Nanosphere for targeted delivery of siRNA based on SFTSV-Gn and application thereof

Abstract

The invention provides nano microspheres for targeted delivery of siRNA based on SFTSV-Gn and application of the nano microspheres, and belongs to the technical field of antiviral drugs. SiRNA is optimally designed, a nano microsphere delivery system is a nano compound formed by assembling optimally designed siRNA and cell-penetrating nano arginine, and the nano microspheres are used as a delivery carrier of siRNA to protect siRNA from enzymatic degradation, so that the siRNA delivery efficiency is improved, and the siRNA delivery efficiency is improved. And the target cells can be promoted to uptake, the replication of new bunyavirus RNA (Ribonucleic Acid) can be effectively inhibited, the injury of liver and lung tissues can be improved, and the survival rate can be increased. In addition, the surface-modified nano-microspheres have the advantages of high targeting, deep penetration, non-invasiveness and the like, and have wide application prospects in the antiviral field.

Description

Technical Field

[0001] The invention belongs to the technical field of antiviral drugs, and in particular relates to a nano-microsphere for targeted delivery of siRNA based on SFTSV-Gn and an application thereof. Background Art

[0002] The new bunyavirus, the fever and thrombocytopenia syndrome virus (SFTSV), is primarily transmitted by longhorned ticks and causes fever, fatigue, nausea, abdominal pain, thrombocytopenia, and leukopenia. In severe cases, SFTSV can invade lymphocytes, lymphoid tissues, and even lymphoid organs, potentially leading to multi-organ failure and death.

[0003] SFTSV is a spherical, enveloped, segmented, negative-strand RNA virus. The SFTSV genome consists of three segments: large (L), medium (M), and small (S). The L segment encodes RNA-dependent RNA polymerase (RdRp), which is responsible for replicating and transcribing the viral genome; the M segment encodes a glycoprotein precursor (Gp), which can be cleaved by proteases into two antigenic proteins (Gn and Gc); and the S segment mainly encodes viral nucleocapsid protein (N) and non-structural protein (NS). The function of the N protein is to protect RNA transcription and replication, while the NS protein is a key virulence factor in SFTSV infection, inhibiting the innate antiviral immune response by mediating the degradation of cytokines such as RIG-I, TLR3, and STAT. This inhibition of interferon and cytoplasmic nuclear factor signaling pathways enhances the replication potential of SFTSV. Currently, there is still a lack of an effective SFTSV treatment drug. Summary of the invention

[0004] Based on the above problems in the prior art, the present invention provides a nanoparticle for targeted delivery of siRNA based on SFTSV-Gn, characterized in that the sequence of the siRNA is 5'-3': GCCCUACAGAUUGGCUAAATT; 3'-5': UUUAGCCAAUCUGUAGGGCTT.

[0005] The nano-microspheres for targeted delivery of siRNA based on SFTSV-Gn include a shell structure capable of targeted delivery and a core structure capable of silencing viral genes, and the core structure contains the siRNA.

[0006] Wherein, the shell structure is prepared by modifying the synthetic Gn-PEG-DSPE.

[0007] Wherein, the core structure comprises a mixture of the siRNA and a cationic peptide.

[0008] The cationic peptide is nona-arginine, and the mixing ratio is nona-arginine: siRNA=30:1.

[0009] Wherein, the particle size of the nano-microspheres is 95-105nm.

[0010] Among them, the preparation method of the nano-microspheres for targeted delivery of siRNA based on SFTSV-Gn is as follows: after mixing siRNA and nona-arginine in proportion, a nano-complex is prepared by combining the shell structure, the cell membrane of the target cell is extracted, and the nano-complex is coated with the cell membrane of the target cell to form nano-microspheres.

[0011] The present invention also discloses the use of the above-mentioned nano-microspheres for targeted delivery of siRNA based on SFTSV-Gn in the preparation of a drug for treating hyperthermia with thrombocytopenia syndrome.

[0012] The present invention also discloses the use of the above-mentioned nano-microspheres for targeted delivery of siRNA based on SFTSV-Gn in inhibiting the replication of new bunyavirus.

[0013] The present invention also discloses the use of the above-mentioned nano-microspheres for targeted delivery of siRNA based on SFTSV-Gn in reducing the titer of new bunyavirus.

