DNA surface modified carbon quantum dot containing i-motif and application thereof in tumor treatment

Abstract

The invention discloses a preparation method and application of an i-motif-containing DNA surface modified carbon quantum dot, and the carbon dot is prepared by the following steps: synthesizing a carbon dot, and then carrying out DNA modification. The preparation method comprises the following steps: dissolving phenylenediamine in a polymer solution, uniformly mixing, transferring the solution into a polytetrafluoroethylene inner container, putting the inner container into a stainless steel high-pressure kettle, and heating and reacting in a muffle furnace. And after the reaction is finished, cooling a reaction system to room temperature, then polymerizing DNA containing i-motif as a cross-linking agent with CDs, after the polymerization reaction is finished, freeze-drying the solution, taking out the solution, putting the solution into a dialysis bag, and carrying out dialysis purification to obtain DNA-CDs after dialysis is finished. And then carrying out bionic treatment on the MV-DNA-CDs, so as to obtain the MV-DNA-CDs disclosed by the invention. The nano-carrier prepared by the invention is high in yield and good in stability, has responsiveness and targeting property, and can be used for precise treatment of tumors.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterial science and technology, and relates to carbon quantum dots with DNA surface modification containing i-motif and applications thereof in tumor diagnosis and treatment. Background Art

[0002] Chemotherapy can prolong the survival time of patients and improve their quality of life. It is an indispensable means of clinical treatment or adjuvant treatment of tumors. However, chemotherapy drugs generally have the disadvantage of poor specificity, which leads to serious systemic or local toxic side effects in patients during chemotherapy. In order to solve the above problems, researchers have found in recent years that the delivery of chemotherapy drugs by nano-drug delivery system is one of the most promising methods.

[0003] Carbon quantum dots, also known as carbon dots (CDs), are a new type of fluorescent carbon-based nanomaterials. Due to their unique optical properties, excellent biocompatibility and easy availability, they have been widely used in the biomedical field. Especially in the field of cancer diagnosis and treatment, they are widely used for multi-level resolvable bioimaging of cancer cells or tissues, and even for subcellular organelle localization microimaging. At the same time, the imaging-assisted drug delivery platform (IADDP) derived from CDs has achieved precise tracking of in vivo drug delivery and visualization of the ADME behavior (absorption, distribution, metabolism and excretion) of drug carriers. However, with the deepening of research, CDs-based cancer treatment IADDP has gradually exposed defects, which severely limited the delivery behavior of IADDP in vivo, especially in tumor targeting, release behavior of therapeutic agents, etc. The obstacles are usually mainly reflected in two aspects, namely poor size regulation and ignoring the versatility of fluorescence.

[0004] In order to make CDs-based IADDP tumor-targeted and controllable in drug release, traditional CDs have been improved into CD complexes with special sizes or surface ligand structures through simple physical regulation (charge / hydrophobic interaction) or chemical modification. Through comprehensive comparison and stability verification, compared with physical methods, chemical modification, that is, the introduction of surface ligands or sensitive chemical bonds through chemical reactions, can better achieve size regulation and drug-sensitive release of carbon dot nanocomposites. However, further analysis found that due to the small size of the primary spherical structure of carbon dots, there are fewer functional groups on its surface that can be used for chemical reactions, and it is impossible to achieve particle size regulation, chemical sensitive bond introduction, and targeting ligand modification at the same time. At the same time, complex and multi-step chemical reactions on the surface of synthesized carbon dots can easily cause fluorescence quenching. Summary of the invention

[0005] The purpose of the present invention is to provide a DNA surface-modified carbon quantum dot containing i-motif and its application in tumor diagnosis and treatment in order to address the deficiencies of the prior art. The nanopolymer carrier has acid responsiveness, light responsiveness, targeting and high biocompatibility, and can be used in the precise treatment of tumors.

