Double-lock type viroid fluorescent nanoprobe for monitoring virus infection process as well as preparation method and application of double-lock type viroid fluorescent nanoprobe

Abstract

The invention provides a double-lock type viroid fluorescent nanoprobe for monitoring a virus infection process, and a preparation method and application of the double-lock type viroid fluorescent nanoprobe. The probe is composed of a Japanese encephalitis virus envelope E protein modified polyamidoamine dendrimer, and the polymer is loaded with a vRNA and depurine / depyrimidine endonuclease 1 (APE1) dual activated molecular beacon. The probe can penetrate through a blood brain barrier and enter virus-infected cells to release molecular beacons complementary with vRNA, and the molecular beacons are selectively split by APE1, so that specific fluorescence signal amplification in virus-infected lesions is realized. The double-activation strategy greatly improves the spatial accuracy and sensitivity of virus infection focus imaging. The invention provides a feasible method for monitoring the in-vivo virus infection level in real time and evaluating the effectiveness of antiviral drugs.

Description

Technical Field

[0001] The invention relates to the technical field of nanoprobes, and in particular to a double-locked virus-like fluorescent nanoprobe for monitoring the progress of viral infection, a preparation method and an application thereof. Background Art

[0002] In vivo imaging detection of viral infection lesions can provide more realistic and reliable information for the accurate diagnosis of viral diseases and the evaluation of the viral infection process, which is crucial for establishing effective diagnostic and treatment programs and formulating appropriate prevention and control strategies. However, viral infection in vivo is a complex and highly dynamic process, accompanied by the dissociation, replication and recombination of viral components, and the signal provided is very weak due to the small size of the virus and the rapid changes in the infection process. In addition, due to the complex and variable composition and structure of tissues and organisms, it is still a major challenge to accurately and specifically deliver fluorescent probes to the site of viral infection while avoiding clearance by the host immune system, reticuloendothelial system and lysosomes. Therefore, achieving high-sensitivity and high-specificity imaging detection of viral infection lesions in vivo has always been an urgent and challenging task.

[0003] Since a series of biochemical reactions occur during viral infection, such as viral nucleic acid replication, expression of target proteins and enzymes, the development of probes based on key signal molecules related to the viral infection process has become a common method for detecting and analyzing viral infection. Among them, viral RNA (vRNA) can be used as a very reliable biomarker to assess the degree of infection of various viruses, but the expression level of vRNA in the early stage of viral infection is low, resulting in insufficient signal output and poor signal-to-noise ratio (S / N) in imaging. In order to improve the sensitivity of RNA detection, various amplification strategies have been developed, such as enzyme-activated molecular beacons, rolling circle amplification, and strand displacement amplification, for high-sensitivity RNA detection. However, single-signal detection of vRNA during viral infection often encounters problems such as cross-hybridization or nonspecific binding, resulting in amplification of nonspecific signals in normal tissues, reducing the specificity and sensitivity of imaging of lesion sites. Given the unique advantages of dual-signal response probes in reducing false-positive results and improving detection accuracy, the development of dual-lock probes with spatially selective signal amplification to detect vRNA can improve the specificity and sensitivity of in vivo imaging, thereby achieving accurate imaging and monitoring of the viral infection process.

[0004] Furthermore, it remains a major challenge to effectively and precisely deliver nucleic acid probes to viral infection sites in the complex in vivo environment, as the probes may be cleared by the blood or immune system cells before reaching the target site. Virus-like nanosystems (VMNs), designed to mimic the structure or function of viruses, have recently emerged as a promising approach to achieve this goal. Our research group has developed a design strategy that mimics the viral envelope to obtain virus-like nanocarriers with the natural tissue phagocytosis and lysosomal escape capabilities of the virus, which can precisely and efficiently deliver fluorescent probes to viral infection sites in vivo. Specifically, we designed a reversible ROS-responsive virus-like nanoprobe that can monitor the progression of viral encephalitis in real time. However, despite the excellent targeting ability of VMNs to lesion sites in vivo, the detection of ROS alone in the complex in vivo environment during viral infection still faces challenges such as poor specificity, low sensitivity, and reduced accuracy. Therefore, the development of a design strategy for a dual-locked virus-like nanoprobe that is triggered simultaneously by two signaling molecules during viral infection is expected to achieve high-precision and high-sensitivity imaging of viral infection in vivo. Invention content

[0005] In view of this, the present invention proposes a double-locked virus-like fluorescent nanoprobe, preparation method and application for monitoring the progress of viral infection, which solves the technical problems in the prior art such as the difficulty in targeted delivery of nanoprobes, poor specificity and low sensitivity of in vivo fluorescence imaging.

[0006] According to the first aspect of the present invention, the present invention provides a double-locked viroid fluorescent nanoprobe for monitoring the progress of viral infection, the probe comprising a polyamidoamine dendrimer modified with Japanese encephalitis virus (JEV) envelope E protein, the polymer carrying a molecular beacon that can be dually activated. The double-locked viroid fluorescent nanoprobe can cross the blood-brain barrier and enter virus-infected cells to release molecular beacons complementary to vRNA, and is selectively cleaved by apurinic / apyrimidinic nuclease 1 (APE1), thereby achieving specific fluorescence signal amplification in virus-infected lesions, and achieving ultra-high specificity and high-sensitivity imaging of JEV-infected lesions in vivo.

