Preparation method and application of response type cationized two-dimensional nano catalytic transfection agent

Abstract

The invention discloses a preparation method and application of a response type cationized two-dimensional nano catalytic transfection agent, and belongs to the field of biology. According to the transfection agent disclosed by the invention, two-dimensional MXene (such as niobium carbide) is taken as a matrix, and the surface of the matrix is sequentially modified with a multi-amino cationic polymer (such as dendritic polyethyleneimine) and aldehyde group polyethylene glycol (CHO-PEG-CHO), so that PNb2C (at) PEG with pH responsiveness is formed. The preparation method comprises the steps of Nb2C nanosheet stripping, PEI cationization, PEG shielding modification and the like. Material characterization shows that the potential of the transfection agent is-12.5 mV (shielding positive charges) when the pH value is 7.4, the potential of the transfection agent is converted into + 14.0 mV (responding to a tumor acidic microenvironment) when the pH value is 6.5, and the transfection agent has the capability of catalyzing decomposition of active oxygen and can protect cells from oxidative damage. Compared with the traditional cationic liposome, the transfection efficiency is equivalent, but the toxicity is obviously reduced, the raw material cost is low, the gene load is high, and the cationic liposome is suitable for the field of gene delivery.

Description

Technical Field

[0001] The present invention relates to the field of biology, and in particular to a preparation method and application of a responsive cationic two-dimensional nanocatalytic transfection agent. Background Art

[0002] Transfection agents are tools for introducing exogenous nucleic acids (such as DNA and RNA) into cells and are widely used in basic research and gene therapy. Transfection agents are primarily categorized into viral and non-viral vectors. While viral vectors possess high gene delivery efficiency, they are subject to significant safety concerns, such as strong immunogenicity and the potential for mutation and viral reactivation upon insertion into the host cell genome. These limitations significantly limit their in vivo application. Non-viral vectors, including cationic liposomes (such as Lipofectamine 2000, abbreviated as Lipo2000), cationic polymers, and inorganic nanomaterials, offer advantages over viral vectors, such as high stability, low immunogenicity, and ease of preparation. However, they suffer from limitations such as low transfection efficiency, high toxicity, complex synthesis processes, and high cost. Two-dimensional inorganic nanomaterials possess unique physicochemical properties, such as large surface area, high gene loading and cellular uptake efficiency, and amenable surface functionalization. These materials offer high in vivo circulation stability and targeted targeting, making them an emerging research hotspot in the field of gene delivery.

[0003] The present invention aims to solve the problems of high toxicity, high cost, low loading capacity and limited in vivo application of gene transfection reagents. Summary of the Invention

[0004] In response to the problems existing in the prior art, the present invention provides a preparation method and application of a responsive cationic two-dimensional nanocatalytic transfection agent.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0006] The present invention discloses a cationic two-dimensional nanocatalytic transfection agent, which is characterized by comprising: Two-dimensional MXene nanomaterial matrix; A polyamino cationic polymer layer modified on the surface of the substrate; An acid-responsive PEG layer is coated on the outside of the cationic polymer layer.

[0007] Furthermore, the MXene nanomaterial is a transition metal carbide or nitride, selected from at least one of niobium carbide (Nb2C), titanium carbide (Ti3C2), and tantalum carbide (Ta4C3).

[0008] Furthermore, the polyamino cationic polymer is a branched polyethyleneimine (PEI), ethylene glycol chitosan or a polyamide-amine type dendrimer.

[0009] Furthermore, the acid-responsive PEG is dialdehyde polyethylene glycol (CHO-PEG-CHO) with a molecular weight range of 2000-5000 Da.

[0010] The present invention also discloses a method for preparing the cationic two-dimensional nanocatalytic transfection agent described in any one of the above items, characterized in that it comprises the following steps: (a) Nb2AlC powder was immersed in 50% HF aqueous solution, stirred at room temperature for 48-72 h, and centrifuged to obtain Nb2C nanosheets. (b) Nb2C nanosheets were dispersed in tetrapropylammonium hydroxide (TPAOH) solution, stirred at room temperature for 48–72 h, and centrifuged for washing. (c) mixing the product of step (b) with branched polyethyleneimine in a mass ratio of 1:1, reacting at room temperature for 2 h, and centrifuging and washing to obtain PNb2C; (d) PNb2C and CHO-PEG-CHO were mixed in PBS buffer (pH 7.4) at a mass ratio of 2:1. After reacting for 2 h, the mixture was centrifuged and washed to obtain the transfection agent.

