A dual-targeted near-infrared bionic drug delivery system and its preparation method and application

Abstract

This invention provides a dual-targeting near-infrared biomimetic drug delivery system, its preparation method, and its application. It comprises a erythrocyte membrane and a nanocarrier encapsulated within the cell membrane. The erythrocyte membrane surface is modified with β-D-galactoside. The nanocarrier includes near-infrared responsive upconversion nanoparticles, anti-HBV antisense oligonucleotides, and anti-HBV nucleocapsid inhibitors. The upconversion nanoparticles are surface-modified with mesoporous silica. This dual-targeting near-infrared biomimetic drug delivery system achieves comprehensive treatment of HBV with high targeting, high drug loading capacity, and high biocompatibility.

Description

Technical Field

[0001] This invention relates to the field of biomimetic nanoparticle technology, and in particular to a dual-targeting near-infrared biomimetic drug delivery system, its preparation method, and its application. Background Technology

[0002] Hepatitis B virus (HBV) infection, caused by the hepatitis B virus, results in significant liver-related morbidity and mortality worldwide. According to the World Health Organization (WHO), nearly 300 million people worldwide suffer from long-term HBV infection, and more than 780,000 die annually from its complications, including cirrhosis and liver cancer. The global market for hepatitis B drugs is projected to reach $3 billion by 2024, placing a heavy burden on global healthcare systems. Currently, due to the difficulty in eliminating HBV covalently closed circular DNA (cccDNA), treatment for chronic HBV infection tends to focus on functional therapy, aiming to continuously reduce circulating viral load and HBV surface antigen (HBsAg). In this regard, drugs currently used clinically for the treatment of hepatitis B, such as interferon (IFN) and nucleoside analogues (NUCs), can inhibit hepatitis B virus replication, slow the progression of cirrhosis, and moderately reduce the incidence of hepatocellular carcinoma (HCC). However, long-term treatment with these drugs is also accompanied by drug resistance and adverse reactions, such as bone marrow suppression. It is well known that capsid inhibitors are crucial to the HBV life cycle and are potential targets for direct-acting antiviral drugs to reduce HBV DNA. A recent report on a novel capsid assembly regulator (CAM) shows that a small molecule, C39, can inhibit HBV DNA synthesis by affecting the normal assembly of the HBV nucleocapsid. Increasing evidence from recently published studies suggests that antisense oligodeoxynucleotides (ASOs) can induce targeted cleavage of HBV mRNA, thereby reducing viral protein production.

[0003] Significant progress has been made in understanding the mechanisms of HBV persistence; the development of anti-HBV therapies is no longer limited to directly targeting the virus itself, but rather employs multi-target treatments that target different stages of the viral life cycle. Extensive evidence demonstrates that this novel therapeutic concept, seeking to treat complex diseases with multiple drugs, can effectively inhibit HBV replication. However, both nucleic acids without nanocarriers and highly negatively charged small molecule drugs struggle to enter mammalian cells, posing a significant challenge to drug therapy. Furthermore, the injection of insoluble microparticles into the bloodstream may induce venous thromboembolism, and the potential increase in systemic toxicity due to non-specific drug distribution in the body limits its clinical application.

[0004] In recent years, drug delivery nanoparticles have been used to improve drug delivery efficiency and reduce systemic side effects. However, most nanoparticles cannot distinguish between abnormal and normal cells, often leading to serious side effects. Coupled with accidental release during delivery, the rational use of drug delivery nanoparticles remains a significant challenge.

[0005] Stimulus-responsive nanocarriers, a type of intelligent nanocarrier, have attracted widespread attention in recent years due to their ability to effectively reduce unintended drug release. Photostimulation-responsive nanoparticles can ensure controlled and on-demand release of loaded drugs in both time and space. Their controllability and non-invasiveness highlight their potential applications in drug delivery systems and biomedicine. Most reported photoresponsive nanocarriers to date utilize ultraviolet or visible light to trigger drug delivery; however, the low penetration and phototoxicity of these lights limit their in vivo applications. Near-infrared (NIR) light, due to its ability to penetrate tissue with minimal damage to healthy tissue, has garnered significant attention for non-invasive therapies and is widely used in biomedical research. There is substantial evidence that upconversion nanoparticles (UCNPs) possess advanced optical properties, such as excellent dimensional stability and low toxicity, enabling the conversion of low-energy NIR light into high-energy ultraviolet / visible light emission.

[0006] However, UCNPs exhibit low drug loading capacity and a lack of precise targeting ability. Furthermore, nucleic acid drugs on the surface of nanoparticles are easily degraded by nucleases when exposed to complex physiological and heterogeneous environments. Therefore, there is an urgent need to develop a novel delivery system with high targeting accuracy, strong drug loading capacity, and good biocompatibility. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention provides a dual-targeting near-infrared biomimetic drug delivery system, its preparation method, and its application, which solves the problems of low targeting accuracy, poor drug loading capacity, and low biocompatibility in existing technologies.

[0008] In one aspect, the present invention provides a dual-targeting near-infrared biomimetic drug delivery system, comprising a erythrocyte membrane and a drug-loaded nanocarrier encapsulated within the erythrocyte membrane. The drug-loaded nanocarrier comprises near-infrared responsive upconversion nanoparticles, an anti-HBV antisense oligonucleotide, and an anti-HBV nucleocapsid inhibitor. The surface of the upconversion nanoparticles is coated with mesoporous silica, and the anti-HBV antisense oligonucleotide and the anti-HBV nucleocapsid inhibitor are respectively loaded on the mesoporous silica.

[0009] Furthermore, the anti-HBV nucleocapsid inhibitor is loaded within the mesoporous channels of mesoporous silica, and the anti-HBV antisense oligonucleotide is loaded on the surface of the mesoporous silica. Specifically, some of the anti-HBV antisense oligonucleotide is loaded at the pores of the mesoporous channels to block the release of the anti-HBV nucleocapsid inhibitor within the mesoporous channels.

[0010] Furthermore, the surface of the erythrocyte membrane is modified with a targeting molecule, preferably β-D-galactosidase.

[0011] Furthermore, the near-infrared responsive upconversion nanoparticles have a core-shell structure, Ca 2+ The doped NaYF4:Yb / Tm forms the core, and NaYF4:Yb / Nd forms the shell.

[0012] Furthermore, the anti-HBV antisense oligonucleotide is ASO, and the anti-HBV nucleocapsid inhibitor is C39.

[0013] In another aspect, the present invention provides a method for preparing a dual-targeting near-infrared biomimetic drug delivery system, comprising the following steps:

[0014] 1) Synthesis of UCNP: Preparation of NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd by thermal decomposition;

[0015] 2) Synthesis of UCNP@MSN: Using CTAB as a template agent, NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd prepared in step 1) was reacted sequentially with tetraethyl silicate and 3-aminopropyltriethoxysilane to obtain mesoporous silica-coated UCNP.

[0016] 3) Synthesis of UCNP@MSN-PcASO / C39: PcDNA was modified on the surface of the UCNP@MSN obtained in step 2), and then anti-HBV antisense oligonucleotides and anti-HBV nucleocapsid inhibitors were added to obtain the drug-loaded nanocarrier UCNP@MSN-PcASO / C39.