[0007] Furthermore, the particle size of the double-locked virus-like fluorescent nanoprobe is 200-300nm; and the Zeta potential is 4.3mV.

[0008] According to the second aspect of the present invention, the present invention provides a method for preparing a double-locked viroid fluorescent nanoprobe, comprising the following steps: incubating a polyamidoamine dendrimer modified with phenylboronic acid with Japanese encephalitis virus envelope E protein to obtain a polyamidoamine dendrimer, and incubating the polyamidoamine dendrimer with a molecular beacon dually excited by vRNA and APE1 to obtain a double-locked viroid fluorescent nanoprobe.

[0009] Further, the polyamidoamine dendrimer and 4-(bromomethyl)phenylboronic acid are dissolved in a solvent and reacted at 75-85° C. for 20-28 hours to obtain a polyamidoamine dendrimer modified with phenylboronic acid;

[0010] Preferably, the polyamidoamine dendrimer is a fifth generation polyamidoamine dendrimer;

[0011] Preferably, the solvent is anhydrous dimethyl sulfoxide.

[0012] Furthermore, the polyamidoamine dendrimer modified with phenylboronic acid was dissolved in PBS buffer solution and incubated with Japanese encephalitis virus envelope E protein at room temperature for 30-40 minutes to obtain E protein modified polyamidoamine dendrimer, which was then incubated with molecular beacons dually excited by vRNA and APE1 at room temperature for 30-60 minutes to obtain a double-locked virus-like fluorescent nanoprobe.

[0013] Furthermore, the preparation of a molecular beacon dually excited by vRNA and APE1 includes integrating two base deletion sites and a sequence complementary to vRNA into the hairpin loop of the molecular beacon to obtain a molecular beacon dually excited by vRNA and APE1, which can be complementary to vRNA and selectively cleaved by APE1, thereby achieving specific fluorescence signal amplification in the virus-infected lesions.

[0014] The ratio of the total number of amine groups in the polyamidoamine dendrimer to the total number of negatively charged phosphate groups in the molecular beacon is (2-16):1;

[0015] Preferably, the ratio of the total number of amine groups in the polyamidoamine dendrimer to the total number of negatively charged phosphate groups in the molecular beacon is 4:1.

[0016] The double-locked viroid fluorescent nanoprobe is formed by loading the molecular beacon dually excited by vRNA and APE1 into the polyamidoamine dendrimer modified by phenylboronic acid through electrostatic adsorption.

[0017] The surface of the double-locked virus-like fluorescent nanoprobe is modified with JEV envelope E protein, which enables the probe to cross the blood-brain barrier and achieve specific targeting of JEV-infected lesions.

[0018] According to the third aspect of the present invention, the present invention provides a double-locked viroid fluorescent nanoprobe obtained by the above-mentioned double-locked viroid fluorescent nanoprobe or preparation method, which is used for imaging of Japanese encephalitis virus infection lesions for non-diagnostic purposes, and can be used for ultra-high specificity and high sensitivity imaging of JEV infection lesions.

[0019] According to the fourth aspect of the present invention, the present invention provides the use of a double-locked viroid fluorescent nanoprobe obtained by the above-mentioned double-locked viroid fluorescent nanoprobe or preparation method in the preparation and detection of antiviral drug products and in monitoring the progress of viral infection in vivo. The double-locked viroid fluorescent nanoprobe is used to detect the degree of JEV infection in the brain with high precision, high sensitivity and high specificity, and to evaluate the efficacy of different antiviral drugs.

[0020] Compared with the prior art, the double-locked virus-like fluorescent nanoprobe for monitoring the progress of viral infection, the preparation method and the application of the present invention have the following advantages:

[0021] The present application provides a double-locked virus-like fluorescent nanoprobe, preparation method and application for monitoring the progress of viral infection. The probe is composed of a polyamidoamine dendrimer modified by the E protein of the Japanese encephalitis virus envelope, and the polymer carries a molecular beacon dually activated by vRNA and APE1. The probe can pass through the blood-brain barrier and enter the virus-infected cells to release molecular beacons complementary to vRNA, and is selectively cleaved by APE1, thereby achieving specific fluorescent signal amplification in the virus-infected lesions. The probe requires two related inputs (vRNA and APE1) to generate signal amplification, which greatly improves the spatial accuracy of imaging of viral infection lesions, because the extent of viral infection can be identified by multiple markers, thereby distinguishing it from healthy tissue. Using this probe, fluorescent signals depending on the progress of viral infection were detected in the mouse brain. The fluorescence intensity is related to the viral load in the brain and weakens after antiviral drug treatment. The present application provides a feasible method for real-time monitoring of the level of viral infection in vivo and evaluating the effectiveness of antiviral drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings constituting part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments and descriptions of the present invention are used to explain the present invention and do not constitute an improper limitation on the present invention. In the drawings:

