A method for upconversion nanoparticle phase transition and DNA coupling using amphiphilic DNA

Abstract

The present invention discloses a method for upconversion nanoparticle phase transition and DNA coupling using amphiphilic DNA, comprising the following steps: S1, synthesizing an amphiphilic DNA molecule modified with multiple alkane side chains; S2, dissolving the amphiphilic DNA molecule with DMSO, adding chloroform and ultrasonication; S3, providing an upconversion nanoparticle, and resuspending it with chloroform; S4, uniformly mixing the upconversion nanoparticle with the amphiphilic DNA molecule, ultrasonically dispersing it, and resuspending it with an aqueous buffer solution, combining the alkane side chain of the DNA molecule with the hydrophobic ligand on the surface of the upconversion nanoparticle through the effect of ligand intercalation, obtaining a DNA molecule-modified upconversion nanoparticle, and realizing phase transition and DNA coupling of the upconversion nanoparticle at the same time. According to the present invention, the upconversion nanoparticle can be well converted from the organic phase to the aqueous phase, realizing uniform dispersion of the upconversion nanoparticle in the aqueous phase, and having good biomedical application prospects.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and more specifically to a method for up-conversion nanoparticle phase transition and DNA coupling using amphiphilic DNA. Background Art

[0002] In the field of gene drug delivery, upconversion nanoparticles can be used to load nucleic acid drugs for gene silencing, gene therapy, and gene editing. However, when upconversion nanoparticles are used in vivo or in vitro, they are often susceptible to aggregation in complex biological system environments. Maintaining stable dispersion of upconversion nanoparticles in biological systems is essential to ensure effective drug delivery and target gene expression. Therefore, stable phase transfer of hydrophobic nanoparticles is one of the key challenges in achieving effective nucleic acid drug delivery.

[0003] At present, the methods for phase transfer and surface nucleic acid coupling of upconversion nanoparticles generally include physical adsorption and chemical modification. The oleic acid ligand on the surface of hydrophobic upconversion nanoparticles can be washed off by reagents such as hydrochloric acid. The surface of upconversion nanoparticles without ligand modification is positively charged and can be connected to negatively charged DNA through electrostatic adsorption to improve the water solubility of upconversion nanoparticles. However, this method requires a large amount of DNA, resulting in higher costs. In addition, the spherical nucleic acid of the upconversion nanoparticles obtained by this method is susceptible to the influence of the biological system to produce DNA shedding phenomenon, causing the leakage of loaded drugs, which is not conducive to the controlled release of drugs. The water solubility of nanoparticles can be enhanced by modifying the surface ligands of nanoparticles (such as amino, carboxyl, azide, etc.) through chemical reactions. However, chemical modification often requires special synthesis steps, complicated operations and low efficiency.

[0004] The disadvantages of these methods limit the application of upconversion nanoparticles in nucleic acid drug delivery. Therefore, the need to develop a simpler, more efficient, and non-special method to prepare upconversion nanoparticles with well-dispersed spherical nucleic acids in water has become more urgent. Summary of the invention

[0005] The purpose of the present invention is to provide a method for upconversion nanoparticle phase transition and DNA coupling using amphiphilic DNA, thereby solving the drawbacks of the prior art that hydrophobic particles cannot be evenly dispersed in biological systems or that the DNA coupling steps are complicated and inefficient.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] According to a first aspect of the present invention, a method for upconversion nanoparticle phase transition and DNA coupling using amphiphilic DNA is provided, comprising the following steps: S1, synthesizing an amphiphilic DNA molecule modified with multiple alkane side chains by introducing an alkane side chain at the 2' position of the single-stranded ribose backbone of DNA; S2, dissolving the amphiphilic DNA molecule synthesized in step S1 with a small amount of DMSO solution, and then adding chloroform and ultrasonically dissolving the amphiphilic DNA molecule completely; S3, providing an upconversion nanoparticle uniformly dispersed in cyclohexane, precipitating the upconversion nanoparticle with three times the volume of ethanol, and resuspending with chloroform; S4, uniformly mixing the upconversion nanoparticle obtained in step S3 with the amphiphilic DNA molecule obtained in step S2, ultrasonically dispersing, and resuspending with an aqueous buffer solution, wherein the alkane side chain of the DNA molecule and the hydrophobic ligand on the surface of the upconversion nanoparticle are combined through the action of ligand intercalation to obtain an upconversion nanoparticle modified with an amphiphilic DNA molecule, thereby achieving phase transition and DNA coupling of the upconversion nanoparticle at the same time.