[0017] 4) Synthesis of UCNP@MSN-PcASO / C39-M-Gal: The UCNP@MSN-PcASO / C39 obtained in step 3) was encapsulated in erythrocyte membrane modified with Gal to obtain the drug-loaded nanocarrier UCNP@MSN-PcASO / C39-M-Gal encapsulated in erythrocyte membrane.

[0018] Further, in step 2), the mass-to-volume ratio of NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd to tetraethyl silicate is 1:5, calculated in mg / μL; and the volume ratio of tetraethyl silicate to 3-aminopropyltriethoxysilane is 10:1.

[0019] Furthermore, in step 3), the loading amount of anti-HBV antisense oligonucleotide is 101.5 μmol / g·UCNP@MSN, which, by mass percentage, represents 7.3% of the loading amount of anti-HBV nucleocapsid inhibitor.

[0020] Further, in step 4), the Gal-modified erythrocyte membrane sac is prepared by modifying the erythrocyte membrane sac with distearate phosphatidylethanolamine-polyethylene glycol-galactose.

[0021] In another aspect, the present invention provides the application of a dual-targeting near-infrared biomimetic drug delivery system in the preparation of drugs for treating hepatitis.

[0022] The term "nPcDNA" refers to a single-stranded DNA complementary to ASO, and "PcDNA" refers to nPcDNA with an inserted pc linker (iPCLink). The nucleotide sequences are shown in Table 1. The pc linker is a photolytically cleavable group with the structural formula shown in Formula I.

[0023] Formula I

[0024] The term "ASO" stands for antisense oligonucleotide, whose nucleotide sequence is shown in SEQ ID NO.1; "Pc-ASO" is the complementary double strand formed by pcDNA and ASO; "NIR" stands for near-infrared; "UCNP" stands for lanthanide upconversion nanoparticles; "CM" stands for cell membrane-derived vesicles; "MSN" stands for mesoporous silica nanoparticles; "ASGPR" stands for desialyl glycoprotein receptor; "Gal" stands for β-D-galactopyranoside; "PC" stands for photolysis; "FRET" stands for fluorescence resonance energy transfer; and "BHQ" stands for black hole quencher.

[0025] The technical principle of this invention is as follows: A novel UMAC-responsive nanoparticle, UCNP@MSN-ASO-C39 (or UCNP@MSN-PcASO / C39), was successfully synthesized through surface modification of mesoporous silica (MSN). This nanoparticle is loaded with a large amount of anti-HBV antisense oligonucleotides and anti-HBV nucleocapsid inhibitors, improving biocompatibility. The designed DNA containing PC groups (pclinker) (or PCDNA) can be used to block the pores of the drug C39 payload, thereby achieving controlled release of C39. This delivery system is ultimately encapsulated by Gal-modified erythrocyte membrane vesicles, protecting the nucleic acids from degradation. These vesicles target the salivary glycoprotein receptor (ASGPR), a transmembrane protein specifically expressed on hepatocytes, with high binding affinity, resulting in receptor-mediated endocytosis and accurate liver targeting. Simultaneously, under 808nm NIR radiation, the ultraviolet light emitted by UCNP excitation causes photolysis of the PC groups, triggering the release of ASO and C39, producing a synergistic therapeutic effect and inhibiting HBV replication.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] (1) This invention utilizes a novel photoinduced co-delivery strategy to achieve comprehensive treatment of HBV. By utilizing the immune escape of the cell membrane and the targeting ability of Gal, the nanosystem is successfully delivered to hepatocytes; due to the special photoinducible properties of the PC group, the ultraviolet light emitted by UCNP under near-infrared irradiation successfully triggers the release of ASO and C39; thereby achieving spatial and temporal control of drug release and precise targeting of hepatocytes.

[0028] (2) The two drugs delivered by this nanocarrier achieve functional HBV cure by targeting different stages of the viral life cycle and reducing the secretion of viral antigens, thereby producing a synergistic effect. More importantly, the drug delivery system of this invention achieves functional HBV cure both in vitro and in vivo.

[0029] (3) This invention provides a release strategy for the treatment of other diseases (especially tumors), and this novel controlled release method provides an advanced strategy and new insights for drug delivery. Attached Figure Description

[0030] Figure 1 This is a schematic diagram of Pc-ASO hybridization and photoactivated release in Example 1 of the present invention. a) is a schematic diagram of FRET-labeled Pc-ASO (or FRET-PcDNA) formed by hybridization of ASO (or FAM-ASO / F-ASO) labeled with fluorophore (F, FAM) and PcDNA (or Q-PcDNA) labeled with quencher (Q, BHQ1); b) is a schematic diagram of photoactivated release of FAM-ASO from FRET-PcDNA to destroy the FRET pair, where PC Group represents the photoresponsive group; c) is a schematic diagram of FRET-labeled nPc-ASO (or FRET-nPcDNA) formed by hybridization of ASO (or FAM) labeled with fluorophore (F, FAM) and nPcDNA (or Q-nPcDNA) labeled with quencher (Q, BHQ1); d) is a schematic diagram of photoactivated release of FAM-ASO from nPcDNA to destroy the FRET pair.

[0031] Figure 2 The following is a characterization of Pc-ASO in Example 1 of the present invention, wherein a is the fluorescence spectrum of Pc-ASO; b is the fluorescence spectrum of nPc-ASO; c is the agarose gel electrophoresis analysis of Pc-ASO after ultraviolet irradiation; and d is the agarose gel electrophoresis analysis of nPc-ASO after ultraviolet irradiation.

[0032] Figure 3The images show the SEM images and size distributions of the three groups of nanoparticles in Examples 2 and 3 of this invention, where a is NaYF4:Yb / Tm / Ca, b is NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd, and c is UCNP@MSN.

[0033] Figure 4 This is an HRTEM image of NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd in Embodiment 2 of the present invention.

[0034] Figure 5 This is a schematic diagram of the multidimensional energy transfer path of lanthanide ions in UCNP in Example 2 of the present invention.

[0035] Figure 6 This is the EDS map of UCNP@MSN in Embodiment 3 of the present invention.

[0036] Figure 7 This is the nitrogen adsorption-desorption isotherm of UCNP@MSN in Example 3 of the present invention.

[0037] Figure 8 The image shows the upconversion emission spectra of UCNP@MSN under different power densities at 808 nm in Example 3 of this invention.

[0038] Figure 9 These are the preparation and characterization diagrams of UCNP@MSN and UMAC in Examples 3 and 5 of this invention. In this diagram, a is a schematic diagram of UMAC-M-Gal synthesis; b is NaYF4:Yb / Tm / Ca; c is NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd; d is a TEM image of UCNP@MSN (scale bar = 20 nm); e is the elemental (Na, F, Tm) corresponding to the high-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) image of UCNP@MSN. (Ca, Y, Yb, Nd, Si) mapping image (scale bar = 20 nm); f is the XRD pattern of UCNPs and UCNPs@MSN; g is the surface feature of UCNPs@MSN-NH2 (3D structure of C39 analyzed by Chem3D); h is the FTIR spectrum of each stage of UCNP@MSN-PcASO synthesis; i is the UCL spectrum of UCNPs and UCNPs@MSN (bright field and NIR fluorescence images of UCNPs@MSN); j is the drug release curve of UMAC under 808 nm NIR excitation; k is the specificity of C39 release from UMAC with or without PC groups and NIR excitation.