[0023] Figure 1 Schematic diagram of the design and working principle of the dual-locked virus-like fluorescent nanoprobe (DVMP) (a is a schematic diagram of the fluorescence response of the dual-trigger molecular beacon (DTMB) to viral RNA (vRNA) and apurinic / apyrimidinic endonuclease 1 (APE1), b is a schematic diagram of the preparation of DVMP, and c is a schematic diagram of the working mechanism of DVMP);

[0024] Figure 2Schematic diagram of the design and evaluation of in vitro DTMB dual-activation signal amplification (a is a schematic diagram of the fluorescence response of CMB (no AP site) and DTMB (two AP sites) to vRNA and APE1; b is the fluorescence spectrum of DTMB (50nM) to vRNA (5nM) and APE1 (1U / mL); c is the statistics of the fluorescence response signal intensity of DTMB and CMB to vRNA (5nM) and APE1 (1U / mL); d is the specificity of DTMB (50nM) response to JEV_vRNA (5nM) in the presence of APE1 (1U / mL), IAV_vRNA and JEV_vRNA represent the RNA sequences of influenza A virus and JEV, respectively; e is the fluorescence intensity of DTMB detecting vRNA (5nM) over time in the presence or absence of APE1 (1U / mL), and the data are expressed as mean±SD (n=3));

[0025] Figure 3 Specific ultrasensitive imaging of cells infected with JEV (a is a schematic diagram of DVMP preparation, b is TEM of DVMP. Scale bar, 100 nm; c is a schematic diagram of the Zeta potential of G5, JEV E protein-modified G5 (VMN) and DVMP; d is a confocal fluorescence image of BHK cells (JEV host cells) and non-host MDCK cells incubated with Cy5-labeled DTMB@G5 and DVMP at 4°C for 10 minutes; DTMB@G5 indicates that the nanoprobe is not modified with E protein. Scale bar, 40 μm; e is a flow cytometry detection of MDCK and BHK cells treated with Cy5-labeled DTMB, DTMB@G5 and DVMP, respectively; f is a confocal fluorescence image of JEV-infected BHK cells treated with iFluor488-labeled DVMP (green) and further stained with Lysotracker red (red). Scale bar, 20 μm. BHK cells infected with JEV Confocal fluorescence images (g) and intensity statistics (h) of cells treated with CVMPs or DVMPs after 24 h or without infection. CVMPs represent VMNs encapsulating CMBs. Scale bars, 20 μm. Data are presented as mean ± SD (n = 3).

[0026] Figure 4To evaluate the ability of DVMPs to target viral infection lesions in vivo (a is a schematic diagram of the mouse imaging process; b is a representative fluorescence image of nude mice at different times after injection of Cy5-labeled DTMB@G5 or DVMP; c is the fluorescence intensity statistics obtained from the brain shown in b; d and e are representative fluorescence images of the brain (d) and major organs (heart, liver, spleen, lung and kidney) (e) 1 hour after injection of Cy5-labeled DTMB, DTMB@G5 or DVMP, respectively; f is the corresponding fluorescence intensity statistics obtained from the brain shown in d and the major organs shown in e, and the data are expressed as mean ± SD (n = 3));

[0027] Figure 5 Highly sensitive imaging of virus infection lesions in vivo (a is a schematic diagram of intracerebroventricular injection of JEV and probes in mice; b is a fluorescent image of the JEV-treated mouse brain 12 hours after injection of PBS (left brain) or DVMP (right brain); c is an immunofluorescence image of a brain tissue section obtained from b, with viral RNA (vRNA), E protein, and cell nuclei in red, green, and blue, respectively; d is a schematic diagram of the application of DVMPs in the JEV-infected mouse model via intracerebroventricular injection; e is a representative fluorescent image of the JEV-treated mouse brain at different times after injection of DVMPs or CMB-encapsulated VMNs (CVMPs); f is the fluorescence intensity obtained from the mouse brain in e; g is the fluorescence intensity of the mouse brain 30 minutes after injection of DVMPs or CVMPs, respectively;

[0028] Figure 6 Figure 3 Time-dependent imaging of viral infection in vivo (a is the experimental timeline of intracerebroventricular injection of JEV, tail vein injection of different probes and fluorescence imaging; b is the in vivo fluorescence image of JEV-infected mice changing over time after tail vein injection of DVMP or CVMP; c is the quantification of fluorescence intensity in the mouse brain in b; d is the vRNA level in the mouse brain at different times of viral infection measured by qPCR after intracerebroventricular injection of JEV; e is the time-dependent fluorescence image of JEV-infected mice treated with manidipine; f is the change of fluorescence intensity over time in the brain of mice treated with manidipine and atorvastatin, respectively; g is the quantification of fluorescence intensity in the brain of JEV-infected mice treated with PBS, manidipine and atorvastatin). DETAILED DESCRIPTION

[0029] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0030] The present invention will be described in detail below with reference to the accompanying drawings and in combination with embodiments.

