Preparation method and application of near-infrared absorption electropositive carbon nitride nano-composite PEISECCNs

Abstract

The invention provides a preparation method and application of a near-infrared absorption electropositive carbon nitride nano-composite PEISECCNs, and relates to the technical field of biological medicines.The preparation method comprises the following steps that a nitrogen-containing precursor and a selenium-containing precursor are calcined under nitrogen protection to obtain a mixture, and the mixture is placed in mixed acid to react to obtain-COOH-SeCCNs modified by-COOH groups; and dissolving the obtained-COOH group modified COOH-SeCCNs and a branched chain PEI600, HOBT, HBTU and N, N-diisopropylethylamine in DMF (Dimethyl Formamide), carrying out an acylation reaction, and dialyzing to obtain a final product PEISeCCNs. The invention constructs the electropositive carbon nitride-based nano-composite PEISECCNs capable of absorbing near-infrared light, and the electropositive carbon nitride-based nano-composite PEISECCNs has good biocompatibility, is beneficial to application in the field of biomedicine, and can absorb near-infrared light. The carbon nitride-based nano-composite PEISeCCNs disclosed by the invention is mainly synthesized by high-temperature calcination, acidification and acylation reaction. The synthesis method is simple, good in repeatability and easy for industrial production, and is a brand-new hybrid compound.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a preparation method and application of a near-infrared absorbing positively charged carbon nitride nanocomposite PEISeCCNs. Background Art

[0002] Gene therapy has low cost, good therapeutic effect, and relatively small toxic side effects. Gene therapy is the transfer of foreign genetic material into the patient's body to treat a specific disease. However, due to the negative charge on nucleic acid molecules and the presence of a variety of negatively charged proteins and glycolipids on the cell membrane surface, nucleic acid molecules cannot passively diffuse into cells due to the repulsive effect of the same charge, resulting in a very low transfection rate. In addition, exogenous genes are easily degraded by lysosomes when entering cells, so it is particularly important to construct a gene delivery system with high transfection efficiency.

[0003] With the development of nanotechnology, nanomaterials have been used as drug carriers to specifically target cancer cells and release drugs into the tumor environment. This technology has become an important research hotspot for cancer treatment. Graphitic carbon nitride has excellent physical and chemical properties such as high biocompatibility, stable fluorescence, and wide excitation wavelength, which has attracted great interest in the biomedical field. However, its short excitation wavelength and negative surface charge limit its application as a gene carrier. Summary of the invention

[0004] The purpose of the present invention is to solve the technical problem in the prior art that the nanomaterial has negative charge on its surface at the excitation wavelength end, which limits its application as a gene carrier.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A method for preparing near-infrared absorbing positively charged carbon nitride nanocomposite PEISeCCNs comprises the following steps:

[0007] A nitrogen-containing precursor and a selenium-containing precursor are calcined under nitrogen protection to obtain a mixture, and the mixture is placed in a mixed acid for reaction to obtain -COOH-modified COOH-SeCCNs; the obtained -COOH-modified COOH-SeCCNs are reacted with a branched PEI 600 HOBT, HBTU and N,N-diisopropylethylamine were dissolved in DMF and acylated to obtain the final product PEISeCCNs.

[0008] Preferably, the -COOH-modified COOH-SeCCNs and the branched PEI 600 The mass ratio of HOBT, HBTU and N,N-diisopropylethylamine is 30:90:27:379:52.

[0009] Preferably, in the acylation reaction, 30 mg of -COOH-modified COOH-SeCCNs and 90 mg of branched PEI are added to every 10 mL of DMF. 600 , 27mg HOBT, 379mg HBTU, 52mg N,N-diisopropylethylamine.

[0010] Preferably, the acylation reaction is carried out at room temperature and stirred for 24 hours, and dialyzed for 12 hours after the reaction is completed.

[0011] Preferably, the mass ratio of the nitrogen-containing precursor to the selenium-containing precursor is 10:1.

[0012] The present application also provides a near-infrared absorbing positively charged carbon nitride nanocomposite PEISeCCNs, which is prepared using the above-mentioned preparation method.

[0013] The present application also provides the use of PEISeCCNs as a non-viral nucleic acid carrier, and the PEISeCCNs are prepared using the above-mentioned preparation method.

[0014] Preferably, the PEISeCCNs are used for delivery of DNA.

[0015] Compared with the prior art, this application has the following beneficial effects:

[0016] (1) The present invention constructs a near-infrared light absorbing positively charged carbon nitride-based nanocomposite PEISeCCNs, which has good biocompatibility, is beneficial for application in the biomedical field, and can absorb near-infrared light. The disclosed carbon nitride-based nanocomposite PEISeCCNs constructed by the present invention is mainly synthesized by high-temperature calcination, acidification, and acylation reactions. The synthesis method of the present invention is simple, has good repeatability, and is easy to industrialize.

