Golgi apparatus targeting carbon dot nano-enzyme carrying GP73-siRNA and preparation and application thereof

Abstract

The invention relates to a Golgi apparatus targeting carbon dot nano-enzyme carrying GP73-siRNA. L-cysteine is used as a Golgi apparatus targeting raw material, a Mn-CDs nano-enzyme is prepared from L-cysteine, neutral red and manganese dioxide through a one-step solvothermal method, a PEI-HA complex is coupled to the surface of the Mn-CDs nano-enzyme to prepare a Mn-CDs-PEI-HA nano-enzyme carrier, and the Golgi apparatus targeting carbon dot nano-enzyme carrying GP73-siRNA is prepared through a one-step solvothermal method. And further carrying GP73-siRNA to construct a gene-loaded complex Mn-CDs-PEI-HA / GP73-siRNA, so as to target a liver cancer cell Golgi apparatus under the guidance of fluorescence / magnetic resonance bimodal imaging to carry out gene silencing treatment. The gene-loaded complex constructed by the invention has good fluorescence / magnetic resonance bimodal imaging capability, Golgi apparatus targeting capability and tumor hypoxic microenvironment improvement effect, so that integration of imaging and treatment is realized.

Description

Technical Field

[0001] The present invention belongs to the field of nanomaterials and biomedicine technology, and relates to a composite carbon dot with the functions of improving hypoxia and dual-modal imaging, a gene-carrying complex constructed by using the composite carbon dot to carry GP73-siRNA, and the application of the gene-carrying complex in gene silencing therapy and liver cancer imaging monitoring. Background Art

[0002] Hepatocellular carcinoma (HCC) is a malignant tumor with high morbidity and mortality worldwide. Currently, HCC treatments primarily include surgery (hepatectomy and liver transplantation), transarterial chemoembolization (TACE), hepatic arterial infusion chemotherapy (HAIC), and systemic therapy. Despite significant advances in traditional treatment techniques, the overall survival rate of HCC patients remains low.

[0003] HCC is a relatively insidious disease, with over 70% of patients diagnosed in the advanced stages. According to the Guidelines for the Diagnosis and Treatment of Primary Liver Cancer (2022 edition), local treatments for advanced HCC include transcatheter arterial blood circulation (TACE) and hepatocellular carcinoma (HAIC), both of which can increase local HCC drug concentrations to kill tumor cells. However, the hypoxic microenvironment created by TACE can contribute to HCC proliferation, recurrence, and metastasis. Currently, there is no unified standard for the treatment of HAIC, and the efficacy varies widely. Therefore, exploring novel treatment approaches to improve treatment outcomes is a key focus for future HCC treatment.

[0004] With the development of genome editing technology, small interfering RNA (siRNA) therapy has been widely used in the field of tumor treatment due to its advantages such as strong specificity, few toxic side effects, high silencing efficiency, and easy detection of therapeutic effects. It has great clinical translation value.

[0005] As a nucleic acid-based drug, siRNA, when introduced into target cells, can sequence-specifically bind to and cleave messenger RNA (mRNA), downregulating mRNA expression and thereby reducing the synthesis of specific proteins, ultimately inhibiting tumor cell proliferation. The Golgi apparatus is the primary site for protein modification, sorting, packaging, and transport, and abnormal expression of proteins on its surface is closely associated with tumor development and progression. Therefore, using siRNA to target and inhibit the synthesis of Golgi-related proteins could improve the efficacy of HCC treatment.

[0006] Golgi protein 73 (GP73) is a transmembrane glycoprotein in the Golgi apparatus with a relative molecular mass of 73×10 3Da is primarily expressed in epithelial cells of various human tissues, rarely or not expressed in normal liver tissue, and overexpressed in HCC tissue. Bioinformatics analysis revealed that GP73 expression varies across HCC stages. Compared with the low GP73 expression group, HCC patients in the high GP73 expression group had a shorter overall survival, confirming that GP73 plays a key role in the development and progression of HCC.

[0007] Studies have shown that GP73 overexpression alters glycosphingolipid synthesis and plasma membrane composition, promoting HCC cell mitosis and proliferation. Furthermore, in the hypoxic microenvironment of HCC, overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes GP73 synthesis, enhances the TGF-β / Smad signaling pathway, and triggers epithelial-mesenchymal transition (EMT) in HCC. This downregulates the expression of EMT-related markers N-cadherin and E-cadherin on the HCC cell surface, inhibits cell junctions, and promotes HCC proliferation, invasion, and metastasis.

[0008] Given the potential role of GP73 in the development and progression of HCC, a GP73-siRNA was designed to specifically degrade GP73 mRNA through the RNA-induced silencing complex (RISC), thereby reducing target protein expression. However, the lack of efficient and safe siRNA delivery nanocarriers has become a major bottleneck restricting its clinical application.

[0009] Nanocarriers used to deliver siRNA primarily include viral nanocarriers and non-viral nanocarriers. Non-viral nanocarriers offer advantages over viral vectors in terms of improved siRNA stability, transfection efficiency, and targeting, while also reducing the toxicity of the delivery system. However, their future clinical translation still faces numerous challenges. First, siRNA lacks the ability to cross biological barriers and is easily degraded by enzymes and cleared by the kidneys. Second, siRNA suffers from severe off-target effects. Third, the production process is complex, making large-scale production difficult. Therefore, designing non-viral nanocarriers for precise, targeted delivery of siRNA is crucial for improving the efficacy of gene therapy.

