Bionic nano-gene vector with CT imaging and anti-oxidative stress effects as well as preparation method and application of bionic nano-gene vector

Abstract

The invention belongs to the technical field of nano biological materials, and particularly relates to a bionic nano gene vector with CT imaging and anti-oxidative stress effects as well as a preparation method and application of the bionic nano gene vector. The bionic nano gene vector comprises a gene delivery vector and an HGF plasmid gene loaded on the gene delivery vector, the gene delivery vector comprises protamine sulfate and composite metal-based nanoparticles, and the composite metal-based nanoparticles comprise a combination of at least three of gold, cobalt, platinum, copper, cerium and iron. Three metal elements are compounded in the gene delivery carrier, and the doping ratio of the three metals is strictly controlled, so that the gene delivery carrier has excellent enzyme-like activity, the biomimetic nano gene carrier is endowed with enzyme catalytic activity, and the anti-oxidative stress capability of therapeutic cells can be improved, thereby improving the survival rate and the therapeutic performance of the therapeutic cells. The method can be applied to cell function regulation and visual tracing of therapeutic cells such as stem cells and immune cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanobiomaterials, and particularly relates to a biomimetic nano-gene carrier with CT imaging and antioxidant stress effects, a preparation method thereof, and applications thereof. Background Art

[0002] In recent years, regenerative medicine technologies centered on stem cell therapy have brought hope for intractable diseases in humans. Mesenchymal stem cells (MSCs) have attracted wide attention because they can effectively repair lung injuries due to their abilities of multi-directional differentiation, immunomodulation, and paracrine (Oncotarget 2017, 8, 102600.). In the past two decades, a number of preclinical studies have demonstrated the feasibility and effectiveness of conducting clinical trials of MSCs for treating patients with pulmonary fibrosis. However, there are only more than 40 approved stem cell drugs on the market at home and abroad (Stem Cell Res. Ther. 2020, 11, 477; Stem Cells Transl. Med. 2017, 6, 10061; Journal of Controlled Release 2024, 365, 981 - 1003.). This is mainly attributed to the fact that MSCs cannot adapt to the harsh disease microenvironment of patients, resulting in a rapid decline in the cell viability of transplanted MSCs or even cell death. The research team led by Geoffrey C Gurtner at Stanford University in California found that the survival rate of MSCs transplanted into the body was only 5% after 5 days (Biomaterials 2012, 33, 80.). Therefore, how to improve the survival rate of transplanted MSCs in the body and enhance their efficacy is a key problem that urgently needs to be solved at present.

[0003] To address this challenge, research teams led by Director Yang Junling of the Department of Respiratory Medicine at the Second Hospital of Jilin University and Gregory P Downey of the Department of Respiratory Medicine at the University of Toronto in Canada have demonstrated that intense oxidative stress reactions and a large number of inflammatory factors at the injury site are important factors leading to low survival rates of transplanted MSCs (Stem Cells Dev. 2021, 30, 128; Am. J. Respir. Cell Mol. Biol. 2003, 29, 427; Int. J. Mol. Sci. 2016, 17, 1752.). Therefore, enhancing the anti-inflammatory and antioxidant stress capabilities of transplanted MSCs is an effective means to improve their survival rates. Hepatocyte growth factor (HGF) is an important paracrine factor of MSCs and has functions such as anti-inflammation, anti-apoptosis, and promoting angiogenesis (Proc. Am. Thorac. Soc. 2012, 9, 158.). MSCs overexpressing HGF can effectively regulate the immune system to enhance the anti-inflammatory ability of MSCs. However, this still cannot resist the harm of reactive oxygen species (ROS)-induced oxidative stress. In recent years, nanozymes have been widely used due to their unique advantages such as high structural stability, adjustable catalytic activity, good biocompatibility, and especially excellent ROS scavenging ability (Adv. Mater. 2020, 32, e2003065; Adv. Mater. 2022, e2205324; Adv. Mater. 2021, 34, e2105711.). Therefore, designing a gene delivery vector with enzyme-like activity for intracellular ROS scavenging and HGF gene delivery is expected to enhance the antioxidant stress and anti-inflammatory abilities of transplanted MSCs, improve the survival rates of transplanted MSCs, and thus enhance their therapeutic effects.

[0004] Another challenge faced by stem cell therapy is the unclear biological behavior information of transplanted MSCs in vivo, and the repair of damaged lung tissue by transplanted MSCs is a staged and long-term process. Achieving long-term visualization tracking of transplanted MSCs is crucial for in-depth understanding of their biological behavior and elucidation of their mechanism of action. Therefore, there is an urgent need to propose a reliable non-invasive imaging method for long-term tracking of the homing and migration of therapeutic MSCs. Summary of the Invention

[0005] Aiming at the deficiencies of existing stem cell therapy technologies, the present invention provides a biomimetic nano-gene vector with CT imaging and antioxidant stress effects, as well as its preparation method and applications.

