Application of Virus Targeting Nanocarrier Drug Delivery System in

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Application of virus targeting nano-carrier drug delivery system in virus-induced central nervous system diseases treatment Song Zhu, Aiguo Huang, Fei Luo, Jian Li, Jing Li, Long Zhu, Liang Zhao, Bin Zhu, Fei Ling, and Gaoxue Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b06365 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 9, 2019

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ACS Applied Materials & Interfaces

Application of virus targeting nano-carrier drug delivery system in virus-induced central nervous system diseases treatment

Song Zhu, Ai-Guo Huang, Fei Luo, Jian Li, Jing Li, Long Zhu, Liang Zhao, Bin Zhu, Fei Ling, Gao-Xue Wang*

College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China

*Corresponding author: Gao-Xue Wang Northwest A&F University, Xinong Road 22nd, Yangling, Shaanxi 712100, China. Tel./fax: +86 29 87092102. E-mail address: [email protected] (G-X. Wang).

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Abstract Virus-induced central nervous system (CNS) diseases represent a significant burden to animal health worldwide. The difficulty in treating these diseases is mainly attributable to the elaborate barrier system, which limits the transport of drugs to the infected sites. Therefore, it is necessary to develop smart delivery technologies for these diseases treatment. In the study, viral nervous necrosis disease was studied as a model to evaluate the feasibility of multi-walled carbon nanotubes (MWCNTs) conjugated with virus-specific nanobody and antiviral drug for targeted therapy of virus-induced CNS diseases. The virus (named as PGNNV) was isolated, identified and purified from diseased grouper. A naïve phage-displayed alpaca nanobody library was constructed, and the purified PGNNV was used for biopanning of PGNNV-specific nanobody from the library. The targeted delivery system based on MWCNTs conjugated with polyethylenimine, ribavirin and PGNNV-specific nanobody was constructed, designated as MWCNTs-PEI-R-Nb. Targeting ability and treatment effects of the MWCNTs-PEI-R-Nb were checked both in vitro and in vivo. MWCNTs-PEI-R-Nb showed an increasing distribution in PGNNV-infected cells, and an obvious accumulation in the brain of PGNNV-infected zebrafish larvae. MWCNTs-PEI-R-Nb also showed a strong anti-PGNNV ability both in vitro and in vivo. The mortality of larvae treated with MWCNTs-PEI-R-Nb (equivalent to 100 mg/L ribavirin) was 27% during 10 days post infection, while that was 100% for the control group. The results so far indicated that MWCNTs conjugated with antiviral drugs and viral-specific antibody are effective means for virus-induced CNS diseases targeted therapy. 2


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Keywords: targeted therapy, virus-induced central nervous system diseases, nanobody, carbon nanotubes, phage-displayed alpaca VHH library, nervous necrosis virus

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1. Introduction Increasing viruses have developed unique strategies to gain access into the central nervous system (CNS), where they establish acute or persistent infections.1 In fact, the number virus-induced CNS diseases each year is greater than all bacterial, fungal and protozoa infections combined.2 Many viruses, such as nipah virus, influenza virus, rabies virus and nervous necrosis virus (NNV), have caused serious CNS diseases. The CNS is protected by an elaborate barrier system, such as the blood-cerebral spinal fluid (CSF) barrier and the blood-brain barrier (BBB). The relative impermeability of the barrier system results in the inability of some antiviral drugs to cross the barriers, limiting the amount of drugs reaching the target sites to tackle CNS viral infections.3 Therefore, developments in smart delivery technologies for targeted delivery of antiviral agents play a vital role in resolving difficulties faced by the antiviral therapy.4-5 With the rapid development of nanotechnology, various nanocarriers have been designed, making targeted delivery possible.4 Carbon nanotubes (CNTs) have been regarded as promising carriers in biomedicine attributable to their excellent properties, such as needle-like structure, high carrying capacity and biocompatibility.6-8 As carriers, CNTs are uniquely equipped to carry drugs and other ligands across biological membranes, particularly, they have shown an intrinsic ability to cross the BBB in vitro and in vivo.9-10 In recent years, CNTs-mediated drug delivery has been extensively studied to facilitate the BBB crossing and targeted delivery. 9, 11-12 For targeted delivery, nanocarriers need to conjugate with various targeting ligands, such as antibodies,13 aptamers,14 peptides11 and folic acid.15 Antibodies have 4


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been widely used as the targeting ligands owing to their unique properties, such as multi-targeting ligands, high specificity and affinity, and long-circulating time in bloodstream.13, 16 Among the existing antibody types, nanobodies (Nbs; ~15 kDa) are the smallest intact antigen-binding fragments, which are derived from the variable domain of heavy-chain antibodies (HCAbs) in the camelid (camels, llamas and alpacas).17-18 The small size of Nbs makes their intrinsic accessible into cells, and has little impact on the size of nanocarriers. Furthermore, Nbs also offer special advantages compared to others, such as improved robustness, high affinity to antigens, and easily expressed in various expression hosts.19-21 To date, targeted delivery have been primarily employed in cancer treatment, while few studies have focused on the treatment of viral diseases, especially on the virus-induced CNS diseases. In the study, the disease of viral nervous necrosis (VNN) was studied as a model to evaluate the feasibility of targeted delivery application in virus-induced CNS diseases treatment. The causative agent of VNN is NNV, which classified as a member of the Nodaviridae family.22 The notable advantages of employ VNN as the model including: (1) safety. Aquatic animals constitute the narrow natural host range of NNV, and human is non-susceptible;23 (2) widespread distribution and easy accessibility. NNV has been isolated from at least 120 marine and freshwater species;24 (3) simpleness. NNV is a non-enveloped and icosahedral RNA virus with a diameter of 20-30 nm, and considered as one of the smallest virus. Moreover, due to it has one of the smallest and simplest genomes (4.5 kb in total) among the known viruses, a lot of information about NNV has been detailedly reported. The viral RNA2 (1.4 kb) 5



