A multifunctional nano delivery system integrating gene delivery and CT tracing, and a preparation method and application thereof

Abstract

This invention provides a multifunctional nanodelivery system integrating gene delivery and CT tracking. The multifunctional nanodelivery system includes a gene delivery vector and an HGF plasmid gene loaded on the gene delivery vector; the gene delivery vector comprises gold nanoparticles coated with protamine sulfate. The gene delivery vector AuPS provided by this invention has excellent cell membrane transport and nuclear localization capabilities, enabling the HGF plasmid gene to efficiently enter cells and be expressed in the nucleus. The multifunctional nanodelivery system provided by this invention can be used for cell function regulation and tracking of therapeutic cells such as stem cells.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanobiomaterials, and particularly relates to a multifunctional nano delivery system integrating gene delivery and CT tracing, and a preparation method and application thereof. BACKGROUND

[0002] Mesenchymal stem cells (MSCs) are an important cell population for stem cell therapy in regenerative medicine. MSCs have become an extremely attractive cell source for repairing various tissue injuries due to their self-renewal and differentiation potential, as well as their antioxidant and immunomodulatory abilities (Y. Han, X. Li, Y. Zhang, Y. Han, F. Chang, J. Ding, Cells 2019, 8, 886.).

[0003] However, the therapeutic effect of transplanted MSCs in harsh tissue microenvironments such as ischemia, inflammation or anoikis is poor, which seriously hinders the progress of MSCs-based disease treatment (H. Song, B. W. Song, M. J. Cha, I. G. Choi, K. C. Hwang, Expert Opin. Biol. Ther. 2010, 10, 309.). In recent years, various methods have been used to improve the therapeutic effect of transplanted MSCs, including hypoxia-inducible factor-1α (HIF-1α) pretreatment, hepatocyte growth factor (HGF) lentivirus transduction and liposome-mediated vascular endothelial growth factor (VEGF) transfection (H. Zhang, H. Wang, Y. Xia, N. Qi, Stem Cells Int. 2021, 2021, 6658855; H. Wang, R. T. Sun, Y. Li, Y. F. Yang, F. J. Xiao, Y. K. Zhang, S. X. Wang, H. Y. Sun, Q. W. Zhang, C. T. Wu, L. S. Wang, PLoS One 2015, 10, e0124420; J. Yang, W. Zhou, W. Zheng, Y. Ma, L. Lin, T. Tang, J. Liu, J. Yu, X. Zhou, J. Hu, Cardiology 2007, 107, 17.). Comprehensive studies have shown that therapeutic gene-engineered MSCs can significantly improve the therapeutic effect of transplanted MSCs, opening up a new prospect for MSCs-based treatment of various diseases.

[0004] Development of excellent gene delivery vectors to achieve efficient gene transfection is an important prerequisite for MSCs treatment of diseases. At present, gene delivery vectors can be roughly divided into viral vectors and non-viral vectors. Although the viral vector has a high gene transfection efficiency, its potential carcinogenicity, immunogenicity and complex synthesis process make the safety of gene therapy a wide range of controversy (C.A. Gersbach, S.R. Coyer, J.M. Le Doux, A.J. Garcia, Biomaterials 2007, 28, 5121; A.K. Zaiss, D.A. Muruve, Curr. Gene Ther. 2005, 5, 323.). Non-viral vectors, usually cationic polymers, have attracted widespread attention due to their ease of synthesis, multifunctionality and safety. However, the existing cationic polymer carriers still have many problems to be solved, such as unsatisfactory transfection efficiency, high cytotoxicity, etc. (M.J. Palate, R.T. Raines, J. Am. Chem. Soc. 2012, 134, 6218; L.-H. Peng, S.-Y. Tsang, Y. Tabata, J.-Q. Gao, J. Control. Release 2012, 157, 321; G.S. Oggu, S. Sasikumar, N. Reddy, K.K.R. Ella, C.M. Rao, K.K. Bokara, Stem Cell Rev. Rep. 2017, 13, 725.). Therefore, it is of great significance to develop a high-efficiency, biocompatible gene delivery vector for MSCs gene transfection to enhance the efficacy of transplanted MSCs.

