A composite carbon dot carrier and a carbon dot complex carrying VEGF siRNA

Abstract

The present application relates to a kind of composite carbon dot carrier and carry VEGF siRNA carbon dot complex, it is with cysteine, neutral red and gadolinium diamine by one-step solvothermal method to obtain the Gd-CDs with fluorescence-magnetic resonance dual-mode imaging ability as foundation, PEI and FA molecule are coupled on its surface by EDC / NHS activation to prepare carrier FA-PEI-Gd-CDs, and then carry VEGF siRNA to form gene carrier complex FA-PEI-Gd-CDs@VEGF siRNA.The gene carrier complex constructed in the present application has good dual-mode imaging ability, and also has specific inhibitory effect on liver tumor cells, and can be applied to the preparation of liver tumor cell growth inhibition drug, to achieve good therapeutic effect on liver cancer at nanoscale level.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanomaterials and biomedicine, and relates to a composite carbon dot with a dual-mode imaging function and a gene-carrying complex constructed by using the composite carbon dot to carry VEGF siRNA, and application thereof in liver cancer treatment and imaging monitoring. BACKGROUND

[0002] Liver cancer is one of the common cancers with extremely high mortality. Survey data shows that there were more than 900,000 new cases of liver cancer worldwide in 2020, accounting for 4.69% of global cancer incidence, with an incidence of 9.5 / 100,000. By 2040, the number of new cases and deaths of liver cancer worldwide may increase by 55% compared with now.

[0003] The liver itself lacks nerve wrapping, and early symptoms of liver cancer are more common symptoms such as abdominal distension, indigestion, loss of appetite, etc. Most liver cancer patients (about 70%) are found to be in the advanced stage when they are discovered, and cannot be surgically removed, but can only be treated by other means including liver transplantation, transcatheter arterial chemoembolization (TACE) or systemic chemotherapy. However, the lack of liver supply limits the clinical application of liver transplantation, TACE cannot completely embolize the tumor blood vessels, the postoperative hypoxic microenvironment leads to tumor progression, recurrence and even metastasis, systemic chemotherapy has serious side effects and poor overall prognosis, etc., which will lead to the failure of liver cancer treatment.

[0004] At the same time, the current clinical monitoring of liver cancer mainly relies on traditional examination methods, which has low sensitivity to small lesions, making it difficult to detect early lesions and unable to correctly evaluate tumor proliferation or inhibition to achieve "cancer treatment".

[0005] Therefore, exploring a tumor diagnosis and treatment technology that integrates liver cancer treatment and imaging monitoring can not only accurately diagnose diseases in real time and provide treatment, but also monitor the efficacy of the treatment process in real time and adjust the treatment plan in a timely manner, which can play an important role in improving the efficiency of liver cancer treatment and monitoring.

[0006] Vascular endothelial growth factor (VEGF) is a highly specific pro-vascular endothelial cell growth factor that promotes increased vascular permeability, extracellular matrix degeneration, vascular endothelial cell proliferation and migration, and vascular formation. Studies have found that VEGF-mRNA is overexpressed in liver cancer and is closely related to liver cancer proliferation, invasion and recurrence.

[0007] Gene therapy is a new treatment method, by transferring exogenous double-stranded small interfering RNA (siRNA) to the patient's special receptor cells, forming an RNA-induced silencing complex, specifically silencing the target gene mRNA, inhibiting tumor growth at the genetic level, with high specificity, less side effects, and hereditary effects. The advantages of high specificity, less side effects, and hereditary effects. Taking VEGF as the target, design and synthesis of VEGF-siRNA into hepatocellular carcinoma cells, can inhibit the expression of VEGF-mRNA by siRNA, reduce the synthesis of VEGF, and block the angiogenesis of tumor, so as to improve the long-term efficacy of cancer and prolong the survival of patients.

[0008] However, due to the inherent physical and chemical properties of siRNA itself, such as low charge density, high structural rigidity and rapid enzymatic degradation, the medical application of siRNA is seriously hindered.

[0009] Carbon dots (CDs) can effectively protect genes from degradation due to their surface functionalization, high biocompatibility and stability, and achieve efficient gene silencing. Therefore, a kind of carrier based on CDs can be used to combine with VEGF siRNA to form a gene silencing complex and applied in the gene therapy of hepatocellular carcinoma.

[0010] On the other hand, not only CDs itself is an excellent targeted fluorescent probe, but also gadolinium-doped carbon dots (Gd-CDs) synthesized by modifying CDs with paramagnetic metal ions such as gadolinium ions can be endowed with magnetic resonance imaging (MRI) ability, realizing fluorescence-magnetic resonance dual-mode imaging (FLI-MRI). Dual-mode imaging probes can provide high-resolution macroscopic anatomical information and high-sensitivity microscopic optical signals at the same time, and have the advantages of integration and cross-validation, which can provide protection for early diagnosis of hepatocellular carcinoma.

[0011] However, the emission of most Gd-CDs at present is in the short wavelength region, such as the blue light Gd-CDs prepared by Du et al. (Du Jinglei, et al., Folic acid functionalized gadolinium-doped carbon dots as fluorescence / magnetic resonance imaging contrast agent for targeted imaging of liver cancer[J], Colloids and Surfaces B: Biointerfaces, 2023, 113721.) for targeted imaging of liver cancer FLI-MRI research, not only its optimal emission wavelength is 456nm, and its fluorescence has weak tissue penetration ability, but also is interfered by the body's own fluorescence, which limits its application in tumor diagnosis and treatment.

[0012] Therefore, in order to overcome the defects and drawbacks of liver cancer monitoring and treatment at present, improve the application of nanomaterials in biological medicine, and establish a high-efficiency and safe siRNA delivery system and delivery method by constructing a reasonable long-wavelength emission Gd-CDs carrier system, it is of great significance to realize the integration of diagnosis and treatment of liver cancer by combining bimodal imaging with gene therapy. SUMMARY

[0013] The purpose of the present application is to overcome the shortcomings of the prior art, and to provide a composite carbon dot carrier which not only has a dual-mode imaging function, but also has good gene loading capacity.

[0014] Another object of the present application is to use the composite carbon dot carrier to construct a VEGF siRNA carbon dot complex.

[0015] The composite carbon dot carrier of the present application is based on Gd-CDs prepared by one-step solvothermal method with cysteine, neutral red and gadolinium diamine, and the polyethyleneimine (PEI) and folic acid (FA) molecules are coupled on the surface of the carrier FA-PEI-Gd-CDs by activating amide activator 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS).

[0016] Further, the Gd-CDs are prepared by solvothermal reaction of cysteine, neutral red and gadolinium diamine in an ethanol aqueous solution system at 120-180℃ for 6-8h.

[0017] Further, the application specifically activates the Gd-CDs solution with EDC / NHS, adds PEI for reaction to form PEI-Gd-CDs by chemical bond, and then adds FA, EDC and NHS for reaction in the dark to prepare the composite carbon dot carrier FA-PEI-Gd-CDs by electrostatic action.

[0018] The composite carbon dot carrier FA-PEI-Gd-CDs prepared by the application has positive charge, emits wavelength 604 nm, has fluorescence-magnetic resonance dual-mode imaging capability, and can be used as a fluorescence-magnetic resonance dual-mode imaging probe.