[0039] Figure 10The UV / Vis absorption spectra of UCNP@MSN, UCNP@MSN-PcASO, and UCNP@MSN-PcASO / C39 in Example 4 of this invention are shown.

[0040] Figure 11 These are the standard curves for C39(a) and FAM-ASO(b) in Embodiment 4 of the present invention.

[0041] Figure 12 This describes the release kinetics of C39 in UMC, UMAC, and UMAC-M-Gal in Embodiment 5 of the present invention.

[0042] Figure 13 For the performance verification of UMAC-M-Gal in Example 6 of this invention, a is a SEM image of UMAC-M-Gal; b is a TEM image of UMAC-M-Gal; c is SDS-PAGE protein analysis, with lanes 1 and 5 as markers, lane 2 as RBC membrane, lane 3 as RBC vesicles, and lane 4 as UMAC-M; d is the hydrodynamic size distribution of RM, UMAC, and UMAC-M-Gal; e is the Zeta potential of UCNPs, UCNPs@MSN, UMA, UMA-M-Gal, and RBC vesicles; f is the WB analysis of cell lysates from various cells; g is a CLSM image of HepG2.2.15 cells cultured with UMAC-M-Gal, with DAPI (blue) used to stain cell nuclei, Dil (red) used to stain RM-Gal, and FAM (green) used to label ASO (scale bar is 10 μm).

[0043] Figure 14 The Gal, NHS-PEG-DSPE, and Gal-PEG-DSPE in Embodiment 6 of the present invention 1 H NMR spectrum.

[0044] Figure 15 a represents the stability of UMAC-M-Gal under different physiological conditions over 14 days in Example 6 of this invention; b represents the hydration size of UMAC-M-Gal in FBS at different times; and c represents the polymer dispersion index.

[0045] Figure 16For the immune evasion assessment in Example 7 of this invention, a is a CLSM image (scale bar, 50 μm) of RAW264.7 macrophage-like cells after incubation with various FAM-labeled nanoparticles; b is the flow cytometry quantification of RAW264.7 cells after incubation with various FAM-labeled nanoparticles; c is the quantitative analysis of the absorption of biomimetic nanoparticles with different incubation time periods; d is the biodistribution of various biomimetic nanoparticles 48 hours after injection; e is the in vivo pharmacokinetic curve 48 hours after injection of various biomimetic nanoparticles; (blood retention of nanoparticles 24 hours after injection, data points represent mean ± standard deviation, n=3, NS, * and *** indicate no statistical difference, p<0.05 and p<0.001, respectively).

[0046] Figure 17 This is an example of the in vitro near-infrared photocontrolled release of FRET-labeled (pc)UMAC-M-Gal in Example 7 of the present invention. In this example, a is a CLSM image of HepG2.2.15 cells after incubation with FRET-labeled (pc)UMAC-M-Gal, showing NIR irradiation (NIR+) and no NIR irradiation (NIR-) (808nm, 2W / cm²). 2 b) shows the results of flow cytometry analysis, and c) shows the quantification of HepG2.2.15 cells after incubation with FRET-labeled (pc) UMAC-M-Gal, with and without NIR irradiation (808nm, 2W / cm). 2 ).

[0047] Figure 18 The liver-targeting assessment in Example 7 of this invention includes: a) CLSM images of HepG2.2.15 cells after incubation with various nanoparticles (scale bar = 20 μm); b) flow cytometry images of various non-hepatic cell lines after incubation with FAM-labeled nanoparticles for 3 hours; c) in vivo fluorescence images of HBV Tg mice 24 hours after injection of PBS containing UMAC-M-Gal nanoparticles stained with DiR; and d) in vitro fluorescence images of major organs of HBV-Tg mice sacrificed 24 hours after injection (data points represent mean ± standard deviation, n = 3, NS, * and *** indicate no statistical difference, p < 0.05 and p < 0.001, respectively).

[0048] Figure 19 The FACS analysis in Example 7 of this invention was used to assess the uptake efficiency of different nanoparticles in HepG2.2.15 cells.

[0049] Figure 20 This is a CLSM image of L02 cells taking up UMAC-M-Gal in Example 7 of the present invention.

[0050] Figure 21This is a CLSM image of UMAC-M-Gal cells with or without antibody blocking in Example 7 of the present invention.

[0051] Figure 22 For the liver-targeting assessment in Example 7 of the present invention, a is the CLSM image of various non-hepatic cell lines after incubation with FAM-labeled nanoparticles for 3 hours, b is the flow cytometry quantification of various non-hepatic cell lines after incubation with FAM-labeled nanoparticles for 3 hours, and d is the quantitative analysis of nanoparticle uptake in HepG2.2.15 cells at different incubation time periods.

[0052] Figure 23 This is an agarose gel electrophoresis analysis of UM-PcASO after 808nm NIR irradiation in Example 7 of the present invention.

[0053] Figure 24 For the cell viability assessment in Example 8 of this invention, a represents the viability of HepG2.2.15 and L02 cells incubated with different concentrations of UMAC-M-Gal nanoparticles without NIR irradiation (control), and b represents the viability of HepG2.2.15 and L02 cells under 808nm NIR irradiation (1.0W / cm²) by changing the irradiation time. 2 The viability of HepG2.2.15 and L02 cells under 808 nm NIR light (5 min) by changing the power density is shown in Figure 1. c represents the survival rate of HepG2.2.15 and L02 cells under 808 nm NIR light (1.0 W / cm²) by changing the nanoparticle concentration. 2 (10 minutes) of vitality.

[0054] Figure 25 This document describes the in vitro antiviral activity of UMAC-M-Gal in Example 8 of the present invention. In Example 8, a is a schematic diagram of the anti-HBV replication activity of ASO and C39; b is the experimental procedure for cell treatment using NIR irradiation; dg represents the antigen detection in the supernatant using ELISA; and dg represents the difference in HBV inhibition with and without a delivery system. The detection parameters are intracellular HBV DNA, HBV 3.5kb RNA, HBsAg, and HBeAg. hk represents the difference in HBV inhibition with and without NIR and PC groups. The detection parameters are intracellular HBV DNA, HBV 3.5kb RNA, HBsAg, and HBeAg.

[0055] Figure 26 This illustrates the anti-HBV effect of nanoparticles from different synthesis stages in Example 8 of the present invention.

[0056] Figure 27 The hemolysis rate of different concentrations of UMAC-M-Gal in Example 9 of this invention.

[0057] Figure 28 This is an example of the antiviral effect of UMAC-M-Gal in vivo in Example 9 of the present invention. In the example, a is a schematic diagram of the HBV transgenic mouse experiment; bd shows the difference in the inhibitory effect of different treatments on HBV; e is a schematic diagram of the immunohistochemical analysis of mouse liver; f is a representative immunohistochemical image showing the expression of HBsAg and HBeAg in the liver of sacrificed mice (scale bar = 50µm).

