DNA nanoparticle and preparation method thereof
Metal-free DNA nanoparticles were prepared by high-temperature carbonization of nucleosides and hydrogen bonding with the DNA strand, which solved the problem of metal-free DNA nanoparticles preparation in the prior art, and achieved nanoparticles preparation with high biocompatibility and biosafety.
Patent Information
- Application Number
- CN202411871429.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-18
- Publication Date
- 2025-03-28
AI Technical Summary
In the prior art, it is difficult to achieve metal-free biocompatibility and biosafety, and there is a lack of clear force between non-metal ions or particles and DNA, resulting in a small probability of co-aggregation of DNA nanoparticles.
By carbonizing the nucleoside at high temperature, separation and purification, nucleoside carbon dots were obtained, and reacted with the DNA strand with hydrogen bonding similar to the principle of base complementary pairing. After purification, DNA nanoparticles were obtained. This method realizes the preparation of DNA nanoparticles without introducing metal elements.
The preparation of metal-free DNA nanoparticles has been achieved, with good biocompatibility and biosafety, low cytotoxicity, and can be used for nucleic acid delivery, and has application potential in the field of biomedical medicine.
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Figure CN119700677A_ABST
Abstract
Description
Technical Field
[0001] The present disclosure relates to the technical field of new material preparation, and in particular to a DNA nanoparticle and a preparation method thereof. Background Art
[0002] In the eyes of biologists, DNA is the main genetic material of living organisms, controlling their birth, aging, illness and death; in the eyes of materials scientists, DNA is capable of precise base complementary pairing and has the potential to construct precise materials, and the field of DNA nanomaterials has emerged. At present, DNA nanomaterials mainly include: DNA origami structures, branched DNA, DNA polyhedrons, DNA hybrid nanoparticles, etc., which are widely used in biosensors, drug delivery, disease diagnosis and treatment, etc. Among them, DNA hybrid nanoparticles are nanoparticles formed by electrostatic interactions between DNA and ions or particles. Metal ions are currently commonly used, and nanoparticles are aggregated through the electrostatic interaction between metal ions and negatively charged DNA. First, DNA hybrid nanoparticles have great side effects in subsequent biomedical applications because excessive metal ions have great biological and cytotoxicity; secondly, Cu 2 + , Fe 2+ Metal ions have Fenton reaction activity and can produce reactive oxygen to destroy the structure of DNA, which is not conducive to the stability of DNA nanoparticles.
[0003] Metal-free DNA nanoparticles have good biocompatibility and biosafety, but due to the development of technology, there are no reports on metal-free DNA nanoparticles. This is because there is no clear interaction between non-metal ions or particles and DNA, and the probability of coaggregation into DNA nanoparticles is relatively small. Therefore, the preparation of metal-free DNA nanoparticles is still a great challenge. Summary of the invention
[0004] The present disclosure provides a DNA nanoparticle and a preparation method thereof, so as to at least solve the above technical problems existing in the prior art.
[0005] According to a first aspect of the present disclosure, a method for preparing DNA nanoparticles is provided, comprising the following steps:
[0006] S1: high temperature carbonization of nucleosides, separation and purification to obtain nucleoside carbon dots;
[0007] S2: The nucleoside carbon dots react with the DNA chain through hydrogen bonding similar to the base complementary pairing principle, and are purified to obtain DNA nanoparticles.
[0008] In one embodiment, the nucleoside is adenine, guanosine, cytosine or uridine, all of which are components of RNA, and their CAS numbers are 58-61-7, 118-00-3, 65-46-3 and 58-96-8, respectively.
[0009] In a preferred embodiment, the nucleoside is uridine, referred to as uridine, and its CAS number is 58-96-8.
[0010] In one embodiment, the nucleoside is dissolved in water before the high temperature carbonization to obtain a nucleoside solution. In a specific embodiment, the concentration of the nucleoside solution is 0.1 g / mL.
[0011] In one possible implementation manner, the high temperature carbonization is performed by a hydrothermal reaction in a high temperature and high pressure reactor.
[0012] In a preferred embodiment, the heating temperature of the hydrothermal reaction is 100-240°C, and the reaction time is 8-16 hours. In a specific embodiment, the heating temperature of the hydrothermal reaction is 200°C, and the reaction time is 10 hours.