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

[0007] A method for preparing carbon quantum dots with DNA surface modification containing i-motif comprises the following steps:

[0008] Dissolve phenylenediamine in the polymer solution, mix well, transfer the solution to a polytetrafluoroethylene liner, then place the liner in a stainless steel autoclave and heat it in a muffle furnace for reaction. After the reaction is completed, the reaction system is cooled to room temperature, and then the i-motif-containing DNA is used as a crosslinker to polymerize with CDs. After the polymerization reaction is completed, the above solution is freeze-dried, and the solution is taken out and placed in a dialysis bag for dialysis purification. After the dialysis is completed, DNA-CDs are obtained. Then it is subjected to biomimetic treatment to obtain the final nanomaterial MV-DNA-CDs for tumor treatment.

[0009] Furthermore, the phenylenediamine is one of o-phenylenediamine, m-phenylenediamine and p-phenylenediamine.

[0010] Furthermore, the polymer solution is methacrylic acid, ethacrylic acid, or polyacrylic acid solution; and the optimal solution pH value is 2-9.

[0011] Going further, the two single-stranded sequences of DNA containing the i-motif are ssDNA1: CTGTCATCG GTCACACATTCCTAAGTC TGACCCTAA CCCTAACCC TAACCC; ssDNA2: GACAGTAGC CAGTGTGTA AGGATTCAGACTGGGATT GGGATTGGG ATTGGG; the sequence can be shortened / extended.

[0012] Furthermore, the optimal reaction temperature for the heating reaction is 180-500° C., and the optimal reaction time is 2-8 hours.

[0013] Furthermore, the molecular weight cut-off of the dialysis bag is 500-1000Da, and the dialysis time is 12-48 hours.

[0014] Furthermore, the biomimetic treatment was performed by mixing the carbon dots and the extracted macrophage microbubbles evenly, ultrasonically treating for 2 min, stopping for 1 min, and repeating the above operation 5 times to encapsulate the DNA-CDs in the macrophage microbubbles.

[0015] A DNA surface modified carbon quantum dot containing i-motif is used in the precision treatment of tumors.

[0016] Compared with the prior art, the present invention has the following advantages:

[0017] The synthesis method is simple and easy, and the experimental operation is simple. Carbon dots modified with DNA surface containing i-motif can actively target tumor sites and can respond to light and the tumor microenvironment to achieve controllable disintegration of carbon dots at tumor sites.

[0018] After bionic treatment, nanomaterials can be endowed with high biocompatibility and active tumor targeting, thereby improving the specific distribution of drugs at the tumor site.

[0019] The synthesis method is controllable, and the particle size regulation and controllable disintegration of carbon dots can be achieved by adjusting the DNA sequence and adjusting the reaction conditions (time, temperature).

[0020] After optimization, the nanopolymer carrier synthesized after modifying the carbon dots in the present invention can achieve particle size control, acid responsiveness, light responsiveness, targeting and high biocompatibility, and can be applied to the precise treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0022] Figure 1 This is a schematic diagram of the preparation of Example 1;

[0023] Figure 2 The UV (left side) and fluorescence emission spectra (right side) of the carbon dots prepared in Example 1;

[0024] Figure 3 The transmission electron microscope image (left) and the particle size distribution diagram (right) of the carbon dots prepared in Example 1 show that the synthesized carbon dots basically present a spherical or quasi-spherical structure. The particle size distribution histogram obtained through statistical analysis shows that the average particle size of the carbon dots is 270nm;

[0025] Figure 4 The carbon dots prepared in Example 1 are released responsively under acid treatment and light treatment; DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with embodiments. The embodiments provide detailed implementation methods and specific processes, but the protection scope of the present invention is not limited to the following embodiments.

[0027] The instruments and equipment used in the following examples are as follows:

[0028] Fluorescence spectrophotometer RF-5301 (Shimadzu, Japan); transmission electron microscope JEM-1200EX TEM (JEOL, Japan); X-ray photoelectron spectrometer EscaLab 250Xi (Thermo, USA); small animal in vivo imager VISQUE In vivo Smart-LF (Shanghai, China).

[0029] The carbon dots described in the following examples are MV-DNA-CDs.