[0019] The composite carbon dot carrier has long-wave emission fluorescence imaging effect and high-relaxivity MRI imaging capability, can not only penetrate living tissues for living fluorescence imaging and magnetic resonance imaging, but also can enter cells for cell dual-mode imaging, and has excellent cell transfection capability and biological safety as a carrier.

[0020] Further, the VEGF siRNA-loaded carbon dot complex constructed by the application carries VEGF siRNA as a target gene on the composite carbon dot carrier FA-PEI-Gd-CDs by electrostatic adsorption to form the gene-loaded complex FA-PEI-Gd-CDs@VEGF siRNA.

[0021] The target gene VEGF siRNA is a group of siRNAs with the following nucleotide sequence shown in SEQ ID NO. 5 and SEQ ID NO. 6.

[0022] Sense strand: 5'-GCAGAUCAUGCGGAUCAAA-3';

[0023] Antisense strand: 5'-UUUGAUCCGCAUGAUCUGC-3'.

[0024] In the VEGF siRNA-loaded carbon dot complex constructed by the application, the mass ratio of the composite carbon dot carrier FA-PEI-Gd-CDs to the target gene VEGF siRNA is preferably (4-10):1.

[0025] Further, the optimal mass ratio of the composite carbon dot carrier FA-PEI-Gd-CDs to the target gene VEGF siRNA is 4:1, which can ensure that the FA-PEI-Gd-CDs completely carry VEGF siRNA and can also avoid red blood cell adhesion phenomenon.

[0026] The average particle size of the FA-PEI-Gd-CDs@VEGF siRNA constructed in the application is about 20 nm, and the cell permeability is good.

[0027] The VEGF siRNA carbon dot complex is prepared according to the following method.

[0028] 1) Cysteine, neutral red and gadolinium diamine are added to a mixed solution of ethanol and water, and solvent thermal reaction is carried out at 120-180 DEG C for 6-8 h; the reaction product is filtered, dialyzed and freeze-dried to obtain Gd-CDs powder;

[0029] 2) The Gd-CDs are dissolved in PBS, and amide activators EDC and NHS are added, and the mixture is placed at room temperature for 2-4 h; after the activation is completed, PEI is added, and the mixture is stirred and reacted at 60-80 DEG C for 4-8 h; the reaction product is filtered, dialyzed and freeze-dried to obtain PEI-Gd-CDs powder;

[0030] 3) The PEI-Gd-CDs, FA, EDC and NHS are dissolved in PBS, and the mixture is stirred and reacted at room temperature in the dark for 12-24 h; the reaction product is filtered, dialyzed and freeze-dried to obtain FA-PEI-Gd-CDs solid powder;

[0031] 4) The positively charged FA-PEI-Gd-CDs and the negatively charged VEGF siRNA are mixed and placed, and the gene carrying complex FA-PEI-Gd-CDs@VEGF siRNA is formed by electrostatic interaction.

[0032] Specifically, in the above preparation method, the filtration is preferably carried out by using a disposable filter membrane with a pore size of 0.22 μm.

[0033] More specifically, in the above preparation method, the dialysis is preferably carried out by using a dialysis bag with a molecular weight of 1000 Da.

[0034] The gene carrying complex FA-PEI-Gd-CDs@VEGF siRNA constructed in the application can inhibit the expression of VEGF-mRNA and reduce the synthesis of VEGF by the siRNA carried thereby, and has an excellent gene silencing effect, so that the VEGF siRNA carbon dot complex has good dual-mode imaging ability and specific inhibitory effect on liver tumor cells, and can be used for preparing a drug for inhibiting the growth of liver tumor cells, and realizing good treatment effect on liver cancer at the nanoscale level.

[0035] Based on the good binding capacity of the FA-PEI-Gd-CDs and the VEGF siRNA, the gene carrier complex constructed in the application not only effectively avoids the influence of the enzyme degradation in the body on the VEGF siRNA, but also enables the VEGF siRNA to successfully enter the cells and be dispersed in the cytoplasm, and has excellent transfection capacity, so that the growth and proliferation of the tumor can be better inhibited. Meanwhile, the gene carrier complex of the FA-PEI-Gd-CDs@VEGF siRNA constructed in the application also has low toxicity and good biocompatibility.

[0036] Experiments prove that the gene silencing effect and the tumor inhibition capacity of the gene carrier complex of the FA-PEI-Gd-CDs@VEGF siRNA constructed in the application on the in-vitro cells and the in-vivo tumor model of the tumor-bearing rabbits are excellent, and the activity of the VEGF protein of the tumor cells can be well inhibited. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is an XRD diagram of the Gd-CDs powder.

[0038] Figure 2 is a TEM and HRTEM diagram of the composite carbon dot carrier FA-PEI-Gd-CDs, and the inserted drawing is a particle size statistical diagram and a lattice diagram under an amplification multiple.

[0039] Figure 3 is an FTIR spectrum diagram of the Gd-CDs and the FA-PEI-Gd-CDs.

[0040] Figure 4 is an ultraviolet absorption spectrum diagram of the Gd-CDs, the FA-PEI-Gd-CDs and the FA.

[0041] Figure 5 is a fluorescence emission spectrum diagram of the Gd-CDs, the PEI-Gd-CDs and the FA-PEI-Gd-CDs.

[0042] Figure 6 is an XPS full spectrum diagram of the Gd-CDs.

[0043] Figure 7 is an XPS full spectrum diagram of the FA-PEI-Gd-CDs.

[0044] Figure 8 is a Zeta potential of the Gd-CDs, the PEI, the PEI-Gd-CDs, the FA and the FA-PEI-Gd-CDs.

[0045] Figure 9 is a T1 weighted image of the Gd-CDs, the FA-PEI-Gd-CDs and the gadodiamide.

[0046] Figure 10 Hemolysis rate histogram of different concentration of FA-PEI-Gd-CDs solution.

[0047] Figure 11 Cell survival rate histogram of different concentration of Gd-CDs, FA-PEI-Gd-CDs solution.

[0048] Figure 12 Pathological section of organs of nude mice treated with PBS and FA-PEI-Gd-CDs solution for different time.

[0049] Figure 13 Body weight-time change curve of nude mice in different treatment groups.

[0050] Figure 14 Serum analysis of nude mice treated with PBS and FA-PEI-Gd-CDs solution for different time.

[0051] Figure 15 Laser confocal microscope image and MRI image of FA-PEI-Gd-CDs in VX2 cells.

[0052] Figure 16 In vivo time-dependent imaging of nude mice after FA-PEI-Gd-CDs treatment, ex vivo fluorescence image of organs at different time, and in vivo MRI image of tumor-bearing mice after intravenous injection of FA-PEI-Gd-CDs.

[0053] Figure 17 Evaluation of anti-tumor proliferation ability of different VEGF siRNA sequences.

[0054] Figure 18 TEM image of FA-PEI-Gd-CDs@VEGF siRNA, and the inserted graph is the particle size statistics.

[0055] Figure 19 Agarose gel electrophoresis diagram of different mass ratio of FA-PEI-Gd-CDs@VEGF siRNA.

[0056] Figure 20 Gel electrophoresis diagram of different mass ratio of FA-PEI-Gd-CDs@VEGF siRNA and free siRNA after FBS and RNase for different time.

[0057] Figure 21 Fluorescence imaging and intracellular fluorescence colocalization image of FA-PEI-Gd-CDs@VEGF siRNA in VX2 cells at different wavelengths.

