CN111939132A - Multifunctional nucleic acid nano assembly and preparation method thereof - Google Patents

Abstract

The invention relates to the field of biomedicine, in particular to a multifunctional nucleic acid nanometer assembly and a preparation method thereof. The preparation method includes: using fluorine-modified functional nucleic acid and functional molecules as raw materials, self-assembling into a multifunctional nucleic acid nanometer assembly in an aqueous solution. In the preparation method provided by the present invention, due to the particularity of the fluorine atom, the active site and spatial structure of the nucleic acid structure are changed, so the fluorine-modified nucleic acid can be directly assembled with various types of molecules to form a series of nanostructures. The method is simple, the raw materials are readily available, and has wide versatility, and the obtained nanostructures have high stability and loading efficiency. As a general method, the present invention provides a good reference for the preparation and development of novel nucleic acid drugs.

Description

A kind of multifunctional nucleic acid nano-assembly and preparation method thereof

technical field

The invention relates to the field of biomedicine, in particular to a multifunctional nucleic acid nanometer assembly and a preparation method thereof.

Background technique

Nucleic acid drugs, also known as nucleotide drugs, are various oligoribonucleotides (RNA) or oligodeoxyribonucleotides (DNA) with different functions, mainly at the gene level. It is generally believed that nucleic acid drugs include Aptamer, Antigene, Ribozyme, Antisencenucleic acid, and RNA interference agents. Because of its specificity against pathogenic genes, that is to say, it has a specific target and mechanism of action, nucleic acid drugs, especially small nucleic acid drugs, show excellent application prospects in the field of biological diagnosis and treatment. The synergistic use of functional nucleic acids and functional small molecules, especially some chemotherapeutic drugs, is becoming an important research direction in current clinical practice.

However, since functional nucleic acids are mostly short single-stranded nucleic acids and siRNAs are double-stranded structures, it is difficult for them to directly assemble with functional molecules, especially chemotherapy drugs (such as chemotherapy drugs doxorubicin hydrochloride, cisplatin can only be embedded in nucleic acids) , but in order to achieve synergy, it has to be co-delivered. Therefore, appropriate vectors become the first choice for the delivery of nucleic acids and functional small molecules. The carrier combines with the nucleic acid through positive and negative electric attraction, hydrophilic and hydrophobic switching, and further delivers the functional nucleic acid into the body. Cationic polymers, hydrophobic materials, liposomes, organic mesoporous silica, acid-responsive or glutathione-responsive materials are becoming delivery vehicles. But carrier toxicity and low loading efficiency also limit its effectiveness.

So far, the existing technology of direct assembly of small molecules and functional nucleic acids (Self-Assembled and Size-Controllable Oligonucleotide Nanospheres for Effective Antisense GeneDelivery through an EndocytosisIndependent Pathway) utilizes the self-assembly of thiol monomers and nucleic acids to form nanoparticles. However, the thiol monomer is too toxic, and the in vivo application effect is poor. It has been reported (A BiomimeticCoordination Nanoplatform for Controlled Encapsulation and Delivery of Drug–Gene Combinations) that Fe ²⁺ and nucleic acid are assembled to form nanoparticles, but their stability is too poor, and degradation occurs within 10 minutes in PBS.

Based on the technical problems of the current preparation method of nucleic acid drugs, the method is single, the method is specific, the loading rate is low and the stability is poor, the application of nucleic acid drugs is severely limited. Therefore, it is necessary to develop a scalable and versatile self-assembly engineering system to realize the direct self-assembly of any type of molecules, especially functional small molecules, and functional nucleic acids into nanostructures.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a multifunctional nucleic acid nano-assembly and a preparation method thereof. The preparation method of the multifunctional nucleic acid nano-assembly is simple, green, mild and low-cost.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a preparation method of a multifunctional nucleic acid nano-assembly. The preparation method comprises: using fluorine (F) modified functional nucleic acid and functional molecules as raw materials, and self-assembling into a multifunctional nucleic acid nano-assembly in an aqueous solution.