[0005] Another challenge of stem cell therapy is that the information of transplanted MSCs in vivo is unclear, so it is urgent to propose a reliable non-invasive imaging method for tracking the homing and migration of therapeutic MSCs. SUMMARY

[0006] In view of the shortcomings of the prior art, the purpose of the present application is to provide a multifunctional nano delivery system integrating gene delivery and CT tracking, and a preparation method and application thereof. The multifunctional nano delivery system integrating gene delivery and CT tracking provided by the present application has good membrane transport capacity, releases plasmid genes at the perinuclear site, realizes low-toxicity and high-efficiency gene delivery in cells, and can improve the CT imaging contrast of cells, effectively tracking the distribution, migration and homing of transplanted cells in vivo for a long period of time through CT imaging.

[0007] To achieve the purpose of the present application, the following technical solutions are adopted:

[0008] In a first aspect, the present application provides a multifunctional nano delivery system integrating gene delivery and CT tracing, which comprises a gene delivery carrier and an HGF plasmid gene loaded on the gene delivery carrier; the gene delivery carrier comprises protamine sulfate coated gold nanoparticles.

[0009] In the present application, the gene delivery carrier with CT tracing function comprises protamine sulfate and gold nanoparticles grown with protamine sulfate as a template.

[0010] The multifunctional nano delivery system provided by the present application can significantly improve the intracellular gene delivery efficiency, regulate the function of cells, and realize the non-invasive visual tracing of homing and migration of therapeutic cells.

[0011] The multifunctional nano delivery system provided by the present application has the function of enhancing mesenchymal stem cell therapy for pulmonary fibrosis.

[0012] In the present application, the particle size of the gene delivery carrier is 5-10 nm, for example, it can be 5 nm, 6 nm, 7 nm, 8 nm, 9 nm, 10 nm, etc.

[0013] In the present application, the gene delivery carrier can be completely metabolized by the liver, and has good biological safety.

[0014] Preferably, the gene delivery carrier and the HGF plasmid gene are combined through a binding site of the HGF plasmid gene on the gene delivery carrier.

[0015] Preferably, the binding site of the HGF plasmid gene is derived from protamine sulfate.

[0016] In the present application, the protamine sulfate has good cell membrane transport capacity and nuclear localization function.

[0017] In the present application, the mass ratio of protamine sulfate and gold nanoparticles in the gene delivery carrier is 1:(5-15);

[0018] Wherein, "5-15" can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc.

[0019] Preferably, the mass ratio of the gene delivery carrier and the HGF plasmid gene is (1-60):1, preferably (9-60):1, and further preferably (15-30):1.

[0020] Wherein, "1-60" can be 1, 10, 20, 30, 40, 50, 60, etc.

[0021] "9-60" can be 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, etc.

[0022] "15-30" can be 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc.

[0023] Preferably, the mass ratio of the gene delivery carrier and the HGF plasmid gene is 15:1.

[0024] In the present application, the multifunctional nano delivery system further comprises any one or a combination of at least two of cobalt, platinum, copper or cerium.

[0025] In a second aspect, the present application provides a preparation method of the multifunctional nano delivery system according to the first aspect, the preparation method comprising the following steps: co-incubating a gene delivery carrier with an HGF plasmid gene to obtain the multifunctional nano delivery system.

[0026] The multifunctional nano delivery system according to the present application delivers the plasmid gene and the CT tracer to the targeted area.

[0027] Preferably, the incubation temperature is 25-35℃ (for example, it can be 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, etc.), and the incubation time is 5-15min (for example, it can be 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, etc.).

[0028] In the present application, the preparation method of the gene delivery carrier comprises the following steps: mixing a tetrachloroauric acid trihydrate solution and a protamine sulfate solution, and then performing a reduction reaction to obtain the gene delivery carrier.

[0029] In the present application, the mass percentage content of tetrachloroauric acid trihydrate in the tetrachloroauric acid trihydrate solution is 0.005-0.015% (for example, it can be 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.011%, 0.012%, 0.013%, 0.014%, 0.015%, etc.).

