A carbon dot delivery system for delivering exogenous nucleic acids into bacteria and its gene interference application

Abstract

The present invention relates to the field of biotechnology, and in particular to a carbon dot delivery system for delivering exogenous nucleic acids into bacteria and its gene interference application. The present invention uses a one-step hydrothermal method to synthesize carbon dots (CDs), which can effectively load empA ASO and efficiently deliver it to Vibrio anguillarum to target and regulate the expression of bacterial pathogenic genes and study gene functions, opening up a new way for the prevention and treatment of pathogenic bacteria.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a carbon dot delivery system for delivering exogenous nucleic acid into bacteria and a gene interference application thereof. Background Art

[0002] In the post-antibiotic era, researchers are increasingly using methods to inhibit pathogenicity to prevent and treat bacterial diseases. Fish diseases induced by Vibrio anguillarum infection are caused by the decomposition of mucus proteins in the mucus on the surface of fish by metalloproteinases (EmpA) secreted by Vibrio anguillarum at the protein level. In the late stage of Vibrio anguillarum infection, Vibrio anguillarum can also cause sepsis in fish, damage organs and tissues such as the liver and spleen, and cause systemic diseases. Many studies have also shown that the expression of the empA gene of Vibrio anguillarum is the primary cause of a variety of marine fish diseases. Therefore, inhibiting the expression of its coding gene empA or inactivating EmpA is expected to overcome the diseases caused by Vibrio anguillarum from the root.

[0003] Antisense oligonucleotide (ASO) is a single-stranded oligonucleotide molecule (18-30 nt). As a tool for downregulating gene expression, it has the advantages of high efficiency and targeting. After entering the cell, it can be complementary to the target sequence, causing mRNA degradation under the action of ribonuclease H1 (RNase H1), thereby inhibiting protein expression; or blocking the translation process of ribosomes through steric hindrance effect, regulating gene expression, and achieving the purpose of treating diseases. It is currently widely used in the medical field. However, the application of ASO technology is still limited, mainly because ASO itself is negatively charged and difficult to enter cells through passive diffusion, which makes the delivery efficiency of ASO molecules low. In particular, bacteria have a cell wall structure, which makes it more difficult for ASO to enter the bacteria to exert its effect.

[0004] At present, the commonly used strategies for delivering exogenous nucleic acids into bacteria are electroporation and chemical transformation, but these methods have disadvantages such as complex processes, low editing flux, and limited use occasions. Therefore, a vector that can simply and efficiently deliver ASO into bacterial cells to achieve the silencing effect of target genes is urgently needed to be developed. The applicant previously studied the "Construction and Application of Nanocarrier Systems for Efficient Delivery of Exogenous Nucleic Acids for Bacterial Gene Silencing" (Patent No. 2023100021435), and used a nanogold cluster delivery system to efficiently deliver ligA siRNA into bacterial cells to inhibit bacterial growth. However, the nanogold cluster delivery system is costly. In order to obtain a more economical and efficient delivery system and realize its application in inhibiting the pathogenicity of Vibrio anguillarum, the applicant continued to study new nanocarrier delivery systems and construction methods.

[0005] As a new type of carbon-based nanomaterials with significant fluorescence properties, carbon dots (CDs) have attracted widespread attention from researchers in the biological field. They have the advantages of small size, good biocompatibility, wide raw material sources, high stability, and rich functional groups. They have achieved remarkable research results in many fields such as drug delivery, biosensing, bioimaging, and biocatalysis. As an excellent delivery material, CDs have good application potential in the field of delivering anticancer drugs to treat cancer. For example, by constructing CDs with a hydrophobic carbon core and a hydrophilic shell, a composite material of CD and doxorubicin (DOX) was assembled, showing significant tumor targeting and enhanced antitumor efficacy, and reduced adverse reactions. In addition to DOX, the CDs platform based on cisplatin (Pt(IV)) can enhance the drug therapeutic effect of Pt(IV), demonstrating the potential of CDs as smart nanocarriers. At present, the technology of using carbon dots as carriers to deliver exogenous nucleic acids into bacterial cells has not been reported. The applicant has attempted to synthesize carbon dots using existing methods and use them to deliver antisense oligonucleotides into bacterial cells. However, the carbon dots synthesized by most methods are negatively charged and cannot successfully load antisense oligonucleotides. Therefore, it is of great significance to construct a CDs delivery system that can load antisense oligonucleotides to simply and efficiently deliver the specific antisense oligonucleotides of the present invention to Vibrio anguillarum, specifically regulate the expression of the pathogenic gene empA gene, and effectively inhibit its pathogenicity. Summary of the invention