[0010] Currently, researchers have constructed a variety of non-viral nanocarriers for siRNA delivery, including carbon nanoparticles, liposomes, metal nanoparticles, and cationic polymers. Among these nanocarriers, carbon dots (CDs), a zero-dimensional carbon nanomaterial less than 10 nm in size, have been widely used in tumor therapy and bioimaging due to their low toxicity, good biocompatibility, surface richness in functional groups, ease of modification, and excellent fluorescence properties. Studies have shown that siRNA carried by CDs can effectively silence mRNA expression and reduce the synthesis of the corresponding protein, thereby inhibiting tumor growth. Therefore, developing a CDs-based delivery vehicle, combining it with GP73-siRNA to construct a gene silencing complex, and applying it to gene silencing therapy for HCC is of great value. Summary of the Invention

[0011] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a composite carbon dot nanozyme carrier, which has the advantages of improving the tumor hypoxic microenvironment, dual-modal imaging function and good gene loading ability.

[0012] Another purpose of the present invention is to use the composite carbon dot nanozyme carrier to construct a Golgi apparatus-targeted carbon dot nanozyme carrying GP73-siRNA.

[0013] Specifically, the composite carbon dot nanozyme carrier of the present invention is based on the Mn-CDs nanozyme prepared by a one-step solvothermal method using L-cysteine, neutral red and manganese dioxide. After activation by amide activators 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), polyethyleneimine (PEI) and hyaluronic acid (HA) molecules are coupled on its surface to form the Mn-CDs-PEI-HA nanozyme carrier.

[0014] Furthermore, the Mn-CDs nanozyme is specifically prepared using L-cysteine, neutral red and manganese dioxide as raw materials, and is prepared by solvothermal reaction at 120-160° C. for 6-14 hours in an ethanol and deionized water system.

[0015] Furthermore, the present invention specifically activates PEI and HA with EDC / NHS, chemically bonds them together to form a PEI-HA complex, then activates the Mn-CDs nanozyme with EDC / NHS, adds the PEI-HA complex to react in the dark, and prepares the Mn-CDs-PEI-HA nanozyme carrier through chemical bonding.

[0016] The Mn-CDs nanozyme and Mn-CDs-PEI-HA nanozyme carriers constructed above can both successfully target the Golgi apparatus.

[0017] The Mn-CDs nanozyme and Mn-CDs-PEI-HA nanozyme carriers constructed above have good oxygen production capacity and can be used to improve the hypoxic microenvironment of tumor cells in subsequent treatment.

[0018] The Mn-CDs nanozyme and Mn-CDs-PEI-HA nanozyme carriers constructed above also have low cytotoxicity and good biocompatibility.

[0019] The research in this invention shows that the Mn-CDs nanozyme prepared by doping Mn ions into CDs has hydrogen peroxide nanozyme activity and a high relaxation rate. It can not only enter HCC cells to produce oxygen in situ and improve the hypoxic microenvironment, but also enhance the FL / MR dual-modality efficacy evaluation level and reduce probe cost and toxicity.

[0020] Improving the hypoxic microenvironment of HCC can improve the effect of HCC gene therapy and the level of efficacy evaluation. Therefore, the Mn-CDs nanozyme with hydrogen peroxide nanozyme activity and magnetism of the present invention provides a new strategy and idea for improving the effect of HCC gene therapy and the level of dual-modal efficacy evaluation.

[0021] Furthermore, the composite carbon dot nanozyme carrier prepared by the present invention carries a positive charge, has an emission wavelength of 629nm, has a red light emission fluorescence effect and a high relaxation rate, can successfully target the Golgi apparatus of liver cancer cells, penetrate living tissues into cells for fluorescence imaging and MRI imaging, and be used as a fluorescence-magnetic resonance dual-modal imaging probe.

[0022] The composite carbon dot nanozyme carrier prepared by the present invention also catalyzes the decomposition of hydrogen peroxide in the tumor microenvironment to produce oxygen through its manganese dioxide component, achieving its targeted Golgi apparatus effect of improving the tumor's hypoxic microenvironment. Furthermore, the composite carbon dot nanozyme carrier of the present invention exhibits excellent cell transfection ability and biosafety as a gene delivery vehicle.

[0023] The present invention also further constructs a Golgi apparatus-targeted carbon dot nanozyme carrying GP73-siRNA on the basis of the above-mentioned composite carbon dot nanozyme carrier, which uses GP73-siRNA as the targeting gene and is carried on the composite carbon dot nanozyme carrier by electrostatic adsorption to construct the gene-carrying complex Mn-CDs-PEI-HA / GP73-siRNA;

[0024] The siRNA targeting the gene GP73 is a group of siRNAs having the following nucleotide sequences as shown in SEQ ID NOs. 5 to 6:

[0025] Sense strand: 5′-GCAGGGAAUGACAGAAACAUATT-3′;

[0026] Antisense strand: 5′-UAUGUUUCUGUCAUUCCCUGCTT-3′.

[0027] Furthermore, in the Golgi-targeted carbon dot nanozyme carrying GP73-siRNA constructed by the present invention, the mass ratio of the composite carbon dot nanozyme carrier Mn-CDs-PEI-HA to the target gene GP73-siRNA is preferably (20-50):1.

[0028] Furthermore, the optimal mass ratio of the composite carbon dot nanozyme carrier Mn-CDs-PEI-HA to the target gene GP73-siRNA is 20:1, which can ensure that Mn-CDs-PEI-HA can completely carry GP73-siRNA.

[0029] The Golgi apparatus-targeted carbon dot nanozyme carrying GP73-siRNA constructed above in the present invention, that is, the gene-carrying complex Mn-CDs-PEI-HA / GP73-siRNA, has an average particle size of about 7.73 nm and has good cell permeability.

[0030] Specifically, the Golgi apparatus-targeted carbon dot nanozyme carrying GP73-siRNA of the present invention is prepared according to the following method:

[0031] 1) L-cysteine, neutral red, and manganese dioxide were added to a mixed solution of ethanol and water, and the mixture was solvothermally reacted at 120-160°C for 6-14 hours. The precipitate was discarded by centrifugation, and the Mn-CDs nanozyme powder was obtained by filtration, dialysis, and freeze-drying.