has a single ORF that encodes a 37 or 42 kDa capsid protein, and viral genome is encapsulated by 180 molecules of the capsid protein;24-25 (4) susceptibility of zebrafish. Zebrafish has been used as a powerful model in CNS and BBB research to increase the understanding of brain function, CNS disorders, and genetic and pharmacological modulation in human.26-28 Zebrafish is susceptible to NNV under the suitable conditions,29 thus, it can be used as an animal model. NNV used in the study was isolated from infected pearl gentian grouper (Epinephelus lanceolatus × Epinephelus fuscoguttatus) using snakehead cell line (SSN1), and named as pearl gentian grouper nervous necrosis virus (PGNNV). A naïve phage-displayed alpaca nanobody library was constructed, and the purified PGNNV was used for biopanning of PGNNV-specific nanobody from the library. A targeted delivery system based on multi-walled carbon nanotubes (MWCNTs) conjugated with polyethylenimine (PEI), ribavirin and PGNNV-specific nanobody (Nb) was constructed. Targeting ability and treatment effects of the targeted delivery system were checked both in vitro and in vivo. The present research will expand the targeted therapy to the field of virus-induced CNS diseases, and provide reference for the application of nanotechnology in viral diseases. 2. Experimental section 2.1. Isolation, identification and purification of PGNNV Diseased pearl gentian grouper juveniles (3.0-4.0 cm in total length) were collected from the Maoming Binhai New Area Chenxi Biotechnology Co., Ltd. (Maoming, China). The diseased grouper showed clinical signs like anorexia, lethargy, 6


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darkening of skin pigmentation and abnormal spiral swimming behaviour, which are the typical symptoms of VNN.24-25 Both histopathological examination and PCR analysis using the specific primer pair30 were performed on the brain tissue of diseased grouper for disease diagnosis. The PCR product was analyzed on 1.5% agarose gels. SSN-1 was used to isolate and propagate PGNNV according to the method described by John et al., (2014),31 and 50% tissue culture infective dose (TCID50) of the PGNNV was measured. In order to identify the PGNNV genotype, the full sequence of RNA2 was sequenced by Sangon Biological Engineering Technology Services Co., Ltd (Shanghai, China). The PGNNV was purified according to a previous study.32 The purified PGNNV was examined by SDS-PAGE and transmission electron microscopy (TEM; JEM 1200EX, Japan). 2.2. Construction of a naïve alpaca VHH library A naïve alpaca VHH phage display library was constructed according to a previous study.20 The library size was measured by serial dilutions, and 96 colonies were randomly collected to detect the insertion rate and diversity of the library by PCR and DNA sequencing. Helper phage M13K07 was used for library rescue, and phage titers were checked by counting colony-forming units (cfu). After rescuing, the Nbs were expressed on phage surfaces. 2.3. Biopanning and identification for PGNNV-specific nanobody Biopanning was performed on 96-well plates (Corning, MA) with five panning cycles. The 96-well plates were coated with PGNNV (100 μL, 100 μg/mL) at 4°C overnight, and then blocked by incubation with 3% BSA or 2% ovalbumin (OVA) 7



solution at 37°C for 1 h. In the first cycle of panning, 100 μL of the naïve VHH library (1×1012 cfu/mL) was added into the PGNNV coated well and incubated at 37°C for 1 h, then washed with PBST [0.01 M PBS (pH 7.4), 0.10% Tween-20]. The binding phages were eluted with 100 μL of elution buffer [0.2 M glycine-HCl (pH 2.2), 1 mg/mL BSA] and neutralized with 1 M Tris–HCl (pH 9.0). For subsequent cycles of panning, the concentrations of coating PGNNV were decreased to 75, 50, 25 and 5 μg/mL, respectively. Meanwhile, the concentrations of Tween-20 in elution buffer were increased to 0.20%, 0.30%, 0.40% and 0.50%, respectively. Following each cycle, the eluted phages were cultured with Escherichia coli TG1 for amplification. Twenty-four colonies were randomly picked from the fifth biopanning cycle and used to infect TG1 with helper phage M13K07 for phage amplification and purification. PGNNV binding activities of the purified phages were tested by phage ELISA. Briefly, 96-well ELISA plates were coated with 100 μL PGNNV solution (10 μg/mL) and blocked with 3% BSA solution at 37°C for 1 h, followed by washed with PBST. Phage supernatant of each clone was added into the wells and incubated at 37 °C for 1 h. After washing with PBST, 100 μL of HRP-conjugated anti-M13 antibody (1:5000; GE Healthcare, UK) was incubated in each well at 37°C for 30 min. TMB substrate (100 μL/well) was introduced into the washed wells, and the absorbance at 450 nm was measured after the reaction terminated by adding H2SO4 (50 μL/well, 2 M). The ELISA positive phage clones were selected and sequenced with M13R-48 (5′-CAGGAAACAGCTATGACC3′) sequencing primer. 2.4. Expression, purification and characterization of nanobody 8