[0013] In one embodiment, the separation and purification in step S1 includes centrifugation, filtration and dialysis.
[0014] In a preferred embodiment, the centrifugation is performed at 12000-25000 g for 1-2 hours. In a specific embodiment, the centrifugation is performed at 22000 g for 1 hour.
[0015] In a preferred embodiment, the filtration is performed using a 0.22 μm filter membrane.
[0016] In a preferred embodiment, the molecular weight cut-off of the dialysis is 500-5000Da, and the time is 24-48 hours. In a specific embodiment, the dialysis is performed on the collected filtrate using a dialysis bag with a molecular weight cut-off of 1000Da, and the time is 24 hours.
[0017] In one embodiment, the DNA chain is a complementary DNA chain complementary to the nucleoside base used in the nucleoside carbon dot or a hybrid of a complementary DNA chain and a functional chain.
[0018] In a preferred embodiment, the length of the complementary DNA chain is 10-60 bases, and the functional chain can be connected to the 3' end or 5' end of the complementary DNA chain. In a specific embodiment, when the nucleoside is uridine, the DNA chain is A10 or A10-PLK1; wherein the sequence of A10 is a single-stranded DNA with 10 adenine bases, and its sequence is 5'-AAAAAAAAAA-3', as shown in SEQ ID NO.1; the A10-PLK1 is an antisense oligonucleotide chain connected to PLK1 at the 5' end of A10, and its sequence is 5'-GCACTTGGCAAAGCCGCCCTTAAAAAAAAAA-3', as shown in SEQ ID NO.2.
[0019] In one embodiment, the conditions for the reaction between the nucleoside carbon dots and the DNA chain through hydrogen bonding similar to the base complementary pairing principle are room temperature standing, room temperature shaking, high temperature cooling or programmed annealing.
[0020] In a preferred embodiment, the reaction conditions are high temperature cooling or programmed annealing. In a specific embodiment, the high temperature cooling is heating at 90-100°C for 5 minutes and then cooling naturally; the program of the programmed annealing is: cooling from 95°C to 25°C. In a specific embodiment, the high temperature cooling is heating at 95°C for 5 minutes and then cooling naturally; the program of the programmed annealing is: 95°C, 2 minutes; 85°C, 5 minutes; 60°C, 5 minutes; cooling to 25°C at a rate of 1°C / min.
[0021] In one embodiment, before the nucleoside carbon dots react with the DNA chain by hydrogen bonding similar to the base complementary pairing principle, the nucleoside carbon dots and the DNA chain are respectively prepared into a nucleoside carbon dot solution with a concentration of 1-1000 mg / mL and a DNA chain solution with a concentration of 1-100 μmol / L, and equal volumes are mixed. In a specific embodiment, the concentration of the nucleoside carbon dot solution is 100 mg / mL, and the concentration of the DNA chain solution is 10 μmol / L.
[0022] In one embodiment, the solvent used to prepare the solution is NaCl solution, 1*TAE-Mg 2+ Buffer solutions and other commonly used buffers for the synthesis of DNA nanomaterials.
[0023] In a preferred embodiment, the solvent is 1*TAE-Mg 2+ Buffer solution. In a specific embodiment, the 1*TAE-Mg 2+The composition and concentration of the buffer solution are: 40mM Tris, 20mM acetic acid, 2mM EDTA, 12.5mM Mg(CH3COOH)2, where mM is mmol / L, the Chinese name of Tris is trishydroxymethylaminomethane, CAS No. 77-86-1, and it can also be replaced by its hydrochloride Tris-HCl, CAS No. 1185-53-1; the CAS No. of acetic acid is 64-19-7; the Chinese name of EDTA is ethylenediaminetetraacetic acid, CAS No. 60-00-4, and its sodium salt EDTA-2Na can also be used, CAS No. 139-33-3; the Chinese name of Mg(CH3COOH)2 is magnesium acetate, CAS No. 142-72-3.