[0032] 2) Dissolve polyethyleneimine (10,000 Da) in PBS, then dissolve hyaluronic acid, amide activator EDC, and NHS in PBS. Mix the two solutions and incubate in the dark for 3–5 h to prepare a PEI-HA complex solution.

[0033] 3) Dissolve Mn-CDs nanozyme powder, EDC, and NHS in PBS, let stand for 3-5 hours, then mix with PEI-HA complex solution, and react in the dark at 60-70°C with stirring for 5-8 hours to obtain Mn-CDs-PEI-HA nanozyme carrier solution. Purify by centrifugation, filtration, and dialysis, and freeze-dry to obtain Mn-CDs-PEI-HA nanozyme carrier powder carrying a positive charge;

[0034] 4) The Mn-CDs-PEI-HA nanozyme carrier solution and the negatively charged target gene GP73-siRNA solution were mixed and allowed to stand, and electrostatic adsorption was used to combine to form the gene-carrying complex Mn-CDs-PEI-HA / GP73-siRNA.

[0035] Specifically, in the above preparation method, the filtration is preferably performed using a disposable microporous filter membrane with a pore size of 0.22 μm.

[0036] More specifically, the dialysis treatment involved in the above preparation method is carried out using a dialysis bag with a molecular weight cut-off of 1000 Da.

[0037] The gene-carrying complex Mn-CDs-PEI-HA / GP73-siRNA constructed by the present invention can inhibit the expression of GP73-mRNA through the siRNA it carries, reducing the synthesis of GP73, and has excellent gene silencing therapeutic effects. Therefore, in addition to having excellent dual-modal imaging capabilities, the Golgi-targeted carbon dot nanozyme carrying GP73-siRNA can also successfully target the Golgi apparatus of liver cancer cells, showing a specific inhibitory effect on liver tumor cells. It can be used as a drug to inhibit the growth of liver tumor cells, thereby achieving a good therapeutic effect on liver cancer at the nanoscale.

[0038] Based on the good binding ability of Mn-CDs-PEI-HA and GP73-siRNA, the gene-carrying complex constructed by the present invention not only effectively avoids the effects of heparin and enzymatic degradation on GP73-siRNA in vivo, but also, through confocal co-localization results, proves that Mn-CDs-PEI-HA encapsulates GP73-siRNA and successfully enters the cell and is dispersed in the cytoplasm, indicating that the Mn-CDs-PEI-HA / GP73-siRNA gene-carrying complex has excellent gene transfection ability and can better inhibit tumor growth and proliferation.

[0039] Experiments have shown that the Mn-CDs nanozyme and Mn-CDs-PEI-HA nanozyme carriers constructed in the present invention can effectively improve the hypoxic microenvironment of tumor cells, and the gene-loaded complex Mn-CDs-PEI-HA / GP73-siRNA can show a good gene silencing therapeutic effect on liver tumor cells, and can inhibit the activity of tumor cell GP73 protein to a certain extent. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the ultraviolet absorption spectrum of Mn-CDs nanozyme and Mn-CDs-PEI-HA nanozyme carrier.

[0041] Figure 2This is the TEM image of Mn-CDs nanozyme, and the inset is its particle size statistics.

[0042] Figure 3 This is the fluorescence emission spectrum of Mn-CDs nanozyme.

[0043] Figure 4 This is the Mn 2p and Mn 3s analysis diagram of Mn-CDs nanozyme in XPS.

[0044] Figure 5 This is the dissolved oxygen curve of Mn-CDs nanozyme at different pH.

[0045] Figure 6 These are the T1-weighted images of Mn-CDs nanozyme under neutral and acidic conditions.

[0046] Figure 7 It is the histogram of hemolysis rate of Mn-CDs nanozyme solution with different concentrations.

[0047] Figure 8 This is a TEM image of the Mn-CDs-PEI-HA nanozyme carrier, and the inset is a statistical diagram of its particle size.

[0048] Figure 9 This is the fluorescence emission spectrum of the Mn-CDs-PEI-HA nanozyme carrier.

[0049] Figure 10 This is the Mn 2p and Mn 3s analysis diagram of the Mn-CDs-PEI-HA nanozyme carrier in XPS.

[0050] Figure 11 This is the dissolved oxygen curve of the Mn-CDs-PEI-HA nanozyme carrier at different pH values.

[0051] Figure 12 It is the Zeta potential of Mn-CDs nanozyme, PEI-HA complex, and Mn-CDs-PEI-HA nanozyme carrier.

[0052] Figure 13 It is the histogram of hemolysis rate of Mn-CDs-PEI-HA nanozyme carrier solution with different concentrations.

[0053] Figure 14 This is the in vitro oxygen production verification diagram of RDPP of Mn-CDs nanozyme and Mn-CDs-PEI-HA nanozyme carriers.

[0054] Figure 15 This is a graph evaluating the anti-tumor proliferation ability of different targeted gene GP73-siRNA sequences.

[0055] Figure 16This is a TEM image of the gene-loaded complex Mn-CDs-PEI-HA / GP73-siRNA, and the inset is a statistical diagram of its particle size.

[0056] Figure 17 This is the agarose gel electrophoresis diagram of the gene-loaded complex Mn-CDs-PEI-HA / GP73-siRNA at different mass ratios.

[0057] Figure 18 These are the fluorescence imaging images of Mn-CDs-PEI-HA / GP73-siRNA in Huh-7 cells at different wavelengths and the intracellular fluorescence co-localization images.