Fluorine-modified nucleic acids are widely used in nucleic acid aptamers and G-quadruplexes because they can improve the stability of nucleic acids. As the most polar atom, fluorine atom can significantly change the physicochemical properties of nucleic acid. The fluorine-modified nucleic acid can destroy the hydrogen bond between bases, reverse the spatial structure of the nucleic acid, increase the active site of the nucleic acid, and thus improve the assembly ability of the nucleic acid. In the present invention, the fluorine-modified functional nucleic acid P-gp (FNA) that reduces cell drug resistance is used as a raw material, and further through the action of molecular electrostatic attraction, molecular stacking and water bridges, and functional molecules are self-assembled to form functional nucleic acid nanomedicines, The direct and high loading rate assembly with functional small molecules such as chemotherapeutic drugs, metal ions, rare earth ions, peptides, fluorescent molecules and carbohydrates has been achieved. The present invention overcomes the limitation of traditional nucleic acid nanostructure preparation, and exhibits a simple and universal method for realizing the direct assembly of any nucleic acid and multifunctional small molecules, thereby having important scientific significance for the development and preparation of new nucleic acid drugs with different functions.

In the present invention, the fluorine-modified functional nucleic acid is not limited, and all types, random sequences, and any single-stranded or double-stranded nucleic acid meet the requirements. Preferably, the nucleic acid in the fluorine-modified functional nucleic acid is one or more of siRNA, mRNA, antisense nucleic acid, oligonucleotide or DNA.

In the specific embodiment provided by the present invention, the nucleic acid in the fluorine-modified functional nucleic acid is an antisense nucleic acid P-gp.

In the present invention, the fluorine of the present invention is modified at the 2' position of the five-membered sugar ring in the base, but the modification position of the fluorine atom on the base and the number of modifications are not limited. Any position, any number, and nucleic acid derivative modification F all meet the requirements.

In the present invention, functional molecules are not limited, and any type of compound, ion, or small molecule chemotherapeutic drug can be assembled. Preferably, the functional molecules are small molecule chemotherapeutics, fluorescent molecules, rare earth ions, metal ions, carbohydrates, polypeptides, and aggregation-induced fluorescent molecules.

In the specific embodiment provided by the present invention, the metal ion is iron ion, the rare earth ion is Yb ³⁺ , the small molecule chemotherapeutic drug is doxorubicin hydrochloride (DOX), and the fluorescent molecule is photosensitizer Ce6 or photothermal molecule indocyanine green (ICG), the carbohydrate is Mannose, the polypeptide is Melittin, and the aggregation-induced fluorescent molecule is AIE.

Preferably, the molar mass ratio of the fluorine-modified functional nucleic acid to the functional molecule is 1:25-1:300.

As preferably, the preparation method specifically comprises the following steps:

(1) Dissolving the fluorine-modified functional nucleic acid in water to obtain a fluorine-modified functional nucleic acid stock solution;

(2) Dissolving functional molecules in water to obtain functional molecule stock solution;

(3) Mix the fluorine-modified functional nucleic acid stock solution and the functional molecule stock solution evenly, place them in a metal bath, and react at 90 to 100°C for 0.1 to 1.5 hours;

(4) centrifuging the reaction product and washing with water to prepare a multifunctional nucleic acid nano-assembly.

Preferably, the molar concentration of the fluorine-modified functional nucleic acid stock solution is 50-200 μM.

In the specific embodiment provided by the present invention, the molar concentration of the fluorine-modified functional nucleic acid stock solution is 100 μM.

Preferably, the concentration of the functional molecule stock solution is 5-25 mM.

In the specific embodiment provided by the present invention, the concentration of the functional molecule stock solution is 10-20 mM.

Preferably, the reaction conditions in step (3) are: 95° C. for 10 min.

Preferably, the centrifugal rotation speed is 5000-10000 rpm, and the time is 4-6 min.

In the specific embodiment provided by the present invention, the centrifugal speed is 8000rpm and the time is 5min.

The present invention also provides the multifunctional nucleic acid nano-assembly prepared by the above preparation method.