[0008] Preferably, in step S1, the length of the DNA single strand is 6 to 200 bases, and the corresponding base sequence can be designed according to actual needs.

[0009] Preferably, in step S1, the alkane side chain is derived from a precursor

[0010] DMTr-2'-O-C16-rA(Bz)-3'-CE-Phosphoramidite.

[0011] Preferably, in step S1, the alkane side chain is hexadecane or octadecane.

[0012] Preferably, in step S1, the alkane side chains are evenly distributed on the DNA single strand at equal intervals by design.

[0013] It should be understood that in step S1, the single-stranded DNA has two properties of an alkane chain and a DNA chain, namely, the hydrophobicity of the alkane chain and the hydrophilicity of the DNA material. Therefore, the present invention regards it as an amphiphilic compound that is dissolved in both the organic phase and the aqueous phase environment.

[0014] Preferably, in step S2, the volume content of the DMSO solution is 3-10%, so as to improve the solubility of the amphiphilic DNA in chloroform.

[0015] Preferably, in step S3, the surface ligand of the upconversion nanoparticles is selected from at least one of oleic acid, oleylamine and 1-octadecene.

[0016] Preferably, step S4 comprises: uniformly mixing the upconversion nanoparticles obtained in step S3 and the amphiphilic DNA molecules obtained in step S2, bathing in a water bath at 30 to 60° C. for 10 to 20 minutes, centrifuging under reduced pressure to volatilize the organic solvent, and resuspending with an aqueous buffer solution, thereby converting the upconversion nanoparticles from the organic phase to the aqueous phase, thereby achieving uniform dispersion of the upconversion nanoparticles in the aqueous phase.

[0017] In step S4, preferably, the DNA-modified upconversion nanoparticles are resuspended in water, 1×TAE buffer, PBS buffer, 25 mM HEPES buffer, 1640 culture medium, and DMEM culture medium, respectively, and the dispersion degree of the upconversion nanoparticles modified with amphiphilic DNA molecules in different biological systems can be observed.

[0018] According to a second aspect of the present invention, there is provided an upconversion nanoparticle modified with an amphiphilic DNA molecule prepared according to the above method.

[0019] According to a third aspect of the present invention, there is provided a method for upconversion nanoparticle phase transition and DNA coupling using amphiphilic DNA for use in the delivery of nucleic acid drugs and the controlled release of nucleic acid drugs.

[0020] Upconversion nanoparticles (UCNPs) are a class of nanoparticles with unique optical properties. They are able to convert low-energy photons (such as near-infrared light) into high-energy photons, usually in the visible or ultraviolet spectrum. This phenomenon is called upconversion luminescence. UCNPs are usually composed of a host material doped with rare earth ions, such as yttrium (Y), ytterbium (Yb), and erbium (Er). These nanoparticles have properties that are very useful in biomedical imaging, biomarkers, and other optical applications. The present invention is not limited to the material composition of the upconversion nanoparticles.

[0021] It is also worth noting that all upconversion nanoparticles of suitable size (10 to 20 nanometers) are suitable for the present invention. According to a preferred embodiment of the present invention, as an example but not a limitation, core-shell structure upconversion nanoparticles NaYF4: 0.05% Tm, 30% Yb@NaYF4 with a particle size of 15 nanometers are selected as verification materials.