[0058] Figure 19 These are the laser confocal images and colocalization curves of Mn-CDs nanozyme colocalized with the Golgi apparatus of Huh-7 cells.

[0059] Figure 20 It is a bar graph showing the cell survival rate of Huh-7 cells treated with Mn-CDs, Mn-CDs-PEI-HA, GP73-siRNA, Mn-CDs-PEI-HA / NC and Mn-CDs-PEI-HA / GP73-siRNA under normoxic (a) and hypoxic (b) conditions. Implementation Method

[0060] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention so that those skilled in the art can better understand and utilize the present invention, but are not intended to limit the scope of protection of the present invention.

[0061] Unless otherwise specified, the production processes, experimental methods or detection methods involved in the embodiments of the present invention are all conventional methods in the prior art, and their names and / or abbreviations are conventional names in the field and are very clear and unambiguous in the relevant fields of use. Those skilled in the art can understand the conventional process steps based on the names and apply the corresponding equipment to implement them according to conventional conditions or the conditions recommended by the manufacturer.

[0062] The various instruments, equipment, raw materials or reagents used in the embodiments of the present invention are not particularly limited in their sources and are all conventional products that can be purchased through regular commercial channels or prepared according to conventional methods well known to those skilled in the art. Example

[0063] Example 1

[0064] Weigh 58 mg of neutral red and dissolve it in 30 mL of ethanol. Then weigh and add 34.77 mg of manganese dioxide and 1.2167 g of L-cysteine, add 10 mL of deionized water, and mix by ultrasonic stirring until there is no precipitation. Transfer the mixture to a stainless steel autoclave with a polytetrafluoroethylene liner, seal it, place it in an oven, heat it to 140°C, react for 8 h, and cool it to room temperature after the reaction is completed.

[0065] The reaction product was removed and centrifuged at 8000 rpm for 10 minutes. The deep red supernatant containing Mn-CDs was collected and filtered through a 0.22 μm hydrophilic microporous filter membrane to remove large particles. The filtrate was rotary evaporated for 30 minutes to remove excess ethanol, and then dialyzed in deionized water for 48 hours using a dialysis bag with a molecular weight cutoff of 1000 Da to remove unreacted small molecules. The water was changed every 12 hours during this period. Finally, the liquid in the dialysis bag was freeze-dried to obtain deep red Mn-CDs nanozyme powder.

[0066] The UV-Vis absorption spectrum (UV-Vis) of Mn-CDs was tested. Figure 1 As shown, there is obvious absorption at 278nm and 540nm.

[0067] Figure 2 Transmission electron microscopy (TEM) was used to observe the morphology, size, and microstructure of Mn-CDs. The results showed that the Mn-CDs were spherical in shape, well dispersed, and had no obvious aggregation. From the particle size statistics in the small window of the figure, it can be seen that the diameter of the Mn-CDs particles ranged from 1.0 to 3.0 nm, with an average particle size of 1.82±0.045 nm.

[0068] The emission spectra of Mn-CDs at different excitation wavelengths were measured using a fluorescence spectrometer. Figure 3 As shown, it is proved that Mn-CDs in solution have excitation-dependent properties, with the optimal excitation wavelength of 510 nm and the optimal emission wavelength of 625 nm.

[0069] Furthermore, X-ray photoelectron spectroscopy (XPS) was used to analyze the 2p and 3s orbitals of Mn in Mn-CDs.

[0070] like Figure 4 As shown in (a), in the high-resolution Mn 2p spectrum of Mn-CDs, there are two main peaks at 641.15 and 652.77 eV, and a characteristic peak at 645.35 eV, which belong to Mn 2p 3 / 2 、Mn 2p 1 / 2 and satellite peaks; Figure 4 In the high-resolution Mn 3s spectrum of Mn-CDs (b), the ΔE between the two peaks is 5.96 eV, indicating that Mn 2+ Successful doping.

[0071] Take Mn-CDs powder and dissolve it in pH=6.5 and pH=7.4 solution respectively, add H2O2, and use dissolved oxygen meter to test the oxygen generated in the reaction system. The results are as follows: Figure 5 As shown in the figure, the oxygen production capacity of Mn-CDs with the same concentration under acidic conditions (pH=6.5) is better than that under neutral conditions (pH=7.4), proving that they can both produce oxygen, laying the foundation for improving the hypoxic microenvironment of tumors.

[0072] The relaxivity of Mn-CDs under different pH conditions was evaluated to verify the feasibility of Mn-CDs as a new MRI contrast agent.

[0073] like Figure 6 As shown in the figure, the MRI signal intensity of Mn-CDs increases with the 2+ The results indicate that the prepared Mn-CDs can be used as new candidates for MRI. Figure 6 It can also be seen that the image signal of Mn-CDs is higher under acidic conditions than under neutral conditions.

[0074] The hemolysis rate of Mn-CDs solutions with different concentrations was quantitatively analyzed by UV-Vis detection to investigate their biocompatibility. Figure 7 As shown in (a), as the concentration of Mn-CDs increased from 3.125 μg / mL to 200 μg / mL, no hemolytic reaction occurred. Figure 7 As shown in (b), the hemolysis rates are 1.3%, 0.8%, 1.1%, 2%, 2.9%, 3.3% and 3.8%, respectively, indicating that Mn-CDs as a carrier has good blood compatibility.

[0075] Example 2

[0076] Weigh 20 mg of PEI (10,000 Da) and dissolve it in 50 mL of PBS. Ultrasonicate and dissolve for 20 minutes.

[0077] 10 mg of HA, 26 mg of amide activator 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), and 16 mg of N-hydroxysuccinimide (NHS) were weighed and dissolved in 10 mL of PBS and sonicated for 20 min.

[0078] The two solutions were mixed and kept in the dark for 3 h to prepare a PEI-HA complex solution.