[0058] Figure 29 This is an H&E staining image of the main organs in different treatment groups in Example 9 of the present invention.

[0059] Figure 30 This is a systemic toxicity evaluation of the biomimetic delivery system UMAC-M-Gal in Example 9 of the present invention, wherein a is the body weight of mice in different treatment groups during 15 days, and serum biochemical parameters b is BUN, c is ALT and d is AST level in different treatment groups. Detailed Implementation

[0060] The technical solutions of this invention are further described below with reference to the accompanying drawings and embodiments. Unless otherwise specified, the reagents and experimental materials used in this invention are commercially available. The DNA oligonucleotide sequences in this invention were synthesized and purified by Sangon Biotech Co. (Shanghai, China); the HBV transgenic mice were obtained from the Key Laboratory of Molecular Biology of Infectious Diseases, Ministry of Education, Chongqing Medical University.

[0061] Example 1 Synthesis of Pc-ASO

[0062] To examine the UV-activated properties of Pc-ASO, Foster resonance energy transfer (FRET) pairs were used as fluorescent reporters. A single-stranded PcDNA carrying BHQ1 (black hole quenching group) was hybridized with a FAM (fluorescein)-labeled ASO strand (FAM-ASO) to form a double-stranded structure. A negative control was established: a single-stranded PcDNA without the photoresponsive group (nPcDNA) labeled with BHQ1 was hybridized with the FAM-labeled ASO strand (FAM-ASO) to form a double-stranded nPc-ASO. The nucleotide sequences of each sequence are shown in Table 1.

[0063] Table 1 DNA oligonucleotide sequences

[0064]

[0065] Hybridization and photoactivation properties of photoactivated Pc-ASO and nPc-ASO, as follows Figure 1 As shown. By Figure 1It can be seen that FAM-ASO is released from the double-chain structure due to the destruction of the PC group. Real-time monitoring of the UV activation process yielded the following results: Figure 2 As shown in figure a, the fluorescence emission of FAM is significantly reduced, almost disappearing, indicating that energy is transferred from FAM to BHQ1 with the formation of the double-stranded structure, confirming the formation of Pc-ASO. Under ultraviolet light irradiation, the fluorescence emission of Pc-ASO gradually recovers, indicating that the FRET pair disrupts the structure.

[0066] Depend on Figure 2 As shown in b, the fluorescence of nPc-ASO was quenched by FRET, consistent with the results for Pc-ASO. However, the fluorescence emission of nPc-ASO did not increase with ultraviolet irradiation. Agarose gel electrophoresis was used to analyze the specificity of the PC group. Figure 2 c, d) indicate that the UV activation properties depend on the PC group.

[0067] Example 2: Synthesis of NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd (UCNPs)

[0068] 1. Synthesis of nuclear NaYF4:Yb / Tm / Ca

[0069] A mixture was prepared by thermal decomposition in a three-necked flask containing 12 mL oleic acid (OA) and 30 mL 1-octadecene (ODE). The mixture consisted of 1.6 mmol LnCl3·6H2O, 1.112 mmol YCl3·6H2O, 0.32 mmol YbCl3·6H2O, 0.008 mmol TmCl3·6H2O, and 0.16 mmol CaOA. The mixture was heated to 120 °C for 15 minutes under a nitrogen atmosphere. Then, it was heated to 160 °C for 1 hour with stirring, and then cooled to room temperature. 10 mL of a methanol solution containing 4 mmol NaOH and 7.2 mmol NH4F was added, and the mixture was stirred at 50 °C for 30 minutes. The methanol was then removed by heating to 100 °C for 30 minutes. The mixture was then heated to 300 °C under vacuum for 1 hour, and then slowly cooled to room temperature. Ethanol precipitation was performed, and the mixture was centrifuged to obtain NaYF4:Yb / Tm / Ca. SEM images and size distribution of NaYF4:Yb / Tm / Ca are shown below. Figure 3 As shown in a, by Figure 3 As can be seen from a, the diameter of NaYF4:Yb / Tm / Ca is approximately 30 nm.

[0070] 2. Synthesis of core-shell NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd (UCNP)

[0071] 0.8 mmol LnCl3·6H2O, 0.32 mmol YCl3·6H2O, and 0.4 mmol NdCl3·6H2O were added to a three-necked flask containing 6 mL oleic acid (OA) and 15 mL 1-octadecene (ODE). The mixture was heated to 120 °C for 15 minutes under a nitrogen atmosphere. Then, it was heated with stirring for 1 hour, and then cooled to room temperature. 2.5 mL of the core NaYF4:Yb / Tm / Ca prepared in step 1 was added dropwise, and the mixture was stirred at room temperature for 30 minutes, then at 80 °C for 30 minutes. After cooling to room temperature, 5 mL of a methanol solution containing 2 mmol NaOH and 3.6 mmol NH4F was added, and the mixture was stirred at 50 °C for 30 minutes. The methanol was then removed by heating to 100 °C for 30 minutes. The mixture was heated to 300 °C under vacuum for 1 hour, and then slowly cooled to room temperature. Ethanol precipitation and centrifugation yielded the core-shell NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd. SEM images and size distribution of NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd are shown below. Figure 3 As shown in b, by Figure 3 b indicates that the average diameter of NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd is approximately 42 nm. High-resolution transmission electron microscopy (HRTEM) is shown below. Figure 4 As shown in the figure. The results show that the lattice spacing of UCNPs is 0.52 nm, and the diffraction peaks of UCNPs correspond to β-NaYF4 (JCPDSCard No. 28-1192), indicating that UCNPs have a hexagonal phase.

[0072] like Figure 5 As shown, compared with individual Nd 3+ Compared to sensitizers, Nd-doped Nd 3+ The sensitizer reduces the strong water absorption of biological tissues at 980 nm and enables UCNPs to be excited at 808 nm, thereby increasing the penetration depth of UCNPs in biological tissues.

[0073] Example 3 Synthesis of UCNP@MSN

[0074] 20 mg of UCNP prepared in Example 2 was dissolved in 2 mL of chloroform and added dropwise to 10 mL of aqueous solution containing 360 mg of cetyltrimethylammonium bromide (CTAB). The solution was ultrasonically treated with a probe ultrasonic disruptor at 400 W for 1 h to obtain a transparent UCNPs / CTAB solution. The mixture was centrifuged and repeated three times. The precipitate was collected and dispersed in ddH2O, and 15 μL of 2M NaOH was added. The mixture was stirred for 1 h. 100 μL of tetraethoxysilane (TEOS) was added under ultrasonic conditions, and the mixture was stirred at room temperature for 12 h. 10 μL of 3-aminopropyltriethoxysilane (APTES) was added, and the mixture was stirred at room temperature for 6 h. The product was purified by centrifugation, and extracted with 1% NaCl methanol solution at 40 °C for 24 h to remove the template CTAB. The resulting white powder was washed three times with water and ethanol and dried under vacuum for 12 h. The SEM images and size distribution of UCNP@MSN are shown below. Figure 3 As shown in c, by Figure 3 As can be seen from c, the average diameter of UCNP@MSN is approximately 57 nm.