[0014] The present invention has the beneficial effects: The nano-microsphere delivery system disclosed in the present invention is composed of an optimized siRNA and a cell-penetrating nano-arginine assembly to form a nano-complex, and the nano-microspheres are used as siRNA delivery carriers to protect siRNA from enzymatic degradation and promote its uptake by target cells, which can effectively inhibit the replication of new bunyavirus RNA, improve liver and lung tissue damage, and improve survival rate. In addition, the surface-modified nano-microspheres have the advantages of high targeting, deep penetration and non-invasiveness, and have broad application prospects in the antiviral field. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is a schematic diagram of the design, synthesis and expected functions of the nano-microspheres provided by the present invention.

[0016] Figure 2 It is the structural formula of the synthetic cationic peptide nonaarginine (R9).

[0017] Figure 3 This is the HPLC result of nonaarginine purity verification.

[0018] Figure 4 This is the MALDI-TOF / TOF mass spectrometry verification result of nonaarginine.

[0019] Figure 5It is the result of agarose gel electrophoresis used to characterize the nanospheres.

[0020] Figure 6 The results of characterizing the nanospheres were obtained using dynamic light scattering (DLS) and zeta potential (ζ potential).

[0021] Figure 7 Transmission electron microscopy (TEM) characterization of the nanospheres (R9 / siRNA = 30:1).

[0022] Figure 8 This is the SDS-PAGE verification result of Gn-PEG-DSPE.

[0023] Fig. 9 This is the indirect immunofluorescence result of the targeted delivery siRNA nanoparticles based on SFTSV-Gn combined with the new Bunyavirus natural NP protein.

[0024] Fig.10 Agarose gel results used to evaluate the stability of nanospheres.

[0025] Fig.11 is the DLS evaluation result, which is used to evaluate the stability of nanospheres.

[0026] Fig.12 is the CCK-8 result used to evaluate the biosafety of nanospheres (48 h).

[0027] Fig.13 is the RT-qPCR result used to evaluate the inhibitory ability of nanospheres against SFTSV.

[0028] Fig.14 is the Western blot result used to evaluate the inhibitory ability of nanospheres against SFTSV.

[0029] Fig.15 is the immunofluorescence result used to evaluate the inhibitory ability of nanospheres on SFTSV.

[0030] in Fig. 9 and Fig.15 This is the result of immunofluorescence. The colors in the figure are part of the result. A color map is used to accurately display the experimental results. DETAILED DESCRIPTION

[0031] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0032] Example 1: A nanoparticle for targeted delivery of siRNA based on SFTSV-Gn and its application.

[0033] As attached Figure 1As shown, the present invention provides a nano-microsphere for targeted delivery of siRNA based on SFTSV-Gn, which includes a shell structure capable of targeted delivery and a core structure capable of silencing viral genes, wherein the shell structure is prepared by modification of synthetic Gn-PEG-DSPE; the core structure includes an optimized self-designed siRNA and nona-arginine as a cationic peptide, and is prepared by mixing nona-arginine and siRNA in a mixing ratio (nona-arginine: siRNA=30:1), and the sequence of the siRNA is: 5'-3': GCCCUACAGAUUGGCUAAATT; 3'-5': UUUAGCCAAUCUGUAGGGCTT.

[0034] The preparation method of nano-microspheres is as follows: after mixing siRNA and nona-arginine in proportion, a nano-complex is formed by combining the shell structure, and then the cell membrane of the target cell is extracted, and the nano-complex is coated with the cell membrane of the target cell to form a nano-microsphere with a particle size of 100nm. The nano-microspheres can be used in the preparation of drugs for treating hyperpyrexia with thrombocytopenia syndrome, and can also be used in inhibiting the replication of new bunyavirus and reducing the titer of new bunyavirus.

[0035] RNA interference (RNAi) is an emerging antiviral therapeutic technology, in which microRNA (miRNA) and small interfering RNA (siRNA) are most widely used. siRNA has become an important tool for antiviral therapy by controlling the expression of specific target genes by mediating post-transcriptional gene silencing. However, since siRNA is a negatively charged macromolecule, it is easily degraded by nucleases in serum and its cellular uptake efficiency is low. In order to improve the intracellular delivery efficiency of siRNA, delivery via a nanosphere delivery system can prevent nuclease degradation and enhance targeted delivery to infected cells or viruses.