[0079] Example 3

[0080] 100 mg of the Mn-CDs powder prepared in Example 1, 250 mg of EDC, and 100 mg of NHS were weighed and dissolved in 25 mL of PBS to obtain a Mn-CDs solution with a concentration of 4 mg / mL. After standing for 1 hour, the solution was mixed with the PEI-HA complex solution prepared in Example 2 and magnetically stirred at a constant speed in a constant temperature water bath at 60°C for 5 hours to obtain a Mn-CDs-PEI-HA nanozyme carrier solution.

[0081] The obtained Mn-CDs-PEI-HA nanozyme carrier solution was centrifuged, filtered with a disposable filter membrane (pore size 0.22 μm), dialyzed for 24 hours using a dialysis membrane with a molecular weight cutoff of 10,000 Da, and finally freeze-dried to obtain a brown powder of the Mn-CDs-PEI-HA nanozyme carrier.

[0082] Take a small amount of Mn-CDs-PEI-HA powder and disperse it in deionized water. Test its UV-Vis absorption spectrum (UV-Vis). The results are as follows: Figure 1 As shown in the figure, Mn-CDs-PEI-HA and Mn-CDs have the same absorption at 278 nm and 540 nm, indicating that the connection to the PEI-HA complex does not affect its ultraviolet absorption.

[0083] Figure 8 The TEM morphology, size and microstructure of Mn-CDs-PEI-HA are also provided, showing that its morphology is spherical, well dispersed, and there is no obvious aggregation phenomenon. From the particle size statistics in the small window in the figure, it can be seen that the particle diameter is between 2.0 and 6.0 nm, and the average particle size is 3.56±0.103 nm.

[0084] The emission spectra of Mn-CDs-PEI-HA at different excitation wavelengths were measured using a fluorescence spectrometer. Figure 9 It was proved that the Mn-CDs-PEI-HA solution was excitation-dependent, with the optimal excitation wavelength being 490 nm and the optimal emission wavelength being 625 nm.

[0085] Furthermore, the 2p and 3s orbitals of Mn in Mn-CDs-PEI-HA were analyzed using X-ray photoelectron spectroscopy (XPS).

[0086] like Figure 10 As shown in (a), in the high-resolution Mn 2p spectrum of Mn-CDs-PEI-HA, there are two main peaks at 641.42 and 653.05 eV, and a characteristic peak at 645.03 eV, which belong to Mn 2p 3 / 2 、Mn 2p 1 / 2 and satellite peaks; such as Figure 10As shown in (b), in the high-resolution Mn 3s spectrum of Mn-CDs-PEI-HA, the ΔE between the two peaks is 5.93 eV, indicating that Mn 2+ Successful doping.

[0087] Mn-CDs-PEI-HA powder was dissolved in pH = 6.5 and pH = 7.4 solutions. After ultrasonic dissolution, H2O2 was added and the oxygen generated in the reaction system was tested using a dissolved oxygen meter.

[0088] like Figure 11 As shown in the figure, the oxygen production capacity of Mn-CDs-PEI-HA at the same concentration under acidic conditions (pH=6.5) is better than that under neutral conditions (pH=7.4), which proves that it can produce oxygen and lay the foundation for improving the hypoxic microenvironment of tumors.

[0089] Mn-CDs, PEI-HA, and Mn-CDs-PEI-HA were dispersed in deionized water, and the surface Zeta potential of different particles was measured using laser Doppler microelectrophoresis. Figure 12 As shown in the figure, both Mn-CDs and PEI-HA carry positive charges, and the two are combined by chemical bonds to form Mn-CDs-PEI-HA carrying positive charges, laying the foundation for the subsequent combination with the negatively charged GP73-siRNA.

[0090] UV-Vis detection was used to quantitatively analyze the hemolysis rate of Mn-CDs-PEI-HA solutions with different concentrations to investigate their biocompatibility. Figure 13 As shown in (a), as the concentration of Mn-CDs-PEI-HA increased from 3.125 μg / mL to 200 μg / mL, no hemolytic reaction occurred. Figure 13 As shown in (b), the hemolysis rates are 1.1%, 0.9%, 1.5%, 1.1%, 2.5%, 2.7% and 2.9%, respectively, indicating that Mn-CDs-PEI-HA has good blood compatibility as a carrier.

[0091] Example 4

[0092] Verify / evaluate the ability of the Mn-CDs nanozyme prepared in Example 1 and the Mn-CDs-PEI-HA nanozyme carrier prepared in Example 3 to improve the hypoxic microenvironment in tumor cells.

[0093] [Ru(dpp)₃]Cl₂ (RDPP) is an oxygen-sensitive, self-luminescent probe. Its fluorescence is quenched when oxygen is present in the environment. Therefore, the intensity of RDPP fluorescence after cellular uptake can be used to determine the oxygen content within cells co-incubated with carbon dots.

[0094] Huh-7 cells in good condition were obtained, digested with trypsin, and diluted to 1×10 5 The cell suspension of 100 cells / mL was evenly inoculated into a 6-well plate, 2 mL was added to each well, and the plate was placed in a constant temperature incubator at 37°C and 5% CO2 for 24 h.

[0095] After culturing for 12 hours in a hypoxic environment, Mn-CDs and Mn-CDs-PEI (200 μg / mL) were dissolved in DMEM high-glucose medium containing RDPP (10 μg / mL) and incubated for another 6 hours. The cells were observed and photographed under a fluorescence microscope.

[0096] like Figure 14 As shown in the figure, the RDPP fluorescence intensity of cells incubated with Mn-CDs and Mn-CDs-PEI-HA was significantly weakened compared with the normal group, indicating that RDPP has been oxidized by oxygen produced in the cells and the fluorescence disappeared, proving that Mn-CDs and Mn-CDs-PEI-HA can produce oxygen in the cells, thereby alleviating the hypoxic microenvironment of the tumor.