[0006] One object of the present invention is achieved through the following technical solutions:

[0007] A biomimetic nano-gene carrier with CT imaging and antioxidant stress functions. The biomimetic nano-gene carrier includes a gene delivery carrier (TBNCs) and an HGF plasmid gene loaded on the gene delivery carrier. The gene delivery carrier includes protamine sulfate and a composite metal-based nanoparticle, and the composite metal-based nanoparticle includes a combination of at least three of gold, cobalt, platinum, copper, cerium, and iron.

[0008] More preferably, the composite metal-based nanoparticle is composed of gold, platinum, and cobalt. Further compounding gold in platinum and cobalt elements can endow the gene delivery carrier with excellent enzyme-like activity and endow the biomimetic nano-gene carrier with enzyme catalytic activity, improving the antioxidant stress ability of therapeutic cells.

[0009] Preferably, the molar ratio of gold, platinum, and cobalt in the composite metal-based nanoparticle is (1-4):(1-4):(1-4), where "1-4" can be 1, 2, 3, 4, etc.

[0010] More preferably, the molar ratio of gold, platinum, and cobalt is 4:4:1 or 1:4:4. When the molar ratio of gold, platinum, and cobalt in the composite metal-based nanoparticle is the above, it has better enzyme-like activity.

[0011] Preferably, the gene delivery carrier uses protamine sulfate (PS) as a template, and a composite metal-based nanoparticle grows on the protamine sulfate to improve the cell membrane transport and nuclear localization ability of the biomimetic nano-gene carrier.

[0012] Preferably, the plasmid gene binds to the binding site on the composite metal-based nanoparticle.

[0013] Preferably, the binding site on the composite metal-based nanoparticle is derived from protamine sulfate.

[0014] Preferably, the particle size of the biomimetic nano-gene carrier is 1-20 nm, such as 1 nm, 2 nm, 3 nm, 4 nm, 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, 11 nm, 12 nm, 13 nm, 14 nm, 15 nm, 16 nm, 17 nm, 18 nm, 19 nm, 20 nm, etc.

[0015] More preferably, the particle size of the biomimetic nano-gene carrier is 2-8 nm.

[0016] Preferably, the SOD-like enzyme activity of the gene delivery carrier is 50-90%, and the CAT-like enzyme activity is 15-90%.

[0017] More preferably, the SOD-like enzyme activity of the gene delivery carrier is 60-85%, and the CAT-like enzyme activity is 65-80%.

[0018] The concentration of the gene delivery vector is directly proportional to the antioxidant capacity, and the antioxidant capacity can be expressed by the scavenging ability of the gene delivery vector against hydroxyl radicals.

[0019] Preferably, when the concentration of the gene delivery vector is 50 - 60 μg / mL, the scavenging rate of the gene delivery vector against hydroxyl radicals is ≥ 90%.

[0020] The biomimetic nano - gene vector provided by the present invention can safely and efficiently label therapeutic cells, effectively deliver plasmid genes into therapeutic cells to enhance the antioxidant stress and therapeutic performance of therapeutic cells, and achieve non - invasive visualization and tracing of the homing and migration of therapeutic cells. Secondly, the biomimetic nano - gene vector of the present invention has the ability to enhance the antioxidant stress ability and anti - fibrosis ability in the treatment of pulmonary fibrosis by mesenchymal stem cells, so as to improve the survival rate of transplanted stem cells and their therapeutic effect on pulmonary fibrosis. And the biomimetic nano - gene vector can be completely metabolized by the liver and has good biosafety.

[0021] Preferably, the mass ratio of protamine sulfate to composite metal - based nanoparticles in the gene delivery vector is 1:(1 - 15), and "1 - 15" can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0022] Preferably, the mass ratio of the gene delivery vector to the plasmid gene is (1 - 60):1, and "1 - 60" can be 1, 5, 10, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, etc. Further preferably, the mass ratio of the gene delivery vector to the plasmid gene is (9 - 60):1; still further preferably, the mass ratio of the gene delivery vector to the plasmid gene is (12 - 30):1.

[0023] The second object of the present invention is achieved by the following technical solution:

[0024] A preparation method of a biomimetic nano - gene vector with CT imaging and antioxidant stress effects, the preparation method of the biomimetic nano - gene vector includes the following steps: co - incubating a gene delivery vector with an HGF plasmid gene to obtain the biomimetic nano - gene vector.

[0025] Preferably, the incubation temperature is 25 - 35 °C, for example, it can be 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, etc.

[0026] Preferably, the incubation time is 5 to 15 minutes, for example, it can be 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, etc.

[0027] Preferably, the method for preparing the gene delivery vector comprises the following steps: mixing a chloroauric acid solution, a chloroplatinic acid solution, a cobalt chloride solution, and a protamine sulfate solution, and then undergoing a reduction reaction to obtain the gene delivery vector.

[0028] Preferably, the mass percentage content of the chloroauric acid solution is 0.01 to 0.1%, for example, it can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, etc.

[0029] Preferably, the mass percentage content of the chloroplatinic acid solution is 0.01 to 0.1%, for example, it can be 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, etc.