[0011] Furthermore, the centrifugation conditions in steps (a), (b), (c), and (d) are all 20,000 rpm for 10 min, and the washing solvents are deionized water and anhydrous ethanol for (a), (b), and (c), respectively; and deionized water for (d).

[0012] Furthermore, the molecular weight of the CHO-PEG-CHO in step (d) is 2000 Da.

[0013] The present invention also discloses a use of any one of the above cationic two-dimensional nanocatalytic transfection agents in the preparation of products related to gene delivery.

[0014] Furthermore, the gene is siRNA, shRNA or plasmid DNA.

[0015] Furthermore, the transfection agent triggers PEG stripping and charge reversal in a slightly acidic environment with a pH of ≤6.5.

[0016] Compared with the prior art, the present invention has the following beneficial effects.

[0017] The present invention provides a preparation method and application of a responsive cationic two-dimensional nanocatalytic transfection agent. Experiments have shown that the transfection agent disclosed in the present invention, on the basis of the gene delivery function, realizes the decomposition of intracellular ROS, protects cells from oxidative stress damage during the transfection process, ensures cell activity, and has significant advantages. The present invention grafts PEG on the surface to shield its positive charge, which can effectively resist nonspecific adsorption of proteins in the body circulation, prevents it from being degraded by nucleases during the delivery process, and can respond to the local acidic microenvironment of the tumor to remove PEG, promote cell uptake, and improve the accuracy of gene delivery therapy. In addition, the present invention also has the characteristics of low raw material cost and can be prepared in batches; large specific surface area and higher gene loading capacity. In summary, the present invention can not only improve the efficiency and targeting of gene delivery, but also reduce damage and toxic side effects to normal cells of the body, and has good clinical transformation value and application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Figure 1 shows material characterization. Figure A shows a transmission electron microscopy image of a material with a diameter of approximately 200 nm, exhibiting a dispersed, two-dimensional, flake-like structure. Figure B shows a dynamic light scattering experiment, which reveals that the particle size distribution peaks for Nb2C, PNb2C, and PNb2C@PEG are located at 205.1 nm, 315.4 nm, and 372.6 nm, respectively. Figure C shows the Zeta potential measurement of the surface charge of the materials. The potentials of the three materials, Nb2C, PNb2C, and PNb2C@PEG in water are -43.6 mV, +42.9 mV, and +34.5 mV, respectively. Figure D shows the Zeta potential measurement of PNb2C@PEG in solutions of different pH values. At pH 7.4, the material's Zeta potential is -12.5 mV, successfully shielding the material's positive charge in its physiological state (blood circulation). At pH 6.5, it reaches +14.0 mV, achieving charge reversal.

[0019] Figure 2 The transfection efficiency and toxicity of this material were evaluated. Figure A shows RT-qPCR assays to measure the expression of circular RNA PUM1 in ovarian cancer cells, evaluating the transfection efficiency of free siRNA, lipo2000-transfected circular RNA PUM1 siRNA, and this material loaded with circular RNA PUM1 siRNA. Figure B shows a CCK8 assay to examine the effects of different working concentrations of lipo2000 on cell viability. Figure C shows a CCK8 assay to examine the effects of different working concentrations of this material on cell viability.

[0020] Figure 3 Flow cytometry was used to quantitatively analyze the effects of lipo2000 and this material on the generation of intracellular reactive oxygen species. DETAILED DESCRIPTION

[0021] The present invention is further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the present invention to the following embodiments, as all technologies implemented based on the present invention fall within the scope of the present invention.

[0022] Unless otherwise specified, all reagents and materials used in the present invention are commercially available.

[0023] Example 1: Preparation of transfection agent PNb2C@PEG.