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Plasmid DNA was extracted from the positive clones and ligated into expression vector pET-25b (+) after digestion by restriction enzymes Nco I and Not I. The ligation products were transformed into Escherichia coli Rosetta (DE3) cells, and then cultured in LB medium (containing 100 μg/mL ampicillin) at 37 °C by shaking to OD600~0.60. Afterwards, 0.20 mM IPTG was added into the culture, and incubation at 30°C for 6 h with shaking. Expression of Nb was analyzed by SDS-PAGE. Nb contains 6×His tag was purified with Ni-chelating affinity chromatography, further purification was performed on a chromatography system (AKTA; GE Healthcare) with an anionexchange column. The purified Nb was further analyzed by SDS-PAGE. PGNNV binding activity of purified Nb was checked by indirect ELISA, which was similar to the phage ELISA (as described above). HRP-conjugated anti-His antibody instead of HRP-conjugated anti-M13 antibody was used to detect the PGNNV binding activity. 2.5. Synthesis and characterization of constructs Pristine MWCNTs (P-MWCNTs) were purchased from Chengdu Organic Chemicals Co., Ltd., Chinese Academy of Sciences (Chengdu, China) and oxidized by H2SO4/HNO3 mixture (3:1, v/v) to form carboxyl groups on the surface of MWCNTs (O-MWCNTs).33 Modification of MWCNTs with PEI to obtain MWCNTs-PEI was performed according to a previous study.34 Conjugation of MWCNTs-PEI with ribavirin to form MWCNTs-PEI-R was according to Zhu et al., (2015).35 Unbound ribavirin was collected and determined by Hitachi L2000 HPLC System (Hitachi, Tokyo, Japan). Ribavirin loading efficiency (%) is defined as (weight of ribavirin loaded on MWCNTs-PEI-R/ weight of MWCNTs-PEI-R) × 100. 9



MWCNTs-PEI-R (1.0 g) and butanedioic anhydride (1.0 g) were ultrasonically dispersed in 30 mL N, N-dimethylformamide (DMF), and the mixture was stirred for 24 h at 70°C. N-hydroxysuccinimide (NHS; 1.0 g) and diisopropylcarbodiimide (DIC; 2.0 mL) were added into the mixture and stirred for 24 h at room temperature. The resulting mixture was filtered and washed thoroughly with excess PBS (pH 7.4). The product (0.50 g) was ultrasonically dispersed in 200 mL PBS, then the purified Nb (0.20 g) was gradually introduced into the suspension and stirred for 48 h at 4°C. The mixture was filtered and thoroughly washed, and then dried using a freeze dryer (FD5-3, GOLD-SIM) to obtain MWCNTs-PEI-R-Nb. Unbound Nb in the filtrate was determined using a BCA protein assay kit (ComWin Biotech Co., Ltd., Beijing, China). Nb loading efficiency (%) is defined as (weight of Nb loaded on MWCNTs-PEI-RNb/weight of MWCNTs-PEI-R-Nb) × 100. For conjugation of fluorescein isothiocyanate (FITC), MWCNTs-PEIR/MWCNTs-PEI-R-Nb (0.50 g) was ultrasonically dispersed in PBS (pH 5.0), FITC (0.20 g) was then introduced and continued sonicating for 2 h in the dark. The mixture was filtered and thoroughly washed, and then dried using the freeze dryer to obtain MWCNTs-PEI-FITC-R/MWCNTs-PEI-FITC-R-Nb. Amount of the unbounded FITC was determined using a spectrophotometer (Hitachi, Japan) at 520 nm. FITC loading efficiency (%) is defined as [(weight of FITC loaded on MWCNTs-PEI-FITCR/MWCNTs-PEI-FITC-R-Nb)/(weight of MWCNTs-PEI-FITC-R/MWCNTs-PEIFITC-R-Nb)] × 100. The P-MWCNTs and MWCNTs-PEI-R-Nb were characterised by the TEM and 10


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high resolution TEM (HR-TEM; Tecnai G2 F20, USA), respectively. An X-ray photoelectron spectroscopy (XPS; PHI-5600, Russia) was used to analyze elemental compositions of P-MWCNTs, O-MWCNTs, MWCNTs-PEI and MWCNTs-PEI-R-Nb. Thermo-gravimetric analysis (TGA; Mettler Toledo, Switzerland) was carried out to further qualitatively or quantitatively characterize the modification of MWCNTs. Particle size (nm) and zeta potential (mV) of P-MWCNTs, O-MWCNTs, MWCNTsPEI, MWCNTs-PEI-R and MWCNTs-PEI-R-Nb were determined by dynamic light scattering (DLS) analysis (ZEN3600, Malvern, UK). UV-vis absorption spectra of OMWCNTs, MWCNTs-PEI and MWCNTs-PEI-FITC-R-Nb were recorded on a 3900H UV-vis spectroscopy (Hitachi, Japan). 2.6. In vitro evaluation of PGNNV targeting SSN-1 cells were maintained at 25°C in Lebovitz-15 medium (L-15; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; ZETA LIFE, USA). To check the targeting ability of targeted delivery system to PGNNV in vitro, SSN-1 cells were cultured on cover slips in 12-well plates and grown to a monolayer. Following infected with PGNNV (103 TCID50) for 24 h, the cells were treated with FITC, MWCNTs-PEIFITC-R or MWCNTs-PEI-FITC-R-Nb with the same FITC concentration (5.0 ug/ml) for 4 h, respectively. Free FITC treatment was conducted as the control. After incubation, the cells were washed with PBS (pH 7.4) three times and fixed with 4.0% paraformaldehyde. Subsequently, cell membrane and nucleus were respectively dyed with 5.0 mg/mL Dil and 1.0 mg/L DAPI (Beyotime, China). After thoroughly washed with PBS, the cells were observed under a confocal microscopy (Nikon, Japan). 11