[0036] Example 2: Synthesis of nona-arginine and siRNA.

[0037] The peptides were synthesized using a standard solid phase peptide synthesis (SPPS) protocol with 2-chlorotrityl chloride resin and N-Fmoc protected amino acids. Figure 2 The Nona-arginine (R9) shown is as follows: The resin was first swollen in anhydrous dichloromethane (DCM) for 5 min, and then the first arginine was loaded on the resin at the C-terminus of the resin using an Fmoc-protected amino acid (1.1 equiv.) and N,N-diisopropylethylamine (DIPEA) in DCM for 1 h.

[0038] After washing with DMF, the resin was stirred with blocking solution (DCM:MeOH:DIPEA=16:3:1) for 20 minutes to inactivate unreacted sites. The resin was then treated with 20% piperidine (in DMF) for 20 minutes to remove the protecting groups, and then Fmoc-protected amino acids (3 equivalents) were coupled to the free amino groups on the resin using HBTU as a coupling agent. These two steps were repeated to extend the peptide chain, which was performed by a standard Fmoc-SPPS protocol. The resin was washed 3 to 5 times with DMF after each step. The peptide was then cleaved with 95% TFA (2.5% ddH2O and 2.5% triisopropylsilane) to obtain a crude product, which was then purified by reverse phase HPLC and lyophilized to obtain a white powder. The results of HPLC are shown in Figure 3 shown.

[0039] As attached Figure 4 As shown, calculated using MALDI-TOF MS: 1423.7160 [M + H] + , the result is: 1423.2580.

[0040] siRNA was synthesized on a K&a H-8 DNA / RNA synthesizer.

[0041] Conventional CPG was used to synthesize the guide and passenger strands of siRNA. After standard solid phase synthesis, CPG cleavage and deprotection, siRNA was purified by HPLC and washed with CH3CN / H2O (0.1 M CH3COONH + ) as the eluent (CH3CN from 2% to 40% in 30 min). The collected product was desalted and lyophilized. Then, siRNA was dissolved in DEPC-treated H2O and its concentration was determined using NanoDrop 2000 (Thermo Scientific).

[0042] Example 3: siRNA functional evaluation.

[0043] To screen functional siRNAs, Vero cells were seeded in 12-well plates and, after 80% confluence, transfected with different siRNAs using Lipofectamine 2000 (11668019, Thermo Fisher Scientific) according to the manufacturer's instructions. The final concentration of siRNA was 100 nM.

[0044] After 5 hours, SFTSV at a multiplicity of infection (MOI) of 0.05 was incubated with Vero cells for 2 hours at 35°C. Unbound virus was removed and replaced with MEM containing 2% FBS and incubated at 35°C for 48 hours. Cells and supernatants were subjected to RNA extraction for reverse transcription real-time PCR (RT-qPCR) analysis.

[0045] The results showed that siRNA-3057 showed a higher inhibitory effect.

[0046] Example 4: Extraction of cell membrane and loading of Gn.

[0047] Cell membranes were obtained from Vero cells according to the following method: The collected cells were washed three times with PBS, and then they were dispersed in a membrane protein extraction buffer solution (P0033, Beyotime) and cooled in an ice bath for 15 min. The solution was subsequently frozen, thawed, and the freeze-thaw cycle was repeated three times. The mixture was then centrifuged at 700 g for 10 min at 4 °C, and the resulting cell suspension was further centrifuged at 14,000 g for 30 min at 4 °C, and the pellet was resuspended in deionized water and stored at -80 °C.

[0048] The Gn protein was coupled to the Vero cell membrane using a DSPE-PEG-NHS linker as follows: Gn (100 μg) was conjugated to DSPE-(PEG)2000-NHS (10 mg) in 2 mL PBS (10 mM, pH 7.4) overnight at 4° C. Unbound DSPE-(PEG)2000-NHS was removed by centrifugation and washing using an Amicon centrifugal filter unit with a molecular weight cutoff of 30 kDa (Millipore, UFC801096, three washes with 10 mM PBS, 4° C, 4500 g, 10 min each wash).