[0030] Preferably, the mass concentration of protamine sulfate in the protamine sulfate solution is 5-15mg / mL (for example, it can be 5mg / mL, 6mg / mL, 7mg / mL, 8mg / mL, 9mg / mL, 10mg / mL, 11mg / mL, 12mg / mL, 13mg / mL, 14mg / mL, 15mg / mL, etc.).

[0031] Preferably, the volume ratio of the tetrachloroauric acid trihydrate solution to the protamine sulfate solution is (90-110):(0.05-0.15).

[0032] Preferably, the volume ratio of the tetrachloroauric acid trihydrate solution to the protamine sulfate solution is (90-110):(0.05-0.15).

[0033] Preferably, the volume ratio of the tetrachloroauric acid trihydrate solution to the protamine sulfate solution is (90-110):(0.05-0.15).

[0034] Preferably, the reducing agent used in the reduction reaction is a sodium borohydride solution, the molar concentration of the sodium borohydride solution is 3-8 mM (for example, it can be 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, etc.), and the volume ratio of the tetrachloroauric acid trihydrate solution to the sodium borohydride solution is (90-110):1.

[0035] Preferably, the volume ratio of the tetrachloroauric acid trihydrate solution to the protamine sulfate solution is (90-110):(0.05-0.15).

[0036] Preferably, the temperature of the reduction reaction is 25-35℃ (for example, it can be 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, 35℃, etc.), and the time is 20-40 min (for example, it can be 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 32 min, 34 min, 36 min, 38 min, 40 min, etc.).

[0037] In the present application, the preparation method further comprises the following step: concentrating the gene delivery carrier solution obtained after the reduction reaction by ultrafiltration to obtain the gene delivery carrier.

[0038] Preferably, the molecular weight cut-off of the ultrafiltration tube used in the ultrafiltration concentration is 8-12 kD (for example, it can be 8 kD, 9 kD, 10 kD, 11 kD, 12 kD, etc.), and the volume is 10-20 mL (for example, it can be 10 mL, 12 mL, 14 mL, 16 mL, 18 mL, 20 mL, etc.).

[0039] Preferably, the rotation speed of the ultrafiltration concentration is 3500-4500 rpm (for example, it can be 3500 rpm, 3700 rpm, 3900 rpm, 4100 rpm, 4300 rpm, 4500 rpm, etc.), and the time is 10-20 min (for example, it can be 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, etc.).

[0040] As a preferred technical scheme of the present application, the preparation method of the multifunctional nano delivery system comprises the following steps:

[0041] (1) mixing a tetrachloroauric acid trihydrate solution and a protamine sulfate solution to obtain a mixed solution;

[0042] wherein the mass percentage of tetrachloroauric acid trihydrate in the tetrachloroauric acid trihydrate solution is 0.005-0.015%, the mass concentration of protamine sulfate in the protamine sulfate solution is 5-15 mg / mL; the volume ratio of the tetrachloroauric acid trihydrate solution to the protamine sulfate solution is (90-110):(0.05-0.15);

[0043] (2) reducing the mixed solution obtained in step (1) to obtain a gene delivery carrier solution;

[0044] wherein the reducing agent used in the reduction reaction is a sodium borohydride solution, the molar concentration of the sodium borohydride solution is 3-8 mM, and the volume ratio of the tetrachloroauric acid trihydrate solution to the sodium borohydride solution is (90-110):1;

[0045] (3) ultrafiltration concentration of the gene delivery carrier solution obtained in step (2) to obtain a gene delivery carrier;

[0046] wherein the ultrafiltration tube used in the ultrafiltration concentration has a molecular weight cut-off of 8-12 kD and a volume of 10-20 mL; the rotation speed of the ultrafiltration concentration is 3500-4500 rpm, and the time is 10-20 min;

[0047] (4) co-incubation of the gene delivery carrier obtained in step (3) with an HGF plasmid gene to obtain the multifunctional nano delivery system;

[0048] wherein the incubation temperature is 25-35°C, and the incubation time is 5-15 min.