[0006] The present invention provides a carbon dot delivery system for delivering exogenous nucleic acids into bacteria and its gene interference application. Carbon dots (CDs) are synthesized by a one-step hydrothermal method, and empA ASO is designed so that the prepared carbon dots can effectively load empA ASO and efficiently deliver it to Vibrio anguillarum, thereby targetedly regulating the expression of pathogenic genes of Vibrio anguillarum, opening up a new way for the prevention and treatment of pathogenic bacteria in fish and solving the problems existing in the prior art.

[0007] One of the technical solutions adopted by the present invention is:

[0008] A carbon dot delivery system for delivering exogenous nucleic acid into bacteria is constructed, comprising the following steps:

[0009] (1) Preparation of carbon dots

[0010] Dissolve citric acid and polyethyleneimine in ultrapure water and heat to react to prepare carbon dots CDs;

[0011] (2) Loading exogenous nucleic acid

[0012] The CDs are co-incubated with exogenous nucleic acids in a certain ratio to obtain CDs loaded with exogenous nucleic acids.

[0013] Furthermore, the carbon dots CDs in step (1) have a positive charge on their surface and a diameter of 3-4 nm, and bind to the exogenous nucleic acid by electrostatic adsorption; the exogenous nucleic acid in step (2) is ASO, and the sequence is shown in SEQ ID No. 1; the mass ratio of CDs to ASO is ≥5, and the incubation time is 1-5 h.

[0014] Furthermore, the ASO is empA ASO, and the sequence is: SEQ ID No. 1: TTACAAGGTACGCGAAGC. The sequence is completely phosphorothioated (the non-bridging oxygen atoms of the sulfodiester bonds of the single-stranded DNA are all replaced with sulfur atoms): 5′-T*T*A*C*A*A*G*G*T*A*C*G*C*G*A*A*G*C-3′ (* represents phosphorylation), so that the ASO can be better protected from enzyme degradation. The ASO sequence was synthesized by Shanghai Bioengineering Co., Ltd.

[0015] Furthermore, the sequence of the empA ASO is designed with the empA gene sequence of Vibrio anguillarum as the target chain, and the empA gene sequence is shown in SEQ ID No2.

[0016] Furthermore, the mass ratio of CDs to ASO is 5-50:1; and the incubation time is 2 h.

[0017] Furthermore, the mass ratio of CDs to ASO was 20:1.

[0018] Furthermore, in step (1), the mass ratio of citric acid to polyethyleneimine is 1:0.5 to 1:1.5, the heating temperature is 180 to 220° C., and the heating cycle is 0.5 h to 4 h.

[0019] Furthermore, the specific method for preparing carbon dots in step (1) is as follows:

[0020] 1.0 g of citric acid (CA) and 2.0 g of polyethyleneimine (PEI) were dissolved in 10 mL of ultrapure water and ultrasonically treated for 10 min; then, the mixed solution was transferred to a polytetrafluoroethylene-lined reactor for hydrothermal reaction at 200 ° C for 2 h; after the reaction was completed, it was cooled to room temperature, and then the yellow solution was dialyzed in ultrapure water with a dialysis bag (1000 Da) for 24 h; finally, the filtrate was vacuum freeze-dried to obtain yellow CD powder. The size of the CDs was 3.12 nm.

[0021] Furthermore, the bacterium is the Gram-negative bacterium Vibrio anguillarum.