[0024] 1. Preparation method

[0025] 5g of Nb2AlC powder was immersed in 70mL of 50% HF aqueous solution and stirred at room temperature for 2-3 days. The resulting black precipitate was then centrifuged and washed four times with water / ethanol before being dispersed in 70mL of 25% TPAOH and stirred at room temperature for 2-3 days. Finally, the product was washed twice with deionized water and twice with anhydrous ethanol by centrifugation at 20,000rpm for 10 minutes, respectively, to obtain the target product, Nb2C nanosheets. The Nb2C nanosheets were mixed with dendritic polyethyleneimine (PEI) in deionized water at a mass ratio of 1:1. After stirring at room temperature for 2 hours, the mixture was washed three times with centrifugation at 20,000rpm for 10 minutes to obtain PNb2C. PNb2C and CHO-PEG-CHO were mixed in a mass ratio of 2:1, stirred and reacted in 1×PBS buffer at pH = 7.4 at room temperature for 2 h, centrifuged at 20,000 rpm for 10 min with deionized water, and washed three times to obtain PNb2C@PEG (cationic two-dimensional nanocatalytic transfection agent).

[0026] 2. Material characterization (TEM; DLS; Zeta potential).

[0027] Transmission electron microscopy (TEM) showed that the material had a diameter of about 200 nm and was a dispersed flake-like two-dimensional structure ( Figure 1 A). Dynamic light scattering (DLS) was used to further evaluate the particle size distribution, dispersibility, and aggregation state of the materials. The results showed that the particle size distribution peaks of Nb2C, PNb2C, and PNb2C@PEG materials were located at 205.1nm, 315.4nm, and 372.6nm, respectively. That is, Nb2C nanosheets were grafted onto PEI to obtain PNb2C, and PNb2C was further modified with CHO-PEG-CHO to obtain PNb2C@PEG. The particle sizes of the three materials increased in sequence, indicating that the above modifications were successful ( Figure 1 B).

[0028] The Zeta potential was used to measure the surface charge of the materials, reflecting the stability of the particles in solution. The results showed that the potentials of the three materials, Nb2C, PNb2C, and PNb2C@PEG, in water were -43.6mV, +42.9mV, and +34.5mV, respectively, indicating that PEI grafting successfully achieved cationization of Nb2C nanosheets, which is beneficial for gene loading and promoting cellular uptake ( Figure 1 C). PNb2C@PEG was further placed in solutions with different pH values, and the zeta potential was measured to evaluate the stability of the material in the in vivo environment.

[0029] A solution with a pH of 7.4 was used to simulate blood circulation, and a solution with a pH of 6.5 was used to simulate the slightly acidic environment of tumor tissue. The results showed that the Zeta potential of the material was -12.5mV at pH 7.4, successfully shielding the positive charge of the material in a physiological state (in blood circulation). That is, the steric effect was used to reduce the possibility of protein adsorption and non-specific binding in blood circulation, thereby reducing damage to normal cells in the body and toxic side effects. As the pH decreased, its Zeta potential gradually became positive and reached +14.0mV at pH 6.5, indicating that the material can respond to the acidic microenvironment of the tumor, undergoing "acid response" peeling of PEG, exposing the underlying cationic surface, achieving charge reversal in the tumor microenvironment, significantly enhancing cellular internalization, and promoting the efficient and precise delivery of negatively charged nucleic acids such as siRNA to tumor cells ( Figure 1 D).

[0030] Example 2: Transfection efficiency and toxicity evaluation.

[0031] Taking the transfection of siRNA targeting the knockdown of circular RNA PUM1 (has_circ_0000043) as an example (siRNA sequence: sense 5'-3': UCUCAACAACAGGGCCCAATT; antisense 5'-3': UUGGGCCCUGUUGUUGAGATT), free siRNA was added to ovarian cancer OVCAR3 cells; 50nM circular RNA PUM1 siRNA was transfected using lipo2000; and the present material was loaded with an equal amount of circular RNA PUM1 siRNA (i.e., 5μg / mL PNb2C and 50nM siRNA were mixed and vortexed for 2 minutes). RT-qPCR was used to detect the expression level of circular RNA PUM1 in each group of cells to evaluate the transfection efficiency of the present material. The results showed that free siRNA had no effect, and both lipo2000 and PNb2C transfection of siRNA could significantly reduce the expression level of the gene in the cells, and the transfection efficiency of the present material was not significantly different from that of lipo2000 ( Figure 2A). CCK8 experiments were further used to detect the effects of different working concentrations of lipo2000 and this material on cell viability. The results showed that with the increase of lipo2000 working concentration, cell viability decreased significantly ( Figure 2 B); however, this material had no significant effect, and even when the concentration of the material was increased to 100 μg / mL (20 times the working concentration), the cells still showed good cell activity after 72 hours of continuous treatment, indicating that this material has reliable biosafety ( Figure 2 C).