[0049] In a third aspect, the present application provides a multifunctional nano delivery system according to the first aspect for use in cell gene delivery and / or cell CT tracking.

[0050] Preferably, the cells are therapeutic cells.

[0051] Preferably, the cells include any one or a combination of at least two of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, neural stem cells, human umbilical cord stem cells, or lymphocytes.

[0052] Preferably, the CT tracking time is ≥ 30 days.

[0053] Compared with the prior art, the present application has the following beneficial effects:

[0054] (1) The present application uses protamine sulfate (PS) as a template, and gold nanoparticles (Au) are grown on the protein in one step to obtain an AuPS gene delivery carrier. HGF plasmid genes are combined on the protamine sulfate, and the gold nanoparticles can be used as a CT tracer to achieve simultaneous delivery of plasmid genes and a CT tracer. The protamine sulfate is located in the nucleus, and the plasmid genes are released in the perinuclear region. The problem of efficient delivery of plasmid genes, which is difficult to achieve by traditional methods, is overcome, and a new method is provided for cell apoptosis, survival, directional differentiation, paracrine function, and tracing of therapeutic cells.

[0055] (2) The multifunctional nano delivery system provided by the present application has the following effects: a, efficient HGF plasmid gene delivery is achieved, the genes are expressed in the perinuclear region, and better effects are achieved; b, the high-resolution characteristics of CT imaging can be used to realize real-time visualization tracking of the distribution, migration and homing of transplanted labeled stem cells; c, the small size of the nano system can be completely metabolized by the liver in vivo, and has excellent biological safety.

[0056] (3) The present application helps to establish a general plasmid gene and CT tracer simultaneous delivery strategy, and provides a new method for plasmid gene and CT tracer labeled cells. BRIEF DESCRIPTION OF DRAWINGS

[0057] Figure 1 It is a transmission electron microscope image of the AuPS gene delivery carrier.

[0058] Figure 2 It is a preparation schematic diagram of the multifunctional nano delivery system AuPS@HGF plasmid gene which integrates gene delivery and CT tracing.

[0059] Figure 3a It is a CT imaging performance diagram of the multifunctional nano delivery system AuPS@HGF plasmid gene which integrates gene delivery and CT tracing.

[0060] Figure 3b It is a relationship diagram of the attenuation of Hounsfield value and Au concentration of the multifunctional nano delivery system AuPS@HGF plasmid gene which integrates gene delivery and CT tracing.

[0061] Figure 4a It is a transfection effect diagram of the multifunctional nano delivery system AuPS@HGF plasmid gene which integrates gene delivery and CT tracing.

[0062] Figure 4b It is a CT imaging performance of the multifunctional nano delivery system AuPS@HGF plasmid gene which integrates gene delivery and CT tracing.

[0063] Figure 4c A graph showing the relationship between the decay of Hounsfield values ​​and Au concentration in AuPS@HGF plasmid gene-labeled stem cells, a multifunctional nanodelivery system integrating gene delivery and CT tracing.

[0064] Figure 5a This is a diagram showing the induction of myofibroblast apoptosis by labeled mesenchymal stem cells in Example 3.

[0065] Figure 5b This is a diagram illustrating the inhibition of epithelial-mesenchymal transition by labeled mesenchymal stem cells in Example 3. Detailed Implementation

[0066] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0067] The sources of each component in the following preparation examples and embodiments are shown below:

[0068]

[0069] Preparation Example 1

[0070] This preparation example provides an AuPS gene delivery vector, the preparation method of which includes the following steps:

[0071] (1) Mix 100 mL of 0.01% tetrachloroauric acid trihydrate solution with 100 μL of 10 mg / mL protamine sulfate solution to obtain a mixed solution;

[0072] (2) Stir the mixed solution obtained in step (1) at 1000 rpm at room temperature, quickly add 1 mL of freshly prepared 5 mM sodium borohydride solution, react at room temperature for 30 min, and obtain a wine-red AuPS gene delivery vector solution.