[0024] In one embodiment, the purification in step S2 is to use an ultrafiltration tube with a molecular weight cutoff of 10-100kDa to centrifuge at 5000-10000g for 10-60min, collect the liquid in the ultrafiltration tube, wash, and then use an ultrafiltration tube with a molecular weight cutoff of 10-100kDa to centrifuge at 5000-10000g for 10-60min, and collect the liquid in the ultrafiltration tube; in a specific embodiment, the purification in step S2 is to use an ultrafiltration tube with a molecular weight cutoff of 30kDa to centrifuge at 7500g for 30min, collect the liquid in the ultrafiltration tube, wash 3 times, and then use an ultrafiltration tube with a molecular weight cutoff of 30kDa to centrifuge at 7500g for 30min, collect the liquid in the ultrafiltration tube.
[0025] In a preferred embodiment, the cleaning solution is the above-mentioned 1*TAE-Mg 2+ Buffer solution.
[0026] According to a second aspect of the present disclosure, a DNA nanoparticle is provided, which is prepared according to the above-mentioned method for preparing the DNA nanoparticle.
[0027] According to an implementable embodiment of the present disclosure, at least the following beneficial effects are achieved:
[0028] 1. The present invention adopts the principle of complementary base pairing to prepare new DNA nanoparticles, which has a simple operation process and low cost.
[0029] 2. The present invention uses biological molecule nucleosides as raw materials, and prepares nucleoside carbon dots by hydrothermal method, which are combined with DNA chains by hydrogen bonding similar to the principle of base complementary pairing to generate DNA nanoparticles. No metal elements are introduced in the entire preparation process, there is no metal hybridization, the cytotoxicity is small, the biosafety is high, the biocompatibility is good, and nucleic acid delivery can be achieved, which has great application potential in the biomedical field.
[0030] It should be understood that the content described in this section is not intended to identify the key or important features of the embodiments of the present disclosure, nor is it intended to limit the scope of the present disclosure. Other features of the present disclosure will become easily understood through the following description. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] The above and other objects, features and advantages of the exemplary embodiments of the present disclosure will become readily understood by reading the detailed description below with reference to the accompanying drawings. In the accompanying drawings, several embodiments of the present disclosure are shown in an exemplary and non-limiting manner, in which:
[0032] In the drawings, the same or corresponding reference numerals represent the same or corresponding parts.
[0033] Figure 1 A transmission electron microscope image of uridine carbon dots-A10 DNA nanoparticles synthesized by programmed annealing in Example 1 of the present disclosure is shown;
[0034] Figure 2 A transmission electron microscope image of uridine carbon dots-A10 DNA nanoparticles synthesized by high temperature cooling in Example 2 of the present disclosure is shown;
[0035] Figure 3 A transmission electron microscope image of uridine carbon dots-A10-PLK1 functional DNA nanoparticles synthesized by programmed annealing in Example 3 of the present disclosure is shown;
[0036] Figure 4 A transmission electron microscope image of uridine carbon dots-A10-PLK1 functional DNA nanoparticles synthesized by high temperature cooling in Example 4 of the present disclosure is shown;
[0037] Figure 5 The cytotoxicity comparison results of the uridine carbon dot-A10 DNA nanoparticles of Example 1 and the uridine carbon dot-A10-PLK1 functional DNA nanoparticles of Example 3 are shown;
[0038] Figure 6 The cytotoxicity comparison results of the uridine carbon dots-A10 DNA nanoparticles of Example 2 and the uridine carbon dots-A10-PLK1 functional DNA nanoparticles of Example 4 of the present disclosure are shown. DETAILED DESCRIPTION
[0039] In order to make the purpose, features, and advantages of the present disclosure more obvious and easy to understand, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below in conjunction with the drawings in the embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, not all of the embodiments. Based on the embodiments in the present disclosure, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present disclosure.
[0040] The present disclosure provides a method for preparing DNA nanoparticles, comprising the following steps:
[0041] S1: high temperature carbonization of nucleosides, separation and purification to obtain nucleoside carbon dots;
[0042] S2: reacting the nucleoside carbon dots with the DNA chain through hydrogen bonding similar to the base complementary pairing principle, purifying, and obtaining DNA nanoparticles.
[0043] The nucleosides described in the embodiments of the present disclosure are adenine nucleoside, guanosine nucleoside, cytidine nucleoside and uridine nucleoside, all of which are components of RNA. As an example, the nucleoside is uridine nucleoside, which is abbreviated as uridine, and its CAS number is 58-96-8.