[0032] Example 3: Characterization of catalytic active oxygen decomposition.

[0033] Based on the fact that H2DCFDA can be oxidized by intracellular ROS into fluorescent DCF (2',7'-dichlorofluorescein), the fluorescence intensity is positively correlated with the intracellular ROS content. The flow cytometry FITC detection method was used to quantitatively analyze the intracellular reactive oxygen species to explore the effects of Lipo2000 and the present invention on the intracellular ROS level. Working concentrations of Lipo2000 or PNb2C were added to ovarian cancer cells, respectively. After incubation for 24 hours, the H2DCFDA probe was added, and the cells were collected for flow cytometry. The results showed that the DCF fluorescence intensity of the cells in the Lipo2000-treated group was significantly higher than that of the control group, indicating that Lipo2000 induced a large amount of ROS in the cells. The fluorescence intensity of the nanomaterial-treated group was significantly lower than that of the Lipo2000-treated group, indicating that the present invention can protect cells from oxidative damage to a certain extent ( Figure 3 ).

[0034] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. A cationic two-dimensional nanocatalytic transfection agent, characterized in that: include: Two-dimensional MXene nanomaterial matrix; A polyamino cationic polymer layer modified on the surface of the substrate; An acid-responsive PEG layer is coated on the outside of the cationic polymer layer.

2. The transfection agent according to claim 1, characterized in that: The MXene nanomaterial is a transition metal carbide or nitride, and is selected from at least one of niobium carbide (Nb2C), titanium carbide (Ti3C2), and tantalum carbide (Ta4C3).

3. The transfection agent according to claim 1, wherein: The polyamino cationic polymer is a branched polyethyleneimine (PEI), ethylene glycol chitosan or a polyamide-amine type branched polymer.

4. The transfection agent according to claim 1, wherein: The acid-responsive PEG is a dialdehyde polyethylene glycol (CHO-PEG-CHO) with a molecular weight range of 2000-5000 Da.

5. A method for preparing the cationic two-dimensional nanocatalytic transfection agent according to any one of claims 1 to 4, characterized in that: The following steps are involved: (a) Nb2AlC powder was immersed in 50% HF aqueous solution, stirred at room temperature for 48-72 h, and centrifuged to obtain Nb2C nanosheets. (b) Nb2C nanosheets were dispersed in tetrapropylammonium hydroxide (TPAOH) solution, stirred at room temperature for 48–72 h, and centrifuged for washing. (c) mixing the product of step (b) with branched polyethyleneimine in a mass ratio of 1:1, reacting at room temperature for 2 h, and centrifuging and washing to obtain PNb2C; (d) PNb2C and CHO-PEG-CHO were mixed in PBS buffer (pH 7.4) at a mass ratio of 2:

1. After reacting for 2 h, the mixture was centrifuged and washed to obtain the transfection agent.

6. The method according to claim 5, characterized in that: The centrifugation conditions in steps (a), (b), (c), and (d) are all 20,000 rpm for 10 min, and the washing solvents are deionized water and anhydrous ethanol for (a), (b), and (c), respectively; and deionized water for (d).

7. The method according to claim 5, characterized in that: The molecular weight of the CHO-PEG-CHO in step (d) is 2000 Da.

8. Use of the cationic two-dimensional nanocatalytic transfection agent according to any one of claims 1 to 4 in the preparation of products related to gene delivery.

9. The use according to claim 8, characterized in that: The gene is siRNA, shRNA or plasmid DNA.

10. The use according to claim 8, characterized in that: The transfection agent triggers PEG stripping and charge reversal in a slightly acidic environment with a pH of ≤6.5.

Images