For flow cytometry analysis, SSN-1 cells were seeded in 6-well plates and grown to a monolayer. Similar treatments to the confocal microscopy observation were performed without staining with Dil or DAPI. The treated cells were resuspended in PBS and analyzed using a BD FACSAria III flow cytometer (BD, USA) at 488 nm. 2.7. In vivo evaluation of PGNNV targeting Adult wild-type AB zebrafish (Danio rerio) were obtained from China Zebrafish Resource Center (Wuhan, China) and maintained at 28°C. The adult zebrafish and eggs used for the experiments were NNV free, as validated by PCR using the specific primer pair. Naturally spawned eggs were kept in petri dishes with a density of 100 eggs in 100 ml water at 28°C. After hatching, larvae were transferred to 12-well plates (10 larvae/mL). Larvae at the age of four days post-fertilization (dpf) were infected by immersion in water containing 105 TCID50/mL PGNNV at 25°C for 12 h. Following infection, larvae were exposed to FITC, MWCNTs-PEI-FITC-R or MWCNTs-PEIFITC-R-Nb with the same FITC concentration (10 ug/ml) for 12 h, respectively. Free FITC treatment was conducted as the control. After thoroughly washed with water, the larvae were observed using the confocal microscopy. For tissue section observation, larvae were collected from each treatment and fixed in 4.0% paraformaldehyde. Paraffin-embedded larvae were processed and cut into 5 μm-thick sections. The sections were stained with 5.0 mg/mL Dil, and then observed under a fluorescence stereomicroscope (Leica DM5000B, Germany). 2.8. Anti-PGNNV activity in vitro SSN-1 cells were seeded in 12-well plates and grown to a monolayer. Following 12


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infected with PGNNV (103 TCID50) for 2 h, the cells were treated with ribavirin, MWCNTs-PEI-R or MWCNTs-PEI-R-Nb with the same ribavirin concentrations (2.5, 5.0 and 10 ug/mL). Free ribavirin treatment was conducted as the control. After treatment for 48 h, cells were collected and thoroughly washed with PBS. Total mRNA was extracted from SSN-1 cells with Trizol (TaKaRa, Japan) and reverse transcribed into cDNA. Expression of PGNNV RNA2 was evaluated by reverse transcription quantitative real-time PCR (RT-qPCR) using the specific primer pair (forward: 5′CTGCTAGAATCTTCCAGCGATA-3′

and

reverse:

5′-

TGTCAGTTGGATCAGGCAGGAA-3′). β-actin of SSN-1 cell was conducted as internal

standard

using

the

following

primer

pair:

forward

(5′-

CACTGTGCCCATCTACGAG-3′) and reverse (5′-CCATCTCCTGCTCGAAGTC3′). The PCR protocol consisted of an initial denaturation step at 95°C for 5 min followed by 40 cycles of 95°C for 15 s, 57°C for 30 s and 72°C for 20 s. Relative expression was calculated by using the 2-∆∆Ct method36 and normalized to the expression of the internal standard gene in the same sample. 2.9. Anti-PGNNV activity in vivo Twenty larvae (4 dpf) was maintained in each well of 12-well plates containing 2 ml water with 105 TCID50/mL PGNNV at 25°C for 12 h. After infection, larvae were treated with ribavirin, MWCNTs-PEI-R or MWCNTs-PEI-R-Nb with the same ribavirin concentrations (25, 50 and 100 ug/mL). Free ribavirin treatment was conducted as the control. One hundred and eighty larvae were used in each treatment. Larvae were fed twice a day with paramecium or artemia, and the compounds and water 13



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was replaced daily throughout the experimental period. Larvae were collected and sacrificed by fast freezing at -70°C. Five larvae were taken together for RNA extraction. Expression of PGNNV RNA2 was evaluated by RT-qPCR as described above. β-actin of zebrafish was selected as internal standard with the primer pair: forward (5′ATGGATGAGGAAATCGCTG-3′)

and

reverse

(5′-

ATGCCAACCATCACTCCCTG-3′). Same treatments were performed for mortality test. The larvae were monitored daily for a period of 10 days post infection. 2.10. Statistical analysis All of the treatments were carried out at least three times, and the data were expressed as mean ± standard deviation (SD). To perform statistical analysis, the SPSS 15.0 software (SPSS Inc., USA) was used. Data were analyzed for differences between the controls and treatments using one-way ANOVA followed by Tukey’s test, where p < 0.05 is considered significant. 3. Results and discussion 3.1. Isolation, identification and purification of PGNNV The CNS is protected by a highly complex barrier system, yet a wide variety of viruses still manage to gain access and induce diseases. VNN belongs to a member of virus-induced CNS diseases, and is considered as one of the most serious viral threats for aquatic organisms.24 Larvae and juvenile fish are especially susceptible to VNN with up to 100% mortality. The causative agent of VNN is NNV, which mainly infect the CNS of hosts, causing vacuolation and necrosis of brain and retina. 22, 24 NNV is a non-enveloped icosahedral RNA virus, and the virions (~25 nm) consist of 180 14