[0044] The high-temperature carbonization of nucleosides described in the embodiments of the present disclosure is as follows: dissolving nucleosides in water to obtain a nucleoside solution; transferring the nucleoside solution to a high-temperature and high-pressure reactor for hydrothermal reaction, wherein the conditions of the hydrothermal reaction are 100-240°C for 8-16h. As an example, the heating temperature may be 100°C, 130°C, 150°C, 170°C, 190°C, 200°C, 210°C or 240°C, etc., and the reaction time may be 8 hours, 10 hours, 12 hours, 14 hours or 16 hours, etc.
[0045] The separation and purification described in the embodiments of the present disclosure is to centrifuge, filter and dialyze the high-temperature carbonization product, wherein the centrifugation is to centrifuge at 12000-25000g for 1-2h, the filtration is to filter the supernatant collected by centrifugation with a 0.22μm filter membrane; the dialysis is to dialyze the filtrate collected by filtration with a dialysis bag with a molecular weight cutoff of 500-5000Da for 24-48h. As an example, the centrifugal force of the centrifugation can be 12000g, 15000g, 18000g, 21000g, 22000g or 25000g, etc., the centrifugation time can be 1 hour, 1.2 hours, 1.5 hours, 1.8 hours or 2 hours, etc., the retention molecular weight can be 500Da, 1000Da, 2000Da, 3000Da, 4000Da or 5000Da, etc., and the dialysis time can be 24 hours, 30 hours, 36 hours, 40 hours or 48 hours, etc.
[0046] In the present disclosure, carbon dots, also called carbon quantum dots, are spherical nanoparticles with a size of less than 20 nm; nucleoside carbon dots are carbon dots with a size rich in nucleoside residues that retain the ability of base complementary pairing.
[0047] In the disclosed embodiments, the nucleoside carbon dots react with the DNA chain by hydrogen bonding similar to the base complementary pairing principle, wherein the uridine carbon dots and the DNA chain are diluted into a 1-1000 mg / mL uridine carbon dot solution and a 1-100 μmol / L DNA chain solution; equal volumes of the uridine carbon dot solution and the DNA chain solution are mixed and then allowed to stand at room temperature, shaken at room temperature, cooled at high temperature, or subjected to programmed annealing reaction. As an example, the concentration of the uridine carbon dot solution can be 1 mg / mL, 100 mg / mL, 300 mg / mL, 500 mg / mL, 700 mg / mL, 900 mg / mL or 1000 mg / mL, etc., and the concentration of the DNA chain solution can be 1 μmol / L, 10 μmol / L, 30 μmol / L, 50 μmol / L, 70 μmol / L, 90 μmol / L or 100 μmol / L, etc.; the high temperature cooling is heating at 95°C for 5 minutes and then cooling naturally; the program annealing procedure is: 95°C, 2 minutes; 85°C, 5 minutes; 60°C, 5 minutes; and decreasing to 25°C at a rate of 1°C / min. As an example, the solvent used to dilute the uridine carbon dots and DNA chains is 1*TAE-Mg 2+ Buffer solution, the 1*TAE-Mg 2+ The composition and concentration of the buffer solution are: 40mM Tris, 20mM acetic acid, 2mM EDTA, 12.5mM Mg(CH3COOH)2, where mM is mmol / L, the Chinese name of Tris is trishydroxymethylaminomethane, CAS No. 77-86-1, and it can also be replaced by its hydrochloride Tris-HCl, CAS No. 1185-53-1; the CAS No. of acetic acid is 64-19-7; the Chinese name of EDTA is ethylenediaminetetraacetic acid, CAS No. 60-00-4, and its sodium salt EDTA-2Na can also be used, CAS No. 139-33-3; the Chinese name of Mg(CH3COOH)2 is magnesium acetate, CAS No. 142-72-3.
