aging medicament.

12. Use according to claim 11, characterized in that: the medicine is a medicine for promoting hair growth.