[0097] Example 5

[0098] The sequence NM_016548.4 encoding human GP73 cDNA / GOLM1 cDNA (Homo sapiens golgi membrane protein 1A) was searched in Gene Bank as the target gene. Using siRNA design software, common RNAi targets targeting different splice variants of GP73 mRNA were designed. Three sequences that were conducive to siRNA binding to form silencing complexes were selected: GP73 siRNA-241, GP73 siRNA-671, and GP73 siRNA-1274. Three groups of siRNA fragments from different positions on the target gene mRNA were synthesized.

[0099] hGP73 siRNA-241 (SEQ ID NO.1~2):

[0100] Sense strand: 5′-CAUCAUCGUCUUGGGCUUCAATT-3′;

[0101] Antisense strand: 5′-UUGAAGCCCAAGACGAUGAUGTT-3′.

[0102] hGP73 siRNA-671 (SEQ ID NO.3~4):

[0103] Sense strand: 5′-GAACAGUGUGAGGAGCGAAUATT-3′;

[0104] Antisense strand: 5′-UAUUCGCUCCUCACACUGUUCTT-3′.

[0105] hGP73 siRNA-1274 (SEQ ID NO.5~6):

[0106] Sense strand: 5′-GCAGGGAAUGACAGAAACAUATT-3′;

[0107] Antisense strand: 5′-UAUGUUUCUGUCAUUCCCUGCTT-3′.

[0108] At the same time, a group of NC-GP73 siRNAs were designed, which had no obvious homology with human mRNA but had the same GC composition.

[0109] NC-GP73 siRNA (SEQ ID NO.7~8):

[0110] Sense strand: 5′-UUCUCCGAACGUGUCACGUTT-3′;

[0111] Antisense strand: 5′-ACGUGACACGUUCGGAGAATT-3′.

[0112] The above siRNAs were synthesized with the assistance of Shanghai Sangon Biotechnology Co., Ltd., and the base TT was added to the 3' end of each single strand.

[0113] Using NC-GP73 siRNA as negative, the anti-tumor proliferation ability of different siRNAs designed above was evaluated at the in vitro cell level.

[0114] Huh-7 cells in good growth condition were taken and 2×10 5 Cells were seeded into 6-well plates, incubated overnight in an incubator, and then replaced with serum-free DMEM medium for 2 h before transfection.

[0115] The experiment was divided into 5 groups, group A was the normal cell group, group B was the NC-GP73 siRNA negative control group, group C was the GP73 siRNA-241 group, group D was the GP73 siRNA-671 group, and group E was the GP73 siRNA-1274 group.

[0116] After grouping, each group was transfected: 10 μL of each group's siRNA and 5 μL of transfection reagent were diluted with 100 μL of serum-free DMEM medium, and after standing at room temperature for 5 minutes, the two solutions were mixed and stood for 20 minutes to obtain a mixed solution.

[0117] 200 μL of each mixed solution was added to the above 6-well plate, and the cells were cultured in the incubator for 6 hours, and then replaced with normal culture medium for further culture.

[0118] Total RNA was extracted from each group and reverse transcribed into cDNA. Real-time fluorescence quantitative PCR was used to detect mRNA in each group. -ΔΔCt The relative expression level was calculated by Figure 15 The bar chart shown.

[0119] Depend on Figure 15 The PCR results showed that compared with the normal cell group and the NC-GP73 siRNA negative control group, GP73siRNA-241 and GP73 siRNA-671 both had a certain inhibitory effect on tumors, but GP73 siRNA-1274 had the strongest tumor inhibition effect, with a gene silencing effect of 89.1%, and a good gene silencing effect on liver cancer cells, which was statistically significant.

[0120] Therefore, the present invention screened out GP73 siRNA-1274 (SEQ ID NO. 5-6) from the three groups of gene sequences as the target gene GP73-siRNA for constructing the final gene-carrying complex of the present invention.

[0121] Example 6

[0122] 500 μg of the Mn-CDs-PEI-HA nanozyme carrier solid powder prepared in Example 3 was weighed, dissolved in 1 mL of DEPC water, and filtered through a 0.22 μm filter membrane to obtain a Mn-CDs-PEI-HA solution.

[0123] 40 μg of the GP73-siRNA constructed in Example 5 was weighed into an EP tube, and the above-mentioned Mn-CDs-PEI-HA solution was added and mixed. The mixture was allowed to stand on crushed ice for 1 h to allow it to fully combine, thereby preparing the Golgi-targeted carbon dot nanozyme carrying GP73-siRNA, that is, the gene-carrying complex Mn-CDs-PEI-HA / GP73-siRNA.

[0124] TEM observation of the morphology and size of the prepared Mn-CDs-PEI-HA / GP73-siRNA, as shown in Figure 16 As shown in the figure, its morphology is approximately ellipsoidal and there is no aggregation. The average particle size is approximately 12.84±0.17nm. Compared with Mn-CDs-PEI-HA, its particle size is significantly increased, indirectly indicating that Mn-CDs-PEI-HA carries GP73-siRNA to form a gene-carrying complex Mn-CDs-PEI-HA / GP73-siRNA.

[0125] Example 7

[0126] According to the method in Example 6, the Mn-CDs-PEI-HA solution was mixed with different masses of GP73-siRNA to prepare Mn-CDs-PEI-HA / GP73-siRNA with a mass ratio of Mn-CDs-PEI-HA to GP73-siRNA of 2:1, 4:1, 8:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1 and 50:1, respectively.