[0073] (3) The AuPS gene delivery vector solution obtained in step (2) is concentrated by ultrafiltration at room temperature to obtain the AuPS gene delivery vector;

[0074] The ultrafiltration concentration includes the following steps: centrifugation at 4000 rpm for 15 min using a 15 mL Millipore ultrafiltration tube with 10 kD MWCO; and storage of the AuPS stock solution at 4°C for later use.

[0075] like Figure 1 As shown, the transmission electron microscope image of the AuPS gene delivery vector shows that the particle size of the AuPS gene delivery vector is approximately 8 nm.

[0076] Example 1

[0077] The present embodiment provides a multifunctional nano delivery system integrating gene delivery and CT tracing, and a preparation method of the multifunctional nano delivery system includes the following steps:

[0078] The AuPS gene delivery carrier obtained in Preparation Example 1 is mixed with the HGF plasmid gene in a certain ratio, and is uniformly placed at room temperature for 10 minutes, to obtain the multifunctional nano delivery system AuPS@HGF plasmid gene.

[0079] The specific steps are as follows:

[0080] Different amounts of AuPS (1, 3, 6, 9 μg) are mixed with the HGF plasmid gene (1 μg diluted in 100 μL sodium acetate buffer solution (25 mg NaAc, pH 5)) for 10 minutes, and then analyzed by 1% agarose gel (TAE buffer zone, 110 V, 0.5 hours) to freely control the HGF plasmid gene at 1 μg / loading. DNA staining is performed by goldview II, and the loading capacity of AuPS for the HGF plasmid gene is determined.

[0081] The optimal reaction mass ratio of AuPS and the HGF plasmid gene is screened by agarose gel electrophoresis, and the results show that the optimal reaction mass ratio of the two is 15.

[0082] As shown in Figure 2 The preparation method of the multifunctional nano delivery system AuPS@HGF plasmid gene integrating gene delivery and CT tracing includes the following steps: first, a tetrachloroauric acid trihydrate solution and a protamine sulfate solution are mixed to obtain a mixed solution; then, sodium borohydride reduction is performed to obtain an AuPS gene delivery carrier; the AuPS gene delivery carrier is mixed with the HGF plasmid gene to obtain the AuPS@HGF plasmid gene.

[0083] Example 2

[0084] The CT imaging performance of the multifunctional nano delivery system AuPS@HGF plasmid gene integrating gene delivery and CT tracing prepared in Example 1 is evaluated

[0085] The evaluation method: in vitro CT imaging is performed by a Micro-CT imager (Hiscan XM). The scanning parameters include: 60 kV, 133 μA, single exposure time 1.2 s, scanning resolution 50 μm, scanning angle interval 0.5°, and scanning circle 360°. The reconstruction software and analysis software are provided by Hiscan.

[0086] Performance characterization: as shown in Figure 3aAs shown, the CT image of AuPS@HGF plasmid gene gradually brightened with the increase of Au concentration. As shown in Figure 3b As shown, the attenuation of Hounsfield value (HU) of AuPS@HGF plasmid gene was linearly related to Au concentration.

[0087] Example 3

[0088] Evaluation of the gene delivery effect of the multifunctional nanodelivery system AuPS@HGF plasmid gene integrating gene delivery and CT tracing prepared in Example 1 and its CT imaging ability of labeling stem cells

[0089] Evaluation method: 100 μg mL -1 AuPS@HGF plasmid gene was incubated with mesenchymal stem cells for 24 hours, then the labeled stem cells were fixed with paraformaldehyde and subjected to immunofluorescence specific staining to verify the expression of HGF. At the same time, mesenchymal stem cells were cultured with AuPS@HGF plasmid gene at different gold concentrations (20, 40, 80, 100 μg mL -1 ) for 4 hours, then the labeled stem cells were washed and collected at the bottom of 100 μL tube for Micro-CT imaging. Then the sample was completely dissolved in aqua regia. Then the concentration of Au was determined by ICP-MS, and the uptake of Au in each cell was calculated.