[0030] Preferably, the mass percentage content of the cobalt chloride solution is 0.001 - 0.01%, for example, it can be 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, etc.

[0031] Preferably, the mass concentration of protamine sulfate in the protamine sulfate solution is 1 to 20 mg / mL, for example, it can be 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, etc.

[0032] Preferably, the volume ratio of the chloroauric acid solution, the chloroplatinic acid solution, the cobalt chloride solution, and the protamine sulfate solution is (10 - 200) : (10 - 200) : (20 - 100) : (1 - 10);

[0033] Among them, "100 to 200" can be 110, 130, 140, 150, 160, 170, 180, 190, 200, etc.; "20 to 100" can be 20, 30, 40, 50, 60, 70, 80, 90, 100, etc.; "1 to 10" can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0034] Preferably, the molar ratio of chloroauric acid, chloroplatinic acid, and cobalt chloride in the mixed solution is (1 to 4):(1 to 4):(1 to 4).

[0035] More preferably, the molar ratio of chloroauric acid, chloroplatinic acid, and cobalt chloride in the mixed solution is 4:4:1 or 1:4:4.

[0036] Preferably, the ratio of protamine sulfate to chloroauric acid in the mixed solution is 1 g:(0.5 to 20) mmol.

[0037] Preferably, the reducing agent used in the reduction reaction is a sodium borohydride solution.

[0038] Preferably, the molar concentration of the sodium borohydride solution is 10 to 100 mM, and can be, for example, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, etc.

[0039] Preferably, the molar ratio of sodium borohydride to chloroauric acid is 100:(1 to 20).

[0040] Preferably, the reduction reaction temperature is 25 to 35 °C, and can be, for example, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, etc.

[0041] Preferably, the reduction reaction time is 20 to 40 min, and can be, for example, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, etc.

[0042] Preferably, the method for preparing the gene delivery vector further includes the following step: subjecting the gene delivery vector solution obtained after the reduction reaction to ultrafiltration concentration to obtain the gene delivery vector.

[0043] Preferably, the cut-off molecular weight of the ultrafiltration tube used for ultrafiltration concentration is 8 to 12 kD, and can be, for example, 8 kD, 9 kD, 10 kD, 11 kD, 12 kD, etc.

[0044] Preferably, the rotation speed for ultrafiltration concentration is 3000 - 5500 rpm, for example, it can be 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm, 5000 rpm, 5500 rpm, etc.

[0045] Preferably, the time for ultrafiltration concentration is 10 - 20 min, for example, it can be 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, etc.

[0046] Preferably, the method for preparing the biomimetic nano - gene vector includes the following steps:

[0047] (1) Mix chloroauric acid solution, chloroplatinic acid solution, cobalt chloride solution and protamine sulfate solution to obtain a mixed solution;

[0048] (2) Conduct a reduction reaction on the mixed solution to obtain a gene delivery vector solution;

[0049] (3) Ultrafilter and concentrate the gene delivery vector solution to obtain a gene delivery vector;

[0050] (4) Co - incubate the gene delivery vector with HGF plasmid gene to obtain the biomimetic nano - gene vector.

[0051] The third object of the present invention is achieved by the following technical solutions:

[0052] The application of a biomimetic nano - gene vector with CT imaging and antioxidant stress effects in cell gene delivery and / or cell antioxidant stress and / or cell CT tracing.

[0053] Preferably, the cell is a therapeutic cell.

[0054] Preferably, the cell includes one or more of adipose - derived mesenchymal stem cells, bone marrow - derived mesenchymal stem cells, neural stem cells, human umbilical cord - derived mesenchymal stem cells or lymphocytes.

[0055] Preferably, the time for cell CT tracing is ≥ 45 days, and more preferably 45 - 60 days.

[0056] Preferably, the survival time of the cell in vivo is ≥ 14 days, and more preferably 14 - 30 days.

[0057] Compared with the prior art, the present invention has the following beneficial effects:

[0058] 1. In the bionic nano-gene carrier with CT imaging and antioxidant stress functions provided by the present invention, three metal elements, namely gold, platinum, and cobalt, are compounded in the gene delivery carrier. Compared with traditional single-metal-based nano-carriers, it endows the bionic nano-gene carrier with excellent enzyme catalytic activity, effectively scavenges intracellular ROS in therapeutic cells, improves the antioxidant stress ability of therapeutic cells, and thus increases their survival rate.

[0059] 2. In the bionic nano-gene carrier provided by the present invention, the molar ratios of the three metals on the gene delivery carrier are strictly controlled, enabling the bionic nano-gene carrier to have higher enzyme-like activity and further enhancing the antioxidant stress performance and therapeutic performance of therapeutic cells.

[0060] 3. In the bionic nano-gene carrier provided by the present invention, the gene delivery carrier is grown from gold-platinum-cobalt composite metal-based nanoparticles using protamine sulfate as a template. Protamine sulfate can be localized in the nucleus, enabling efficient delivery of HGF plasmid genes, releasing genes for expression around the nucleus, and achieving better therapeutic effects.