[0049] The supernatant was then collected, treated with non-reducing loading buffer, and analyzed by 10% SDS-PAGE. Figure 8 As shown. The gel was stained with a Coomassie Brilliant Blue staining kit according to the manufacturer's instructions. The resulting Gn-PEG-DSPE was then allowed to bind to the cell membrane for 24 h at 4°C. Unbound Gn-PEG-DSPE was concentrated and washed three times at 4°C in an Amicon centrifugal filter (Millipore, UFC810096, 30 kDa molecular weight cutoff) with cold PBS buffer (10 mM, pH 7.4, 4500 g, 10 min each wash).

[0050] To further purify the product, the resulting solution was subjected to ultracentrifugation at 100,000 g for 2 h at 4 °C, then washed once with PBS buffer (10 mM, pH 7.4) and resuspended in 2 mL PBS buffer (10 mM, pH 7.4).

[0051] Example 5: Preparation of nanoparticles.

[0052] R9 and siRNA-3057 (100 pmol) were mixed at different molar ratios of 5:1, 10:1, 20:1, 30:1, 40:1, and 50:1 to obtain nanospheres. After standing at room temperature for 30 min, they were analyzed by 2% agarose gel stained with gel red (D0140, Beyotime) and naked siRNA was used as a control. The results are shown in Figure 5 shown.

[0053] Nanosphere solutions (0.5 mL) with different molar ratios and Gn-loaded cell membrane solutions (from 107 cells, 0.5 mL) were mixed and the mixture was sonicated for 5 min at a frequency of 40 kHz and a power of 100 W using a Fisher Scientific FS30D bath sonicator. After standing for 2 h at 4°C, the mixture was centrifuged at 10,000 rpm for 5 min at 4°C and the pellet was resuspended in PBS to obtain cell membrane-coated nanospheres (nanospheres). They were analyzed by 2% agarose gel stained with Gel Red (D0140, Beyotime) and naked siRNA was used as a control. The results are shown in Fig.10 shown.

[0054] Zeta Potential and Dynamic Light Scattering (DLS): R9 and siRNA-3057 (100 pmol) were mixed at different molar ratios of 5:1, 10:1, 20:1, 30:1, 40:1, and 50:1 and then allowed to stand at room temperature for 30 min. The samples were diluted to 500 μL with ddH2O for dynamic light scattering (DLS) and Zeta potential analysis. The results are shown in Figure 6 , 7 and 11.

[0055] Cell viability assay: Vero cells were seeded in 96-well plates at a density of 5000 cells per well. After 12 h, the cells were treated with different concentrations of nanospheres. After 48 h of incubation, the cytotoxicity of the nanospheres was detected using a cell counting kit-8 (CCK-8) (C0038, Beyotime) according to the manufacturer's instructions. 10 μL of CCK-8 reagent was added to each well of the 96-well plate, and the cells were incubated at 37 °C for 1 h. The absorbance was read at a wavelength of 450 nm using a microplate reader (Bio-Rad), and the absorbance of cells without any treatment (OD control) was used as a control. The assay was performed at least three times independently, and the results are shown in Table 1. Fig.12 shown.

[0056] Example 6: SFTSV transfection and nanosphere transfection of cells.

[0057] To determine the effect of nanospheres on SFTSV, Vero cells were seeded in 12-well plates and pretreated with nanospheres at different concentrations for 4 hours before virus infection after reaching about 80% confluence, followed by incubation with SFTSV virus (MOI = 0.05) for 2 hours. Then, the virus-containing supernatant was replaced with nanospheres or medium containing nanospheres until the end of the experiment. RNA was extracted from cells and supernatants for subsequent reverse transcription real-time PCR (RT-qPCR) analysis, and proteins were extracted for western blot analysis.

[0058] RT-qPCR analysis: Total RNA was isolated using the RNeasy Mini Kit (74104, Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using the HiScript III RT SuperMix for qPCR Kit (R323, Vazyme Biotech), and qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme Biotech) according to the manufacturer’s instructions.

[0059] Primer: SFTSV Forward: 5'-AAGAGTGCGTTCATTATTG-3', reverse: 5′-ATTGCCTRAGGACATTGGTGAG-3′; Primer: GAPDH, served as internal control.

[0060] Forward: 5'-AGCCTTCCCAGTGGGTGAAGAC-3', Reverse: 5′-CGGAGTCACACGGATTTGGTCG-3′.

[0061] The PCR amplification process was as follows: 30 seconds at 95°C, followed by 40 cycles of 10 seconds at 95°C and 30 seconds at 60°C. The dose-response curve of viral RNA copies and drug concentration was plotted using GraphPad Prism 8 software. The results are shown in Fig.13 shown.