[0048] The DNA chain in the disclosed embodiment is a complementary DNA chain complementary to the nucleoside base used in the nucleoside carbon dot or a hybrid of a complementary DNA chain and a functional chain, the length of the complementary DNA chain is 10-60 bases, and the functional chain can be connected to the 3' end or 5' end of the complementary DNA chain. As an example, when the nucleoside is uridine, the DNA chain is A10 or A10-PLK1; wherein the sequence of A10 is a single-stranded DNA with 10 adenine bases, which can be combined with uridine by hydrogen bonding similar to the base complementary pairing principle, and its sequence is 5'-AAAAAAAAAA-3', as shown in SEQ ID NO.1; the A10-PLK1 is an antisense oligonucleotide chain connected to PLK1 at the 5' end of A10, and its sequence is 5'-GCACTTGGCAAAGCCGCCCTTAAAAAAAAAA-3', as shown in SEQ ID NO.2.
[0049] The purification in the disclosed embodiment is to centrifuge the product after the reaction at 5000-10000g for 10-60min using an ultrafiltration tube with a molecular weight cutoff of 10-100kDa, collect the liquid in the ultrafiltration tube, wash it, and then centrifuge it at 5000-10000g for 10-60min using an ultrafiltration tube with a molecular weight cutoff of 10-100kDa; as an example, the washing is performed using 1*TAE-Mg 2+ The buffer solution is used for washing three times. The molecular weight cutoff can be 10Da, 30Da, 50Da, 70Da or 100Da. The centrifugal force can be 5000g, 7500g or 10000g, etc. The centrifugation time can be 10min, 20min, 30min, 40min, 50min or 60min, etc.
[0050] The technical solution of the embodiment of the present disclosure is further illustrated by specific examples below.
[0051] Example 1
[0052] In this example, DNA nanoparticles were prepared by programmed annealing, and the specific process was as follows:
[0053] (1) Weigh 0.5 g of uridine and dissolve it in 5 mL of deionized water to obtain a uridine solution;
[0054] (2) The uridine solution was transferred to a 25 mL Teflon high temperature and high pressure reactor for hydrothermal reaction at 200 °C for 10 h, and then cooled naturally to obtain a high temperature carbonized product;
[0055] (3) centrifuging the high temperature carbonization product at 22000g for 60 min, filtering the collected supernatant with a 0.22 μm filter membrane, and collecting the filtrate;
[0056] (4) The collected filtrate was dialyzed using a dialysis bag with a molecular weight cutoff of 1000 Da for 24 h, during which the deionized water for dialysis was replaced every 6 h, and the residual liquid in the dialysis bag was freeze-dried to obtain uridine carbon dots;
[0057] (5) Using 1*TAE-Mg 2+ The buffer solution was used to dilute the uridine carbon dots and A10 into 100 mg / mL uridine carbon dots solution and 10 μmol / L A10 solution respectively;
[0058] (6) 25 μL of uridine carbon dot solution and 25 μL of A10 solution were mixed in a PCR centrifuge tube, and the PCR centrifuge tube was placed in a PCR for programmed annealing. The programmed annealing was as follows: 95°C, 2 min; 85°C, 5 min; 60°C, 5 min; and then reduced to 25°C at a rate of 1°C / min. During this process, the uridine carbon dots and A10 reacted with hydrogen bonds similar to the base complementary pairing principle to obtain a programmed annealing product.
[0059] (7) The product after programmed annealing was centrifuged at 7500 g for 30 min using an ultrafiltration tube with a molecular weight cutoff of 30 kDa, and the liquid in the ultrafiltration tube was collected to remove unreacted nucleoside carbon dots and A10;
[0060] (8) Using 1*TAE-Mg 2+ The liquid in the ultrafiltration tube was washed with a buffer solution for 3 times, and after washing, the ultrafiltration tube was centrifuged at 7500g for 30 minutes with an ultrafiltration tube with a molecular weight cutoff of 30 kDa, and the liquid in the ultrafiltration tube was collected to obtain uridine carbon dot-A10 DNA nanoparticles.