[0017] (2) The carbon nitride-based nanocomposite PEISeCCNs of the present invention is positively charged and can effectively condense DNA to form a nanocomposite therewith.

[0018] (3) The self-assembled complex of carbon nitride-based nanocomposite PEISeCCNs and DNA provided by the present invention can achieve DNA delivery and is a new type of gene transfection reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is an X-ray diffractometer (XRD) spectrum of the calcined product SeCCNs in one embodiment of the present invention;

[0020] Figure 2 Transmission electron microscopy mapping analysis (TEM-mapping) of the calcined product SeCCNs in one embodiment of the present invention;

[0021] Figure 3 This is a transmission electron microscope image of the carbon nitride-based nanocomposite PEISeCCNs in one embodiment of the present invention;

[0022] Figure 4 The Zeta potential diagram of the carbon nitride-based nanocomposite PEISeCCNs in one embodiment of the present invention;

[0023] Figure 5 This is a graph showing the agarose gel retardation experiment of the carbon nitride-based nanocomposite PEISeCCNs on DNA;

[0024] Figure 6 The photographs are of the carbon nitride-based nanocomposite PEISeCCNs loaded with eGFP-DNA during cell transfection. DETAILED DESCRIPTION

[0025] The present invention is further described in detail below in conjunction with specific embodiments.

[0026] A method for preparing near-infrared light absorbing positively charged carbon nitride nanocomposite PEISeCCNs comprises the following steps:

[0027] S1: calcining a nitrogen-containing precursor and a selenium-containing precursor under nitrogen protection to obtain a mixture, and placing the mixture in a mixed acid to react to obtain -COOH-modified COOH-SeCCNs;

[0028] In one embodiment, the nitrogen-containing precursor is melamine, the selenium-containing precursor is selenoglucose, and the mass ratio of the melamine to the selenium-containing precursor is 10:1.

[0029] The calcination temperature is 500°C to 600°C; the calcination time is 3 to 5 hours; and the heating rate is 4 to 8°C / min.

[0030] The mixed acid is a mixed acid of 98% concentrated sulfuric acid and 68% concentrated nitric acid, and the volume ratio of 98% concentrated sulfuric acid to 68% concentrated nitric acid in the mixed acid is 1:1.

[0031] The reaction in the mixed acid was carried out at room temperature for 4 h. After the reaction was completed, 1 L of deionized water was used to dilute and wash the mixture obtained by the reaction several times to obtain COOH-SeCCNs modified with -COOH groups.

[0032] S2: COOH-SeCCNs modified with -COOH groups obtained in S1 were mixed with branched PEI 600, HOBT (1-hydroxybenzotriazole), HBTU (O-benzotriazole-tetramethyluronium hexafluorophosphate), and N,N-diisopropylethylamine were dissolved in DMF (N,N-dimethylformamide) and acylated to obtain the final product PEISeCCNs.

[0033] The -COOH-modified COOH-SeCCNs, branched PEI 600 The mass ratio of HOBT, HBTU and N,N-diisopropylethylamine is 30:90:27:379:52.

[0034] Specifically, in one embodiment, 30 mg of -COOH-modified COOH-SeCCNs and 90 mg of branched PEI were added to each 10 mL of DMF. 600 , 27mg HOBT, 379mg HBTU, 52mg N,N-diisopropylethylamine.

[0035] In one embodiment, the acylation reaction is carried out at room temperature and stirred for 24 hours, and then dialyzed for 12 hours after the reaction is completed.

[0036] The present application also provides an application of near-infrared light absorbing positively charged carbon nitride nanocomposite PEISeCCNs as a non-viral nucleic acid carrier, wherein the near-infrared light absorbing positively charged carbon nitride nanocomposite PEISeCCNs is used for DNA delivery.

[0037] The following is an explanation of the above content in combination with specific verification experiments:

[0038] Embodiment 1:

[0039] Preparation of a near-infrared light absorbing positively charged carbon nitride-based nanocomposite PEISeCCNs:

[0040] S1: Weigh 1 equivalent of melamine molecule and 0.1 equivalent of selenoglucose, grind them evenly, and put them in a 60℃ oven to dry for 24 hours. The dried mixture was calcined at 550℃ for 4 hours in a tube furnace under nitrogen protection, with a heating rate of 5℃ / min. The calcined product SeCCNs was finally obtained;

[0041] See also Figure 1 and Figure 2 The calcined product SeCCNs obtained in S1 successfully doped Se and C elements into carbon nitride, and the crystal form of the nanosheets changed. The calcined product can be excited by infrared light.