The invention provides a multifunctional nucleic acid nanometer assembly and a preparation method thereof. The preparation method comprises: using fluorine (F) modified functional nucleic acid and functional molecules as raw materials, and self-assembling into a multifunctional nucleic acid nanometer assembly in an aqueous solution. The technical effects that the present invention has are as follows:

The invention provides a simple and general preparation method of a fluorine-modified functional nucleic acid drug. Due to the particularity of the fluorine atom, the active site and spatial structure of the nucleic acid structure are changed. Therefore, fluorine-modified nucleic acids can be directly assembled with various types of molecules to form a series of nanostructures (Figure 1). The method is simple, the raw materials are readily available, no carrier is added, and the method has wide versatility, and the obtained nanostructure has high stability and loading efficiency. As a general method, the present invention provides a good reference for the preparation and development of novel nucleic acid drugs;

The nanoparticles provided by the present invention are non-toxic and harmless, and do not involve any organic solvent;

The nanoparticles provided by the invention have high stability, can circulate well in the body, and are enriched in the target area;

The method for preparing the multifunctional nucleic acid nano-assembly of the present invention is simple, green, mild and low in cost.

Description of drawings

Fig. 1 is the reaction flow diagram of constructing multifunctional nucleic acid nano-assembly from fluorine-modified nucleic acid according to the present invention;

Figure 2 is a transmission electron microscope (a), a scanning electron microscope (b) of the FNA-DOX nanostructure prepared by using the fluorine-modified antisense nucleic acid P-gp(FNA) as a model, and the functional small molecule is doxorubicin hydrochloride DOX. ), potential change diagram (c) and particle size distribution diagram (d);

Fig. 3 is a series of nucleic acid/functional molecule nanostructures prepared in the example:

Iron ion/fluorine modified nucleic acid (Fe ²⁺ -FNA),

Rare earth ion/fluorine modified nucleic acid (Yb ³⁺ -FNA),

Doxorubicin hydrochloride/fluorine-modified nucleic acid (DOX-FNA),

photosensitizer Ce6/fluorine modified nucleic acid (Ce6-FNA),

Photothermal Molecular Indocyanine Green ICG/Fluorine Modified Nucleic Acid (ICG-FNA),

Carbohydrate Mannose Mannose/Fluorine Modified Nucleic Acid (Mannose-FNA),

Polypeptide poison melittin/fluorine modified nucleic acid (Melittin-FNA),

Aggregation-induced fluorescent AIE/fluorine-modified nucleic acid (AIE-FNA);

Figure 4 is the stability study of Fe ²⁺ and unmodified nucleic acid (Fe-DNA) and Fe ²⁺ and fluorine-modified nucleic acid (Fe-FNA) prepared in the example to form nanoparticles, showing that different nanoparticles are in phosphate buffer solution degradation in

Figure 5 shows the different nanoparticle loading efficiencies prepared in the Examples.

Detailed ways

The present invention discloses a multifunctional nucleic acid nano-assembly and a preparation method thereof, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The multifunctional nucleic acid nano-assembly provided by the present invention and the nucleic acid drugs, functional molecules or instruments used in the preparation method thereof can be purchased from the market.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1

Original sequence: ATCCATCCCGACCTCATCCATCCCGACCTC;

F modified sequence (FNA): /i2FA/TCCATCCCGACCTCATCCATCCCGACCTC.

FNA was purchased in Shanghai Shenggong.

Example 2 Preparation of DOX-FNA

The preparation method of nucleic acid drug DOX-FNA in the present embodiment is as follows:

(1) Add 527 μL of sterile water to 10 OD FNA, and pipette at room temperature in the dark to disperse the nucleic acid evenly to form a 100 μM stock solution, and store at -20 °C in the dark.

(2) Disperse 57.9 mg of doxorubicin hydrochloride (DOX) in 10 mL of secondary water under the condition of avoiding light at room temperature, and fully disperse it evenly to obtain a 10 mM doxorubicin hydrochloride stock solution, which is stored in the dark.