[0058] Figure 22are qualitative and quantitative analysis graphs of VEGF protein expression after incubation with VX2 under different conditions.

[0059] Figure 23 are cell survival rate histograms after 24h co-incubation with VX2 cells under different conditions.

[0060] Figure 24 are CT image comparisons before and after different treatment conditions and tumor inhibition rates.

[0061] Figure 25 are qualitative and quantitative analysis graphs of VEGF protein expression of tumors after different condition treatments. Embodiment

[0062] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings and examples. The following examples are only used to more clearly illustrate the technical solutions of the present application, so that those skilled in the art can well understand and utilize the present application, and are not intended to limit the protection scope of the present application.

[0063] The production process, experimental method or detection method involved in the embodiments of the present application, if not specifically stated, are conventional methods in the prior art, and the name and / or abbreviation thereof all belong to the conventional names in the art, and are also very clear and explicit in the related use field. Those skilled in the art can understand the conventional process steps and apply the corresponding equipment according to the name, and implement according to the conventional conditions or the conditions recommended by the manufacturer.

[0064] The various instruments, equipment, raw materials or reagents used in the embodiments of the present application do not have special restrictions on the source, and are conventional products that can be purchased through normal commercial channels, or can be prepared according to the conventional methods well known to those skilled in the art. Embodiment

[0065] Embodiment 1

[0066] 1.2167g of cysteine, 58mg of neutral red and 430mg of gadolinium diamine were weighed and sequentially added to a mixed solution composed of 30mL of ethanol and 10mL of deionized water, and then transferred into a 100mL polytetrafluoroethylene-lined stainless steel reaction kettle. The solvent was heated at 140℃ for 8h in a closed state to obtain a light orange transparent solution. The reaction solution was filtered through a 0.22μm filter membrane, and then subjected to dialysis purification treatment. Finally, the dialysis product was freeze-dried to prepare Gd-CDs powder.

[0067] The crystalline properties of Gd-CDs were analyzed by X-ray diffraction (XRD), Figure 1The XRD pattern shows a broad diffraction peak centered at 2θ = 25.6°, belonging to the (002) crystal plane of graphite, indicating the presence of a partially disordered carbon structure in Gd-CDs, attributed to the presence of nitrogen-, sulfur-, and oxygen-containing functional groups. Furthermore, a small diffraction peak near 2θ = 42.6° corresponds to the (100) crystal plane of graphite. The lattice distances of Gd-CDs on the (002) and (100) planes are approximately 0.37 and 0.21 nm, respectively.

[0068] 20 mg of Gd-CDs was dissolved in 20 mL of PBS to prepare a 1 mg / mL Gd-CDs solution. 50 mg of EDC and 20 mg of NHS were added, and the solution was allowed to stand at room temperature for 2 hours after dissolution. After activation, PEI was added to dissolve the PEI solution to achieve a concentration of 5 mg / mL. The solution was then stirred in a 60°C water bath for 5 hours to prepare a PEI-Gd-CDs solution. The solution was filtered through a 0.22 μm filter, dialyzed, and freeze-dried to obtain a PEI-Gd-CDs solid powder.

[0069] Weigh 24 mg FA, 12 mg PEI-Gd-CDs, 9 mg EDC and 9 mg NHS, dissolve them in 30 mL PBS, stir and react at room temperature in the dark for 24 h, filter through a 0.22 μm filter membrane, dialyze and freeze dry to prepare composite carbon dot carrier FA-PEI-Gd-CDs solid powder.

[0070] 1 mg of FA-PEI-Gd-CDs powder was dispersed in anhydrous ethanol. 10 µL of the ethanol was added dropwise onto a special copper grid. After evaporation and drying, the morphology, size, and microstructure of FA-PEI-Gd-CDs were observed using a transmission electron microscope (TEM). The results are as follows: Figure 2 As shown in Figure (a), the FA-PEI-Gd-CDs exhibit a near-spherical morphology, good dispersibility, and no obvious aggregation. The particle size distribution in the small window of the figure shows that the particle diameter ranges from 2.0 to 6.0 nm, with an average particle size of 3.71 ± 0.88 nm. Further analysis... Figure 2 High-resolution transmission electron microscopy (HRTEM) observation in (b) shows that its lattice fringes are 0.20 nm.

[0071] from Figure 3 The Fourier transform infrared (FTIR) spectra clearly show that the FTIR spectra of FA-PEI-Gd-CDs are significantly different from those of Gd-CDs at 1072 cm⁻¹. -1 A strong absorption peak appeared at 1467–1647 cm⁻¹, attributed to CN stretching of the amine groups (primary and secondary groups), indicating that PEI was grafted onto the Gd-CDs surface. -1 Within range and 2954cm -1The characteristic peak of FA appeared at 3375 cm⁻¹, corresponding to the aromatic ring stretching and CH of aminobenzoic acid and pyridine molecules; while at 3375 cm⁻¹... -1 The nearby broad peak and 1647cm -1 The peaks represent the NH and C=O extension absorptions of the amide, respectively, indicating that FA is linked through covalent linkage with the amide.

[0072] Gd-CDs, FA, and FA-PEI-Gd-CDs powders were dispersed in PBS, and their respective UV-Vis absorption spectra were measured. The results are as follows: Figure 4 As shown, in addition to the absorption peak of Gd-CDs, FA-PEI-Gd-CDs also exhibit a characteristic absorption peak of FA molecules at 364 nm. Furthermore, compared to Gd-CDs, its absorption peak shows a red shift, which may be due to the π-π electronic transition of the C=C bond and the n-π electronic transition of the C=O bond. This further proves that FA is bound to the surface of PEI-Gd-CDs through electrostatic interactions, verifying the successful loading of PEI and FA.

[0073] Gd-CDs, PEI-Gd-CDs, and FA-PEI-Gd-CDs powders were dispersed in PBS, and their emission spectra were measured using a fluorescence spectrometer. Figure 5 Figures (a), (b), and (c) show the emission spectra of Gd-CDs, PEI-Gd-CDs, and FA-PEI-Gd-CDs at different excitation wavelengths, demonstrating that each solution exhibits excitation-independent properties. The optimal emission wavelength for FA-PEI-Gd-CDs is 604 nm, which is longer than the optimal emission wavelength of 456 nm in the background technique.

[0074] Furthermore, X-ray photoelectron spectroscopy (XPS) was used to analyze the elemental composition of Gd-CDs and FA-PEI-Gd-CDs, respectively.

[0075] like Figure 6 As shown in (a), the full spectrum of Gd-CDs contains binding energies for C1s, N1s, O1s, S2p and Gd4d, indicating that they contain C, N, O, S and Gd elements. Figure 6 (b) is the high-resolution energy spectrum of C1s, where the peaks at 284.6, 287.1, and 288.0 eV correspond to CC / C=C, CO / CN, and C=O, respectively; Figure 6 (c) The high-resolution energy spectrum of N1s shows that N exists in two chemical states, located at 398.4 and 399.1 eV, corresponding to pyridine N and pyrrole N, respectively; from the high-resolution energy spectrum of O1s of Gd-CDs ( Figure 6(d) It can be seen that the peaks at 530.2 and 531.0 eV correspond to C-O and C=O, respectively; Figure 6 (e) is the high-resolution spectrum of S2p, in which the peaks at 162.9 and 163.8 eV confirm the existence of C=S and C-S; the high-resolution spectrum of Gd4d Figure 6 (f) shows two peaks centered at 148.2 and 141.7 eV, which are similar to the spectral patterns of Gd (2p 5 / 2 ) and Gd (2p 3 / 2 ) in Gd-DTPA.