[0075] The mesoporous properties of UCNP@MSN are as follows: Figure 9 As shown in Figure d, the thickness of the mesoporous silicon layer is approximately 10 nm. The EDS elemental analysis diagram and spectrum of UCNP@MSN are shown below. Figure 9 e, Figure 6 As shown, the successful fabrication of UCNP@MSN is confirmed. XRD imaging is as follows. Figure 9 As shown in f, a broad diffraction peak was observed at 22°, indicating the successful preparation of UCNP@MSN. The porosity characteristics of UCNP@MSN were measured using an automated surface area and porosity analyzer, as shown in f. Figure 9 g, Figure 7 As shown, the results indicate that the specific surface area, pore size, and total pore volume of UCNPs@MSN are approximately 2.74 nm, 373.89 nm, and 100 nm, respectively. 2 g −1 , and 0.352cm 3 g −1 Analysis using Chem3D software estimated the size of drug molecule C39 to be 1.49 nm, indicating that drug molecule C39 can be loaded into UCNP@MSN. Fourier transform infrared spectroscopy (FTIR) was then performed. Figure 9 h) at 3428cm −1 1089cm −1 and 800cm −1 Characteristic peaks were observed, confirming the successful surface modification of MSN. Upconversion emission spectra of UCNP and UCNP@MSN (…) Figure 9 i, Figure 8 This indicates that UCNP@MSN can effectively convert 808nm NIR into light with emission peaks of 345nm and 361nm.

[0076] Example 4 Synthesis of UCNP@MSN-PcASO / C39(UMAC)

[0077] 1. Synthesis of UCNP@MSN-PcDNA

[0078] PcDNA was modified onto the surface of UCNP@MSN using an EDC / NHS chemical method: PcDNA, 50 mg EDC, and 25 mg NHS were added to 1 mL PBS (pH=5.5) and incubated at 37 °C for 1 h. Immediately afterwards, UCNP@MSN-NH2 was added, and the pH was adjusted to 8. Incubation was then carried out overnight at 4 °C (PcDNA: 20 μM; UCNP@MSN-NH2: 2 mg / mL). -1 The obtained nanoparticles UCNP@MSN-PcDNA were centrifuged and washed to remove unreacted PcDNA, and then redispersed in DMSO for later use (the control sample was prepared in the same way, except that Pc was replaced with nPc).

[0079] Compared with UCNP@MSN, another characteristic peak (NH2≈874cm) was also observed. -1 This indicates that the single-stranded PcDNA is linked to the -NH2 in UCNP@MSN via an EDC / NHS condensation reaction through the -COOH group.

[0080] 2. Synthesis of UCNP@MSN-PcASO / C39

[0081] 10 mg of the UCNP@MSN-PcDNA prepared in step 1 was soaked in a DMSO solution containing 5 mg C-39 for 12 hours, followed by the addition of ASO. The mixture was then stirred for 12 hours to obtain UCNP@MSN-PcASO / C39. The UCNP@MSN-PcASO / C39 nanoparticles were then centrifuged and washed with PBS (100 mM, pH 7.4) to remove unreacted C-39.

[0082] UV-Vis absorption spectroscopy indicates successful preparation. Figure 10 The difference method confirmed that ASO and C39 were successfully loaded onto UCNP@MSN, with loading contents of 101.5 μmol / g·UCNP@MSN and 7.3% (C39), respectively. Figure 11 Zeta potentials showed that the charge on UCNP@MSN-PcASO / C39 (or UMAC) decreased from -28 mV to -35 mV, demonstrating the successful modification of the UCNP@MSN surface with PcASO. Figure 13 e).

[0083] Example 5: Controlled-release test of the UMAC drug delivery system

[0084] The UV absorbance and fluorescence values ​​of the UMAC drug delivery system were measured using a Nano-500 spectrophotometer and a Biotech microplate reader under 808 nm NIR or no 808 nm NIR conditions, and the C39 drug release curve was plotted. Figure 9 j).

[0085] In UMAC without 808nm NIR, only about 8.1% of C39 was released after 48 h, indicating that the designed photoactivated Pc-ASO structure blocked the mesoporous channels of MSN. Therefore, these drugs were contained within the nanoparticles. In the 808nm NIR-treated group, C39 release increased significantly, with approximately 55.7% of C39 released after 24 h, indicating that NIR significantly triggered C39 release. Figure 9 The results showed that after continuous irradiation, the drug release of UMAC reached 56.8%. The drug retention capacity of UMAC was evaluated by measuring the drug leakage rate in different solutions. After 24 hours of incubation without irradiation, very little drug release was observed, indicating that the prepared nanoparticles have good drug retention capacity. Figure 12 ).

[0086] Example 6 Synthesis of UMAC-Vessels (UMAC-M-Gal)

[0087] 1. Extraction of red blood cell membranes

[0088] Whole blood samples were collected from healthy C57BL / 6 mice and placed in anticoagulant tubes. Fresh whole blood was diluted with pre-chilled phosphate-buffered saline (PBS, pH 7.4), centrifuged at 4000 rpm for 5 minutes at 4°C, and washed three times with cold PBS. Red blood cells were collected. The washed red blood cells were dispersed in isotonic solution, and 4 volumes of hypotonic solution were added and mixed. The mixture was centrifuged at 800g at 4°C for 10 minutes. The precipitate was collected and washed with 0.25×PBS until the supernatant was clear. The RBC membrane was collected and stored at 4°C.

[0089] 2. Synthesis of distearylphosphatidylethanolamine-polyethylene glycol-galactose (DSPE-PEG2000-Gal)

[0090] 4-Aminophenyl β-D-galactopyranoside and NHS-PEG2000-DSPE were dissolved separately in PBS (pH=7.4). The 4-aminophenyl β-D-galactopyranoside solution was added dropwise to the DSPE-PEG2000-NHS solution at a molar ratio of 5:2, and the mixture was stirred under vacuum for 24 hours. The reaction mixture was dialyzed against a dialysis membrane for 24 hours to remove excess Gal.

[0091] 1 H nuclear magnetic resonance (H nuclear magnetic resonance) 1The structures of free Gal, free DSPE-PEG2000-NHS, and Gal-PEG-DSPE were detected by 1H NMR spectroscopy, and the results are as follows: Figure 14 As shown in the results, chemical shifts of Gal (4.85, 3.93, 6.78, and 6.97 ppm) were observed in the Gal-PEG-DSPE spectrum, indicating successful preparation. The NHS band showed significant changes, indicating that the substitution of the N-hydroxysuccinimide group by Gal led to the disappearance of the 3.18 ppm band. Other CH2 bands in the Gal-PEG-DSPE NHS-PEG-DSPE ranged from 3.36 to 3.14, 2.73 to 2.66, and 1.26 to 1.24 ppm, respectively, which may be due to the high electronegativity of the aromatic groups.

[0092] 3. Preparation of Gal-modified erythrocyte membrane sacs (RM-Gal)

[0093] The RBC membrane prepared in step 1 was dispersed in PBS (pH=7.4), and the DSPE-PEG2000-Gal prepared in step 2 was added. The mixture was stirred at 37°C for 1 hour. Afterwards, it was centrifuged at 800g for 5 minutes and washed three times with PBS to obtain RM-Gal. The RM-Gal was then resuspended in PBS (pH 7.4) for later use.