[0061] 4. In the bionic nano-gene carrier provided by the present invention, after the gold-platinum-cobalt triple-metal-based gene delivery carrier binds to the HGF plasmid gene, gold and platinum elements can serve as CT tracers. Utilizing the high-resolution characteristics of CT imaging, the distribution, migration, and homing of transplanted labeled stem cells can be visualized and tracked in real time.

[0062] 5. For the preparation method of a bionic nano-gene carrier with CT imaging and antioxidant stress functions provided by the present invention, the raw materials can be directly mixed and reacted using a one-pot method to obtain protamine sulfate grown with composite metal-based nanoparticles; then, by incubating with plasmid genes at room temperature, the bionic nano-gene carrier can be obtained. This preparation method is simple and efficient, does not require complex equipment, and the raw materials are easily available, which is conducive to promotion.

[0063] 6. The bionic nano-gene carrier with CT imaging and antioxidant stress functions provided by the present invention combines the localization function of protamine sulfate and the CT tracing and efficient ROS scavenging effects of the triple-metal-based gene delivery carrier, overcoming the problems of traditional methods that are difficult to achieve efficient plasmid gene delivery and anti-ROS oxidative stress effects. It realizes the simultaneous delivery of high-efficiency plasmid genes, CT tracers, and antioxidants, providing a new method for the functions of cell survival, directional differentiation, paracrine, etc., and the tracing of therapeutic cells. At the same time, the small-sized nano-system can be completely metabolized by the liver in the body, having excellent biosafety.

[0064] 7. The bionic nano-gene carrier with CT imaging and antioxidant stress functions provided by the present invention can be applied to gene delivery of therapeutic cells and / or antioxidant stress and / or CT tracing, providing a new method for improving the survival rate of therapeutic cells, enhancing their efficacy, and non-invasively visualizing them. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 It is a diagram for measuring the enzyme-like activity of the TBNCs gene delivery vectors with different doping ratios of three metals in Preparation Examples 1-7.

[0066] Among them, Figure 1 (A) is a schematic diagram of the enzyme-like activity of TBNCs; Figure 1 (B) is a list of the reaction molar ratios of Au, Pt, and Co in Preparation Examples 1-7; Figure 1 (C) is the SOD-like enzyme activity of TBNCs; Figure 1 (D) is the CAT-like enzyme activity of TBNCs.

[0067] Figure 2 It is a transmission electron microscope image of the TBNCs gene delivery vector in Preparation Example 6.

[0068] Figure 3 It is a CT imaging performance diagram of the TBNCs gene delivery vector in Preparation Example 6.

[0069] Figure 4 It is a diagram showing the relationship between the attenuation of the Hounsfield value and the concentration of the TBNCs gene delivery vector in Preparation Example 6.

[0070] Figure 5 It is a diagram showing the scavenging ability of the TBNCs gene delivery vector in Preparation Example 6 for hydroxyl radicals (·OH).

[0071] Figure 6 It is a diagram showing the scavenging ability of the TBNCs gene delivery vector in Preparation Example 6 for superoxide anions (·O 2- )

[0072] Figure 7 It is a loading effect diagram of HGF plasmid gene by TBNCs in the bionic nano-gene carrier TBNCs@pDNA in Examples 1-6.

[0073] Figure 8 It is a plasmid gene transfection effect diagram of TBNCs@pDNA in Example 5.

[0074] Figure 9 It is a CT imaging performance diagram of TBNCs@pDNA in Example 5 for labeling stem cells.

[0075] Figure 10Relationship diagram of attenuation of Hounsfield value and concentration of TBNCs@pDNA-labeled stem cells in Example 5.

[0076] Figure 11 Effect diagram of scavenging of intracellular ROS in stem cells by TBNCs in TBNCs@pDNA of Example 5.

[0077] Figure 12 Effect diagram of protection of stem cell mitochondria by TBNCs in TBNCs@pDNA of Example 5 in ROS microenvironment. Detailed implementation mode

[0078] The technical solutions of the present invention will be further described and illustrated below in conjunction with specific examples and drawings. It should be understood that the specific examples described herein are only for illustrating the present invention and are not used to limit the scope of the present invention. Moreover, the drawings used herein are only for better illustrating the content disclosed by the present invention and do not have a limiting effect on the protection scope. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0079] The sources of each component in the following preparation examples and examples are as follows:

[0080]

[0081]

[0082] Preparation Example 1

[0083] This preparation example provides a TBNCs gene delivery vector. The preparation method of the TBNCs gene delivery vector includes the following steps:

[0084] (1) Add an aqueous solution of HAuCl4·4H2O (10 mM, 0.3 mL), H2PtCl6·6H2O (10 mM, 0.3 mL), and CoCl2 (10 mM, 0.3 mL) to 100 mL of pure water, stir at 1500 rpm at 25°C, and then add 100 μL of a 10 mg / mL protamine sulfate solution to obtain a mixed solution;

[0085] (2) Stir the mixed solution at 1500 rpm at room temperature, quickly add 2 mL of freshly prepared 50 mM sodium borohydride solution, and react at room temperature for 30 min to obtain a TBNCs gene delivery vector solution;

[0086] (3) Ultrafilter and concentrate the TBNCs gene delivery vector solution at room temperature to obtain a TBNCs gene delivery vector, named N1;

[0087] Among them, the ultrafiltration concentration includes the following steps: using a 15 mL Millipore ultrafiltration tube with a MWCO of 10 kD and centrifuging at 4000 rpm for 15 min; storing the TBNCs stock solution at 4 °C for standby.