[0076] As shown in (a) of Figure 7 , the binding energies of C1s, N1s, O1s, S2p and Gd4d also exist in the full spectrum of FA-PEI-Gd-CDs, in which the N1s content increases from 8.01% to 9.80%, indirectly proving the loading of PEI. Comparing the high-resolution spectra of FA-PEI-Gd-CDs in (b-f) with Gd-CDs, the increase of C-O / C-N content in FA-PEI-Gd-CDs is due to the amide bond produced by the covalent combination of Gd-CDs and FA, further proving the loading of FA. Figure 7

[0077] Gd-CDs, PEI, PEI-Gd-CDs, FA and FA-PEI-Gd-CDs were dispersed in aqueous solution, respectively, and the surface Zeta potential of different particles was measured by laser Doppler microelectrophoresis method. As shown in Figure 8 , Gd-CDs and PEI both carry positive charges, and the combination of the two by chemical bond forms PEI-Gd-CDs, which then combines with negatively charged FA through electrostatic interaction, ultimately preparing the positively charged composite carbon dot carrier FA-PEI-Gd-CDs, which lays a foundation for subsequent combination with negatively charged cell membranes and siRNA.

[0078] Example 2

[0079] 1.2167 g of cysteine, 86.6 mg of neutral red and 430 mg of gadolinium diamine were weighed and added into a mixed solution composed of 30 mL of ethanol and 10 mL of deionized water, and then transferred into a 100 mL polytetrafluoroethylene-lined stainless steel reaction kettle. The solvent was heated at 140℃ for 8 h to obtain a light orange transparent solution. The reaction solution was filtered through a 0.22 μm filter membrane, then purified by dialysis, and finally the dialysis product was freeze-dried to prepare Gd-CDs powder.

[0080] ​Take 20 mg Gd-CDs and dissolve in 20 mL PBS to prepare a Gd-CDs solution with a concentration of 1 mg / mL. Add 50 mg EDC and 20 mg NHS, and let the solution stand at room temperature for 4 h after dissolution. Then add PEI to make the concentration of PEI in the solution reach 5 mg / mL, and place the solution in a 60°C water bath for stirring reaction for 8 h to prepare a PEI-Gd-CDs solution. Filter the solution through a 0.22 μm filter membrane, dialyze, and freeze-dry to prepare a PEI-Gd-CDs solid powder.

[0081] Take 24 mg FA, 24 mg PEI-Gd-CDs, 9 mg EDC, and 9 mg NHS, and dissolve them in 30 mL PBS. Stir the solution at room temperature and avoid light for 24 h. Filter the solution through a 0.22 μm filter membrane, dialyze, and freeze-dry to prepare a FA-PEI-Gd-CDs solid powder.

[0082] Example 3

[0083] Take 1.2167 g cysteine, 58 mg neutral red, and 480 mg gadolinium diamine, and add them to a mixed solution composed of 30 mL ethanol and 10 mL deionized water in sequence. Transfer the solution into a 100 mL polytetrafluoroethylene-lined stainless steel reaction kettle, and perform solvent thermal reaction at 140°C for 6 h to obtain a light orange transparent solution. Filter the reaction solution through a 0.22 μm filter membrane, and then perform dialysis purification treatment. Finally, freeze-dry the dialysis product to prepare a Gd-CDs powder.

[0084] Take 40 mg Gd-CDs and dissolve them in 20 mL PBS to prepare a Gd-CDs solution with a concentration of 2 mg / mL. Add 50 mg EDC and 20 mg NHS, and let the solution stand at room temperature for 2 h after dissolution. Then add PEI to make the concentration of PEI in the solution reach 5 mg / mL, and place the solution in a 60°C water bath for stirring reaction for 4 h to prepare a PEI-Gd-CDs solution. Filter the solution through a 0.22 μm filter membrane, dialyze, and freeze-dry to prepare a PEI-Gd-CDs solid powder.

[0085] Take 36 mg FA, 18 mg PEI-Gd-CDs, 9 mg EDC, and 9 mg NHS, and dissolve them in 30 mL PBS. Stir the solution at room temperature and avoid light for 12 h. Filter the solution through a 0.22 μm filter membrane, dialyze, and freeze-dry to prepare a FA-PEI-Gd-CDs solid powder.

[0086] Example 4

[0087] The present embodiment first evaluates the relaxation rate of Gd-CDs and FA-PEI-Gd-CDs, and compares it with the relaxation rate of the commercial product gadodiamide, to verify the feasibility of Gd-CDs and FA-PEI-Gd-CDs as new MRI contrast agents.

[0088] As shown in Figure 9 Fig. (a), the MRI signal intensity of Gd-CDs, FA-PEI-Gd-CDs and gadodiamide all increases with the increase of Gd 3+ concentration, indicating that the prepared Gd-CDs and FA-PEI-Gd-CDs can be used as new candidates for MRI, and from Figure 9 Fig. (b), it can be seen that the image signal of Gd-CDs and FA-PEI-Gd-CDs is higher than that of gadodiamide.

[0089] From the linear relationship diagram of transverse relaxation rate (1 / T1) and Gd concentration plotted in the figure, the relaxation rate (slope) values of Gd-CDs, FA-PEI-Gd-CDs and gadodiamide are calculated to be 7.97, 3.45 and 2.88 mM -1 s -1 , indicating that Gd-CDs and FA-PEI-Gd-CDs have high relaxation rate and can be used for MRI, providing a basis for bimodal imaging in vivo.

[0090] Secondly, the present embodiment uses UV-Vis detection to quantitatively analyze the hemolysis rate of FA-PEI-Gd-CDs solutions with different concentrations, to investigate the biocompatibility thereof, and the results are shown in Figure 10 As the concentration of FA-PEI-Gd-CDs increases from 3.125 μg / mL to 200 μg / mL, the hemolysis rate is 1.2%, 1%, 0.6%, 2.4%, 2.9%, 2.9% and 3% in turn, and the measured values are all less than 5%, indicating that FA-PEI-Gd-CDs as a carrier has good blood compatibility.

[0091] The present embodiment also uses the standard CCK-8 method to evaluate the in vitro cytotoxicity of Gd-CDs and FA-PEI-Gd-CDs.

[0092] HepG2 (human liver cancer cells), VX2 (rabbit liver cancer cells) and HL-7702 cells (human normal liver cells) in good growth state were taken, trypsinized, and then diluted to 1×10 5 / mL, 100 μL per well was inoculated into a 96-well plate, and the cells were cultured in a cell incubator until they adhered. Different concentrations (0, 25, 50, 100, 200 μg / mL) of Gd-CDs and FA-PEI-Gd-CDs solutions were prepared in advance, and the HepG2, VX2, and HL-7702 cells were incubated with them for 24 h. After the incubation, the cells were washed twice with PBS, and a CCK-8 solution prepared in advance was added to each well. After incubation for 1 h, the OD value of the solution was determined using a multifunctional enzyme label meter.