[0094] 4. Preparation of UCNP@MSN-PcASO / C39-M-Gal (UMAC-M-Gal)

[0095] The 1 mL UMAC (2 mg·mL) prepared in Example 4 was used. -1 The mixture was combined with 200 nm RM-Gal or RM (unmodified erythrocyte membrane) and extruded multiple times through a 200 nm polycarbonate porous membrane to obtain UMAC-M-Gal or UMAC-M. The UMAC-M-Gal nanoparticles were centrifuged at 13,000 rpm for 5 minutes, washed three times with PBS (pH=7.4), and lyophilized. SEM images of UMAC-M-Gal are shown below. Figure 13 As shown in a; TEM image as shown Figure 13 As shown in b. SDS-PAGE protein analysis and Coomassie brilliant blue staining are as follows. Figure 13 As shown in Figure c, the results indicate that erythrocyte membrane proteins are extensively retained on the nanoparticles, demonstrating successful encapsulation of UMAC by the erythrocyte membrane. Dynamic light scattering (DLS) was used to monitor the size changes of the nanoparticles before and after erythrocyte membrane encapsulation, and the results are shown in Figure c. Figure 13 As shown in d, the results indicate that the size of UMAC-M-Gal is approximately 90 nm, consistent with the results from SEM and TEM. The final biomimetic nanoparticles exhibited a 30 nm increase in hydrodynamic diameter; the zeta potential increased to a level similar to that of RBC vesicles. Figure 13e). The stability of UMAC-M-Gal is as follows: Figure 15 As shown in a-15c, the results showed that UMAC-M-Gal was stable in water, phosphate-buffered saline (PBS), 100% fetal bovine serum (FBS), and media containing 10% fetal bovine serum, even for up to 14 days in FBS.

[0096] Example 7: Biological characteristics of UMAC-M-Gal

[0097] 1. Assess the cellular uptake of UMAC-M-Gal (ASO chain labeled with FAM) by liver-derived cells.

[0098] First, the specific expression of ASGPR in hepatocytes was detected by immunoblotting analysis, indicating that this receptor is expressed almost exclusively in hepatocytes compared to cells from other tissue sources. Figure 13 f). Then, HepG2.2.15 cells were co-incubated with UMAC-M-Gal (RM-Gal labeled with Dil and ASO labeled with FAM), and the HepG2.2.15 cells were analyzed. It was observed that most of the Dil signal (red) overlapped with the FAM signal (green) around the cell nucleus, confirming that the RBC membrane successfully enveloped the UMAC surface (f). Figure 13 g).

[0099] 2. Immune Escape Capacity Detection

[0100] The antiphagocytic ability of UMAC-M-Gal was detected using RAW264.7 mouse macrophages. Cells were co-incubated with nanoparticles from the UMAC, UMAC-M, and UMAC-M-gal groups, respectively, and imaging was performed using confocal laser scanning microscopy (CLSM). The results are shown below. Figure 16 As shown in Figure a. The mean fluorescence intensity was quantitatively detected using flow cytometry, and the results are as follows. Figure 16 As shown in b. The results showed that the UMAC group exhibited brighter green fluorescence and a higher average fluorescence intensity than the UMAC-M and UMAC-M-gal groups, indicating that coating the erythrocyte membrane can effectively reduce immune clearance.

[0101] Macrophage uptake assays were performed using ICP-MS to detect the Si content in the cytoplasm. The results are as follows: Figure 16 As shown in Figure c. The results showed that, compared with uncoated RBC nanoparticles, the biomimetic nanoparticles (UMAC-M and UMAC-M-Gal) with RBC membranes had lower uptake. This indicates that cell membrane protection can enhance the ability of nanoparticles to resist immune clearance.

[0102] In vivo studies were conducted on blood samples at different time points after intravenous injection. ICP-MS analysis of silicon content showed that the retention capacity of the nanoparticles encapsulating the RBC membrane in the blood was significantly enhanced. Figure 16 e). Since the phagocytes of the reticuloendothelial system (RES) are mainly distributed in the liver and spleen, a large number of nanoparticles accumulate during treatment. Detection showed that UMAC-M significantly reduced Si accumulation in both the liver and spleen, indicating that cell membrane encapsulation can, to some extent, reduce the uptake of nanoparticles by the RES.

[0103] 3. Testing of temporal and spatial controlled release capabilities

[0104] HepG2.2.15 cells were co-incubated with UMAC-M-Gal (PcASO) or UMAC-M-Gal (nPcASO) containing FRET-labeled molecules. After 3 hours of co-culture, the cells were treated with 808nm (1.5W / cm²) solution. 2 The cells were irradiated with a laser. The results were as follows: Figure 17 As shown in Figure ac, the results indicated almost no fluorescence signal in cells without laser irradiation. Significantly stronger green fluorescence was detected under near-infrared (NIR) irradiation, suggesting that NIR triggered the release of the ASO-FAM chain. Figure 17 a). Flow cytometry quantification and agarose gel electrophoresis showed consistent results. Figure 17 b、 Figure 23 ).

[0105] In contrast, the green fluorescence intensity of HepG2.2.15 cells incubated with FRET labeled UMAC-M-Gal(nPcASO) showed almost no change. Figure 17 The presence of PC groups (ac) indicates that PC groups are crucial for biomimetic drug delivery systems.

[0106] like Figure 18 As shown in Figure a, a weak green fluorescence signal was observed when incubated with free ASO chains, because naked DNA cannot penetrate the cell membrane. In contrast, cells co-incubated with UMAC showed a stronger fluorescence signal, indicating that the nanoparticles can promote ASO chain entry into cells. CLSM analysis and flow cytometry showed that UMAC-M-Gal had the highest internalization efficiency, suggesting that Gal-modified biomimetic membrane vesicles are crucial for improving the internalization of UMAC drug delivery systems. Figure 19 Almost all of the fluorescence signal was located in the intracellular region, indicating that UMAC-M-Gal is absorbed via endocytosis.

[0107] Colocalization studies were conducted to evaluate the internalization of UMAC-M-Gal in L02 cells. After 2 hours of incubation, UMAC-M-Gal showed good colocalization with lysosomes labeled with a red fluorescent lysosomal probe, indicating that UMAC-M-Gal is more likely to enter the cell via endocytosis. Figure 20 ).

[0108] 4. Evaluate the targeting ability of biomimetic nanoparticles

[0109] Antibody blocking assays were performed on L02 cells using anti-ASGPR receptor antibody. The results showed that UMAC-M-Gal exhibited a weak fluorescent signal in the cytoplasm after blocking. Figure 21 ).

[0110] Confocal analysis in various non-hepatic cell lines (A549, HeLa, and MCF7 cells) showed that the fluorescence accumulation of UMAC-M-Gal in non-hepatic cell lines was significantly weaker compared to the strong fluorescence signals in L02 (normal hepatocytes) and HepG2.2.15 cells (a cell model in which the HBV genome has been integrated into chromosomal DNA). Figure 22 a). Flow cytometry analysis showed that, after incubation, the number of UMAC-M-Gal cells bound to hepatocytes was significantly higher than that of non-hepatocyte cell lines (a). Figure 18 b, Figure 22 b). ICP-MS results confirmed that the Gal-modified biomimetic nanoparticles exhibited significantly more silicon accumulation ( Figure 22 c). These results indicate that UMAC-M-Gal has high hepatocyte targeting specificity.