[0022] Furthermore, the Vibrio anguillarum is Vibrio anguillarum NBRC 13266.

[0023] The second technical solution adopted by the present invention is:

[0024] The carbon dot delivery system is used to efficiently deliver exogenous nucleic acids into bacterial cells to achieve the application of inhibiting bacterial pathogenicity. The carbon dot delivery system is prepared by the construction method as described above; the bacterial gene is the Vibrio anguillarum empA gene; the exogenous nucleic acid is empA ASO, and the sequence is:

[0025] TTACAAGGTACGCGAAGC.

[0026] Furthermore, as described above, empA ASO is loaded onto carbon dot CDs at a specific ratio and delivered into the cells of Vibrio anguillarum by incubating the carbon dot CDs with the Vibrio anguillarum for 1 to 5 hours, thereby achieving targeted silencing of empA in a short period of time and reducing the pathogenicity of Vibrio anguillarum.

[0027] The third technical solution adopted by the present invention is:

[0028] A drug comprises carbon dot CDs loaded with empA ASO and a pharmaceutically acceptable carrier, wherein the drug targets Vibrio anguillarum and reduces the pathogenicity of Vibrio anguillarum.

[0029] Beneficial effects of the present invention:

[0030] 1. The present invention constructs a carbon dot delivery system for bacteria for the first time, providing an excellent platform for efficient targeted delivery of exogenous nucleic acids into bacteria. There is no such report so far.

[0031] 2. By adopting the technical solution of the present invention, the prepared carbon dots can efficiently deliver empA ASO into the cells of Vibrio anguillarum, and effectively silence empA in a short time, thereby inhibiting the production of metalloproteinases and reducing the pathogenicity of Vibrio anguillarum, providing a new way to solve the problem of Vibrio anguillarum infection.

[0032] 3. The carbon dots loaded with empA ASO constructed by the present invention can achieve efficient delivery of empA ASO of a specific sequence into Vibrio anguillarum, which is economical and has high delivery efficiency, and is of great significance for achieving targeted silencing or elimination of the empA gene of Vibrio anguillarum and preparing corresponding drugs to reduce the pathogenicity of Vibrio anguillarum. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Flow chart for the preparation of ASO-loaded CDs delivery system;

[0034] Figure 2 Characterization of the ASO carbon dot delivery carrier CDs prepared by the present invention;

[0035] Figure 3 Agarose gel electrophoresis was used to detect the loading capacity of carbon dot delivery carriers CDs for ASO;

[0036] Figure 4This is a diagram of the delivery efficiency of CDs to ASO;

[0037] Figure 5 To optimize the uptake conditions of ASO / CDs by Vibrio anguillarum;

[0038] Figure 6 The silencing effect of the multiple ASO chains designed for the present invention on the empA gene of Vibrio anguillarum;

[0039] Figure 7 To analyze the transcription level changes of empA gene using fluorescence quantitative PCR technology;

[0040] in, Figure 2 The three figures in the middle are, from left to right, the transmission electron microscope image, particle size and X-ray photoelectron spectrum of the carbon dots of the present invention;

[0041] Figure 5 The two figures in the middle are the results of flow cytometry evaluation of the amount of Cy3-ASO delivered to Vibrio anguillarum at different incubation times and different CDs / ASO mass ratios. DETAILED DESCRIPTION

[0042] In order to clearly illustrate the technical features of this solution, the present invention is described in detail below through specific implementation methods and in combination with the accompanying drawings. In the embodiments of the present invention, unless otherwise specified, all parts and percentages are weight units, and the equipment and raw materials used can be purchased from the market or are commonly used in the art. The experimental methods, detection methods, etc. involved in the following embodiments, unless otherwise specified, are all conventional experimental methods, detection methods, etc. in the prior art.