[0093] Figure 11 Fig. 6 shows the survival rates of different cells after incubation with different concentrations of Gd-CDs and FA-PEI-Gd-CDs for 24 h. As shown in (a) and (b), even when the concentrations of Gd-CDs and FA-PEI-Gd-CDs were 200 μg / mL, the survival rates of the three types of cells were still greater than 80%, indicating that Gd-CDs and FA-PEI-Gd-CDs had low cytotoxicity and good biocompatibility.

[0094] In addition, the experimental results also showed that under some low concentration conditions, the cell viability was more than 100%, indicating that low concentrations of Gd-CDs and FA-PEI-Gd-CDs had a certain effect on promoting cell proliferation.

[0095] Finally, 6-8-week-old BALB / c nude mice were selected as experimental animals to further verify the biological safety of FA-PEI-Gd-CDs as a carrier.

[0096] Using physiological saline as the control group and FA-PEI-Gd-CDs solution as the experimental group, the injection dose was 10 mg / Kg, and the BALB / c nude mice were fed for 0, 1, 14, and 28 days, respectively, and then dissected. Brain, heart, liver, spleen, lung, kidney, and bladder tissue sections were prepared, and the tissue sections were observed under a microscope to evaluate the pathological differences between the experimental and control groups. At the same time, by monitoring the body weight of BALB / c nude mice in the two groups at different times (1, 3, 7, 14, 21, and 28 days), the biological safety of FA-PEI-Gd-CDs was indirectly verified.

[0097] Figure 12 Fig. 8 shows the pathological differences between the control group (a) and the experimental group (b). No significant changes in cell state and inflammatory infiltration were found in the control and experimental groups, indicating that FA-PEI-Gd-CDs had excellent biocompatibility.

[0098] In addition to the microscopic morphology of the tissues, the body weight growth curve is also an important indicator for testing the biological safety in vivo. As shown in Fig. 9, the body weight growth trends of the BALB / c nude mice in each group were similar within 28 days. Figure 13

[0099] ​The biological safety of FA-PEI-Gd-CDs can be further clarified by performing serum marker analysis. The blood of BALB / c nude mice injected with saline and FA-PEI-Gd-CDs solution 1, 3, 14, and 28 days, respectively, was collected by eyeball after deep anesthesia, and blood routine and blood biochemical analysis were performed to evaluate the white blood cell (WBC), hemoglobin concentration (HGB), and platelet (PLT) content; the liver function and kidney function parameters, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (CREA), and urea nitrogen (UREA), were monitored.

[0100] Figure 14 In the present embodiment, the WBC, PLT, and HGB of the PBS and FA-PEI-Gd-CDs treated BALB / c nude mice showed no significant difference in the short and long term after injection; the AST, ALT, CREA, and UREA indicators slightly increased on the 3rd day after injection of FA-PEI-Gd-CDs, but then decreased, which may be related to the body's clearance mechanism. No BALB / c nude mice died during the observation period, indicating that FA-PEI-Gd-CDs have high in vivo biological safety, laying a foundation for their use as carriers for in vivo studies.

[0101] Example 5

[0102] In this embodiment, VX2 cells were used for in vitro cell imaging studies to observe the cell transfection effect of FA-PEI-Gd-CDs using a confocal laser scanning microscope (CLSM) to evaluate the bioimaging and cell labeling ability of FA-PEI-Gd-CDs.

[0103] VX2 cells in good growth state were taken and dispersed into a cell suspension of 1×10 4 mL was taken with a pipette gun and transferred to a special culture dish. After 12 h of culture in the incubator, the culture medium was discarded, washed with PBS, and then the medium containing 2% fetal bovine serum (FBS) was added for starvation culture for 1 h. The medium was discarded, washed with PBS, and complete medium containing FA-PEI-Gd-CDs (100 µg / mL) was added. Under the same conditions, the cells were cultured for 4 h, fixed with paraformaldehyde for 15 min, and imaged using CLSM. The results are shown in Figure 15 (a).

[0104] Wherein, Bright is the cell distribution under the bright field; FA-PEI-Gd-CDs is the cell distribution in the fluorescence field, and the orange-red fluorescence in the cells can be obviously observed; Merge is the image after the superposition of the bright field and the fluorescence field, thus it can be proved that the FA-PEI-Gd-CDs has entered the cells and is mainly distributed in the cytoplasm, which indicates that the FA-PEI-Gd-CDs has excellent cell transfection capacity.

[0105] The MRI system is used to scan the cell liquid after the VX2 cells are co-cultured with the FA-PEI-Gd-CDs for 4 hours. The VX2 cells in good growth state are taken, and the 6-hole plate is cultured overnight, and different concentrations (0, 0.375, 0.75, 1.5, 3 mg / mL) of the FA-PEI-Gd-CDs solution are added in each hole for 4 hours of co-incubation. The cells are washed with PBS, and after trypsin digestion, the EP tube is centrifuged, and the cells are re-dispersed in the 1% agarose solution for gelation, and then the MRI is performed on the small animal magnetic resonance imaging instrument.

[0106] As shown in Fig. Figure 15 As shown in Fig.

[0107] The small animal live imaging instrument is used for observation, so as to verify that the FA-PEI-Gd-CDs with long-wavelength emission of the application can penetrate the living body tissue for fluorescence imaging, and the metabolic mode of the FA-PEI-Gd-CDs after entering the body is observed.

[0108] The FA-PEI-Gd-CDs solution is injected into the BALB / c nude mice at a dose of 10 mg / Kg, and the small animal live imaging instrument is used to record the fluorescence signal in the body at 0, 0.5, 1, 2, 4, 8, 16 and 24 hours. As can be seen from Fig. Figure 16 As can be seen from Fig.

[0109] Further, the in-vivo biodistribution is also a key factor for evaluating the in-vivo biocompatibility of the FA-PEI-Gd-CDs. The BALB / c nude mice are killed after different time of injection of the FA-PEI-Gd-CDs, and the main organs of brain, heart, liver, spleen, lung, kidney and bladder in each time period are collected, and the fluorescence imaging of the ex-vivo tissue is used to study the biodistribution.

[0110] As Figure 16 As shown in (b), the brain tissue began to fluoresce at 0.5h after injection, the fluorescence intensity reached the strongest at 2h, and the fluorescence gradually disappeared after 24h, which indicated that the FA-PEI-Gd-CDs could penetrate the blood-brain barrier of BALB / c nude mice and enter the brain tissue. Meanwhile, a small part of the FA-PEI-Gd-CDs could penetrate different biological barriers and enter the liver and lung at 2h after injection, but the accumulation rate was relatively low. The fluorescence of the kidney and bladder began to appear at 1h after injection, and significantly decreased or even disappeared at 24h, which indicated that the FA-PEI-Gd-CDs could be removed through the rapid kidney metabolism, further proving that the metabolism was good and the biocompatibility was strong.