[0088] Preparation Example 2

[0089] The difference between Preparation Example 2 and Preparation Example 1 is only that in step (1) of this preparation example, an aqueous solution of HAuCl4·4H2O (10 mM, 0.6 mL), H2PtCl6·6H2O (10 mM, 0.3 mL), and CoCl2 (10 mM, 0.6 mL) was added to 100 mL of pure water, and the rest was the same as in Preparation Example 1, obtaining a TBNCs gene delivery vector named N2.

[0090] Preparation Example 3

[0091] The difference between Preparation Example 3 and Preparation Example 1 is only that in step (1) of this preparation example, an aqueous solution of HAuCl4·4H2O (10 mM, 0.6 mL), H2PtCl6·6H2O (10 mM, 0.6 mL), and CoCl2 (10 mM, 0.3 mL) was added to 100 mL of pure water, and the rest was the same as in Preparation Example 1, obtaining a TBNCs gene delivery vector named N3.

[0092] Preparation Example 4

[0093] The difference between Preparation Example 2 and Preparation Example 1 is only that in step (1) of this preparation example, an aqueous solution of HAuCl4·4H2O (10 mM, 0.3 mL), H2PtCl6·6H2O (10 mM, 0.6 mL), and CoCl2 (10 mM, 0.6 mL) was added to 100 mL of pure water, and the rest was the same as in Preparation Example 1, obtaining a TBNCs gene delivery vector named N4.

[0094] Preparation Example 5

[0095] The difference between Preparation Example 2 and Preparation Example 1 is only that in step (1) of this preparation example, an aqueous solution of HAuCl4·4H2O (10 mM, 1.2 mL), H2PtCl6·6H2O (10 mM, 0.3 mL), and CoCl2 (10 mM, 1.2 mL) was added to 100 mL of pure water, and the rest was the same as in Preparation Example 1, obtaining a TBNCs gene delivery vector named N5.

[0096] Preparation Example 6

[0097] The difference between Preparation Example 2 and Preparation Example 1 is only that in step (1) of this preparation example, an aqueous solution of HAuCl4·4H2O (10 mM, 1.2 mL), H2PtCl6·6H2O (10 mM, 1.2 mL), and CoCl2 (10 mM, 0.3 mL) was added to 100 mL of pure water, and the rest was the same as in Preparation Example 1, obtaining a TBNCs gene delivery vector named N6.

[0098] Preparation Example 7

[0099] The difference between Preparation Example 2 and Preparation Example 1 is only that in step (1) of this preparation example, an aqueous solution of HAuCl4·4H2O (10 mM, 0.3 mL), H2PtCl6·6H2O (10 mM, 1.2 mL), and CoCl2 (10 mM, 1.2 mL) was added to 100 mL of pure water, and the rest was the same as in Preparation Example 1, obtaining a TBNCs gene delivery vector named N7.

[0100] The TBNCs gene delivery vectors of Preparation Examples 1 to 7 were subjected to the determination of enzyme-like activity, and the determination results are as Figure 1 .

[0101] Figure 1 It is a diagram showing the determination of the enzyme-like activity of the TBNCs gene delivery vectors with different doping ratios of three metals in Preparation Examples 1 to 7. Among them, Figure 1 A is a schematic diagram of the principle of the enzyme-like activity of TBNCs; Figure 1 B is a list of the reaction molar ratios of Au, Pt, and Co in Preparation Examples 1 to 7; Figure 1 C is the SOD-like enzyme activity of TBNCs; Figure 1 D is the CAT-like enzyme activity of TBNCs.

[0102] Combined with Figure 1 Comparing and analyzing the experimental data, it can be seen that the TBNCs gene delivery vectors N1 to N7 complexed with three metal elements of gold, platinum, and cobalt all showed varying degrees of enzyme-like activity: the SOD-like enzyme activity was 50 - 90%, and the CAT-like enzyme activity was 15 - 90%.

[0103] It was further found that the TBNCs gene delivery vectors N6 and N7 showed excellent SOD-like and CAT-like enzyme activities: the SOD-like enzyme activity was 60 - 85%, and the CAT-like enzyme activity was 65 - 80%.

[0104] It can be known that when the molar ratio of Au:Pt:Co in the TBNCs gene delivery vector is 4:4:1 or 1:4:4, the obtained gene delivery vector has higher enzyme-like activity. Therefore, the molar ratio of Au:Pt:Co in the TBNCs gene delivery vector was set to 4:4:1 (Preparation Example 6) as the feeding ratio for subsequent tests and examples.