[0061] Example 2
[0062] This example uses high temperature cooling to prepare DNA nanoparticles, and the specific process is as follows:
[0063] (1) Weigh 0.5 g of uridine and dissolve it in 5 mL of deionized water to obtain a uridine solution;
[0064] (2) The uridine solution was transferred to a 25 mL Teflon high temperature and high pressure reactor for hydrothermal reaction at 200 °C for 10 h, and then cooled naturally to obtain a high temperature carbonized product;
[0065] (3) centrifuging the high temperature carbonization product at 22000g for 60 min, filtering the collected supernatant with a 0.22 μm filter membrane, and collecting the filtrate;
[0066] (4) The collected filtrate was dialyzed using a dialysis bag with a molecular weight cutoff of 1000 Da for 24 h, during which the deionized water for dialysis was replaced every 6 h, and the residual liquid in the dialysis bag was freeze-dried to obtain uridine carbon dots;
[0067] (5) Using 1*TAE-Mg 2+ The buffer solution was used to dilute the uridine carbon dots and A10 into 100 mg / mL uridine carbon dots solution and 10 μmol / L A10 solution respectively;
[0068] (6) 25 μL of uridine carbon dot solution and 25 μL of A10 solution were mixed in a PCR centrifuge tube, and the centrifuge tube was heated at 95° C. for 5 min and then naturally cooled. In this process, the uridine carbon dots and A10 reacted by hydrogen bonding similar to the principle of base complementary pairing to obtain a product after high temperature cooling;
[0069] (7) The product after high temperature cooling was centrifuged at 7500 g for 30 min using an ultrafiltration tube with a molecular weight cutoff of 30 kDa, and the liquid in the ultrafiltration tube was collected to remove unreacted nucleoside carbon dots and A10;
[0070] (8) Using 1*TAE-Mg 2+ The liquid in the ultrafiltration tube was washed with a buffer solution for 3 times, and after washing, the ultrafiltration tube was centrifuged at 7500g for 30 minutes with an ultrafiltration tube with a molecular weight cutoff of 30 kDa, and the liquid in the ultrafiltration tube was collected to obtain uridine carbon dot-A10 DNA nanoparticles.
[0071] Example 3
[0072] In this example, functional DNA nanoparticles were prepared by programmed annealing, and the specific process was as follows:
[0073] (1) Weigh 0.5 g of uridine and dissolve it in 5 mL of deionized water to obtain a uridine solution;
[0074] (2) The uridine solution was transferred to a 25 mL Teflon high temperature and high pressure reactor for hydrothermal reaction at 200 °C for 10 h, and then cooled naturally to obtain a high temperature carbonized product;
[0075] (3) centrifuging the high temperature carbonization product at 22000g for 60 min, filtering the collected supernatant with a 0.22 μm filter membrane, and collecting the filtrate;
[0076] (4) The collected filtrate was dialyzed using a dialysis bag with a molecular weight cutoff of 1000 Da for 24 h, during which the deionized water for dialysis was replaced every 6 h, and the residual liquid in the dialysis bag was freeze-dried to obtain uridine carbon dots;
[0077] (5) Using 1*TAE-Mg 2+ The buffer solution was used to dilute the uridine carbon dots and A10-PLK1 into 100 mg / mL uridine carbon dots solution and 10 μmol / L A10-PLK1 solution, respectively;
[0078] (6) 25 μL of uridine carbon dot solution and 25 μL of A10-PLK1 solution were mixed in a PCR centrifuge tube, and the PCR centrifuge tube was placed in a PCR for programmed annealing. The programmed annealing was as follows: 95°C, 2 min; 85°C, 5 min; 60°C, 5 min; and then reduced to 25°C at a rate of 1°C / min. During this process, the uridine carbon dots reacted with A10 through hydrogen bonding similar to the principle of complementary base pairing to obtain a programmed annealed product.
[0079] (7) The product after programmed annealing was centrifuged at 7500 g for 30 min using an ultrafiltration tube with a molecular weight cutoff of 30 kDa, and the liquid in the ultrafiltration tube was collected to remove unreacted nucleoside carbon dots and A10-PLK1;
[0080] (8) Using 1*TAE-Mg 2+ The liquid in the ultrafiltration tube was washed with a buffer solution for three times. After washing, the ultrafiltration tube was centrifuged at 7500g for 30 minutes with an ultrafiltration tube with a molecular weight cutoff of 30 kDa. The liquid in the ultrafiltration tube was collected to obtain uridine carbon dots-A10-PLK1 functional DNA nanoparticles.