[0031] All DNA sequences were synthesized by Qingke Biotechnology Co., Ltd. (Beijing, China) and purified by HPLC. Apurinic / apyrimidinic endonuclease 1 (APE1) was purchased from New England Biolabs (Massachusetts, USA). LysoTrackerRed was purchased from YEASEN, and Hoechst 33242 was purchased from Life Technologies. Anti-JEV E protein antibody (GTX125867) and anti-APE1 antibody (rabbit mAb, catalog number: ab189) were purchased from GeneTeX and Abcam, respectively. JEV E protein was provided by Sangon Biotech Co. Ltd. (Shanghai, China). TaKaRa MiniBEST Viral DNA / RNA Extraction Kit and PrimeScript TM The Ⅱ1st Strand cDNA Synthesis Kit was purchased from Weike Biotechnology Co., Ltd. (Tianjin, China). All chemical reagents were of analytical grade and used without further purification.

[0032] Cell culture;

[0033] BHK and MDCK cells were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum, penicillin (100 mg / mL) and streptomycin (100 mg / mL) at 37°C in an atmosphere of 5% CO 2 . Before the experiment, the cells were cultured on a glass-bottom 35 mm confocal culture dish (Cellvis) or a 6-well plate (NEST) for 18 to 24 hours.

[0034] JEV infection triggers the nuclear and cytoplasmic translocation of APE1 in BHK cells;

[0035] BHK cells were cultured in 35 mm glass-bottom confocal culture dishes for 18-24 hours and then infected with JEV. After infection at 37°C for 24 hours, the cells were washed with phosphate buffered saline (PBS, 0.01 M) and then fixed with 4% paraformaldehyde for further immunofluorescence staining. Immunofluorescence analysis was performed using an antibody against APE1.

[0036] APE1-triggered vRNA signal amplification in vitro;

[0037] DTMB is prepared using the existing technology (C. Sheng et al., Spatially resolved in vivo imaging of inflammation-associated mRNA via enzymatic fluorescence amplification in amolecular beacon. Nat. Biomed. Eng. 6, 1074-1084 (2022)).

[0038] DTMB was prepared in annealing buffer (pH 7.4, 20 mM HEPES, 150 mM NaCl, 5 mM MgCl2); the solution was incubated at 95°C for 5 min, then slowly cooled to room temperature and stored at 4°C until use.

[0039] vRNA detection was performed in 100 μL reaction buffer (pH 7.4, 50 mM HEPES, 50 mM NaCl, 10 mM MgCl2, and 0.1 mg / mL BSA). To monitor the opening of the molecular beacon, 50 nM DTMB (with two AP sites) or CMB (traditional molecular beacon, no AP site) was incubated with 50 nM JEV vRNA in the absence of APE1, followed by fluorescence monitoring. In the fluorescence comparison assay, 50 nM DTMB or CMB was incubated with 50 nM JEV vRNA and 1 U / mL APE1 at 37°C for 40 minutes.

[0040] Excitation was at 620 nm and emission spectra were collected from 640 nm to 800 nm. The fluorescence response of various types of RNA (50 nM) to DTMB (50 nM) was also tested.

[0041] Dual activation signal amplification of DTMB in vitro;

[0042] APE1 can recognize and cleave DNA at the AP site during base excision repair (BER), and it has been reported that it is highly expressed in inflammatory cells and translocates from the nucleus to the cytoplasm. Here, we observed for the first time the translocation of APE1 from the nucleus to the cytoplasm in JEV-infected cells by immunostaining experiments, confirming that APE1 can be used as another marker for detecting viral infection in addition to vRNA. Therefore, in normal cells, DTMB labeled with a fluorophore and a quencher group at each stem end has only a low fluorescence background due to fluorescence resonance energy transfer (FRET), but in JEV-infected cells, APE1 transported from the nucleus to the cytoplasm can selectively cleave DTMB complementary to vRNA, thereby achieving specific fluorescence signal amplification of the virus infection foci, such as Figure 2 As shown in a.

[0043] Next, the feasibility of DTMB dual-activation signal amplification was verified in vitro. Fluorescence spectroscopy analysis showed that after the addition of vRNA, the fluorescence of DTMB increased slightly, while when vRNA and APE1 were added at the same time, the fluorescence intensity increased by about 11 times ( Figure 2 b and Figure 1 In addition, APE1-assisted signal amplification was not observed for conventional MB (CMB) with the same sequence as DTMB but without the AP site in the stem-loop ( Figure 2 c and Figure 1 ), which confirms the key role of the AP site in this design. In addition, in the presence of APE1, DTMB also showed high specificity in distinguishing JEV vRNA from other types of RNA ( Figure 2 d). The fluorescence intensity curve over time shows that DTMB activated by APE1 can detect the target vRNA with a high response rate. The signal is gradually and significantly amplified over time. In contrast, in the absence of APE1, DTMB only shows a slight increase in fluorescence intensity when detecting the target vRNA, and this intensity does not change over time ( Figure 2 e).