[0105] The TBNCs gene delivery vector prepared in Preparation Example 6 was subjected to electron microscopy scanning and CT imaging performance tests, and the test results are shown in Figures 2 to 4 .

[0106] Test method: In vitro CT imaging was performed using a Micro-CT imager (Hiscan XM); the scanning parameters included: 60 kV, 133 μA, single exposure time of 1.2 s, scanning resolution of 50 μm, scanning angle interval of 0.5°, and 360° scanning circle; the reconstruction software and analysis software were provided by Hiscan.

[0107] Figure 2 Figure 9 is a transmission electron micrograph of the TBNCs gene delivery vector prepared in Preparation Example 6. It can be seen that TBNCs was successfully prepared, and the particle size of the TBNCs gene delivery vector characterized by electron microscopy was 3 nm.

[0108] Figure 3 Figure 13 is a CT imaging performance diagram of the TBNCs gene delivery vector prepared in Preparation Example 6. It can be seen that as the concentration of TBNCs increased, the CT image of the TBNCs solution gradually became brighter.

[0109] Figure 4 Figure 17 is a graph showing the relationship between the attenuation of the Hounsfield value and the concentration of the TBNCs gene delivery vector prepared in Preparation Example 6. It can be seen that the attenuation of the Hounsfield value (HU) of the TBNCs solution was linearly related to the concentration.

[0110] The TBNCs gene delivery vector prepared in Preparation Example 6 was subjected to a free radical scavenging ability test, and the test results are shown in Figures 5 to 6 .

[0111] Test method: First, the TBNCs gene delivery vector prepared in Preparation Example 6 was formulated into solutions with concentrations of 0, 4.75, 14.5, 19, and 58.5 μg / mL;

[0112] Hydroxyl radical scavenging ability test: Hydroxyl radicals were generated through the Fenton reaction, that is, 1361 μL of aqueous solution contained 11 μL of H2O2 (30%), 300 μL of FeSO4 (5 mg / mL), 50 μL of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), and 100 μL of TBNCs at different concentrations (0, 14.5, 58.5 μg / mL). After incubation for 5 minutes, it was tested using an electron paramagnetic resonance spectrometer;

[0113] Scavenging ability of superoxide anion: Superoxide anion is generated by the xanthine and xanthine oxidase system, i.e., in 320 μL of aqueous solution, there are 50 μL of methanol solution, 100 μL of xanthine (10 mM), 100 μL of xanthine oxidase (1 U / mL), 20 μL of DMPO, and 50 μL of TBNCs with different concentrations (0, 4.75, 19 μg / mL). Incubate for 10 minutes and detect using an electron paramagnetic resonance spectrometer.

[0114] Figure 5 For the hydroxyl radical (·OH) scavenging ability diagram of the TBNCs gene delivery vector in Preparation Example 6, it can be seen that as the concentration increases, the hydroxyl radical scavenging ability of TBNCs increases, and when the TBNCs concentration is 58.5 μg / mL, the hydroxyl radical scavenging rate of TBNCs approaches 100%.

[0115] Figure 6 For the superoxide anion (·O 2- ) scavenging ability diagram of the TBNCs gene delivery vector in Preparation Example 6, it can be seen that as the concentration increases, the superoxide anion scavenging ability of TBNCs increases.

[0116] Example 1

[0117] The preparation method of a biomimetic nano gene vector with CT imaging and antioxidant stress effects in this example includes the following steps: Mix 1 μg of the TBNCs gene delivery vector N6 obtained in Preparation Example 6 with the HGF plasmid gene solution (pDNA) evenly. The HGF plasmid gene solution is diluted by adding 1 μg of HGF plasmid gene to 100 μL of sodium acetate buffer solution (containing 25 mg of NaAc, pH value of 5), and let it stand at room temperature for 10 minutes to obtain the biomimetic nano gene vector TBNCs@pDNA.

[0118] Example 2

[0119] The difference between Example 2 and Example 1 is only that in this example, 3 μg of the TBNCs gene delivery vector N6 obtained in Preparation Example 6 is mixed evenly with the HGF plasmid gene solution, and the rest is the same as in Example 1 to obtain the biomimetic nano gene vector TBNCs@pDNA.

[0120] Example 3

[0121] The difference between Example 3 and Example 1 is only that in this example, 6 μg of the TBNCs gene delivery vector N6 obtained in Preparation Example 6 is mixed evenly with the HGF plasmid gene solution, and the rest is the same as in Example 1 to obtain the biomimetic nano gene vector TBNCs@pDNA.

[0122] Example 4

[0123] Example 4 is only different from Example 1 in that in this example, 9 μg of the TBNCs gene delivery vector N6 obtained in Preparation Example 6 was uniformly mixed with the HGF plasmid gene solution, and the rest was the same as in Example 1 to obtain the biomimetic nano gene vector TBNCs@pDNA.