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molecules of a single-capsid protein that encapsulates a bipartite genome, RNA1 (~3.1 kb) and RNA2 (~1.4 kb). RNA1 encodes a non-structural RNA-dependent RNA polymerase (110 kDa), and RNA2 encodes a virus capsid protein (37 or 42 kDa). Therefore, NNV is considered as one of the smallest and simplest virus.25, 37 Based on the RNA2 T4 variable region, NNV were classified into four genotypes, including striped jack nervous necrosis virus (SJNNV), barfin flounder nervous necrosis virus (BFNNV), tiger puffer nervous necrosis virus (TPNNV) and red-spotted grouper nervous necrosis virus (RGNNV).38 In the study, NNV was isolated from infected pearl gentian grouper, named as PGNNV. As shown in Figure 1A, diseased grouper showed darker coloration than healthy grouper. By histopathological examination, conspicuous vacuolation was observed in the brain of diseased grouper (Figure 1B). Combined with the PCR diagnosis (Figure 1C), the diseased grouper was diagnosed with VNN. After inoculation with brain homogenate, SSN-1 showed obvious cytopathic effects (CPE), such as rounding of cells, vacuolation and detachment from the growth surface (Figure 1D). Similar CPE were reported by previous study.31 Virus titer in the infected SSN-1 cell suspension after 2 days infection was 107.4 TCID50/mL. Full-length of RNA2 was sequenced and deposited under the Accession No. MG637439 in the GenBank, NCBI. Sequence analysis of RNA2 showed that there is a high degree (98.46%) of homology with the coat protein gene of seven-band grouper nervous necrosis virus strain (Accession No. AB373029), which belongs to the RGNNV genotype. The sequence combined with others (38 NNV isolates) available in GenBank were used to determine 15



the phylogenetic relationships among the main betanodavirus types (Figure S1). The analysis indicated that PGNNV belongs to the RGNNV genotype. PGNNV was purified by ultracentrifugation using a cesium chloride density gradient and checked by SDS-PAGE. As shown in Figure 1E, PGNNV was concentrated in a narrow area following ultracentrifugation. SDS-PAGE analysis of purified PGNNV structural proteins revealed that the molecular weight of capsid protein is approximately 37 kDa, which is consistent with the calculated molecular weight (37.059 kDa) and other studies.39-40 According to the TEM observation (Figure 1F), PGNNV appeared icosahedral in morphology, and the average diameter was 25 nm.

Figure 1. (A) Image of grouper. Diseased grouper showed darker coloration than healthy grouper. (B) Histological section of brain of healthy and diseased grouper. (C) PCR diagnosis performed on the brain of diseased grouper using the NNV-specific primer. (D) Isolation of PGNNV using SSN-1 cells. The infected cells showed obvious cytopathic effects. (E) Purification of PGNNV by ultracentrifugation using 20-40% 16


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CsCl gradients. SDS-PAGE analysis revealed that the molecular weight of capsid protein is approximately 37 kDa. (F) TEM image of purified PGNNV. 3.2. Biopanning, expression and purification of PGNNV-specific nanobody Hamers-Casterman et al.18 (1993) found the HCAbs that devoid of light chains are existed in camelids. The antigen-binding site of HCAbs is comprised in one single domain (variable domain of HCAbs), referred to as VHHs or Nbs.18, 41 DNA encoding VHHs can readily be cloned and expressed in microbes to express protein that retain the antigen-combining properties of HCAbs. Nbs (~15 kDa) have a smaller size compared with others [such as mAbs (~150 kDa) and scFv (~30 kDa)], making their intrinsic ability to access into cells. In addition to the smaller size, several other significant advantages have been found, such as more stable, higher affinity and improved solubility compared with others.19, 42 All the advantages indicate that Nbs are excellent targeting ligands for targeted delivery. In the study, an anti-PGNNV Nb was selected from a naïve alpaca VHH phage display library and used as the targeting ligand. The schematic presentation of the naïve alpaca VHH library construction, PGNNV-specific Nb biopanning, expression, purification and characterization is present in Figure 2A. The obtained naïve alpaca VHH library contained approximately 1.7×109 individual clones, indicating that the library had a high sequence diversity. The phage titer was 1.3×1012 cfu/mL. After 5 panning rounds, a phage clone was selected as the PGNNV-specific Nb, which showed the highest binding activity to PGNNV identified by phage ELISA. The phage clone was sequenced, and the deduced amino acid sequence of Nb was further compared using the web-based program 17



IMGT/3Dstructure-DB.43 Similar to the variable domain of the heavy chain (VH) of conventional antibodies, the Nb is also composed of four framework regions (FRs), surrounding three complementarity determining regions (CDRs) (Figure S2). However, Nbs have larger sizes in CDR1 and CDR3 than that of VH. The large sizes of CDR not only provide a sufficiently large antigen interacting surface but also form a variety of paratope structures to recognize special antigenic epitope.44 Besides, FRs of the Nb contain more hydrophilic amino acids than that of VH, making Nb has improved solubility.21 VHH fragment from the positive clone was subcloned into the expression vector pET-25b (+), and then transformed into Escherichia coli Rosetta (DE3) cells for the expression of 6×histidine-tagged Nb. An obvious band (approximately 18 kDa) was detected in both supernatant and sediment fractions of the ruptured cells induced by IPTG (Figure 2B). Nb in the supernatant fraction was purified with Ni-chelating affinity chromatography. As shown in Figure 2C, several bands were identified due to the nonspecific binding. Thus, further purification was performed on a chromatography system (Figure 2D). Molecular weight of the recombinant Nb is approximately 18 kDa analyzed by SDS-PAGE (Figure 2C and D). As shown in Figure 2E, the purified Nb showed a high binding activity to PGNNV, indicating that the Nb can be used as a targeting ligand for PGNNV targeting.