[0043] Example 1 Preparation of Carbon Dot Delivery Carrier CDs

[0044] 1.0 g of citric acid (CA) and 2.0 g of polyethyleneimine (PEI) were dissolved in 10 mL of ultrapure water and ultrasonically treated for 10 min. Then, the mixed solution was transferred to a polytetrafluoroethylene-lined reactor for hydrothermal reaction at 200 ° C for 2 h. After the reaction was completed, it was cooled to room temperature, and then the yellow solution was dialyzed in ultrapure water for 24 h using a dialysis bag (1000 Da). Finally, the filtrate was vacuum freeze-dried to obtain a yellow CD powder for later use. The preparation flow chart of the complete ASO-loaded CDs delivery system is shown in the figure. Figure 1 shown.

[0045] Transmission electron microscopy (TEM), Fourier transform infrared spectrometer (FT-IR), X-ray photoelectron spectrometer (XPS), Malvern nanoparticle size potentiostat (NanoZS) and other instruments are used to characterize the size, morphology, functional groups, potential and other related properties of CDs. Figure 2 As shown, the size of CDs prepared by the present invention is about 3.12nm.

[0046] Example 2 Gel electrophoresis analysis of the loading capacity of CDs loaded with ASO

[0047] CDs were incubated with ASO at different mass ratios (1:1, 2:1, 3:1, 4:1, 5:1 and 6:1) for 30 min. The ASO loading capacity of CDs was determined by analyzing the brightness of the electrophoretic bands using 1% agarose gel electrophoresis.

[0048] The results are as follows Figure 3 As shown: the free ASO band is clear, and with the addition of CDs, the band gradually weakens, which shows that CDs can effectively load ASO. When the mass ratio of CDs: ASO is 5:1, ASO can be fully loaded. The mass ratio of CDs to ASO is at least 5:1.

[0049] Example 3 Analysis of the delivery efficiency of CDs to ASO

[0050] ASO was modified with a Cy3 fluorescent probe, and it was co-incubated with CDs to obtain an ASO / CDs complex. The final concentration of Cy3-ASO was maintained at 500 nM. The prepared complex was co-incubated with Vibrio anguillarum in the logarithmic growth phase in a 28°C incubator for 2 hours. After the incubation, the complex was centrifuged at 6000 rpm for 6 minutes and washed with PBS. Hoechst 33342 fluorescent dye was added to the precipitate and protected from light for 30 minutes. The bacteria were collected by centrifugation and observed under the conditions of orange-red fluorescence and blue fluorescence under a fluorescence microscope. If there is an overlap between the blue fluorescence and the orange-red fluorescence, it indicates that the ASO can be delivered into the cell.

[0051] The results are as follows Figure 4 As shown: the CDs prepared by the present invention can effectively deliver ASO into bacterial cells, while ASO itself cannot effectively enter bacterial cells, and the delivery efficiency of ASO is related to the amount of carbon dots used and the incubation time.

[0052] The conditions for the uptake of ASO / CDs by Vibrio anguillarum were further optimized: flow cytometry was used to analyze (a) the uptake rate of ASO / CDs with different mass ratios (CD:ASO=0:1, 5:1, 10:1, 20:1, 50:1, 100:1) by Vibrio anguillarum after 2 h of incubation; (b) the uptake rate of Vibrio anguillarum at different incubation times (0, 0.5, 1, 2, 3 and 5 h).

[0053] (1) Investigation of CDs dosage: ASO / CDs complex (100 μL) was mixed with a 10 6CFU / mL of Vibrio anguillarum suspension was cultured together in LB liquid medium (900 μL) at 37 ° C for 1 h. The final concentration of Cy3-ASO was 500 nM, and the final concentration of CDs varied between 0, 14.5, 29, 58, 145 and 290 μg / mL. After incubation, the Vibrio anguillarum suspension was centrifuged (6000 rpm, 6 min), washed 3 times with PBS containing 0.1% Triton X-100 to remove the bacterial surface adsorption complex of Cy3-ASO / CDs, and finally resuspended in PBS. Flow cytometry was used to evaluate the amount of Cy3-ASO delivered to Vibrio anguillarum.