[0081] Example 4
[0082] This example uses high temperature cooling to prepare functional DNA nanoparticles, and the specific process is as follows:
[0083] (1) Weigh 0.5 g of uridine and dissolve it in 5 mL of deionized water to obtain a uridine solution;
[0084] (2) The uridine solution was transferred to a 25 mL Teflon high temperature and high pressure reactor for hydrothermal reaction at 200 °C for 10 h, and then cooled naturally to obtain a high temperature carbonized product;
[0085] (3) centrifuging the high temperature carbonization product at 22000g for 60 min, filtering the collected supernatant with a 0.22 μm filter membrane, and collecting the filtrate;
[0086] (4) The collected filtrate was dialyzed using a dialysis bag with a molecular weight cutoff of 1000 Da for 24 h, during which the deionized water for dialysis was replaced every 6 h, and the residual liquid in the dialysis bag was freeze-dried to obtain uridine carbon dots;
[0087] (5) Using 1*TAE-Mg 2+ The buffer solution was used to dilute the uridine carbon dots and A10-PLK1 into 100 mg / mL uridine carbon dots solution and 10 μmol / L A10-PLK1 solution, respectively;
[0088] (6) 25 μL of uridine carbon dot solution and 25 μL of A10-PLK1 solution were mixed in a PCR centrifuge tube, and the centrifuge tube was heated at 95° C. for 5 min and then naturally cooled. In this process, the uridine carbon dots and A10-PLK1 reacted with hydrogen bonds similar to the base complementary pairing principle to obtain a product after high temperature cooling;
[0089] (7) The product after high temperature cooling was centrifuged at 7500 g for 30 min using an ultrafiltration tube with a molecular weight cutoff of 30 kDa, and the liquid in the ultrafiltration tube was collected to remove unreacted nucleoside carbon dots and A10-PLK1;
[0090] (8) Using 1*TAE-Mg 2+ The liquid in the ultrafiltration tube was washed with a buffer solution for three times. After washing, the ultrafiltration tube was centrifuged at 7500g for 30 minutes with an ultrafiltration tube with a molecular weight cutoff of 30 kDa. The liquid in the ultrafiltration tube was collected to obtain uridine carbon dots-A10-PLK1 functional DNA nanoparticles.
[0091] Test example
[0092] 1. Structural analysis of DNA nanoparticles
[0093] The DNA nanoparticles obtained in Examples 1-4 were observed using a transmission electron microscope (TEM). Figure 1-4 As shown, the results show that the DNA nanoparticles obtained in Examples 1-4 are all spherical, but the sizes are uneven, distributed between 50nm-1000nm, and there is no obvious difference between the nanoparticles formed by programmed annealing and high-temperature cooling. However, the edges of the uridine carbon dot-A10-PLK1 functional DNA nanoparticles in Examples 3 and 4 have burrs, which is presumably caused by the antisense oligonucleotide chains of PLK-1 exposed outside.
[0094] 2. Cytotoxicity test
[0095] The cytotoxicity of the DNA nanoparticles obtained in Examples 1-4 was compared by cell experiments. The specific steps were as follows: 5×10 3 cells, cultured for 10 h, and then the culture medium was replaced; different concentrations of DNA nanoparticles obtained in Examples 1-4 were added ( 0, 300nM, 600nM, 900nM respectively) ， Three parallels were set up for each group. After incubation for 24 hours, the culture medium was discarded; 100 μL of CCK-8 solution was added, and incubation was continued for 2 hours. The absorbance at 450 nm was collected to calculate the cell viability.
[0096] The results are as follows Figure 5 and 6As shown, the results show that: there is no significant difference in the effects of different concentrations of DNA nanoparticles obtained by the two methods of programmed annealing and high-temperature cooling on cell functions, and the biosafety is high; the uridine carbon dot-A10 DNA nanoparticles of Examples 1 and 2 have no obvious biotoxic effects on cells and have good biocompatibility, while the uridine carbon dot-A10-PLK1 functional DNA nanoparticles of Examples 3 and 4 have obvious biotoxicity compared with the uridine carbon dot-A10 DNA nanoparticles of Examples 1 and 2, and the biotoxicity comes from the antisense oligonucleotide chain of PLK-1, indicating that the DNA nanoparticles disclosed in the present invention can successfully deliver PLK-1 into cells.