[0044] Preparation and characterization of DVMP;

[0045] 4-(Bromomethyl)phenylboronic acid-modified PAMAM G5 dendrimer (PBA-G5) was prepared using existing technology (C. Liu et al., A boronic acid-rich dendrimer with robust and unprecedented efficiency for cytosolic protein delivery and CRISPR-Cas9 gene editing. Sci. Adv. 5, eaaw8922).

[0046] Table 1 DNA sequence list

[0047]

[0048] / idSp / indicates AP (apurine-free pyrimidine) site, * represents phosphorothioate;

[0049] Cy5 is a fluorescent dye and BHQ3 is a fluorescence quencher;

[0050] C* is a thio-modification of the phosphate group of C; G* is a thio-modification of the phosphate group of G; A* is a thio-modification of the phosphate group of A; and T* is a thio-modification of the phosphate group of T.

[0051] Then, PBA-G5 dispersed in phosphate buffer solution (PBS, pH 7.4) was mixed with the E protein of Japanese encephalitis virus (JEV) and incubated at room temperature for 30 minutes. Finally, the E protein-modified G5 was incubated with the beacon of dual trigger molecules (DTMB) at a N / P (N is the total number of amine groups of PAMAM, P is the total number of negatively charged DNA phosphate groups) ratio of 4:1 at room temperature for 30 minutes. Subsequently, the obtained dual-locked viroid fluorescent nanoprobes (DVMPs) were used for in vitro and in vivo experiments. The water and particle size and zeta potential of DVMPs were tested using a DLS instrument (Malvern ZetasizerNano-2S, Cambridge, UK). The morphology of DVMPs was observed using a transmission electron microscope (JEOL, Japan, 80kV).

[0052] Agarose gel retardation test;

[0053] A 2% agarose gel containing GelRed (1:10,000) was prepared for further use. PBA-G5 with or without modified JEV E protein was incubated with DTMB at the specified N / P ratio (0, 1, 2, 4, 6, 10, 12, 16, and 20) to obtain DVMPs and DTMB@G5. Then, DVMPs and DTMB@G5 were mixed with 6× DNA loading buffer, respectively. These mixtures were added to each lane of the agarose gel. Electrophoresis was performed in 1× TAE buffer at a voltage of 110 V for 30 min. The gel was detected by the BioRad imaging system. The acquired images were analyzed using Image Lab 5.2 software.

[0054] Specific and highly sensitive imaging of JEV-infected cells;

[0055] To achieve in situ fluorescence imaging of virus-infected lesions in vivo, we further constructed a virus-like nanocarrier (VMN) consisting of a PAMAM G5 dendrimer modified with JEV envelope E protein on its surface ( Figure 3 a). VMN can completely bind DTMB through electrostatic interaction at an N / P (N is the total number of amine groups in G5, P is the total number of negatively charged DNA phosphate groups) ratio of 4 to form uniform particles (DVMPs) of approximately 230 nm in size ( Figure 3 b). The shift of the zeta potential of VMN (12.4 mV) to a lower value (4.3 mV for DVMP) confirmed that DTMBs were loaded into the virus-like nanocarriers ( Figure 3 c).

[0056] Then, the ability of DVMPs to target JEV-infected host cells (BHK) was examined by confocal imaging. Compared with the nanoprobe without E protein modification (DTMB@G5), DVMPs had strong binding to BHK cells, but weak binding to non-host MDCK cells ( Figure 3 d), which confirmed that DVMP can specifically target JEV host cells. Confocal fluorescence images and flow cytometry results ( Figure 3 e) also showed that the fluorescence intensity of BHK cells incubated with DVMP increased significantly compared with DTMB@G5, indicating that the E protein played a key role in the effective intracellular delivery of the nanoprobe. Next, we investigated the ability of DVMP to achieve nucleic acid release through lysosomal escape in host cells. We incubated DVMP loaded with iFluor 488-labeled DTMB with BHK cells at 37°C for 4 h, and stained the nucleus and lysosomes with Hoechst 33342 and Lysotracker red dye, respectively. Three-color imaging showed that the fluorescently labeled DTMB signal could be clearly seen outside the lysosomes after 4 hours, indicating that DVMPs do have the ability to escape from endosomal tracts and can achieve nucleic acid release, similar to the JEV virus ( Figure 3 f). These results suggest that DVMPs have virus-like abilities to specifically target host cells and release their genomes via endosomal escape, thereby enabling efficient targeted delivery of nucleic acid probes for viral infection imaging.

[0057] After confirming the probe-specific cellular internalization ability, the signal amplification ability of DVMPs in detecting vRNA in JEV-infected cells was further evaluated. Figure 3 As shown in g, 24 hours after BHK cells were infected with JEV, the use of CVMP (VMN encapsulating CMB) caused the cells to emit weak fluorescence. In contrast, cells treated with DVMPs showed a significantly higher fluorescence signal. Fluorescence quantification results showed that when BHK cells were infected with JEV and then cultured with DVMP, the cell fluorescence signal increased by 9.6 times compared with the fluorescence signal of cells not infected with JEV ( Figure 3 h), which confirms that DVMP has ultra-high specificity and sensitivity in amplifying signals for monitoring viral infection. These results all demonstrate that DVMP has greatly improved the sensitivity of vRNA imaging in JEV-infected cells.