[0042] S2: 0.1 g of the calcined product obtained in S1 was added to a mixture of 98% concentrated sulfuric acid (20 mL) and 68% concentrated nitric acid (20 mL), reacted at room temperature for 4 h, and then diluted and washed with 1 L of deionized water several times to obtain a mixture;

[0043] S3: Take 10 mL of DMF, add 30 mg of the mixture in S2 and 90 mg of branched PEI 600 , 27 mg HOBT, 379 mg HBTU, and 52 mg N,N-diisopropylethylamine were added and stirred at room temperature for 24 h to obtain a clear solution. The solution was dialyzed using a dialysis bag for 12 h to remove small molecule products and obtain PEISeCCNs.

[0044] See also Figure 3 and Figure 4 , modified PEI 600 The edges of the material become blurred and electrically positive.

[0045] Example 2: Agarose gel blocking experiment comparison:

[0046] PEISeCCNs and puc18-DNA (9 μg / mL) with different mass ratios were incubated in a 37°C water bath for 0.5 h, and then the gel retardation results of PEISeCCNs with different mass ratios were obtained by agarose gel retardation experiment.

[0047] See also Figure 5 , Figure 5 The value marked on the right is the mass ratio of PEISeCCNs to DNA, Figure 5 It can be seen that the PEISeCCNs prepared in Example 1 of the present application can completely block the migration of DNA in agarose at a mass ratio of 1:2 (5 μg / mL:10 μg / mL), that is, the PEISeCCNs prepared in the present application can effectively condense DNA for use as a non-viral nucleic acid carrier.

[0048] Example 3: Gene transfection verification:

[0049] 50 μg / mL of PEISeCCNs obtained in Example 1 was incubated with eGFP-DNA for 30 min, then added to the cells and cultured for 4 h, then the medium was changed, 808 nm light was irradiated for 10 min, and the culture was continued for 48 h. Then, the culture medium was aspirated, washed with PBS 3-5 times, and photographed by laser confocal microscopy, and the light-protected group was used as the control.

[0050] In summary, the present invention constructs a near-infrared light absorbing positively charged carbon nitride-based nanocomposite PEISeCCNs, which has good biocompatibility, is beneficial for application in the biomedical field, and can absorb near-infrared light. The disclosed carbon nitride-based nanocomposite PEISeCCNs constructed by the present invention is mainly synthesized by high-temperature calcination, acidification, and acylation reactions. The synthesis method of the present invention is simple, has good repeatability, and is easy to industrialize.

Claims

1. A method for preparing near-infrared absorbing positively charged carbon nitride nanocomposite PEISeCCNs, characterized in that: The following steps are involved: A nitrogen-containing precursor and a selenium-containing precursor are calcined under nitrogen protection to obtain a mixture, and the mixture is placed in a mixed acid for reaction to obtain -COOH-modified COOH-SeCCNs; the obtained -COOH-modified COOH-SeCCNs are reacted with a branched PEI 600 HOBT, HBTU and N,N-diisopropylethylamine were dissolved in DMF and acylated to obtain the final product PEISeCCNs.

2. The method for preparing a near-infrared absorbing positively charged carbon nitride nanocomposite PEISeCCNs according to claim 1, characterized in that: The -COOH group-modified COOH-SeCCNs and the branched PEI 600 The mass ratio of HOBT, HBTU and N,N-diisopropylethylamine is 30:90:27:379:

52.

3. The method for preparing a near-infrared absorbing positively charged carbon nitride nanocomposite PEISeCCNs according to claim 1, characterized in that: In the acylation reaction, 30 mg of -COOH-modified COOH-SeCCNs and 90 mg of branched PEI were added to every 10 mL of DMF. 600 , 27mg HOBT, 379mg HBTU, 52mg N,N-diisopropylethylamine.

4. The method for preparing a near-infrared absorbing positively charged carbon nitride nanocomposite PEISeCCNs according to claim 1, characterized in that: The acylation reaction was carried out at room temperature and stirred for 24 h, and then dialyzed for 12 h.

5. The method for preparing a near-infrared absorbing positively charged carbon nitride nanocomposite PEISeCCNs according to claim 1, characterized in that: The mass ratio of the nitrogen-containing precursor to the selenium-containing precursor is 10:

1.

6. A near-infrared absorbing positively charged carbon nitride nanocomposite PEISeCCNs, characterized in that: The method is prepared by any one of claims 1 to 5.

7. Application of PEISeCCNs as non-viral nucleic acid carriers, characterized in that: The PEISeCCNs are prepared using the preparation method of claims 1-5.

8. The use of PEISeCCNs as a non-viral nucleic acid carrier according to claim 7, characterized in that: The PEISeCCNs are used for DNA delivery.

Images