[0062] WB analysis: For western blot analysis, proteins were extracted using standard RIPA buffer (R0010, Solarbio) containing 1% PMSF. Total protein was quantified using a BCA protein assay kit (A55864, Thermo Scientific). A total of 20 mg of protein was denatured in 1× SDS loading buffer, resolved by 12% SDS-PAGE, and then transferred to a polyvinylidene difluoride (PVDF) membrane and blocked in 5% skim milk in TBST buffer containing 0.05% Tween 20. The PVDF membrane was then stained with primary antibodies against SFTSV Gn (01-05-118, Cambridge Bioproducts), SFTSV-NP (01-05-0130, Cambridge Bioproducts), and GAPDH (ab8245, Abcam). After washing, the membrane was further incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies and finally visualized using ECL reagents. The results are shown in Figure 2. Fig.14 shown.

[0063] Indirect immunofluorescence analysis: Vero cells were seeded in 12-well plates. After reaching about 80% confluence, SFTSV virus (MOI = 0.05) was added to the cells for infection for 2 hours, and then the virus-containing supernatant was replaced with different doses of medium containing nanospheres until the end of the experiment. All samples were washed three times with pre-cooled PBS, fixed with 4% paraformaldehyde (P0099, Beyotime) for 15 minutes, and permeabilized with 0.1% Triton-X for 10 minutes. After blocking with 2% bovine serum albumin (BSA, A95115G, Sigma) at room temperature for 1 hour, anti-NP primary antibody (SFTSV) was incubated at 37°C for 1 hour. After washing with 2% BAS, goat anti-mouse IgG h&L secondary antibody (AlexaFluor 488) (1:200; ab150113, Abcam) (1:2000; ab150113 Abcam) was incubated at 37°C for one hour. Cells were stained with mounting medium containing DAPI (ab104139, Abcam). Samples were examined and images were captured using a laser scanning confocal microscope system (Leica DMi8). Fig. 9 and 15 shown.

[0064] The above-mentioned embodiments only express various implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A nanoparticle for targeted delivery of siRNA based on SFTSV-Gn, characterized in that: The sequence of the siRNA is 5'-3': GCCCUACAGAUUGGCUAAATT; 3'-5': UUUAGCCAAUCUGUAGGGCTT.

2. A nanoparticle for targeted delivery of siRNA based on SFTSV-Gn according to claim 1, characterized in that: The invention comprises a shell structure capable of targeted delivery and a core structure capable of silencing viral genes, wherein the core structure contains the siRNA.

3. A nanoparticle for targeted delivery of siRNA based on SFTSV-Gn according to claim 2, characterized in that: The shell structure is prepared by modifying the synthesized Gn-PEG-DSPE.

4. A nanoparticle for targeted delivery of siRNA based on SFTSV-Gn according to claim 2, characterized in that: The core structure includes a mixture of the siRNA and a cationic peptide.

5. A nanoparticle for targeted delivery of siRNA based on SFTSV-Gn according to claim 4, characterized in that: The cationic peptide is nona-arginine, and the mixing ratio is nona-arginine: siRNA=30:

1.

6. A nanoparticle for targeted delivery of siRNA based on SFTSV-Gn according to claim 5, characterized in that: The particle size of the nano-microspheres is 95-105 nm.

7. The nanoparticles for targeted delivery of siRNA based on SFTSV-Gn according to claim 6, characterized in that: The preparation method is as follows: after mixing siRNA and nona-arginine in proportion, a nanocomposite is prepared by combining the shell structure, the cell membrane of the target cell is extracted, and the nanocomposite is coated with the cell membrane of the target cell to form a nano-microsphere.

8. Use of a nanoparticle for targeted delivery of siRNA based on SFTSV-Gn as claimed in any one of claims 1 to 6 in the preparation of a drug for treating hyperthermia with thrombocytopenia syndrome.

9. Use of a nanoparticle for targeted delivery of siRNA based on SFTSV-Gn as claimed in any one of claims 1 to 6 in inhibiting the replication of new bunyavirus.

10. Use of the nanoparticles for targeted delivery of siRNA based on SFTSV-Gn as claimed in any one of claims 1 to 6 in reducing the titer of new bunyavirus.

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