[0111] Further, after the FA-PEI-Gd-CDs solution with an injection dose of 10mg / Kg was injected into the BALB / c tumor-bearing mice intravenously, the tumor-bearing mice were scanned by using a 3.0T magnetic resonance scanner. As shown in (c), after the FA-PEI-Gd-CDs were injected intravenously, the short T1 high signal shadow could be seen in the subcutaneous tumor area of the tumor-bearing mice, while the long T1 low signal shadow was seen in the subcutaneous tumor area of the tumor-bearing mice without injection of the FA-PEI-Gd-CDs, which indicated that the FA-PEI-Gd-CDs of the application not only could penetrate the living tissues, but also had the characteristics of targeting the liver cancer tissues for MRI imaging. Figure 16

[0112] Example 6

[0113] The sequence NM_001025366.3 of the human VEGF cDNA (Homo sapiens vascular endothelial growth factor A) was searched in the Gene Bank as the target gene, the siRNA design software was applied to design the RNAi action public target point for the VEGF mRNA different splicers, three sequences beneficial to the siRNA combination to form the silence complex were selected: VEGF siRNA-599, VEGF siRNA-817, VEGF siRNA-921, and 3 groups of siRNA fragments from different positions of the mRNA of the target gene were synthesized.

[0114] VEGF siRNA-599:

[0115] Sense strand: 5'-CCUCCGAAACCAUGAACUU-3';

[0116] Antisense strand: 5'-AAGUUCAUGGUUUCGGAGG-3'.

[0117] VEGF siRNA-817:​

[0118] Chain of Justice: 5'-GAGUACAUAUUCAAGCCUU-3';

[0119] Antisense chain: 5'-AAGGCUUGAAUAUGUACUC-3'.

[0120] VEGF siRNA-921:

[0121] Chain of Justice: 5'-GCAGAUCAUGCGGAUCAAA-3';

[0122] Antonym chain: 5'-UUUGAUCCGCAUGAUCUGC-3'.

[0123] Meanwhile, NC-VEGF siRNA was designed as a negative control, which showed no obvious homology with human mRNA, but had the same GC composition.

[0124] NC-VEGF siRNA:

[0125] Chain of Justice: 5'-UUCUCCGAACGUGUCACGU-3';

[0126] Antonym chain: 5'-ACGUGACACGUUCGGAGAA-3'.

[0127] All the above siRNAs were synthesized with the assistance of Shanghai Sangon Biotech Co., Ltd., and the 3' end of each single strand was augmented with the base TT.

[0128] Using NC-VEGF siRNA as a control, the in vitro cellular level evaluation of the anti-tumor proliferation capacity of different siRNAs designed above was performed.

[0129] Take VX2 cells in good growth condition, at 2×10 5 Inoculate one cell per well into a 6-well plate, incubate overnight in an incubator, and then replace with serum-free DMEM medium for 2 hours before transfection.

[0130] The experiment was divided into 5 groups: Group A was the normal cell group, Group B was the NC-VEGF siRNA control group, Group C was the VEGF siRNA-599 group, Group D was the VEGF siRNA-817 group, and Group E was the VEGF siRNA-921 group.

[0131] After grouping, each group was transfected:

[0132] Dilute 10 μL of each group of siRNA and 5 μL of transfection reagent with 100 μL of serum-free DMEM medium. After standing at room temperature for 5 min, mix the two solutions and stand for 20 min to obtain a mixed solution.

[0133] Add 200 μL of each group's mixture to the above 6-well plate, and continue culturing the cells in an incubator for 6 hours. Then, replace the culture medium with normal culture medium and continue culturing.

[0134] Total RNA was extracted from each group, reverse transcribed into cDNA, and mRNA from each group was detected using real-time quantitative PCR. -ΔΔCt The relative expression level was calculated using the method to obtain... Figure 17 The bar chart shown.

[0135] from Figure 17 The PCR results showed that, compared with the normal cell group and the NC-VEGF siRNA control group, VEGF siRNA-599 and VEGF siRNA-877 did not have a significant gene silencing effect, but VEGF siRNA-921 had the strongest tumor-inhibiting effect, with a gene silencing effect of 62.4%, which was statistically significant and showed good gene silencing effect on liver cancer cells.

[0136] Therefore, this invention screened VEGF siRNA-921 from three sets of gene sequences as the VEGF siRNA for constructing the final gene-carrying complex of this invention.

[0137] Example 7

[0138] Weigh 160 μg of the FA-PEI-Gd-CDs composite carbon dot carrier solid powder prepared in Example 1, dissolve it in 1 mL of DEPC water, and filter it through a 0.22 μm filter membrane to obtain an FA-PEI-Gd-CDs solution.

[0139] Weigh 40 μg of the VEGF siRNA constructed in Example 6 into an EP tube, add the above FA-PEI-Gd-CDs solution and mix. Let it stand on crushed ice for 1 h to allow it to fully bind, and then prepare the carbon dot complex FA-PEI-Gd-CDs@VEGF siRNA carrying the VEGF siRNA gene.

[0140] The prepared FA-PEI-Gd-CDs@VEGF siRNA was dropped onto a special copper grid, evaporated and dried, and its morphology and size were observed by TEM. The results are as follows. Figure 18 As shown, the morphology of FA-PEI-Gd-CDs@VEGF siRNA is approximately ellipsoidal, and no aggregation was observed. The average particle size is approximately 19.52 nm, which is significantly larger than that of FA-PEI-Gd-CDs. This indirectly indicates that FA-PEI-Gd-CDs carrying VEGF siRNA form FA-PEI-Gd-CDs@VEGF siRNA.

[0141] Example 8

[0142] Following the method in Example 7, FA-PEI-Gd-CDs solution was mixed with different masses of VEGF siRNA to prepare FA-PEI-Gd-CDs@VEGF siRNA with mass ratios of FA-PEI-Gd-CDs to VEGF siRNA of 0.25:1, 0.5:1, 1:1, 2:1, 4:1, 8:1 and 10:1 respectively.

[0143] After mixing the FA-PEI-Gd-CDs@VEGF siRNAs with DNA loading at different mass ratios, the mixtures were added to the sample wells for gel electrophoresis and imaging with a gel imaging system to confirm the binding ability between the composite carbon dot vector and the gene in the FA-PEI-Gd-CDs@VEGF siRNAs at different mass ratios, proving that the gene can be completely loaded onto the vector to construct the gene-loaded carbon dot complex.

[0144] Figure 19 As the mass ratio gradually increased from 0.25:1 to 10:1, the bands in the sample wells gradually brightened, while the migration bands gradually darkened. Starting from a mass ratio of 4:1, FA-PEI-Gd-CDs were able to completely retain VEGF siRNA in the sample wells, indicating that FA-PEI-Gd-CDs have good gene binding ability, and also proving that a mass ratio of 4:1 is the critical complexation ratio.

[0145] Example 9

[0146] FA-PEI-Gd-CDs@VEGF siRNA solutions with mass ratios of 4:1, 6:1, 8:1, and 10:1 were used as experimental groups, while untreated free VEGF siRNA was used as the control group, and FBS-treated free VEGF siRNA was used as the blank group. The protective effect of FA-PEI-Gd-CDs on VEGF siRNA in the presence of FBS was evaluated.

[0147] Except for the control group, equal volumes of FBS (fetal bovine serum) were added to the solutions of other groups. After incubation for different times (0, 6, 12, 24, 48, 72 h), the solutions were removed, and 1 mg / mL heparin sodium solution was added. The mixtures were then reacted in a metal bath at 37°C for 1 h. Gel electrophoresis was performed on the samples after the reaction.