[0058] Cell-targeted analysis;

[0059] Before the experiment, MDCK cells and BHK cells were seeded into 35 mm thick glass bottom culture dishes (NEST) and cultured in DMEM supplemented with 10% (v / v) FBS and 1% (v / v) penicillin-streptomycin for 18 to 24 hours at 37°C and 5% CO2. To remove excess mucus secretion, cells were washed three times with PBS and incubated with 100 μL DVMPs and DTMB@G5 (equivalent to 50 nM Cy5-labeled DTMBs) at 4°C for 10 minutes, and then washed before confocal imaging.

[0060] Cellular uptake and endosomal escape studies. BHK cells were cultured at a rate of approximately 2 × 10 5 The cells were seeded at a density of 100 / well in 35 mm glass-bottom confocal culture dishes (for confocal imaging, Cellvis) or six-well plates (for flow cytometry, NESTBiotechnology) overnight. Afterwards, BHK cells were cultured with fresh culture medium containing DVMPs or DTMB@G5 (equivalent to 50 nM Cy5-labeled DTMBs) for 4 hours. To study the intracellular localization of the probe, BHK cells were stained with LysoTracker Red according to the manufacturer's instructions. In addition, the cells in the six-well plates were collected into test tubes for flow cytometric analysis to further evaluate the cellular uptake. Intracellular localization analysis was performed using Image-Pro Plus 6.

[0061] Signal amplification imaging of vRNA in JEV-infected BHK cells;

[0062] BHK cells (about 2×10 5 Cells / well) were seeded in 35 mm glass-bottom confocal culture dishes. After 18 h of culture, BHK cells were washed twice with PBS (pH 7.4), then infected with JEV for 24 h (control group without virus), and then treated with CVMPs (G5 dendrimers modified with JEV E protein on the surface, encapsulating CMBs) or DVMPs (equivalent to 50 nM MBs) for 4 h.

[0063] The ability of DVMPs to target viral infection foci in vivo;

[0064] In order to successfully achieve specific targeting of JEV infection lesions, the targeting ability of DVMPs was evaluated in vivo. The nanoprobes (Cy5-labeled DTMB@G5 or DVMP) were injected into the tail vein of BALB / c nude mice, and fluorescent images of the mice were collected at different time points after injection ( Figure 4 a). Figure 4As shown in (b), 30 minutes after the injection of the probe, obvious fluorescence signals were observed throughout the mouse body. However, compared with mice injected with DTMB@G5, mice injected with DVMPs showed higher fluorescence signal enrichment in the brain. Even after 30 minutes, there was still obvious fluorescence signal enrichment in the brain of mice injected with DVMP, while the fluorescence signal in the body had basically disappeared. In addition, at 60 minutes after injection, the signal enrichment in the mouse brain was still obvious, and the fluorescence intensity was similar to that at 45 minutes after injection, indicating that DVMP had a stronger retention ability in the mouse brain. This also confirms that the virus-mimicking nanoprobe can effectively achieve targeted delivery to the mouse brain. However, 45 minutes and 60 minutes after the injection of DTMB@G5, the fluorescence signal in the mouse body was still strong, while the fluorescence signal in the brain decreased significantly, indicating that DTMB@G5 had a poor targeting ability to the mouse brain. The fluorescence quantification results of the mouse brain further demonstrated that the virus-like nanoprobe we designed has good brain targeting efficiency ( Figure 4 c). In addition, the fluorescence images of the brain and five major organs, including heart, lung, kidney, spleen, and liver, of mice injected with DTMB, DTMB@G5, or DVMP 1 hour later also confirmed that DVMP does have good brain targeting ability ( Figure 4 d-4f). The results showed that DVMPs designed to mimic the function of JEV have good potential for in vivo lesion-targeted imaging.

[0065] Animal models;

[0066] In vivo experiments were performed in accordance with the Regulations on the Administration of Experimental Animals in China, and all procedures were approved by the Animal Experiment Committee of Nankai University (SYXK (Tianjin) 2019-0003). BALB / c nude mice (female, 6-8 weeks) were purchased from Charles River Laboratories. Both sexes were allowed and they were free of any disease or infection during the experiment. Before intracerebroventricular (ICV) and tail vein (IV) injections, mice were anesthetized with 1.2% Avertin (0.02 mL / g body weight, Energy Chemical Company) by intraperitoneal injection. During ICV injection, the solution was injected 2.5 mm into each lateral ventricle of the brain. The injection site was 1.5 mm lateral to the midline and 0.6 mm caudal to the bregma. During bioimaging, mice were intravenously injected with 200 μL of probe. When virus-induced encephalitis was induced, 10 μL of 10 6 PFU / mL of JEV was injected intravenously into the mouse brain.

[0067] In vivo imaging;

[0068] BALB / c nude mice (female, 6-8 weeks) were randomly divided into different groups (n=4) and intravenously injected with DTMBs@G5, CVMPs and DVMPs (MBs dose was 2.5 nmol / kg). The fluorescence signals of each group were recorded at predetermined time intervals using an in vivo near-infrared-II imaging system (Invivo master).