[0030] Embodiment 1:

[0031] 0.025g of o-phenylenediamine was dissolved in methacrylic acid and initiator dilution solution, and after mixing evenly, the solution was transferred to a polytetrafluoroethylene liner, and then the liner was placed in a stainless steel autoclave and heated in a muffle furnace for reaction. After the reaction was completed, the reaction system was cooled to room temperature to obtain the secondary structure CDs, and then the DNA sequences containing the motif were ssDNA1: CTGTCATCG GTCACACAT TCCTAAGTC TGACCCTAA CCCTAACCC TAACCC; ssDNA2: GACAGTAGC CAGTGTGTA AGGATTCAG ACTGGGATT GGGATTGGG ATTGGG. as a crosslinker to polymerize with CDs. After the polymerization reaction was completed, the above solution was freeze-dried, and the solution was taken out and placed in a dialysis bag for dialysis purification. DNA-CDs were obtained after the dialysis was completed. Then it was subjected to biomimetic treatment to obtain the final nanomaterial MV-DNA-CDs for tumor treatment.

[0032] Embodiment 2:

[0033] Example 1 describes the application of carbon quantum dots modified with DNA surface containing i-motif in tumor treatment. The particle size regulation and controllable disintegration of carbon dots were detected by transmission electron microscopy. The results showed that the carbon dots have adjustable particle size, acid responsiveness and light responsiveness.

[0034] The above disclosure is only the preferred embodiment of the present invention, which certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. Preparation of carbon quantum dots with DNA surface modification containing i-motif, characterized in that ,Phenylenediamine was dissolved in the polymer solution, and after mixing evenly, the solution was transferred to a polytetrafluoroethylene liner, and then the liner was placed in a stainless steel autoclave and heated in a muffle furnace for reaction. After the reaction is completed, the reaction system is cooled to room temperature, and then the i-motif-containing DNA is used as a cross-linking agent to polymerize with CDs. After the polymerization reaction is completed, the above solution is freeze-dried, and the solution is taken out and placed in a dialysis bag for dialysis purification. After the dialysis is completed, DNA-CDs are obtained. It is then biomimetic treated to obtain the final nanocarrier MV-DNA-CDs for tumor treatment.

2. The method for preparing DNA-CDs according to claim 1, characterized in that: The phenylenediamine is one of o-phenylenediamine, m-phenylenediamine and p-phenylenediamine.

3. The method for preparing DNA-CDs according to claim 1, characterized in that: The polymer solution is methacrylic acid, ethacrylic acid, or polyacrylic acid solution; the optimal solution pH value is 2-9.

4. The method for preparing DNA-CDs according to claim 1, characterized in that: The optimal reaction temperature is 180-500° C., and the optimal reaction time is 2-8 hours.

5. The method for preparing DNA-CDs according to claim 1, characterized in that: The two single-stranded sequences of DNA containing i-motif are ssDNA1: CTGTCATCG GTCACACAT TCCTAAGTC TGACCCTAA CCCTAACCC TAACCC; ssDNA2: GACAGTAGC CAGTGTGTA AGGATTCAG ACTGGGATT GGGATTGGG ATTGGG. Different DNA sequences can be designed by shortening / extending to meet different needs.

6. The method for preparing DNA-CDs according to claim 1, characterized in that: The molecular weight cutoff of the dialysis bag for dialysis is 500-1000Da, and the dialysis time is 12-48 hours.

7. The method for preparing MV-DNA-CDs according to claim 1, characterized in that: The biomimetic treatment is to mix the carbon dots and the extracted macrophage microbubbles evenly, perform ultrasonic treatment for 2 minutes, stop for 1 minute, and repeat the above operation 5 times to encapsulate the DNA-CDs in the macrophage microbubbles.

8. Use of MV-DNA-CDs according to any one of claims 1 to 7 in biological imaging, characterized in that: A DNA-surface-modified carbon quantum dot polymer carrier containing an i-motif has adjustable particle size, acid responsiveness, and light responsiveness for precise tumor treatment.

9. The use of MV-DNA-CDs in tumor treatment according to claim 7, characterized in that The nanocarriers are endowed with active targeting and high biocompatibility, thereby improving their specific distribution at the tumor site.

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