[0148] like Figure 20As shown in (a), the free VEGF siRNA in the control group migrated at all time points, while the migration brightness of the free VEGF siRNA in the blank group gradually decreased after 12 hours of treatment and disappeared after 48 hours, indicating that VEGF siRNA without vector protection is easily degraded by nucleases in FBS. However, the VEGF siRNA in the experimental group bound with different mass ratios of FA-PEI-Gd-CDs still showed obvious brightness in the wells after 24 hours of treatment, and the brightness did not completely disappear until 72 hours later. Moreover, the smaller the mass ratio, the earlier the brightness disappeared.

[0149] The above experimental results indicate that FA-PEI-Gd-CDs@VEGF siRNA can effectively reduce the influence of serum enzymes on VEGF siRNA.

[0150] Furthermore, FA-PEI-Gd-CDs@VEGF siRNA solutions with mass ratios of 4:1, 6:1, 8:1, and 10:1 were used as experimental groups, while untreated free VEGF siRNA was used as the control group, and RNase A-treated free VEGF siRNA was used as the blank group. The protective effect of FA-PEI-Gd-CDs on VEGF siRNA in the presence of RNase A was evaluated.

[0151] Except for the control group, 1 U of RNase A was added to the solutions of other groups, and the mixture was shaken and reacted in a metal bath at 37°C for 0, 0.5, 1, 2, and 3 hours. Then, an RNase inhibitor was added, and the reaction was continued in a metal bath at 70°C for 10 minutes. The samples were then subjected to gel electrophoresis after the reaction was completed.

[0152] like Figure 20 As shown in (b), the control group formed a bright migration band when completely free, but the blank group did not show a migration band at the beginning of the experiment, and no band brightness was observed in the sample wells, proving that the free VEGF siRNA had been degraded by RNase A enzyme. In each experimental group, the FA-PEI-Gd-CDs@VEGF siRNA at a mass ratio of 4:1 still showed brightness in the sample wells after 0.5 h of reaction, indicating that the VEGF siRNA was not completely degraded; after 1 h of reaction, the brightness in the sample wells at mass ratios of 4:1 and 6:1 had disappeared, and after 2 h, the brightness in the sample wells at a mass ratio of 8:1 also gradually disappeared, but after 3 h of reaction, the sample wells at a mass ratio of 10:1 still showed brightness, indicating that the VEGF siRNA was not completely degraded.

[0153] This demonstrates that FA-PEI-Gd-CDs have a good binding effect with VEGF siRNA, which can effectively prevent degradation by enzymes in vivo.

[0154] Example 10

[0155] FAM-VEGF siRNA with a fluorescent label at the 5' end was used, with an excitation wavelength of 492 nm and an emission wavelength of 518 nm. Fluorescently labeled FA-PEI-Gd-CDs@VEGF siRNA was constructed with FA-PEI-Gd-CDs. The cell transfection capability of the gene-carrying carbon dot complex was investigated by laser confocal microscopy.

[0156] Take VX2 cells in good growth condition and disperse them in complete culture medium at a concentration of 1×10⁻⁶. 4 Cell suspension of cells / mL was pipetted into a dedicated culture dish, incubated for 12 hours, then the culture medium was discarded. After washing with PBS, the cells were starved again with medium containing 2% fetal bovine serum (FBS) for 1 hour, then the culture medium was discarded. After washing with PBS, complete culture medium containing FA-PEI-Gd-CDs@FAM-VEGF siRNA (100µg / mL) was added, and the cells were cultured under the same conditions for 4 hours. Cells were fixed with paraformaldehyde for 15 minutes, and the cell transfection effect of FA-PEI-Gd-CDs@FAM-VEGF siRNA was observed under a laser confocal microscope.

[0157] Figure 21 In the image, Bright represents the cell distribution under bright field; the composite carbon dot carrier FA-PEI-Gd-CDs in FA-PEI-Gd-CDs@FAM-VEGF siRNA exhibits orange-red fluorescence (excitation wavelength 530 nm, emission wavelength 604 nm); FAM-VEGF siRNA exhibits green fluorescence (excitation wavelength 492 nm, emission wavelength 518 nm); Merge1 is the image after fluorescence field superposition; Merge2 is the image after bright field and fluorescence field superposition.

[0158] As can be seen, FA-PEI-Gd-CDs@FAM-VEGF siRNA is dispersed in the cytoplasm, and the colocalization analysis diagram in the lower right corner further shows that FA-PEI-Gd-CDs encapsulate VEGF siRNA and successfully enters the cell, exhibiting good cell internalization ability.

[0159] Example 11

[0160] This embodiment evaluates the in vitro antitumor effect of FA-PEI-Gd-CDs@VEGF siRNA by detecting changes in intracellular VEGF protein expression using Western blotting (WB).

[0161] The experiment consisted of 5 groups: A was the blank group; B was the FA-PEI-Gd-CDs group; C was the VEGF siRNA group; D was the FA-PEI-Gd-CDs@NC control group; and E was the FA-PEI-Gd-CDs@VEGF siRNA experimental group.

[0162] Take VX2 cells in good growth condition and prepare a 1×10⁻⁶ concentration in complete culture medium. 5 2 mL of cell suspension per well was seeded into 6-well plates. After incubation overnight, the culture medium was discarded and replaced with low-concentration serum medium for starvation culture for 1 h. The plates were then washed three times with PBS.

[0163] Except for the blank group, the other groups were transfected with fresh culture medium containing FA-PEI-Gd-CDs, VEGF siRNA, FA-PEI-Gd-CDs@NC, and FA-PEI-Gd-CDs@VEGF siRNA, respectively. After 4 hours of transfection, culture medium with a higher concentration of FBS was added and cultured for another 20 hours. RIPA lysis buffer was added and the proteins were extracted for 10 minutes and their concentrations were calculated.

[0164] After denaturation, electrophoresis, and transfer of the protein to a membrane, it was incubated overnight at 4°C with the primary antibody and then incubated with the secondary antibody at room temperature for 1.5 h. The protein level was detected by the 3,3-diaminobenzidine (DAB) substrate colorimetric assay. The target band and internal control A value were analyzed using ImageJ software, and the protein expression level was expressed as the A ratio of the target protein to the β-actin internal control to assess the VEGF expression level.

[0165] Using intracellularly stable β-actin protein as an internal reference index to compare silencing effects, qualitative analysis of protein bands is as follows: Figure 22 As shown in (a), FA-PEI-Gd-CDs@VEGF siRNA showed the lowest VEGF protein expression, demonstrating that it can effectively inhibit VEGF protein expression.

[0166] Quantitative analysis results as follows Figure 22 As shown in (b), compared with the control group containing only culture medium, the gene silencing effects of the FA-PEI-Gd-CDs group, VEGF siRNA group, and FA-PEI-Gd-CDs@NC group were not significant and had no statistical significance. However, compared with the VEGF siRNA group and the FA-PEI-Gd-CDs@NC group, the FA-PEI-Gd-CDs@VEGF siRNA group showed a significant gene silencing efficiency. P <0.05).

[0167] This demonstrates that the gene-carrying complex FA-PEI-Gd-CDs@VEGF siRNA of the present invention can inhibit the expression of VEGF-mRNA and reduce VEGF synthesis through its siRNA, thus exhibiting excellent gene silencing effect.

[0168] Furthermore, the CCK-8 assay was used to investigate the inhibitory effect of FA-PEI-Gd-CDs@VEGF siRNA on the proliferation of HL-7702 and VX2 cells.