[0054] (2) Incubation time: ASO / CDs complex (100 μL) was incubated with 6 CFU / mL of Vibrio anguillarum were cultured together in LB liquid medium (900 μL) at 28°C for different durations (0, 0.5, 1, 2, 3 and 5 h). After incubation, the bacteria were centrifuged (6000 rpm, 6 min), washed 3 times with PBS containing 0.1% Triton X-100 to remove the bacterial surface adsorption complex of Cy3-ASO / CDs, and finally resuspended in PBS. Then, flow cytometry was used to evaluate the amount of Cy3-ASO delivered to Vibrio anguillarum.

[0055] Results analysis: see Figure 5 In the middle right figure, when the mass ratio of CDs to ASO is 0:1, no fluorescence signal of Cy3 is observed, and the uptake rate of ASO / CDs is 0. When the mass ratio of CDs (58 μg / mL) to ASO is 20:1, the fluorescence signal of Cy3 is the strongest, and the absorption of ASO / CD can reach 75.4%. As the dosage of CDs increases further, the uptake efficiency of ASO / CDs by Vibrio anguillarum gradually decreases. This result can be attributed to the fact that high concentrations of CDs (290 μg / mL) can inhibit bacterial activity and affect the permeability of bacterial membranes, resulting in a decrease in the uptake of ASO / CDs by bacteria.

[0056] See also Figure 5 In the left figure, when the incubation time is 0.5h, the fluorescence intensity of Cy3 is weak, and the bacterial uptake rate of ASO / CDs is 25.4%. When the incubation time is 2h, the fluorescence intensity of Cy3 is the strongest, and the uptake rate of ASO / CDs is 75.4%. As the incubation time increases further, the uptake efficiency of ASO / CDs decreases slightly. This may be attributed to the number of ASO / CDs fixed in the incubation system. As the incubation time increases, Vibrio anguillarum continues to proliferate, resulting in a decrease in the proportion of bacteria that can absorb ASO / CDs.

[0057] Example 4 Analysis of changes in transcription levels of the empA gene using fluorescent quantitative PCR technology

[0058] 10 was prepared by using Vibrio anguillarum NBRC13266 6 CFU / mL of Vibrio anguillarum suspension. ASO / CDs complex (CD:ASO=20:1 (mass ratio)) was added to the Vibrio anguillarum suspension (106CFU / mL). In the total system, the concentrations of ASO and CDs were 500nM and 58μg / mL, respectively. The mixture was incubated at 28°C for 2h. The control group added water, ASO, CDs and CR-ASO / CDs to the same Vibrio anguillarum suspension. After incubation, each system was centrifuged (6000rpm, 6min) and washed, and finally total RNA was extracted using Trizol reagent. The total RNA was then reversely transcribed into cDNA using the HiScript III All-in-one RT SuperMix Perfectfor qPCR kit, where the total reaction system was 20μL, the reaction system was fully mixed, placed in a PCR instrument for reverse transcription, and the reverse reaction system was shown in Table 1 below. The reaction procedure: 50°C for 15min, 85°C for 5s. The cDNA concentration was measured by Nanodrop and recorded, and diluted to 100 ng / μL. Finally, upstream and downstream primers were added, and amplification was performed using the ChamQ SYBR Colour qPCR kit. The amplification reaction system is shown in Table 2 below. The amplification conditions were: 95°C for 3 min, 95°C for 10 s, and 60°C for 35 s, for a total of 45 cycles. Finally, 95°C was maintained for 15 s and 60°C for 1 min to end the program. The cycle threshold (Ct) value was measured using qPCR. The relative expression of the target gene (empA) was quantified using the 2-ΔΔCt method, using 16S rRNA as the control gene.

[0059] Table 1 Reverse transcription reaction system

[0060]

[0061] Table 2 qRT-PCR reaction system (10 μL per well)

[0062]

[0063] The empA gene sequence of the target Vibrio anguillarum of the present embodiment is:

[0064]

[0065] In this embodiment, a total of 4 ASOs were designed to verify the gene silencing effect. The sequences of the 4 ASOs are shown in Table 3. Figure 6 , which is the silencing effect of 4 different chains on the empA gene of Vibrio anguillarum. Therefore, in this example, ASO-250 (ASO of SEQ ID No. 1) is selected as the chain with the best effect.