[0022] In the commonly used synthesis method of upconversion nanoparticles, upconversion nanoparticles can be synthesized by thermal decomposition of rare earth ion organic salts (such as yttrium acetate, thulium acetate, etc.) in high boiling point solvents. During the high temperature thermal decomposition of rare earth ion organic salts at about 300°C, long-chain hydrocarbons and related functional groups (such as COOH in oleic acid solvent) are often used as high boiling point synthesis solvents to prevent the aggregation of nanoparticles. Therefore, the surface of the synthesized upconversion nanoparticles is evenly coated with oleic acid ligands and has good dispersibility in cyclohexane, so cyclohexane is often used as a storage solvent for upconversion nanoparticles.

[0023] According to the method provided by the present invention, its working principle is: in the organic phase, the upconversion nanoparticles are uniformly dispersed due to the surface oleic acid ligand wrapping, and when contacting the hydrophobic alkane chain of the amphiphilic DNA, since the alkane chain and the oleic acid ligand have similar molecular structures (oleic acid molecular formula: C18H34O2, alkane chain molecular formula: C16H33), according to the principle of like dissolves like, the hydrophobic alkane chain can be connected to the particle surface by ligand insertion, while the hydrophilic end of the DNA chain faces the outside. After the solvent is replaced, the particles are uniformly dispersed into the water phase under the wrapping of the hydrophilic DNA, and the uniform dispersion of the nanoparticles is maintained in the salt environment.

[0024] It is noteworthy that the sequence information of the single-stranded DNA used in this method has almost no effect on the phase transition of the nanoparticles, and any fragment of amphiphilic DNA is suitable for the present invention.

[0025] According to a preferred embodiment of the present invention, a method for preparing stably dispersed upconversion nanoparticles in a biological system using amphiphilic DNA is provided, comprising the following steps: 1) using three times the volume of ethanol to precipitate the upconversion nanoparticles dispersed in cyclohexane, collecting the nanoparticles by centrifugation, and resuspending them with chloroform; 2) mixing the amphiphilic DNA molecules dissolved in chloroform (containing 5% DMSO as a solubilizing agent) with the upconversion nanoparticles in 1), ultrasonically dispersing them, and incubating them in a water bath at 50° C. for 10 minutes; 3) placing the reaction solution in 2) in a 45° C. environment and centrifuging under reduced pressure to volatilize the organic solvent, and ultrasonically resuspending them with 0.5×TBE; 4) checking whether there is DNA on the surface of the particles by agarose gel electrophoresis, characterizing the aggregation state of the particles in the above reaction process by TEM imaging, checking the absorption of DNA on the surface of the particles by ultraviolet absorption spectroscopy, and judging whether the surface of the particles is modified with DNA molecules; 5) replacing the dispersion system of the upconversion nanoparticles by multiple centrifugation, placing them in a variety of commonly used biological system buffers or cell culture media for ultrasonic dispersion, and observing the dispersion of the particles by TEM imaging.

[0026] Wherein, the nanoparticle solvent replacement method described in step 1) is a conventional technical means in the art. The same volume of ethanol solution is added to the nanoparticles uniformly dispersed in cyclohexane three times, and the solution is shaken and mixed after the addition. According to the difference in polarity between ethanol and cyclohexane, the nanoparticles can be precipitated from the cyclohexane, and the precipitation is collected under the centrifugal action of 9000rpm. The method is simple to operate and can reduce the centrifugal loss of nanoparticles.

[0027] Among them, the agarose gel electrophoresis detection method described in step 4) is a conventional technical means in the art. Take out 20 μL of the system in step 3) and load it into the 1% agarose gel well, set the electrophoresis voltage to be constant at 90V, use 0.5×TBE as the gel running buffer, run the electrophoresis for 45 minutes, use GelRed nucleic acid dye for DNA staining and tracing, observe the band position of the nanoparticles before and after DNA modification, and judge the surface DNA loading situation.