[0124] Example 5

[0125] Example 5 is only different from Example 1 in that in this example, 12 μg of the TBNCs gene delivery vector N6 obtained in Preparation Example 6 was uniformly mixed with the HGF plasmid gene solution, and the rest was the same as in Example 1 to obtain the biomimetic nano gene vector TBNCs@pDNA.

[0126] Example 6

[0127] Example 6 is only different from Example 1 in that in this example, 15 μg of the TBNCs gene delivery vector N6 obtained in Preparation Example 6 was uniformly mixed with the HGF plasmid gene solution, and the rest was the same as in Example 1 to obtain the biomimetic nano gene vector TBNCs@pDNA.

[0128] The loading capacity of the biomimetic nano gene vectors TBNCs@pDNA of Examples 1 to 6 was tested, and the test results are shown in Figure 7 。

[0129] Test method: Analyze with 1% agarose gel (TAE buffer, 110 V, 0.5 h), and then use goldviewⅡ for DNA staining to examine the loading capacity of TBNCs for the HGF plasmid gene.

[0130] Figure 7 It is the loading effect diagram of TBNCs on the HGF plasmid gene in the biomimetic nano gene vectors TBNCs@pDNA of Examples 1 to 6. It can be seen from screening the biomimetic nano gene vectors of TBNCs and the HGF plasmid gene with different reaction mass ratios by agarose gel electrophoresis that ratios greater than 12:1 are all good for complete loading. Therefore, the best reaction mass ratio of the two is 12:1 in Example 5. So Example 5 was set as the feeding ratio for subsequent tests.

[0131] The gene delivery effect of the biomimetic nano gene vector TBNCs@pDNA of Example 5 and its CT imaging ability to label stem cells were tested, and the test results are shown in Figures 8 to 10 。

[0132] Test method: After co-incubating 50 μg / mL TBNCs@pDNA with mesenchymal stem cells for 24 h, the labeled stem cells were fixed with 4% paraformaldehyde and subjected to immunofluorescence specific staining to verify the expression of HGF. Meanwhile, TBNCs@pDNA was formulated into solutions with concentrations of 6.25, 12.5, 25, 50, and 100 μg / mL. Then, mesenchymal stem cells were cultured with different concentrations of TBNCs@pDNA solutions for 4 h. The labeled stem cells were rinsed at the bottom of 100 μL tubes and collected for Micro-CT imaging.

[0133] Figure 8 It is the plasmid gene transfection effect diagram of TBNCs@pDNA in Example 5. It can be seen that the superposition of two kinds of fluorescence, green (EGFP) and red (HGF), produces yellow staining on the merged image of mesenchymal stem cells, indicating that the HGF plasmid gene is effectively transfected into mesenchymal stem cells through TBNCs@pDNA and highly expressed.

[0134] Figure 9 It is the CT imaging performance diagram of TBNCs@pDNA-labeled stem cells in Example 5. It can be seen that as the concentration of TBNCs@pDNA increases, the CT images of the labeled stem cells gradually become brighter.

[0135] Figure 10 It is the relationship diagram between the attenuation of the Hounsfield value of TBNCs@pDNA-labeled stem cells in Example 5 and the concentration. It can be seen that there is a good linear relationship between the attenuation of the Hounsfield value (HU) of the labeled stem cells and the concentration of TBNCs@pDNA.

[0136] The biomimetic nano-gene carrier TBNCs@pDNA in Example 5 was used to label mesenchymal stem cells, and the antioxidant stress ability was tested and its mitochondrial morphology was observed. The test results are shown in Figures 11 to 12 。

[0137] Test method: Mesenchymal stem cells were seeded in a 35 mm glass-bottom confocal dish at a density of 10 5 cells / dish. When the cell confluence reached 80%, the adherent cells were treated with 50 μg / mL TBNCs for 4 h (i.e., Labeled hMSC was obtained). Then, the cells were cultured with a conditioned medium containing 600 μM H2O2 for 6 h to obtain a control group (Control), a positive control group (hMSC + H2O2), and an experimental group (Labeled hMSC + H2O2). 2',7'-Dichlorofluorescein diacetate (DCFH) probe was added to the culture medium and incubated in the dark at 37 °C for 0.5 h in an incubator. Subsequently, the cells were washed three times with PBS, and the intracellular reactive oxygen species were observed with a laser confocal microscope. Meanwhile, mesenchymal stem cells were seeded at a density of 10 5Cells were seeded at a density of

[0138] Figure 11 cells / well in a 6-well plate and incubated in fresh medium for 12 h to promote cell adhesion. Then, the adherent cells were treated with H2O2, and H2O2 and TBNCs@pDNA for 6 h, respectively. After the cells were collected, centrifuged, and washed, they were immersed in glutaraldehyde fixative (prepared from 500 μL glutaraldehyde, 5 mL PBS, and 4.5 mL H2O) for 60 min, embedded, sectioned, and finally observed for mitochondrial morphology using a biological transmission electron microscope.