18


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Figure 2. (A) Schematic presentation of the naïve alpaca VHH library construction, PGNNV-specific Nb biopanning, expression, purification and characterization. (B) Recombinant expression of Nb. Lane M: standard protein marker; Lane 1 and 2: supernatant (1) and sediment (2) fraction of mock cells [transfection of pET-25b (+) vector] induced by IPTG; Lane 3 and 4: supernatant (3) and sediment (4) fraction of non-induced cells; Lane 5 and 6: supernatant (5) and sediment (6) fraction of IPTGinduced cells. SDS-PAGE analysis of Nb that purified using Ni-chelating affinity chromatography (C) and chromatography system (D). Molecular weight of recombinant Nb is approximately 18 kDa. (E) Binding activity of purified Nb to PGNNV identified by ELISA. 3.3. Construction and characterization of targeted delivery system CNTs have emerged as promising carriers in biomedicine attributable to their excellent properties, such as needle-like structure, high carrying capacity and 19



biocompatibility.6-7 The intercellular diffusion rate of CNTs is higher than globular particles with similar weight due to the needle-like structure and high aspect ratio of CNTs.45 Drugs can be conjugated with CNTs by covalent or noncovalent interaction. Chemical compounds with extended π-structures, such as FITC, can be easily bound to CNTs by strong π-π interactions. Besides, CNTs-drug conjugates equipped with targeting ligands can not only improve their internalization efficiency, but also minimize the potential toxic side effects.6-7, 34 On the other hand, CNTs have also shown intrinsic therapeutic action by reduction in neuronal damage without carrying any drugs.46 In the present work, MWCNTs were covalently functionalized with PEI, and then conjugated with anti-NNV drug (ribavirin), PGNNV-specific Nb and FITC to construct a targeted delivery system (Figure 3A). Water-dispersibility and biocompatibility of MWCNTs can be significantly improved after modification with PEI. Furthermore, the PEI amines can be further modified with different surface functional groups for biomedical applications.47 For MWCNTs-PEI-R construction, ribavirin succinimide active ester was firstly obtained by reaction ribavirin with butanedioic anhydride,35 and then conjugated with the PEI amines. To reduce the effects on Nb binding activity, Nb was linked on the outermost layer of the targeted delivery system. FITC was conjugated by π-π interactions. As shown in Figure 3B, the P-MWCNTs were fibrous with varying lengths (1030 μm). O-MWCNTs (average length: 183 nm) have shorter lengths compared with the P-MWCNTs. The conjugation of PEI, ribavirin and Nb with O-MWCNTs was visually 20


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using HR-TEM as a layer around MWCNTs surface (Figure 3C). Besides, further confirmation of the synthetic constructs was performed using XPS (Figure 3D). The XPS spectrum of P-MWCNTs shows that MWCNTs surface consisted mainly of carbon (284 eV), as well as small amounts of oxygen (532 eV) and cobalt (782 eV). For O-MWCNTs, only the peaks of carbon and oxygen can be identified. Surface oxygen contents for P-MWCNTs and O-MWCNTs were 2.73 and 11.26 atomic %, respectively, indicating that the oxidation of MWCNTs was sufficient and the impurities were removed. Peak of nitrogen (399 eV) can be identified from the XPS spectrum of MWCNTs-PEI. Surface nitrogen contents for MWCNTs-PEI and MWCNTs-PEI-R-Nb were 5.44% and 6.19%, respectively, indicating the constructs were successfully synthesized. Loading efficiencies of ribavirin and Nb were 35.74% and 14.86% (50.60% in total), respectively. Conjugation of FITC onto the MWCNTs was confirmed via UV-vis spectroscopy, the absorption peak at 495 nm is related to the FITC typical absorption peak (Figure 3E), suggesting the successful conjugation. Loading efficiencies of FITC were 13.63% for MWCNTs-PEI-FITC-R and 11.04% for MWCNTs-PEI-FITC-R-Nb. As shown in Figure 3F, P-MWCNTs showed a better thermostability than OMWCNTs. Similar result was reported by Zhao et al., (2018), who demonstrated that weight loss of O-MWCNTs increased with increasing oxidization time due to MWCNTs were cut into shorter pieces with more defects of the graphitization structure (such as carboxyl groups) in both the ends and the sidewalls.48 PEI was absolutely degraded at 430°C. Base on the weight loss of O-MWCNTs (8.81%) and MWCNTs21



PEI (17.92%) at 430°C, the loading efficiency of PEI on MWCNTs was about 9.11%. Wu et al., (2014) constructed a MWCNTs-PEI compound and demonstrated the PEI grafting amount was about 9%,34 which is similar to our result. MWCNTs-PEI-R-Nb exhibited 72.21% mass loss at 800°C, while that was 24.06% for MWCNTs-PEI. The data indicate the total loading efficiency of ribavirin and Nb was approximately 48.15%, which is closed to the sum of ribavirin and Nb loading efficiency (50.60%, as described above). The particle size and zeta potential of the constructs were also measured, and the results were displayed in Figure 3G and Table S1. The average sizes for P-MWCNTs, O-MWCNTs, MWCNTs-PEI, MWCNTs-PEI-R and MWCNTs-PEI-R-Nb were 844.89, 264.53, 157.28, 230.51 and 253.06 nm, respectively. The data indicate that PMWCNTs were easily aggregated in water due to the hydration and reduction of electrostatic repulsion.49 After oxidization and PEI conjugation, dispersibility were significantly improved, similar results have also been reported in previous study.47 The sizes were gradually increased following ribavirin and Nb conjugation, proving that the targeted delivery system was successfully constructed. Zeta potential analysis (Table S1) revealed a negative surface charge (-19.50 ± 1.54 mV) for P-MWCNTs which decrease to -37.10 ± 5.77 mV following oxidization. MWCNTs-PEI have a zeta potential of 31.90 ± 3.45 mV, suggesting the successful conjugation of PEI. After ribavirin and Nb conjugation, the zeta potentials of MWCNTs-PEI-R (18.30 ± 3.64 mV) and MWCNTs-PEI-R-Nb (-32.30 ± 4.18 mV) were decreased, once again proved the successful construction of targeted delivery system. 22