(3) 5 μL of FNA, 5 μL of DOX and 20 μL of secondary water were thoroughly mixed, then placed in a metal bath, reacted at 95° C. for 10 min, and cooled to room temperature.

(4) The sample was taken out, centrifuged at 8000 rpm for 5 min, and washed twice with water.

2 is a transmission electron microscope (a), a scanning electron microscope (b), a potential change diagram (c) and a particle size distribution diagram (d) of the nucleic acid drug DOX-FNA prepared in this example. It can be observed from the figure that the nanoparticles formed by the composite of FNA and DOX are uniform in size, the particle size is concentrated at 200 nm, and the surface charge of the nanoparticles is -13.7mV.

Example 3 Preparation of Fe ²⁺ -FNA

(1) 12 μL of 100 μM FNA, 2 μL of 20 mM FeCl ₂ and 26 μL of secondary water were thoroughly mixed, then placed in a metal bath, reacted at 95°C for 10 min, and cooled to room temperature.

(2) The sample was taken out, centrifuged at 8000 rpm for 5 min, and washed twice with water. Fe ²⁺ -FNA nanospheres were obtained.

Example 4 Preparation of Yb ³⁺ -FNA

(1) 5 μL of 100 μM FNA, 5 μL of 20 mM YbCl ₃ and 50 μL of secondary water were mixed thoroughly, then placed in a metal bath, reacted at 95° C. for 10 min, and cooled to room temperature.

(2) The sample was taken out, centrifuged at 8000 rpm for 5 min, and washed twice with water. Yb ³⁺ -FNA nanospheres were obtained.

Example 5 Preparation of DOX-FNA

(1) 5 μL of 100 μM FNA, 5 μL of 10 mM DOX and 20 μL of secondary water were thoroughly mixed, then placed in a metal bath, reacted at 95° C. for 10 min, and cooled to room temperature.

(1) The sample was taken out, centrifuged at 8000 rpm for 5 min, and washed twice with water. DOX-FNA nanospheres were obtained.

Example 6 Preparation of Ce6-FNA

(1) 5 μL of 100 μM FNA, 5 μL of 20 mM Ce6 and 15 μL of secondary water were mixed thoroughly, then placed in a metal bath, reacted at 95°C for 10 min, and cooled to room temperature.

(2) The sample was taken out, centrifuged at 8000 rpm for 5 min, and washed twice with water. Ce6-FNA nanospheres were obtained.

Example 7 Preparation of ICG-FNA

(1) 5 μL of 100 μM FNA, 5 μL of 20 mM ICG and 25 μL of secondary water were thoroughly mixed, then placed in a metal bath, reacted at 95° C. for 10 min, and cooled to room temperature.

(2) The sample was taken out, centrifuged at 8000 rpm for 5 min, and washed twice with water. ICG-FNA nanospheres were obtained.

Example 8 Preparation of Mannose-FNA

(1) 5 μL of 100 μM FNA, 15 μL of 10 mM Mannose and 50 μL of secondary water were thoroughly mixed, then placed in a metal bath, reacted at 95° C. for 10 min, and cooled to room temperature.

(2) The sample was taken out, centrifuged at 8000 rpm for 5 min, and washed twice with water. Mannose-FNA nanospheres were obtained.

Example 9 Preparation of Melittin-FNA

(1) 5 μL of 100 μM FNA, 10 μL of 10 mM Melittin and 30 μL of secondary water were thoroughly mixed, then placed in a metal bath, reacted at 95° C. for 10 min, and cooled to room temperature.

(2) The sample was taken out, centrifuged at 8000 rpm for 5 min, and washed twice with water. Melittin-FNA nanospheres were obtained.

Example 10 Preparation of AIE-FNA

(1) 5 μL of 100 μM FNA, 10 μL of 10 mM AIE and 15 μL of secondary water were thoroughly mixed, then placed in a metal bath, reacted at 95° C. for 10 min, and cooled to room temperature.

(2) The sample was taken out, centrifuged at 8000 rpm for 5 min, and washed twice with water. AIE-FNA nanospheres were obtained.