[0028] The TEM imaging in steps 4) and 5) is a conventional technique in the art. 10 μL of nanoparticle solutions at different stages are dripped onto the surface of a common carbon support film copper mesh, and transmission electron microscopy imaging is performed to observe the dispersion of nanoparticles in different systems.

[0029] The UV absorption spectrum characterization of the DNA on the particle surface in step 4) is a conventional technical means in the art. Excess DNA is removed by multiple centrifugation, the precipitate is resuspended, and the resuspended particles are tested for the characteristic peak of DNA UV absorption at 260 nm to confirm the coupling of DNA on the particle surface.

[0030] According to the method provided by the present invention, a method for upconversion nanoparticle phase transfer and DNA coupling using amphiphilic DNA is provided, which has a positive improvement effect compared with the prior art in that: the present invention proposes for the first time a method for upconversion nanoparticle phase transfer using amphiphilic DNA, wherein the spherical nucleic acid of the upconversion nanoparticle prepared by the method is simpler than the synthesis process of other methods, and at the same time, through the principle of like dissolves like, the surface ligand properties of the nanoparticle are not changed, and the phase transfer of the nanoparticle is achieved only through the action of the surface nucleic acid. In practical applications, according to the research of the present invention, it is found that the DNA modified upconversion nanoparticles have good dispersibility in common buffers and culture media of various biological systems.

[0031] In summary, the present invention proposes a method for upconversion nanoparticle phase transition and DNA coupling using amphiphilic DNA, which can greatly improve the dispersion stability of nanoparticles in biological systems; more importantly, the method described in the present invention can give play to the optical properties of upconversion nanoparticles, realize the delivery of nucleic acid drugs and optically controllable drug release in biological systems, and has good biomedical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Flow chart (a) and principle diagram (b) of upconversion nanoparticle phase transition and DNA coupling using amphiphilic DNA;

[0033] Figure 2 Agarose gel electrophoresis characterization of amphiphilic DNA, DNA-modified upconversion nanoparticles, and upconversion nanoparticles;

[0034] Figure 3 Transmission electron microscopy imaging characterization of the reaction process of upconversion nanoparticles and DNA-modified upconversion nanoparticles in different organic phases and aqueous phases (scale bar: 50 nm);

[0035] Figure 4 is the UV / visible absorption curve of DNA modified upconversion nanoparticles;

[0036] Figure 5 Transmission electron microscopy imaging characterization of the dispersion of DNA-modified upconversion nanoparticles in different biological system buffers and culture media (scale bar: 50 nm). DETAILED DESCRIPTION

[0037] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional operations in the art, or according to the experimental methods recommended by the kit and instrument manufacturers. The reagents and materials used in the examples can be obtained from commercial sources unless otherwise specified.

[0038] Example 1

[0039] This embodiment provides a method for upconversion nanoparticle phase transition and DNA coupling using amphiphilic DNA, comprising the following steps:

[0040] (1) Preparation of amphiphilic DNA-modified upconversion nanoparticles. This example uses core-shell upconversion nanoparticles NaYF4 with a particle size of 15 nanometers: 0.05% Tm, 30% Yb@NaYF4 to carry out particle phase transfer and DNA modification experiments. The DNA sequence used is 5'-ATCGAGCTATCGACGTACTGA-3' (SEQ ID No. 1), in which each adenine is modified with a 16C alkane chain. The DNA sequence used in this example has one 16C alkane chain connected to the adenine (A) base, for a total of 6 chains. However, it should be understood that the alkane chain can be connected to any base. In this example, adenine is selected mainly to make the alkane chain evenly distributed on the DNA chain.