[0090] Performance evaluation: the superposition of green (EGFP) and red (HGF) fluorescence on the merged image of hMSCs produced yellow staining, indicating that HGF was effectively transferred to stem cells by AuPS@HGF plasmid gene carrier and expressed efficiently (as shown in Figure 4a As shown, the CT image of AuPS@HGF plasmid gene gradually brightened with the increase of Au concentration. As shown in Figure 4b The attenuation of Hounsfield value (HU) of labeled stem cells was linearly related to Au concentration, and the Au concentration in cells was estimated to be 445 pg / cell, which was much higher than that of existing Au-based nanotracer (as shown in Figure 4c

[0091] Example 4

[0092] Evaluation of the function of labeled mesenchymal stem cells in treating pulmonary fibrosis in vitro in Example 3

[0093] Evaluation method: muscle fibroblasts were cultured with ordinary culture medium, normal mesenchymal stem cell (hMSC) conditioned medium or labeled mesenchymal stem cell (hMSC / HGF) conditioned medium to evaluate the effect of labeled mesenchymal stem cell conditioned medium on inducing apoptosis of muscle fibroblasts.

[0094] ​Wherein, TD is the bright field, Annexin-v-FITC is early apoptotic cells, and PI is necrotic cells.

[0095] Meanwhile, human normal lung epithelial cells were seeded in 24-well plates at a density of 1 x 10 4 cells per well. After the cells reached 80% confluence, the cells were cultured with normal medium, normal mesenchymal stem cell conditioned medium containing 2.5 ng / mL -1 TGFβ or labeled mesenchymal stem cell conditioned medium for 24 h. Then the cells were fixed with 4% paraformaldehyde for 30 min, and then incubated in PBS containing 3% goat serum and 0.1% triton for 1 h at room temperature to prevent non-specific binding. The primary antibody α-smooth muscle actin (α-SMA, 1:250 dilution), collagen I (Col I, 1:200 dilution), and fibronectin (FN, 1:200 dilution) were incubated overnight at 4°C. Alexa 555-labeled secondary antibody (1:400 dilution) was added at room temperature for 1 h, and DAPI staining was performed for 5 min, and specific binding was observed. Laser confocal microscopy was used to observe the expression of specific proteins.

[0096] Performance evaluation: confocal fluorescence imaging was used to observe the results of induced apoptosis of myofibroblasts, and the labeled mesenchymal stem cell conditioned medium significantly induced apoptosis of myofibroblasts (as shown in Figure 5a Immunofluorescence staining results showed that the labeled mesenchymal stem cell conditioned medium significantly inhibited epithelial mesenchymal transition (as shown in Figure 5b ).

[0097] The applicant declares that the process of the present application is illustrated by the above examples, but the present application is not limited to the above process steps, that is, it does not mean that the present application must rely on the above process steps to be implemented. Those skilled in the art should understand that any improvement of the present application, equivalent replacement of the raw materials selected by the present application, addition of auxiliary ingredients, selection of specific methods, etc. fall within the protection scope and disclosure scope of the present application.

Claims

1. A multifunctional nanodelivery system integrating gene delivery with CT tracking, characterized in that, The multifunctional nano delivery system comprises a gene delivery carrier and an HGF plasmid gene loaded on the gene delivery carrier; the gene delivery carrier is a protamine sulfate coated gold nanoparticle; The particle size of the gene delivery carrier is 5-10 nm; The gene delivery carrier is combined with the HGF plasmid gene through a binding site of the HGF plasmid gene located in the protamine sulfate; The mass ratio of protamine sulfate and gold nanoparticles in the gene delivery carrier is 1:(5-15); The mass ratio of the gene delivery carrier and the HGF plasmid gene is (1-60):

1.

2. The multifunctional nanodelivery system according to claim 1, wherein, The mass ratio of the gene delivery carrier and the HGF plasmid gene is (9-60):

1.

3. The multifunctional nanodelivery system according to claim 1, wherein, The mass ratio of the gene delivery carrier and the HGF plasmid gene is (15-30):

1.

4. The multi-functional nanodelivery system according to claim 1, wherein, The mass ratio of the gene delivery carrier and the HGF plasmid gene is 15:

1.