[0111] The biodistribution of UMAC-M-Gal in vivo was tracked in real time using an in vivo fluorescence imaging system. Twenty-four hours post-injection, a biomimetic drug delivery system labeled with the lipophilic fluorescent dye DiR detected a significant accumulation of UMAC-M-Gal in the liver. Figure 18 (c, d). To investigate the biodistribution of different nanoparticles in vivo, the content of nanoparticles in major organs of mice was analyzed by detecting Si content. Consistent with in vivo fluorescence imaging, Gal-modified nanoparticles exhibited good liver-targeting ability. Figure 16 d). Meanwhile, the low accumulation of UMAC-M and UMAC-M-Gal in the spleen also confirms that erythrocyte membrane encapsulation enables nanoparticles to evade immune responses. Furthermore, the high accumulation of UMAC-M-Gal in the liver may be attributed to the targeting ability of Gal-modified cell membranes.

[0112] Example 8: NIR light-triggered in vitro anti-HBV effect of UMAC-M-Gal

[0113] 1. Temperature changes were assessed by detecting the effects of NIR laser-induced thermal effects in DMEM and UMAC-M-Gal solutions containing 10% fetal bovine serum under 808 nm NIR irradiation. Results showed that at concentrations of 800 μg / mL and 5 W / cm², the effects of NIR laser-induced thermal effects were minimal. 2 No high thermal effect was detected after 10 minutes of near-infrared irradiation.

[0114] 2. L02 and HepG2.2.15 cells were used to evaluate the in vitro cell viability of UMAC-M-Gal, where the potential cytotoxicity of the nanoparticles was negligible. First, the cells were divided into four groups: the first group was used to determine the effect of different concentrations of nanoparticles on cell viability; the second group was irradiated with 808 nm for 30 min; the third group was irradiated with 808 nm at different powers; and the fourth group was used to evaluate the cytotoxicity of different concentrations of UMAC-M-Gal at fixed power and irradiation time. The results showed that the nanoparticles had no effect on cell viability. Figure 24 a). Even when the laser power reaches 5W / cm² 2 Irradiate for 5 minutes or 1 W / cm 2 After 10 minutes of irradiation, the cells in the irradiated group did not show significant cytotoxicity, indicating that near-infrared laser had no effect on the survival rate of hepatocytes. Figure 24 b, 24c). Co-incubated with 800 μg / mL UMAC-M-Gal and heated with 1.0 W / cm 2 After 10 minutes of near-infrared light irradiation, over 90% of HepG2.2.15 and L02 cells survived, demonstrating its high biocompatibility and potential value in biomedical applications. Figure 24 d).

[0115] 3. The inhibitory effect of UMAC-M-Gal on in vitro HBV replication.

[0116] HepG2.2.15 cells were used to evaluate the inhibitory effect of UMAC-M-Gal on HBV in vitro. Its mechanism of action is as follows: Figure 25 As shown in a, the operation method is as follows: Figure 25 As shown in b. The results are as follows. Figure 25 As shown in dg, the results indicate that the combined use of ASO and C39 enhanced the inhibitory effect on HBV. Simultaneously, the biomimetic drug delivery system significantly improved antiviral efficacy.

[0117] HBV 3.5kb RNA and intracellular HBV DNA were detected by qRT-PCR. The primer sequences used are shown in Table 2 and SEQ ID NO. 3-8. The levels of HBV viral antigens (such as HBsAg and HBeAg) in the supernatant were detected by enzyme-linked immunosorbent assay (ELISA). The procedure is as follows: Figure 25 As shown in c.

[0118] Table 2 RT-qPCR Primer

[0119]

[0120] Due to the absence of the PC group, UMAC-M-Gal had no significant effect on HBV replication under NIR irradiation, which may be due to its ability to inhibit drug release through hybridization with nPcDNA. Figure 25 In contrast, the HBV 3.5kb RNA, intracellular HBV DNA, and HBV viral antigen in the PC-modified nanoparticle group were significantly reduced under NIR irradiation, indicating that the HBV inhibitory activity of UMAC-M-Gal is induced by NIR-mediated activation. Figure 25 jk).

[0121] The effects of biomembranes and Gal on the inhibitory effect of biomimetic nanodelivery systems on HBV replication. Results showed that erythrocyte membrane encapsulation and Gal modification could promote the delivery of biomimetic nanoparticles. However, the effect was not significant. Figure 26 Further verification is needed in in vivo experiments.

[0122] Example 9: In vivo anti-HBV effect of NIR light-triggered UMAC-M-Gal

[0123] Transgenic HBV mice (HBV-Tg C57BL / 6) encoding 1.2 excessively long copies of the HBV genome (serotype awy) were used as an animal model to evaluate the ability of the UMAC-M-Gal drug delivery system to inhibit HBV replication in vivo. Hemolysis assays showed that the biotoxicity of the nanoparticles was negligible. Figure 27 ).

[0124] like Figure 28 As shown in Figure a, on day 15 after using biomimetic nanoparticles and light treatment, parameters related to HBV replication in the serum of HBV-Tg mice are as follows. Figure 28 As shown in bf, the results indicate that all relevant parameters have decreased significantly. Figure 28 b shows that under near-infrared irradiation, serum HBV DNA in mice treated with ASO decreased by only 15%, while serum HBV DNA in mice treated with ASO and C39 NPs decreased by only 1.3%, indicating that biomimetic nanoparticles have the potential for functional cure. After treatment with biomimetic nanoparticles and NIR, the changes in HBsAg and HBeAg levels were largely consistent with the changes in HBV DNA levels, but no decrease was observed in HBV-Tg mice exposed only to NIR irradiation. Figure 28c, 28d). On day 15 after UMAC-M-Gal nanoparticle and NIR irradiation, immunohistochemical staining was used to investigate the expression levels of viral antigens (HBcAg and HBsAg) in the liver of HBV-Tg mice. Figure 28 f), its expression level decreased significantly. Furthermore, histopathological analysis (H&E staining) of various organs showed no organ damage. Figure 29 Meanwhile, weight during treatment ( Figure 30 a) and post-treatment blood biochemical parameters ( Figure 30 There were no significant changes in either bd or d. These experimental data confirm that UMAC-M-Gal has good biocompatibility and properties, and no systemic toxicity in vivo, showing great potential for functional treatment of chronic HBV infection.