[0041] 0.05 mg of upconversion nanoparticles dissolved in cyclohexane were added to an equal volume of ethanol in batches, oscillated to mix, centrifuged at 9000 rpm for 10 minutes, and the precipitate was resuspended by ultrasound with 50 μL of chloroform solution. The amphiphilic DNA dry powder was fully dissolved with 5% DMSO, and then chloroform solution was added to a concentration of 100 μM and dispersed by ultrasound. 0.125 nmol DNA was added to the 0.05 mg upconversion nanoparticle solution and dispersed by ultrasound. Incubated at a constant temperature of 50°C in a water bath for 10 minutes, and then centrifuged under reduced pressure at 45°C to evaporate the organic solvent. Finally, 100 μL of 0.5×TBE buffer was added to the precipitate, and it was evenly dispersed by ultrasound to obtain DNA-modified upconversion nanoparticles ( Figure 1 ).

[0042] (2) Agarose gel electrophoresis to verify the DNA coupling on the particle surface. Take 20 μL of the resuspension in (1), add 6× loading buffer and oscillate to mix. Set up the same concentration of upconversion nanoparticles without DNA and DNA without upconversion nanoparticles as the control group, and load them into the gel wells together with the DNA marker. The agarose gel used in the experiment has a mass concentration of 1% (containing GelRed nucleic acid dye), the electrophoresis buffer is 0.5×TBE, the electrophoresis voltage is set to 90V, and the electrophoresis is carried out at room temperature for 45 minutes ( Figure 2 ).

[0043] (3) Characterization of the dispersion of nanoparticles during coupling by transmission electron microscopy. At each stage in (1), 10 μL of sample was taken and dropped onto a common carbon support film. The dispersion of the upconversion nanoparticles in cyclohexane, chloroform, chloroform after adding DNA, and aqueous resuspension after evaporation of organic solvent was characterized by transmission electron microscopy ( Figure 3 ).

[0044] (4) Characterization of DNA loading on the surface of nanoparticles by UV / visible absorption spectroscopy. The resuspended solution in (1) is centrifuged multiple times. After each centrifugation, the UV / visible absorption spectrum of free DNA in the supernatant is measured. The DNA concentration in the supernatant can be calculated based on its absorption value at 260 nanometers using the Lambert-Beer law. The nanoparticles are then resuspended in new 0.5×TBE buffer, centrifuged and washed again, and the DNA concentration in the supernatant of each wash is measured. When the characteristic peak of DNA cannot be detected in the supernatant of the last wash, the bottom precipitate is resuspended, and the UV / visible absorption spectrum of the resuspended material is measured again. If the characteristic absorption peak of DNA at 260 nanometers appears in the bottom resuspended solution, it proves that the particle surface has DNA loading ( Figure 4 ).

[0045] (5) Transmission electron microscopy imaging characterizes the stability of DNA-modified upconversion nanoparticles in different biological system buffers. The resuspended solution in (1) was centrifuged several times to replace the buffer. Water, 1×TAE buffer (containing 12.5 mM Mg 2+ ), 1×TAE buffer (containing 5 mM Mg 2+ ), PBS buffer, 25mM HEPES buffer, 1640 culture medium, and DMEM culture medium to resuspend the DNA modified upconversion nanoparticles. The sample was dropped onto a common carbon support film for transmission electron microscopy imaging, and the dispersion of the particles was observed to determine their dispersibility in the biological system ( Figure 5 The 1×TAE buffer (containing 12.5 mM Mg 2+ ) is a universal buffer for assembling DNA origami structures, 1×TAE buffer (containing 5 mM Mg 2+ ) is a commonly used buffer for double-stranded DNA hybridization, PBS buffer is a commonly used buffer for cell culture, and 1640 and DMEM medium are commonly used culture media for cell culture.