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Figure 3. (A) Schematic presentation of the targeted delivery system construction. (B) Representative transmission electron microscopy images of P-MWCNTs (B) and MWCNTs-PEI-R-Nb (C). Characterization of the constructs using X-ray photoelectron spectroscopy (D), UV-vis spectroscopy (E), thermo-gravimetric analysis (F), and dynamic light scattering analysis (G). 3.4. PGNNV targeting For targeted delivery, nanocarriers conjugate with various targeting ligands, making most of the therapeutic agents distribute at the target site. In the study, targeting ability of the targeted delivery system was checked both in vitro and in vivo. The internalization of MWCNTs-PEI-FITC-R/MWCNTs-PEI-FITC-R-Nb in SSN-1 cells was assessed using confocal microscopy imaging and flow cytometry. Cells were incubated with free FITC (as the control treatment), FITC (as the mock treatment), MWCNTs-PEI-FITC-R or MWCNTs-PEI-FITC-R-Nb with the same FITC 23



concentration. As shown in Figure 4A, the cell membrane and nucleus were stained with Dil (red fluorescence) and DAPI (blue fluorescence), respectively. The green fluorescence corresponded to the FITC labelling on MWCNTs. For the FITC treatment, little FITC was attached on the cell membrane without entering into the cells. MWCNTs-PEI-FITC-R and MWCNTs-PEI-FITC-R-Nb were found to be internalized inside the cells after incubation for 4 h. A stronger green fluorescence was observed following exposure to MWCNTs-PEI-FITC-R-Nb than that of MWCNTs-PEI-FITCR, indicating that the enriched cellular uptake is correlated to the affinity of Nb to PGNNV. The cellular uptake was quantitatively measured using flow cytometry. As shown in Figure 4B and C, there was no significant difference between the control and mock treatments. In contrast, SSN-1 cells showed significantly higher fluorescence signals following exposure to MWCNTs-PEI-FITC-R-Nb for 4 h as compared with that of MWCNTs-PEI-FITC-R. The percentage of positively labeling cells for MWCNTsPEI-FITC-R-Nb treatment was 78.44%, while it was 32.57% for MWCNTs-PEI-FITCR treatment (Figure 4C). NNV mainly infects the CNS of hosts, causing vacuolation and necrosis of brain.22, 24

The CNS is protected by an elaborate barrier system, such as the CSF barrier and the

BBB. Many drugs can not cross the barriers due to the relative impermeability of barrier system, limiting the number of drugs reaching the target sites to tackle CNS viral infections.3 CNTs have emerged as promising carriers for drug delivery applications to the brain due to they have shown an intrinsic ability to cross the BBB both in vitro and in vivo.9-10 In recent years, many studies on the CNTs-mediated drug delivery have been 24


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performed to facilitate the BBB crossing and targeted delivery to brain.9,

11-12

For

example, Ren et al., (2012) have developed a drug delivery system based on MWCNTs modified with angiopep-2 and doxorubicin for treatment of brain glioma.50 Moreover, Guo et al., (2017) have fabricated dual-modified CNTs by both polyethylene glycol and lactoferrin for targeting dopamine delivery into the brain of parkinsonian mice.9 To date, NNV are known to affect more than 120 different fish and invertebrate species.24 Fish larvae are most sensitive to NNV infection, mortality among NNVinfected fish larvae is almost 100%. Zebrafish have an integrated nervous system, which is proposed to contain homologous brain structures to those found in humans, as well as equivalent cellular and synaptic structure and function.51 Its applications towards the goal of modeling major human CNS diseases have been widely studied to increase the understanding of brain function, CNS disorders, and genetic and pharmacological modulation in human.26-28 Moreover, zebrafish shows a highly conserved nature of both the structure and function of the BBB with mammals, making it as a model for assessing and predicting the permeability of BBB to compounds.27, 52 Danny et al., (2015) investigated the sensibility of zebrafish larvae at different stages to NNV, and demonstrated that larvae were most sensitive to NNV at the 4th dpf.53 Therefore, zebrafish larvae at the 4th dpf were used as the NNV infection model. For checking the targeting ability in vivo, distribution of the constructs in zebrafish was observed by confocal microscopy imaging. As shown in Figure 5A, the larvae showed auto-fluorescence in the control group. Although zebrafish have auto-fluorescence, we can still identify that MWCNTs-PEI-FITC-R mainly distributed in the abdomen of 25



larvae, while MWCNTs-PEI-FITC-R-Nb mainly distributed in the head. To further identification the distribution, tissue section observation was performed. As shown in Figure 5B, no obvious green fluorescence was observed in the control and mock groups. For MWCNTs-PEI-FITC-R exposure, it showed a systemic distribution, and only few distributed in the brain. On the other hand, MWCNTs-PEI-FITC-R-Nb displayed an enriched distribution in the brain, indicating that MWCNTs-PEI-FITC-R-Nb has an excellent targeting ability to PGNNV. Although CNTs are uniquely equipped to carry various ligands across biological membranes, they have no tissue selectivity. Thus, CNTs may enter and distribute in different tissues, causing side effects. CNTs conjugate with targeting ligands can not only improve their internalization efficiency, but also minimize the side effects.6-7, 34 Both in vitro and in vivo results illustrated that using anti-PGNNV Nb as the targeting ligand greatly increased ribavirin accumulation in the infection site.