5. A method for preparing the multifunctional nanodelivery system according to any one of claims 1-4, characterized by, The preparation method comprises the following steps: co-incubating a gene delivery carrier with an HGF plasmid gene to obtain the multifunctional nano delivery system.

6. The production method according to claim 5, wherein The incubation temperature is 25-35℃, and the incubation time is 5-15 min.

7. The preparation method according to claim 5, characterized in that, The preparation method of the gene delivery carrier comprises the following steps: mixing a tetrachloroauric acid trihydrate solution and a protamine sulfate solution, and then performing a reduction reaction to obtain the gene delivery carrier.

8. The preparation method according to claim 7, characterized in that, The mass percentage content of tetrachloroauric acid trihydrate in the tetrachloroauric acid trihydrate solution is 0.005-0.015%.

9. The preparation method according to claim 7, characterized in that, The mass concentration of protamine sulfate in the protamine sulfate solution is 5-15 mg / mL.

10. The preparation method according to claim 7, characterized in that, The volume ratio of the tetrachloroauric acid trihydrate solution to the protamine sulfate solution is (90-110):(0.05-0.15).

11. The preparation method according to claim 7, characterized in that, The reducing agent used in the reduction reaction is a sodium borohydride solution, the molar concentration of the sodium borohydride solution is 3-8 mM, and the volume ratio of the tetrachloroauric acid trihydrate solution to the sodium borohydride solution is (90-110):

1.

12. The method of claim 7, wherein, The temperature of the reduction reaction is 25-35℃, and the time of the reduction reaction is 20-40 min.

13. The preparation method according to claim 7, characterized in that, The preparation method further comprises the following step: concentrating the gene delivery carrier solution obtained after the reduction reaction by ultrafiltration to obtain the gene delivery carrier.

14. The method of claim 13, wherein, The ultrafiltration tube used in the ultrafiltration concentration has a molecular weight cut-off of 8-12 kD and a volume of 10-20 mL.

15. The preparation method according to claim 13, characterized in that, The rotation speed of the ultrafiltration concentration is 3500-4500 rpm, and the time of the ultrafiltration concentration is 10-20 min.

16. The method of making according to any one of claims 5-15, wherein, The preparation method of the multifunctional nano delivery system comprises the following steps: (1) mixing a tetrachloroauric acid trihydrate solution and a protamine sulfate solution to obtain a mixed solution; wherein the mass percentage content of tetrachloroauric acid trihydrate in the tetrachloroauric acid trihydrate solution is 0.005-0.015%, the mass concentration of protamine sulfate in the protamine sulfate solution is 5-15 mg / mL, and the volume ratio of the tetrachloroauric acid trihydrate solution to the protamine sulfate solution is (90-110):(0.05-0.15); (2) performing a reduction reaction on the mixed solution in step (1) to obtain a gene delivery carrier solution; The reducing agent used in the reduction reaction is sodium borohydride solution, the molar concentration of the sodium borohydride solution is 3-8 mM, and the volume ratio of the tetrachloroauric acid trihydrate solution to the sodium borohydride solution is (90-110):1; (3) concentrating the gene delivery carrier solution obtained in step (2) by ultrafiltration to obtain a gene delivery carrier; The molecular weight cut-off of the ultrafiltration tube used in the ultrafiltration concentration is 8-12 kD, and the volume is 10-20 mL; the rotation speed of the ultrafiltration concentration is 3500-4500 rpm, and the time is 10-20 min; (4) co-incubating the gene delivery carrier obtained in step (3) with an HGF plasmid gene to obtain the multifunctional nano delivery system; The incubation temperature is 25-35℃, and the incubation time is 5-15 min.

17. Use of the multifunctional nano delivery system according to any one of claims 1-4 in the preparation of a preparation for cell gene delivery and / or cell CT tracking.

18. The use according to claim 17, characterized in that, The cells are therapeutic cells.

19. The use according to claim 17, characterized in that, The cells include any one or a combination of at least two of adipose mesenchymal stem cells, bone marrow mesenchymal stem cells, neural stem cells, human umbilical cord stem cells, or lymphocytes.

20. The use according to claim 17, characterized in that, The cell CT tracking time is ≥ 30 days.

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