[0139] Figure 12 For the effect diagram of the scavenging of intracellular ROS in stem cells by TBNCs in TBNCs@pDNA of Example 5, it can be seen that confocal fluorescence imaging showed that the content of reactive oxygen species in mesenchymal stem cells labeled with TBNCs was significantly lower than that in the unlabeled mesenchymal stem cell group, indicating that TBNCs can efficiently scavenge intracellular reactive oxygen species.

[0140] All aspects, embodiments, and features of the present invention should be considered illustrative in all respects and not limiting of the present invention. The scope of the present invention is only defined by the claims. Without departing from the spirit and scope of the claimed invention, those skilled in the art will appreciate other embodiments, modifications, and uses.

[0141] In the preparation method of the present invention, the order of each step is not limited to the recited order. For those of ordinary skill in the art, without creative efforts, the changes in the order of each step are also within the protection scope of the present invention. In addition, two or more steps or actions can be carried out simultaneously.

[0142] Finally, it should be noted that the specific embodiments described herein are only illustrative of the present invention and do not limit the implementation mode of the present invention. Those skilled in the art of the present invention can make various modifications or supplements to the described specific embodiments or use similar methods to replace them. It is not necessary and impossible to list all the implementation modes here. And these obvious changes or variations derived from the essential spirit of the present invention still fall within the protection scope of the present invention. Interpreting them as any additional limitation is contrary to the spirit of the present invention.

Claims

1. A biomimetic nano gene carrier with CT imaging and antioxidant stress functions, characterized in that, The bionic nano-gene vector includes a gene delivery vector and an HGF plasmid gene loaded on the gene delivery vector. The gene delivery vector includes protamine sulfate and a composite metal-based nanoparticle, and the composite metal-based nanoparticle includes a combination of at least three of gold, cobalt, platinum, copper, cerium, and iron.

2. The biomimetic nano-gene carrier with CT imaging and antioxidant stress functions according to claim 1, characterized in that, The composite metal-based nanoparticle is composed of gold, platinum, and cobalt; The molar ratio of gold, platinum, and cobalt in the composite metal-based nanoparticle is (1-4):(1-4):(1-4).

3. The biomimetic nano-gene carrier with CT imaging and antioxidant stress functions according to claim 1, wherein, The gene delivery vector uses protamine sulfate as a template, and the composite metal-based nanoparticle grows on the protamine sulfate; And / or, the plasmid gene binds to the binding site on the composite metal-based nanoparticle, and the binding site is derived from protamine sulfate; And / or, the particle size of the bionic nano-gene vector is 1-20 nm.

4. A biomimetic nano-gene carrier with CT imaging and antioxidant stress functions according to claim 1, characterized in that, The SOD-like enzyme activity of the gene delivery vector is 50-90%, and the CAT-like enzyme activity is 15-90%.

5. A biomimetic nano-gene vector with CT imaging and antioxidant stress functions according to claim 1, characterized in that, When the concentration of the gene delivery vector is 50-60 μg / mL, the scavenging rate of the gene delivery vector for hydroxyl radicals is ≥90%; 6. The biomimetic nano-gene vector with CT imaging and antioxidant stress functions according to claim 1, characterized in that, The mass ratio of protamine sulfate to the composite metal-based nanoparticle in the gene delivery vector is 1:(1-15); And / or, the mass ratio of the gene delivery vector to the plasmid gene is (1-60):

1.

7. The preparation method of a biomimetic nano-gene carrier with CT imaging and antioxidant stress functions according to claim 1, wherein, The preparation method includes the following steps: co-incubating the gene delivery vector with the HGF plasmid gene to obtain the bionic nano-gene vector.

8. The preparation method of a biomimetic nano-gene carrier with CT imaging and antioxidant stress effects according to claim 7, characterized in that, The preparation method of the gene delivery vector includes the following steps: mixing a chloroauric acid solution, a chloroplatinic acid solution, a cobalt chloride solution, and a protamine sulfate solution, and then performing a reduction reaction to obtain the gene delivery vector.

9. The preparation method of a biomimetic nano-gene carrier with CT imaging and antioxidant stress effects according to claim 8, characterized in that, The molar ratio of chloroauric acid, chloroplatinic acid, and cobalt chloride in the mixed solution is (1-4):(1-4):(1-4); And / or, the ratio of protamine sulfate to chloroauric acid in the mixed solution is 1 g:(0.5-20) mmol; And / or, the molar ratio of sodium borohydride to chloroauric acid is 100:(1-20); And / or, the reduction reaction temperature is 25-35 °C, and the time is 20-40 min.

10. Use of a biomimetic nano-gene carrier having CT imaging and antioxidant stress functions as described in any one of claims 1 to 5 in cell gene delivery and / or cell antioxidant stress and / or cell CT tracing, characterized in that, The cell is a therapeutic cell; The cell includes one or more of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, neural stem cells, human umbilical cord mesenchymal stem cells, or lymphocytes; The time for CT tracing of the cell is ≥45 days; The survival time of the cell in vivo is ≥14 days.

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