[0066] Table 3

[0067] Primer Name Sequence (5' to 3') Number of bases ASO-80 T*G*A*A*C*A*G*T*G*C*A*C*A*A*T*T*T*C 18 ASO-250 T*T*A*C*A*A*G*G*T*A*C*G*C*G*A*A*G*C 18 ASO-737 C*C*A*A*A*A*T*A*T*G*T*C*T*G*G*T*G*G 18 ASO-1481 C*T*T*C*A*C*C*A*C*A*A*G*T*T*G*A*A*C 18

[0068] The detection primer sequences used in the fluorescent quantitative qPCR of this embodiment are shown in Table 4 below:

[0069] Table 4

[0070]

[0071]

[0072] Fluorescence quantitative qPCR results are as follows Figure 7 As shown, by calculating the relative expression level of the empA gene of Vibrio anguillarum, it was found that the CDs treatment group loaded with ASO was significantly lower than that of other groups, which indicates that the constructed carbon dot carrier successfully delivered ASO into Vibrio anguillarum, achieving effective silencing of the intracellular empA gene, thereby targetedly inhibiting the pathogenicity of Vibrio anguillarum.

[0073] The above is a detailed introduction to the construction of the carbon dot delivery system for delivering exogenous nucleic acids to bacteria provided by the present invention and its application in antibacterial aspects. The above specific implementations cannot be used as limitations on the protection scope of the present invention. For those skilled in the art, any substitution, improvement or change made to the implementation of the present invention falls within the protection scope of the present invention.

[0074] The matters not described in detail in the present invention are all known technologies to those skilled in the art.

Claims

1. A carbon dot delivery system for delivering exogenous nucleic acid into bacteria, characterized in that: The steps are as follows: (1) Preparation of carbon dots Dissolve citric acid and polyethyleneimine in ultrapure water and heat to react to prepare carbon dots CDs; (2) Loading exogenous nucleic acid The CDs are co-incubated with exogenous nucleic acids in a certain ratio to obtain CDs loaded with exogenous nucleic acids; The carbon dots CDs in step (1) have a positive charge on their surface and a diameter of 3-4 nm, and bind to exogenous nucleic acids by electrostatic adsorption; the exogenous nucleic acid in step (2) is ASO, and the sequence is shown in SEQ ID No. 1; the mass ratio of CDs to ASO is ≥5, and the incubation time is 1-5 hours.

2. The carbon dot delivery system for delivering exogenous nucleic acid into bacteria according to claim 1, characterized in that: The mass ratio of CDs to ASO was 5-50:1; the incubation time was 2 h.

3. The carbon dot delivery system for delivering exogenous nucleic acid into bacteria according to claim 1, characterized in that: In step (1), the mass ratio of citric acid to polyethyleneimine is 1:0.5 to 1:1.5, the heating temperature is 180 to 220° C., and the heating cycle is 0.5 h to 4 h.

4. The carbon dot delivery system for delivering exogenous nucleic acid into bacteria according to claim 1, characterized in that: The bacterium is the Gram-negative bacterium Vibrio anguillarum.

5. Application of a carbon dot delivery system to deliver exogenous nucleic acids into bacterial cells to achieve bacterial gene interference for non-diagnostic purposes, characterized in that: The carbon dot delivery system is prepared by the construction method described in any one of claims 1 to 4; the bacterial gene is the Vibrio anguillarum empA gene; the exogenous nucleic acid is empA ASO, and the sequence is: TTACAAGGTACGCGAAGC.

6. The use according to claim 5, characterized in that: The empA ASO was loaded onto carbon dot CDs in a specific ratio and incubated with Vibrio anguillarum for 1 to 5 hours to deliver it into the cells of Vibrio anguillarum, thereby achieving targeted empA silencing in a short period of time and reducing the pathogenicity of Vibrio anguillarum.

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