[0169] Take HL-7702 and VX2 cells in good growth condition and prepare a 1×10⁻⁶ concentration using complete culture medium. 5 Cell suspension of 100 μL / mL was seeded into each well of a 96-well plate and incubated overnight. After washing twice with PBS, 100 μL of each experimental group solution dissolved in complete culture medium was added to each well, and incubation continued for 24 h. After incubation, the cells were washed twice with PBS, and pre-prepared CCK-8 solution was added to each well for 1 h of incubation. The OD value of the solution was measured using a multi-functional microplate reader.

[0170] Figure 23 In the study, for normal HL-7702 cells, no group caused cell death, and the cell survival rates among the groups were similar, all greater than 80%. However, for tumor cells VX2, compared with the other groups which also did not cause cell death, the survival rate of VX2 cells in the FA-PEI-Gd-CDs@VEGF siRNA group was significantly reduced, to only 42.3%.

[0171] This conclusion is consistent with the results of the WB experiment mentioned above, confirming that the gene-carrying complex has a specific inhibitory effect on tumor cells.

[0172] Example 12

[0173] This embodiment evaluates the in vivo antitumor effect of FA-PEI-Gd-CDs@VEGF siRNA.

[0174] Rabbits bearing tumors with successful implantation and a maximum transverse diameter of 1–2 cm were randomly divided into two groups, A and B. Group A consisted of polyvinyl alcohol embolization microspheres, while group B consisted of polyvinyl alcohol embolization microspheres + FA-PEI-Gd-CDs@VEGF siRNA.

[0175] Rabbits bearing tumors were placed under general anesthesia. After skin preparation and disinfection of the surgical area, the right femoral artery was freed, and a 5F arterial sheath was inserted through the femoral artery using the Seldinger puncture technique. A 4F catheter was then inserted through the 5F sheath. Under DSA guidance, celiac artery angiography was performed to clarify the course of the hepatic artery. The hepatic artery was then superselectively angiographically selected at a frame rate of 6 frames per second to clarify the tumor's blood supply. The microcatheter was then superselectively directed to the tumor's feeding artery, and treatment was administered according to the grouping method described above. CT scans were used to monitor tumor growth in the rabbits 2–3 weeks after treatment in each group.

[0176] Figure 24 In Figure (a), CT images of different treatment groups before and after treatment are shown. It can be seen that compared with before treatment, the maximum transverse diameter of the tumors in both groups A and B decreased to varying degrees. Further quantitative analysis of the inhibitory effects of different treatment groups on the tumor yielded the following results: Figure 24 As shown in Figure (b), the tumor inhibition rate of the polyvinyl alcohol embolized microspheres + FA-PEI-Gd-CDs@VEGF siRNA group was higher, indicating that the FA-PEI-Gd-CDs@VEGF siRNA has a good gene silencing effect and can effectively inhibit tumor growth and proliferation.

[0177] Rabbits bearing tumors in each group were sacrificed by air embolization, and tumor tissues were collected. Western blotting was used to detect changes in VEGF expression levels in the tumor tissues.

[0178] Qualitative analysis of protein bands, such as Figure 25 As shown in (a), the silencing effect was compared using β-actin protein, which is stably expressed in the tissue, as an internal reference. The VEGF protein band in group B was significantly lighter, demonstrating that FA-PEI-Gd-CDs@VEGF siRNA can effectively inhibit the expression of VEGF protein. Quantitative analysis results are shown below. Figure 25 As shown in (b), the VEGF protein expression rate in group A was approximately 73.7%, while that in group B was approximately 24.1%, showing a significant difference between the two groups. P <0.1.

[0179] Western blot experiments further verified that the polyvinyl alcohol embolized microspheres + FA-PEI-Gd-CDs@VEGF siRNA group can effectively inhibit the activity of VEGF protein in tumor cells.

[0180] The above embodiments of the present invention do not describe all details exhaustively, nor do they limit the present invention to the embodiments described above. Various changes, modifications, substitutions, and variations made by those skilled in the art to these embodiments without departing from the principles and spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A composite carbon dot support, characterized in that it uses... Based on Gd-CDs prepared from cysteine, neutral red, and gadodiamine in an ethanol-water system at 120–180 °C for 6–8 h using solvothermal reaction, a carrier FA-PEI-Gd-CDs was prepared by coupling polyethyleneimine and folic acid molecules to its surface through activation with amide activators EDC and NHS.

2. The composite carbon dot carrier according to claim 1, characterized in that... First, the Gd-CDs solution was activated with EDC and NHS, and then PEI was added to react and form PEI-Gd-CDs by chemical bonding. Then, FA, EDC and NHS were added simultaneously to react in the dark and chemically bonded to prepare the composite carbon dot carrier FA-PEI-Gd-CDs.

3. The application of the composite carbon dot carrier according to claim 1 in the preparation of fluorescence-magnetic resonance dual-modal imaging probes.

4. A VEGF-carrying siRNA carbon dot complex constructed using the composite carbon dot carrier of claim 1, characterized in that it uses VEGF... The siRNA, as the target gene, is carried on the composite carbon dot carrier FA-PEI-Gd-CDs through electrostatic adsorption, forming the gene-carrying complex FA-PEI-Gd-CDs@VEGF siRNA. The target gene VEGF siRNA is a group of siRNAs with the following nucleotide sequences as shown in SEQ ID NO.5 and SEQ ID NO.6: Chain of Justice: 5'-GCAGAUCAUGCGGAUCAAA-3'; Antonym chain: 5'-UUUGAUCCGCAUGAUCUGC-3'.

5. The VEGF siRNA-carrying carbon dot complex according to claim 4, characterized in that... The mass ratio of the composite carbon dot vector FA-PEI-Gd-CDs to the target gene VEGF siRNA is (4-10):

1.

6. The VEGF siRNA-carrying carbon dot complex according to claim 4 or 5, characterized in that... The mass ratio of the composite carbon dot vector FA-PEI-Gd-CDs to the target gene VEGF siRNA is 4:

1.

7. The method for preparing the VEGF siRNA carbon dot complex according to claim 4, characterized in that: 1) Cysteine, neutral red and gadodiamine were added to a mixed solution of ethanol and water and reacted at 120-180℃ for 6-8 hours. The reaction product was filtered, dialyzed and freeze-dried to obtain Gd-CDs powder. 2) Dissolve Gd-CDs in PBS, add amide activator EDC and NHS, let stand at room temperature for 2-4 hours, add PEI after activation, stir at 60-80℃ for 4-8 hours, filter, dialyze, freeze dry to obtain PEI-Gd-CDs powder; 3) Dissolve PEI-Gd-CDs with FA, EDC and NHS in PBS, stir and react at room temperature in the dark for 12-24 h, filter, dialyze and freeze dry to obtain FA-PEI-Gd-CDs solid powder. 4) Mix positively charged FA-PEI-Gd-CDs with negatively charged VEGF siRNA and let stand. The mixture will combine with electrostatic interaction to form the gene-carrying complex FA-PEI-Gd-CDs@VEGF siRNA.

8. The method for preparing the VEGF siRNA carbon dot complex according to claim 7, characterized in that: Filtration was performed using a disposable filter membrane with a pore size of 0.22 μm, and dialysis was performed using a dialysis bag with a molecular weight of 1000 Da.

9. The use of the VEGF siRNA carbon dot complex according to claim 4 in the preparation of a drug for inhibiting the growth of liver tumor cells.

Images