[0127] The Mn-CDs-PEI-HA / GP73-siRNA at different mass ratios was mixed with DNA loading, added to the sample wells for gel electrophoresis, and imaged on a gel imager to confirm the binding ability between the composite carbon dot carrier and the gene in the gene-loading complex at different mass ratios, proving that the gene can be completely loaded onto the carrier to construct the gene-loading complex.

[0128] Figure 17 In the figure, as the mass ratio of Mn-CDs-PEI-HA to GP73-siRNA gradually increased from 2:1 to 50:1, the bands in the sample wells gradually brightened, while the brightness of the migration band gradually dimmed. When the ratio of Mn-CDs-PEI-HA to GP73-siRNA increased to 20:1, the migrating siRNA band disappeared, indicating that the migration of siRNA complexed with Mn-CDs-PEI-HA toward the positive electrode was completely blocked.

[0129] The above results indicate that an appropriate amount of Mn-CDs-PEI-HA can bind to siRNA to form a stable complex, and therefore may serve as a siRNA carrier. This also indicates that a mass ratio of 20:1 is the minimum critical complex ratio for Mn-CDs-PEI-HA to completely load GP73-siRNA.

[0130] Example 8

[0131] A 5'-end fluorescently labeled FAM-GP73 siRNA was constructed with an excitation wavelength of 492 nm and an emission wavelength of 518 nm. This was then combined with Mn-CDs-PEI-HA to construct a fluorescently labeled Mn-CDs-PEI-HA / GP73-siRNA. Laser confocal microscopy was used to examine the cell transfection ability of the gene-loaded complex.

[0132] Take Huh-7 cells in good growth state and disperse them into 4×10 42 mL of cell suspension was pipetted into a special culture dish. After culturing in the incubator for 12 h, the culture medium was discarded, the cells were washed with PBS, and the culture medium containing 2% fetal bovine serum (FBS) was added again. After starvation culture for 1 h, the culture medium was discarded, the cells were washed with PBS, and complete culture medium containing 200 μg / mL fluorescently labeled Mn-CDs-PEI-HA / GP73-siRNA was added. After culturing under the same conditions for 4 h, the culture medium was discarded, the cells were washed with PBS, and the cells were fixed with paraformaldehyde for 15 min. The cell transfection effect was observed under a laser confocal microscope.

[0133] Figure 18 In the figure, the vector Mn-CDs-PEI-HA in the fluorescently labeled Mn-CDs-PEI-HA / GP73-siRNA exhibits orange-red fluorescence (excitation wavelength 490 nm, emission wavelength 625 nm); FAM-GP73 siRNA exhibits green fluorescence (excitation wavelength 492 nm, emission wavelength 518 nm); Merge is the image after fluorescence field superposition.

[0134] from Figure 18 It can be seen that Mn-CDs-PEI-HA / GP73-siRNA is dispersed in the cytoplasm, and further co-localization analysis on the right shows that Mn-CDs-PEI-HA encapsulated GP73-siRNA successfully entered the cell and has good cellular internalization ability.

[0135] Example 9

[0136] The Golgi targeting ability of Mn-CDs nanozymes was evaluated by co-incubation with the commercial Golgi dye NBD C6-Ceramide-BSA complex.

[0137] Huh-7 cells in the logarithmic growth phase were obtained, digested with trypsin, and dispersed in DMEM high-glucose medium containing 0.1% penicillin-streptomycin mixture and 10% fetal bovine serum. The cells were diluted to 1×10 4 2 mL of cell suspension was drawn and evenly inoculated into a laser confocal imaging culture dish. The cells were incubated at 37°C and 5% CO2 for 12 h. When the cells grew to a coverage rate close to 2 / 3, the imaging culture dish was taken out, the supernatant was removed, the cells were washed three times with PBS buffer solution, 2 mL of culture medium containing 2% fetal bovine serum was added, and the cells were starved and cultured in an incubator for 1 h. The culture medium was discarded and the cells were washed three times with PBS.

[0138] Add 100 μL of 200 μg / mL Mn-CDs nanozyme solution to the cells, incubate in the incubator again for 4 hours, remove the cell culture medium, wash the cells three times with HBSS, remove the washing solution, add freshly prepared NBD C6-Ceramide working solution (5 μM), and incubate at 4°C for 30 minutes.

[0139] The staining solution was aspirated, and the cells were washed three times with ice-cold cell culture medium. Fresh culture medium was then replaced, and the cells were incubated in a 37°C, 5% CO2 incubator for another 30 minutes. The cells were washed once more with fresh culture medium and fixed with paraformaldehyde for 15 minutes. The paraformaldehyde solution was discarded, and the cells were washed with PBS. PBS was then added to wet the cells again, and imaging was performed using a laser confocal imager. The fluorescence emission wavelength was 510 nm, and the excitation wavelength was 625 nm. The NBD fluorescence excitation wavelength was 466 nm, and the emission wavelength was 536 nm.

[0140] Figure 19 In the image, Mn-CDs exhibit orange-red fluorescence, and NBD C6-Ceramide exhibits green fluorescence; Merge is the image after fluorescence field superposition.

[0141] from Figure 19 It can be seen that Mn-CDs are evenly distributed in the cytoplasm, with a Pearson coefficient of 0.84 (Pearson coefficient > 0.5 indicates Golgi targeting ability), and further co-localization analysis shows that it has good Golgi targeting ability.

[0142] Example 10

[0143] According to the method in Example 6, the NC-GP73 siRNA designed in Example 5 was carried by Mn-CDs-PEI-HA to construct the gene-carrying complex Mn-CDs-PEI-HA / NC.

[0144] The CCK-8 method was used to evaluate the inhibitory effects of Mn-CDs, Mn-CDs-PEI-HA, GP73-siRNA, Mn-CDs-PEI-HA / NC and Mn-CDs-PEI-HA / GP73-siRNA on the proliferation of Huh-7 cells under normoxic and hypoxic conditions, respectively.