[0046] Results: Figure 2 As shown in the figure, DNA modified upconversion nanoparticles have a new band that lags behind free DNA, indicating that the upconversion nanoparticles move toward the positive electrode of the power supply as the surface DNA migrates under the drive of voltage, verifying the existence of DNA on the particle surface. Figure 3 As shown in the figure, during the whole process of upconversion nanoparticle phase transition using amphiphilic DNA, the nanoparticles maintained good dispersion and no particle aggregation and adhesion occurred, which verified the stability of the nanoparticles during the experiment. Figure 4 As shown in the figure, in the supernatant of the third wash, no characteristic absorption peak of DNA at 260 nanometers was detected, proving that the free DNA in the system has been washed clean, and the characteristic absorption values ​​of DNA in the precipitate all come from the surface of the upconversion nanoparticles. The particle resuspension after washing showed a curve similar to that of the supernatant of the first wash, and a higher DNA absorption value was detected at 260 nanometers, verifying the existence of DNA on the particle surface. Figure 5 As shown, by replacing the dispersion buffer of the DNA-modified upconversion nanoparticles by centrifugation, it can be observed that the particles maintain a good degree of dispersion in different buffers and two culture media, proving that the upconversion nanoparticle spherical nucleic acid obtained by this method can stably exist in commonly used biological systems, and has good biocompatibility and application potential in biological systems.

[0047] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiment of the present invention can also be modified in various ways. That is, all simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for upconversion nanoparticle phase transfer and DNA coupling using amphiphilic DNA, characterized in that: The following steps are involved: S1, synthesizing an amphiphilic DNA molecule modified with multiple alkane side chains by introducing alkane side chains at the 2' position of the DNA single-stranded ribose backbone; S2, dissolving the amphiphilic DNA molecules synthesized in step S1 with a small amount of DMSO solution, and then adding chloroform and sonicating to completely dissolve the amphiphilic DNA molecules; S3, providing an upconversion nanoparticle uniformly dispersed in cyclohexane, precipitating the upconversion nanoparticle with three volumes of ethanol, and resuspending it with chloroform; S4, the upconversion nanoparticles obtained in step S3 and the amphiphilic DNA molecules obtained in step S2 are evenly mixed, ultrasonically dispersed, and resuspended using an aqueous buffer solution. The alkane side chains of the DNA molecules and the hydrophobic ligands on the surface of the upconversion nanoparticles are combined through the effect of ligand intercalation to obtain an amphiphilic DNA molecule-modified upconversion nanoparticle, which can simultaneously achieve phase transition and DNA coupling of the upconversion nanoparticles.

2. The method according to claim 1, characterized in that In step S1, the length of the DNA single strand is 6 to 200 bases, and the corresponding base sequence can be designed according to actual needs.

3. The method according to claim 1, characterized in that In step S1, the alkane side chain is derived from the precursor DMTr-2'-O-C16-rA(Bz)-3'-CE-Phosphoramidite.

4. The method according to claim 1, characterized in that: In step S1, the alkane side chain is hexadecane or octadecane.

5. The method according to claim 1, characterized in that In step S2, the volume content of the DMSO solution is 3-10%.

6. The method according to claim 1, characterized in that In step S4, the hydrophobic ligand on the surface of the upconversion nanoparticle is selected from at least one of oleic acid, oleylamine, and 1-octadecene.

7. The method according to claim 1, characterized in that Step S4 comprises: uniformly mixing the upconversion nanoparticles obtained in step S3 and the amphiphilic DNA molecules obtained in step S2, bathing in a water bath at 30-60° C. for 10-20 minutes, centrifuging under reduced pressure to volatilize the organic solvent, and resuspending with an aqueous buffer solution to convert the upconversion nanoparticles from the organic phase to the aqueous phase, thereby achieving uniform dispersion of the upconversion nanoparticles in the aqueous phase.

8. The method according to claim 7, characterized in that In step S4, the DNA-modified upconversion nanoparticles are resuspended in water, 1×TAE buffer, PBS buffer, 25 mM HEPES buffer, 1640 culture medium, and DMEM culture medium, respectively, and the dispersion degree of the upconversion nanoparticles modified with amphiphilic DNA molecules in different biological systems or cell culture media can be observed.

9. An upconversion nanoparticle modified with an amphiphilic DNA molecule prepared according to the method of any one of claims 1 to 8.

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