[0097] It should be understood that the various forms of processes shown above can be used to reorder, add or delete steps. For example, the steps recorded in this disclosure can be executed in parallel, sequentially or in different orders, as long as the desired results of the technical solution of this disclosure can be achieved, and this document is not limited here.
[0098] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features.
[0099] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any person skilled in the art who is familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present disclosure, which should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.
Claims
1. A method for preparing DNA nanoparticles, characterized in that: The preparation method comprises the following steps: The nucleoside is carbonized at high temperature, separated and purified to obtain nucleoside carbon dots; The nucleoside carbon dots react with the DNA chain through hydrogen bonding similar to the base complementary pairing principle, and are purified to obtain DNA nanoparticles.
2. The preparation method according to claim 1, characterized in that: The nucleoside is adenosine, guanosine, cytidine or uridine; preferably, the nucleoside is uridine.
3. The preparation method according to claim 1, characterized in that: The high-temperature carbonization is carried out by a hydrothermal reaction in a high-temperature and high-pressure reactor; preferably, the heating temperature of the hydrothermal reaction is 100-240° C., and the reaction time is 8-16 hours.
4. The preparation method according to claim 1, characterized in that: The separation and purification comprises centrifugation, filtration and dialysis; preferably, the centrifugation is centrifugation at 12000-25000g for 1-2 hours; the filtration is filtration using a 0.22 μm filter membrane; the dialysis has a molecular weight cutoff of 500-5000Da and takes 24-48 hours; more preferably, the centrifugation is centrifugation at 22000g for 1 hour; the dialysis is dialysis of the collected filtrate using a dialysis bag with a molecular weight cutoff of 1000Da for 24 hours.
5. The preparation method according to claim 1, characterized in that: The DNA chain is a complementary DNA chain complementary to the nucleoside base used in the nucleoside carbon dots or a hybrid of a complementary DNA chain and a functional chain; Preferably, when the nucleoside is uridine, the DNA chain is A10 or A10-PLK1; wherein the sequence of A10 is shown as SEQ ID NO.1, and the sequence of A10-PLK1 is shown as SEQ ID NO.
2.
6. The preparation method according to claim 1, characterized in that: The conditions for the reaction between the nucleoside carbon dots and the DNA chain by hydrogen bonding similar to the base complementary pairing principle are room temperature standing, room temperature shaking, high temperature cooling or programmed annealing; preferably, the reaction conditions are high temperature cooling or programmed annealing.
7. The preparation method according to claim 6, characterized in that: The high temperature cooling is to heat at 90-100°C for 5 minutes and then cool naturally; the program annealing is: cooling from 95°C to 25°C; preferably, the high temperature cooling is to heat at 95°C for 5 minutes and then cool naturally; the program annealing is: 95°C, 2 minutes; 85°C, 5 minutes; 60°C, 5 minutes; cooling to 25°C at a rate of 1°C / min.
8. The preparation method according to claim 1, characterized in that: Before the nucleoside carbon dots react with the DNA chain by hydrogen bonding similar to the base complementary pairing principle, the nucleoside carbon dots and the DNA chain are respectively prepared into a nucleoside carbon dot solution with a concentration of 1-1000 mg / mL and a DNA chain solution with a concentration of 1-100 μmol / L; preferably, the solvent used for preparing the solution is a NaCl solution or 1*TAE-Mg 2+ Buffer solution.
9. The preparation method according to claim 1, characterized in that: The purification is performed by centrifuging an ultrafiltration tube with a molecular weight cutoff of 10-100kDa at 5000-10000g for 10-60min, collecting the liquid in the ultrafiltration tube, washing, and then centrifuging an ultrafiltration tube with a molecular weight cutoff of 10-100kDa at 5000-10000g for 10-60min to collect the liquid in the ultrafiltration tube; preferably, the purification is performed by centrifuging an ultrafiltration tube with a molecular weight cutoff of 30kDa at 7500g for 30min, collecting the liquid in the ultrafiltration tube, washing 3 times, and then centrifuging an ultrafiltration tube with a molecular weight cutoff of 30kDa at 7500g for 30min to collect the liquid in the ultrafiltration tube.
10. A DNA nanoparticle, characterized in that: The DNA nanoparticles are prepared according to the method for preparing the DNA nanoparticles according to any one of claims 1-9.