[0069] Highly sensitive imaging of viral infection lesions in vivo;

[0070] Inspired by the in vitro amplification performance of DTMB, the applicability of DVMP in highly sensitive imaging of viral infection lesions in vivo was investigated. First, to confirm the ability of the probe to detect viral infection lesions in vivo, a viral infection model was constructed by injecting JEV into the mouse brain via the lateral ventricle ( Figure 5 a). After 12 hours, hematoxylin and eosin staining showed that the cells in the JEV-treated group proliferated significantly, indicating that the injection of JEV into the lateral ventricle caused an in vivo inflammatory response, proving that the construction of the virus infection model was successful. Then, DVMP was injected into the right lateral ventricle, and phosphate-buffered saline (PBS) was injected into the left lateral ventricle as a control, and live imaging was performed 12 hours after infection ( Figure 5 a). Fluorescence images of the mouse brain collected 10 hours after DVMP injection showed that the probe-treated lateral ventricle showed obvious fluorescent signals, while the PBS-treated lateral ventricle showed almost no fluorescent signals ( Figure 5 b). Immunofluorescence images of brain tissue sections further confirmed the ability of DVMP to specifically image viral infection lesions in vivo ( Figure 5 c). Next, we evaluated the feasibility of the nanoprobes for in vivo signal amplification imaging. One day after JEV was injected into the lateral ventricle of the mouse brain, we collected fluorescence images of the mice at different time points after injection of DVMPs or CVMPs ( Figure 5 d). The DVMP-treated brains showed a sustained fluorescence enhancement with the signal reaching a maximum at 30 minutes after injection, whereas no significant fluorescence enhancement was observed in the PBS-treated brains ( Figure 5 e and 5f). Quantitative analysis showed that 60 minutes after DVMPs injection, the fluorescence intensity of the brains pre-injected with JEV was 8.3 times higher than that of the brains pre-injected with PBS ( Figure 5 g). When similar tests were performed with CVMPs, only a slight increase in fluorescence was observed in the JEV-injected brain during imaging ( Figure 5 e-5g). This clearly indicates that the stronger fluorescence signal observed in the DVMP group is due to APE1-mediated signal amplification. These results collectively confirm that DVMP has significantly improved sensitivity in detecting viral infection compared to conventional MB-based strategies.

[0071] Real-time fluorescence quantitative polymerase chain reaction (qPCR);

[0072] vRNA was extracted from mouse brain homogenate and reverse transcribed. When performing real-time fluorescence quantitative qPCR, we first mixed transcription-specific primers and SYBR Green Master with cDNA from each sample in a final volume of 20 μL. Data were collected and analyzed using gene expression analysis software. The relative level of expression was calculated using the comparative method and displayed as fold change.

[0073] Medication

[0074] During infection, 10 μL of 10 6 PFU / mL solution. When using various drugs to treat JEV infection, the drug concentration and administration method were based on the recommendations in published literature. Manidipine (25 mg / kg) was injected intraperitoneally twice a day for the first two days after mice were infected with JEV, and then treated once a day; atorvastatin (5 mg / kg) was injected intraperitoneally once a day after mice were infected with JEV.

[0075] In vivo toxicity and biodistribution of DVMP;

[0076] In the in vivo toxicity study, 24 hours after intravenous injection of DTMB@G5 or DVMPs (DTMBs at a dose of 2.5 nmol / kg), the brain and major organs (heart, liver, spleen, lung and kidney) of mice were collected for biochemical and histological analysis, respectively. For biodistribution analysis, mice were euthanized 1 hour after injection of free DTMB, DTMB@G5 and DVMPs (DTMBs only labeled with Cy5, equivalent dose of 2.5 nmol / kg), and major organs were removed for imaging.

[0077] Time-dependent imaging of viral infection in vivo;

[0078] DVMPs have strong brain targeting ability and ultrasensitive in vivo imaging performance, so the feasibility of this probe to detect viral infection levels related to the progression of viral infection in vivo was further evaluated. DVMPs were intravenously injected into JEV-treated mice at different time points, and CVMPs were used as controls. The mice were imaged 30 minutes later ( Figure 6 a). Fluorescence images of mice were collected at different time points (12, 24, 36, 48, and 72 hours after infection). After mice were infected with JEV, the fluorescence intensity in the brains of mice treated with DVMPs increased significantly over time, indicating that the replication and expression levels of vRNA continued to rise. Obvious fluorescence signals were seen even in the brains of mice 12 hours after infection ( Figure 6However, no obvious signal was observed in the brains of mice treated with CVMPs 12 h after infection, and the fluorescence intensity in the brains of mice increased only slightly with the extension of virus infection time ( Figure 6 b and 6c). The results showed that the dual-activated probe has high specificity and sensitivity for imaging viral infection lesions in vivo and is therefore expected to be used for early diagnosis of viral infection. Next, we quantified vRNA expression using conventional real-time quantitative PCR (qRT-PCR) to assess the extent of viral infection ( Figure 5 d). Notably, the dynamic changes of vRNA at different time points correlated well with the in situ imaging results of DVMPs, confirming that the nanoprobes provide an accurate and effective method for in situ visualization and monitoring of viral infection progression in vivo. In addition, further treatment with the antiviral drugs manidipine and atorvastatin resulted in a statistically significant decrease in the fluorescence signal of DVMPs ( Figure 6 e-6g), suggesting its potential for evaluating the efficacy of antiviral drugs.