[0125] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A dual-targeting near-infrared biomimetic drug delivery system, characterized in that: The invention comprises a erythrocyte membrane and a drug-loaded nanocarrier encapsulated within the erythrocyte membrane. The drug-loaded nanocarrier includes near-infrared responsive upconversion nanoparticles, an anti-HBV antisense oligonucleotide, and an anti-HBV nucleocapsid inhibitor. The surface of the upconversion nanoparticles is coated with mesoporous silica. The anti-HBV antisense oligonucleotide and the anti-HBV nucleocapsid inhibitor are respectively loaded onto the mesoporous silica. The nucleotide sequence of the anti-HBV antisense oligonucleotide is shown in SEQ ID NO.1, and the anti-HBV nucleocapsid inhibitor is C39. The near-infrared responsive upconversion nanoparticles have a core-shell structure. 2+ The preparation steps are as follows: NaYF4:Yb / Tm is the core, NaYF4:Yb / Nd is the shell. A thermal decomposition method was used. In a three-necked flask containing 12 mL oleic acid and 30 mL 1-octadecene, 1.6 mmol LnCl3·6H2O, 1.112 mmol YCl3·6H2O, 0.32 mmol YbCl3·6H2O, 0.008 mmol TmCl3·6H2O, and 0.16 mmol CaOA were added. The mixture was heated to 120 °C for 15 minutes under a nitrogen atmosphere, and then heated to 160 °C for 1 hour with stirring. After cooling to room temperature, 10 mL of a methanol solution containing 4 mmol NaOH and 7.2 mmol NH4F was added, and the mixture was stirred at 50 °C for 30 minutes. The methanol was then removed by heating, and the mixture was heated to 300 °C under vacuum for 1 hour. The mixture was then slowly cooled to room temperature. Ethanol precipitation was performed, and the mixture was centrifuged to obtain NaYF4:Yb / Tm / Ca. 0.8 mmol LnCl3·6H2O, 0.32 mmol YCl3·6H2O, and 0.4 mmol NdCl3·6H2O were added to a three-necked flask containing 6 mL oleic acid and 15 mL 1-octadecene. The mixture was heated to 120 °C for 15 minutes under a nitrogen atmosphere, followed by stirring for 1 hour, and then cooled to room temperature. 2.5 mL NaYF4:Yb / Tm / Ca was added dropwise, and the mixture was stirred at room temperature for 30 minutes, followed by stirring at 80 °C for 30 minutes. After cooling to room temperature, 5 mL of methanol solution containing 2 mmol NaOH and 3.6 mmol NH4F was added, and the mixture was stirred at 50 °C for 30 minutes. The methanol was then removed by heating. The mixture was heated to 300 °C under vacuum and reacted for 1 hour. After slow cooling to room temperature, ethanol was precipitated, and the mixture was centrifuged to obtain core-shell NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd.

2. The dual-targeting near-infrared bionic drug delivery system as described in claim 1, characterized in that: The anti-HBV nucleocapsid inhibitor is loaded within the mesoporous channels of mesoporous silica, and the anti-HBV antisense oligonucleotide is loaded on the surface of the mesoporous silica. Part of the anti-HBV antisense oligonucleotide is loaded in the pores of the mesoporous channels to block the release of the anti-HBV nucleocapsid inhibitor within the mesoporous channels.

3. The dual-targeting near-infrared biomimetic drug delivery system as described in claim 1, characterized in that: The surface of the red blood cell membrane is modified with a targeting molecule, namely β-D-galactosidase.

4. A method for preparing a dual-targeting near-infrared biomimetic drug delivery system, characterized in that: Includes the following steps: 1) Synthesis of UCNP: Preparation of NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd by thermal decomposition; A thermal decomposition method was used. In a three-necked flask containing 12 mL oleic acid and 30 mL 1-octadecene, 1.6 mmol LnCl3·6H2O, 1.112 mmol YCl3·6H2O, 0.32 mmol YbCl3·6H2O, 0.008 mmol TmCl3·6H2O, and 0.16 mmol CaOA were added. The mixture was heated to 120 °C for 15 minutes under a nitrogen atmosphere, and then heated to 160 °C for 1 hour with stirring. After cooling to room temperature, 10 mL of a methanol solution containing 4 mmol NaOH and 7.2 mmol NH4F was added, and the mixture was stirred at 50 °C for 30 minutes. The methanol was then removed by heating, and the mixture was heated to 300 °C under vacuum for 1 hour. The mixture was then slowly cooled to room temperature. Ethanol precipitation was performed, and the mixture was centrifuged to obtain NaYF4:Yb / Tm / Ca. 0.8 mmol LnCl3·6H2O, 0.32 mmol YCl3·6H2O, and 0.4 mmol NdCl3·6H2O were added to a three-necked flask containing 6 mL oleic acid and 15 mL 1-octadecene. The mixture was heated to 120 °C for 15 minutes under a nitrogen atmosphere, followed by stirring for 1 hour, and then cooled to room temperature. 2.5 mL NaYF4:Yb / Tm / Ca was added dropwise, and the mixture was stirred at room temperature for 30 minutes, followed by stirring at 80 °C for 30 minutes. After cooling to room temperature, 5 mL of methanol solution containing 2 mmol NaOH and 3.6 mmol NH4F was added, and the mixture was stirred at 50 °C for 30 minutes. The methanol was then removed by heating. The mixture was heated to 300 °C under vacuum and reacted for 1 hour. After slow cooling to room temperature, ethanol was precipitated, and the mixture was centrifuged to obtain core-shell NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd. 2) Synthesis of UCNP@MSN: Using CTAB as a template agent, NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd prepared in step 1) was reacted sequentially with tetraethyl silicate and 3-aminopropyltriethoxysilane to obtain mesoporous silica-coated UCNP. 3) Synthesis of UCNP@MSN-PcASO / C39: PcDNA was modified on the surface of the UCNP@MSN obtained in step 2), and then an anti-HBV antisense oligonucleotide and an anti-HBV nucleocapsid inhibitor were added to obtain the drug-loaded nanocarrier UCNP@MSN-PcASO / C39; the nucleotide sequence of the anti-HBV antisense oligonucleotide is shown in SEQ ID NO.1, and the anti-HBV nucleocapsid inhibitor is C39; 4) Synthesis of UCNP@MSN-PcASO / C39-M-Gal: The UCNP@MSN-PcASO / C39 obtained in step 3) was encapsulated in Gal-modified erythrocyte membrane sacs to obtain the drug-loaded nanocarrier UCNP@MSN-PcASO / C39-M-Gal encapsulated in erythrocyte membrane. The Gal-modified erythrocyte membrane sacs were prepared by modifying erythrocyte membrane sacs with distearate phosphatidylethanolamine-polyethylene glycol-galactose.

5. The preparation method of the dual-targeting near-infrared biomimetic drug delivery system as described in claim 4, characterized in that: In step 2), the mass-to-volume ratio of NaYF4:Yb / Tm / Ca@NaYF4:Yb / Nd to tetraethyl silicate is 1:5, calculated in mg / μL; the volume ratio of tetraethyl silicate to 3-aminopropyltriethoxysilane is 10:

1.

6. The preparation method of the dual-targeting near-infrared biomimetic drug delivery system as described in claim 4, characterized in that: In step 3), the loading of anti-HBV antisense oligonucleotides was 101.5 μmol / g·UCNP@MSN, and the loading of anti-HBV nucleocapsid inhibitors was 7.3% by mass percentage.

7. The use of the dual-targeting near-infrared biomimetic drug delivery system according to any one of claims 1-3 in the preparation of drugs for treating HBV.

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