[0145] Take Huh-7 cells in good growth state and prepare a concentration of 1×10 5 100 μL of a 5.5 / mL cell suspension was seeded into each well of a 96-well plate and cultured overnight. The cells were washed twice with PBS, and 100 μL of each experimental group solution in complete culture medium was added to each well and incubated for a further 24 hours. After the incubation period, the cells were washed twice with PBS, and the pre-prepared CCK-8 solution was added to each well for a further 1 hour. The OD value of the solution was then measured using a multi-function microplate reader.

[0146] Figure 20 In the figure, compared with the Huh-7 cell control group, Mn-CDs-PEI-HA / NC and GP73-siRNA did not show obvious inhibitory effect on liver cancer cells under normoxic and hypoxic conditions. However, the gene-carrying complex Mn-CDs-PEI-HA / GP73-siRNA after replacing NC with the targeted gene GP73-siRNA had a more significant inhibitory effect on liver cancer cells than Mn-CDs, Mn-CDs-PEI-HA, GP73-siRNA and Mn-CDs-PEI-HA / NC, both under normoxic (a) and hypoxic (b) conditions, and the effect was even more significant under hypoxic conditions, proving that it can improve the tumor hypoxic microenvironment and successfully kill cancer cells.

[0147] The above embodiments of the present invention do not describe all details in detail, nor do they limit the present invention to the above embodiments. Various changes, modifications, substitutions, and variations made by those skilled in the art without departing from the principles and purpose of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A composite carbon dot nanozyme carrier, which is based on Mn-CDs nanozyme prepared by a one-step solvothermal method using L-cysteine, neutral red and manganese dioxide. After activation with amide activators EDC and NHS, polyethyleneimine and hyaluronic acid molecules are coupled on its surface to form an Mn-CDs-PEI-HA nanozyme carrier.

2. The composite carbon dot nanozyme carrier according to claim 1, characterized in that The Mn-CDs nanozyme is prepared by using L-cysteine, neutral red and manganese dioxide as raw materials through a solvothermal reaction at 120-160° C. for 6-14 hours in an ethanol and deionized water solution system.

3. The composite carbon dot nanozyme carrier according to claim 1, characterized in that First, polyethyleneimine and hyaluronic acid are combined under the activation of EDC / NHS to form a PEI-HA complex, then the Mn-CDs nanozyme is activated by EDC / NHS, the PEI-HA complex is added, and the reaction is carried out in the dark to prepare the Mn-CDs-PEI-HA nanozyme carrier.

4. Use of the composite carbon dot nanozyme carrier according to claim 1 as a fluorescence-magnetic resonance dual-modal imaging probe.

5. Use of the composite carbon dot nanozyme carrier according to claim 1 in the preparation of a drug or pharmaceutical composition for improving the hypoxic microenvironment of a tumor, wherein the improvement of the hypoxic microenvironment is achieved by catalyzing the decomposition of hydrogen peroxide in the tumor microenvironment by the manganese dioxide component in the nanozyme carrier to produce oxygen.

6. A Golgi-targeted carbon dot nanozyme carrying GP73-siRNA, wherein GP73-siRNA is used as the targeting gene and is carried on the composite carbon dot nanozyme carrier of claim 1 by electrostatic adsorption to form a gene-carrying complex Mn-CDs-PEI-HA / GP73-siRNA; in, The targeted gene GP73-siRNA is a group of siRNAs having the following nucleotide sequences: Sense strand: 5′-GCAGGGAAUGACAGAAACAUATT-3′; Antisense strand: 5′-UAUGUUUCUGUCAUUCCCUGCTT-3′.

7. The Golgi-targeted carbon dot nanozyme carrying GP73-siRNA according to claim 6, characterized in that The mass ratio of the composite carbon dot nanozyme carrier to the target gene GP73-siRNA is (20-50):

1.

8. The Golgi-targeted carbon dot nanozyme carrying GP73-siRNA according to claim 6 or 7, characterized in that The mass ratio of the composite carbon dot nanozyme carrier to the target gene GP73-siRNA is 20:

1.

9. The method for preparing the Golgi apparatus-targeted carbon-dot nanozyme carrying GP73-siRNA according to claim 6, comprising: 1) L-cysteine, neutral red, and manganese dioxide were added to a mixed solution of ethanol and water, and the mixture was solvothermally reacted at 120-160°C for 6-14 hours. The precipitate was discarded by centrifugation, and the Mn-CDs nanozyme powder was obtained by filtration, dialysis, and freeze-drying. 2) Dissolve polyethyleneimine in PBS, then dissolve hyaluronic acid, amide activator EDC, and NHS in PBS, mix the two solutions, and let them stand for 3-5 hours to prepare the PEI-HA complex solution; 3) Mn-CDs nanozyme powder, EDC, and NHS were dissolved in PBS and allowed to stand for 3-5 hours. The solution was then mixed with the PEI-HA complex solution and stirred at 60-70°C for 5-8 hours to obtain a Mn-CDs-PEI-HA nanozyme carrier solution. The solution was purified by centrifugation, filtration, and dialysis, and then freeze-dried to obtain a positively charged Mn-CDs-PEI-HA nanozyme carrier powder. 4) The Mn-CDs-PEI-HA nanozyme carrier solution and the negatively charged target gene GP73-siRNA solution were mixed and allowed to stand, and electrostatic adsorption was used to combine to form the gene-carrying complex Mn-CDs-PEI-HA / GP73-siRNA.

10. Use of the Golgi apparatus-targeted carbon dot nanozyme carrying GP73-siRNA according to claim 6 in the preparation of a drug for inhibiting the growth of liver tumor cells.

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