26


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Figure 4. (A) Confocal microscopic images of SSN-1 cells incubated with free FITC (as the control treatment), FITC (as the mock treatment), MWCNTs-PEI-FITC-R and MWCNTs-PEI-FITC-R-Nb with the same FITC concentration for 4 h. Cell membrane and nucleus were stained with Dil (red channel) and DAPI (blue channel), respectively. The green fluorescence (green channel) corresponded to the FITC labelling on MWCNTs. Scale bars: 50 μm. (B) Flow cytometry analysis of the uptake of free FITC (as the control), FITC (as the mock treatment), MWCNTs-PEI-FITC-R and MWCNTsPEI-FITC-R-Nb with the same FITC concentration. (C) Percentage of the positively labeling cells calculated according to the flow cytometry analysis. Values are presented 27



as mean ± SD. **P < 0.01.

Figure 5. (A) Confocal microscopic images of zebrafish larvae incubated with free FITC (as the control treatment), FITC (as the mock treatment), MWCNTs-PEI-FITCR and MWCNTs-PEI-FITC-R-Nb with the same FITC concentration for 12 h. (B) Tissue sections of the zebrafish larvae in different groups. The red fluorescence (red channel) corresponded to the Dil, and the green fluorescence (green channel) corresponded to the FITC labelling on MWCNTs. Scale bars: 500 μm. 3.5. Anti-PGNNV activity Ribavirin is a synthetic guanosine nucleotide analog with in vitro and in vivo 28


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inhibitory activity against a broad range of DNA and RNA viruses.54 Morick and Saragovi, (2017) reported that pretreatment of zebrafish larvae with ribavirin reduces the mortality caused by NNV infection during the first 10 days post-infection.55 In the study, ribavirin was used as the anti-PGNNV drug. The anti-PGNNV activities of ribavirin, MWCNTs-PEI-R and MWCNTs-PEI-R-Nb (with the same ribavirin concentration) were evaluated both in vitro and in vivo. As shown in Figure 6A, RNA2 levels of PGNNV showed a significant decrease after exposure to ribavirin, MWCNTsPEI-R and MWCNTs-PEI-R-Nb. MWCNTs-PEI-R-Nb group had the highest antiPGNNV activity among the three constructs. For in vivo evaluation, zebrafish larvae at the 4 dpf were used as the NNV infection model, and the NNV infection was performed under the optimized environmental conditions as reported by Morick et al., (2015).29 As shown in Figure 6B, MWCNTsPEI-R-Nb

showed

a

significantly

stronger

anti-PGNNV

ability

than

ribavirin/MWCNTs-PEI-R after exposure at 100 mg/L for 3 and 5 d. As shown in Figure 6C, exposure of twenty zebrafish larvae to 105 TCID50/mL PGNNV at 25°C for 12 h was sufficient to kill all the larvae, while most (96%) of the uninfected larvae remained alive. The mortality of larvae treated with MWCNTs-PEI-R-Nb (equivalent to 100 mg/L ribavirin) was 27% during the 10 days of the experiment, while that were 58% and 39% for ribavirin and MWCNTs-PEI-R groups, respectively. Morick and Saragovi, (2017) demonstrated that the mortality of larvae pre-treated with ribavirin (100 mg/L) for 8 h before NNV infection was 59.4% during the 10 days of the experiment,55 which is similar to our data. These results clearly indicate that MWCNTs29



PEI-R-Nb can significantly inhibit the PGNNV and decrease the mortality of infected larvae.

Figure 6. Anti-PGNNV activity in vitro (A) and in vivo (B). Relative expression levels of RNA2 of PGNNV after exposure to ribavirin, MWCNTs-PEI-R and MWCNTs-PEIR-Nb (with the same ribavirin concentration) detected by RT-qPCR. The results are expressed as fold changes normalized to RNA2 levels in PGNNV-only control groups. Values are presented as mean ± SD. *P < 0.05; **P < 0.01. (C) Survival curves for zebrafish larvae. The larvae infected with PGNNV for 12 h, and then exposure to ribavirin, MWCNTs-PEI-R and MWCNTs-PEI-R-Nb with the same ribavirin concentration (100 mg/L). The larvae infected without PGNNV were performed as the mock-infected group. The data are representative of those from three independent experiments (n=180 for each group). 4. Conclusion In the study, VNN was studied as a model to evaluate the feasibility of CNTs conjugated with virus-specific nanobody and antiviral drug for targeted therapy of virus-induced CNS diseases. A virus was isolated from diseased pearl gentian grouper and named as PGNNV, which belongs to the RGNNV genotype. The nanobody with high affinity to PGNNV was acquired from a naïve alpaca VHH library. A targeted delivery system based on MWCNTs conjugated with PEI, ribavirin and Nb was 30


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successfully constructed. SSN-1 cells showed a significantly high cellular uptake of MWCNTs-PEI-FITC-R-Nb. Moreover, MWCNTs-PEI-FITC-R-Nb was tended to distribute in the brain, indicating that the targeted delivery system has an excellent targeting ability to PGNNV. MWCNTs-PEI-R-Nb also showed a strong anti-PGNNV ability both in vitro and in vivo, and the mortality of infected larvae was dramatically decreased after exposure to MWCNTs-PEI-R-Nb. Taken together, the results so far indicated that CNTs conjugated with drugs and viral-specific antibody are effective means for virus-induced CNS diseases treatment. The research shows bright future for targeted therapy of viral diseases using nanotechnology.

ASSOCIATED CONTENT Supporting Information Particle size and zeta potential of the constructs displayed in Table S1; Phylogenetic tree deduced from analysis of the RNA2 nucleotide sequences of 38 NNV showed in Figure S1; The amino acid sequence, putative complementary determining region (CDR) and framework region (FR) of nanobody showed in Figure S2.

Author information Corresponding Author *E-mail: [email protected] (G-X. Wang).

31



Acknowledgements This work was supported by the National Natural Science Foundation of China (No. U1701233).

32


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Application of Virus Targeting Nanocarrier Drug Delivery System in

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