Test Example 1

3 is a series of functional molecule/nucleic acid nanostructures prepared in Examples 3-9, and the obtained nanoparticles have uniform morphology. A series of nanostructures with particle sizes of 200 nm for Fe ²⁺ -FNA, 500 nm for Yb ³⁺ -FNA, 500 nm for DOX-FNA, 220 nm for Ce6-FNA, 150 nm for ICG-FNA, 200 nm for Mannose-FNA, and 200 nm for Melittin-FNA is 100 nm and AIE-FNA is 40 nm.

Test Example 2

In this test example, the stability of Fe-FNA nanoparticles prepared in Example 3 and control Fe-DNA nanoparticles incubated in serum for different times was tested.

Wherein, the preparation method of Fe-DNA is:

(1) Thoroughly mix 12 μL of 100 μM DNA, 2 μL of 20 mM FeCl ₂ and 26 μL of secondary water, then place in a metal bath, react at 95°C for 10 min, and cool to room temperature.

(2) The sample was taken out, centrifuged at 8000 rpm for 5 min, and washed twice with water. Fe ²⁺ -DNA nanospheres were obtained.

4 is a TEM image of Fe-DNA and Fe-FNA nanoparticles prepared in Example 3 incubated in serum for different times. It was observed from the figure that Fe-FNA still kept intact morphology after 24h incubation time. It can be seen that the F-modified nucleic acid assembly has higher stability.

Test Example 3

Fig. 5 is the data of the loading efficiency of different nanoparticles prepared in the example. It is observed from the figure that the loading of nucleic acids and small molecules reaches more than 80%. Shows high loading efficiency.

In summary, the present invention provides a simple and general preparation method of fluorine-modified functional nucleic acid drugs. For the first time, it has broken through the limitation of nucleic acid nanostructure assembly, and realized the direct co-assembly of any nucleic acid and any molecule. The method is simple to operate, and the prepared nanostructure has high loading rate and good stability. Ease of commercial and clinical translation.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (10)

1. A method for preparing a multifunctional nucleic acid nano-assembly, the method comprising: the functional nucleic acid and functional molecule modified by fluorine are used as raw materials and self-assembled into the multifunctional nucleic acid nano assembly in aqueous solution.

2. The method according to claim 1, wherein the fluorine-modified functional nucleic acid comprises one or more of siRNA, mRNA, oligonucleotide, and DNA.

3. The method of claim 1, wherein the functional molecule is a small molecule chemotherapeutic agent, a fluorescent molecule, a rare earth ion, a metal ion, a saccharide, a polypeptide, an aggregation-induced fluorescent molecule.

4. The method according to claim 1, wherein the molar mass ratio of the fluorine-modified functional nucleic acid to the functional molecule is 1:25 to 1: 300.

5. The method according to any one of claims 1 to 4, comprising in particular the steps of:

(1) dissolving fluorine modified functional nucleic acid in water to obtain fluorine modified functional nucleic acid stock solution;

(2) dissolving functional molecules in water to obtain a functional molecule stock solution;

(3) uniformly mixing a fluorine modified functional nucleic acid stock solution and a functional molecule stock solution, placing the mixture in a metal bath, and reacting for 0.1-1.5 h at 90-100 ℃;

(4) and centrifuging and washing the reaction product to prepare the multifunctional nucleic acid nano assembly.

6. The method according to claim 5, wherein the molar concentration of the fluorine-modified functional nucleic acid stock solution is 50 to 200. mu.M.

7. The method according to claim 5, wherein the concentration of the stock solution of functional molecule is 5 to 25 mM.

8. The method according to claim 5, wherein the reaction conditions in the step (3) are as follows: the reaction was carried out at 95 ℃ for 10 min.

9. The method according to any one of claims 5 to 8, wherein the centrifugation is performed at 5000 to 10000rpm for 4 to 6 min.

10. The multifunctional nucleic acid nano-assembly prepared by the preparation method of any one of claims 1 to 9.

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