[0079] Immunofluorescence staining;

[0080] The brain tissue sections were washed twice with PBS and permeabilized with 0.3% (v / v) Triton X for 10 minutes, followed by blocking with 5% (w / v) BSA for 30 minutes. These steps were performed at room temperature. Subsequently, the slides were incubated with primary antibody (anti-JEV E protein antibody, dilution 1:300) at 37°C for 2 hours, then washed three times with PBS, and then incubated with secondary antibody (Dylight-488) at 37°C for 1 hour. After washing three times with PBS, the sections were stained with DAPI for nuclei.

[0081] Statistical analysis;

[0082] To compare the significance of the difference in transient diffusion coefficient, the t-test was used for statistical analysis. Significant differences were defined as (***p<0.001).

[0083] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A double-locked virus-like fluorescent nanoprobe for monitoring the progress of viral infection, characterized in that: The probe includes a polyamidoamine dendrimer modified with the Japanese encephalitis virus envelope E protein, and the polymer is loaded with a molecular beacon that can be dually activated by vRNA and apurinic / apyrimidinic nuclease APE1.

2. A double-locked virus-like fluorescent nanoprobe for monitoring the progress of viral infection according to claim 1, characterized in that: The particle size of the double-locked viroid fluorescent nanoprobe is 200-300nm; the Zeta potential of the double-locked viroid fluorescent nanoprobe is 4.3mV.

3. A method for preparing a double-locked virus-like fluorescent nanoprobe for monitoring the progress of viral infection according to claims 1-2, characterized in that: The method comprises the following steps: incubating phenylboronic acid modified polyamide dendrimer with Japanese encephalitis virus envelope E protein to obtain polyamidoamine dendrimer, and incubating polyamidoamine dendrimer with vRNA and APE11 dual excited molecular beacon to obtain double locked virus-like fluorescent nanoprobe.

4. The method for preparing a double-locked virus-like fluorescent nanoprobe for monitoring the progress of viral infection according to claim 3, characterized in that: The preparation method of the polyamide dendrimer modified by phenylboronic acid comprises: dissolving polyamide amine dendrimer and 4-(bromomethyl)phenylboronic acid in a solvent, reacting at 75-85° C. for 20-28 hours, and obtaining the polyamide amine dendrimer modified by phenylboronic acid; Preferably, the polyamidoamine dendrimer is a fifth generation polyamidoamine dendrimer; Preferably, the solvent is anhydrous dimethyl sulfoxide.

5. The method for preparing a double-locked virus-like fluorescent nanoprobe for monitoring the progress of viral infection according to claim 3, characterized in that: The polyamidoamine dendrimer modified with phenylboronic acid was dissolved in PBS buffer solution and incubated with Japanese encephalitis virus envelope E protein at room temperature for 30-40 minutes to obtain E protein modified polyamidoamine dendrimer, which was then incubated with molecular beacons dually excited by vRNA and APE1 at room temperature for 30-60 minutes to obtain a double-locked virus-like fluorescent nanoprobe.

6. The method for preparing a double-locked virus-like fluorescent nanoprobe for monitoring the progress of viral infection according to claim 3, characterized in that: The preparation of the molecular beacon for dual excitation of vRNA and APE1 includes integrating two base deletion sites and a sequence complementary to vRNA into the hairpin loop of the molecular beacon to obtain the molecular beacon for dual excitation of vRNA and APE1.

7. The method for preparing a double-locked virus-like fluorescent nanoprobe for monitoring the progress of viral infection according to claim 3, characterized in that: The ratio of the total number of amine groups in the polyamidoamine dendrimer to the total number of negatively charged phosphate groups in the molecular beacon is (2-16):1; Preferably, the ratio of the total number of amine groups in the polyamidoamine dendrimer to the total number of negatively charged phosphate groups in the molecular beacon is 4:

1.

8. The method for preparing a double-locked virus-like fluorescent nanoprobe for monitoring the progress of viral infection according to claim 3, characterized in that: The double-locked viroid fluorescent nanoprobe is formed by loading a molecular beacon dually excited by vRNA and APE1 into a polyamidoamine dendrimer modified by phenylboronic acid through electrostatic adsorption.

9. The double-locked viroid fluorescent nanoprobe according to claims 1-2 or the double-locked viroid fluorescent nanoprobe prepared by the preparation method according to any one of claims 3-7 is used for imaging of Japanese encephalitis virus infection lesions for non-diagnostic purposes.

10. Use of the double-locked viroid fluorescent nanoprobe according to claims 1-2 or the double-locked viroid fluorescent nanoprobe prepared by the preparation method according to any one of claims 3-7 in the preparation and detection of antiviral drug products.

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