Study On The Nano Targeted Drug Delivery System Against Nervous Necrosis Virus

Fish viral nervous necrosis?VNN?is a worldwide fish epidemic infectious disease caused by nervous necrosis virus?NNV?.At present,more than 120 species of seawater and freshwater fishes from 54 families in 19 orders have been affected by the disease,and the infected species and the damaging degree are increasing.Especially for groupers,the survival rate within one month after hatching is often less than 10%,or even all die.NNV mainly infects the larvae and juveniles of fish.The immune system of fish at the susceptible stages has not been fully developed,and the fish itself cannot effectively produce protective antibodies when immunized with the vaccine,which limits the vaccine's prevention of VNN.Therefore,drug therapy is still an effective measure for the prevention and treatment of VNN.At present,the administration route of fish mainly includes injection,oral administration and bathing.Due to factors such as fry specifications and operation techniques,it is difficult to administer by injection.In addition,the fish larvae mostly feed on plankton,and oral administration is also greatly limited.Therefore,bath administration is the preferred method of VNN prevention and control.NNV mainly infects the nervous system of fish,and the nervous system is protected by a variety of biological tissues and barrier systems.The selective permeability of membrane and barrier systems will limit the transport of drugs,leading to a decline in the effectiveness of drug treatment.Therefore,the development of a new intelligent drug delivery system plays an important role in VNN treatment.Nano targeted drug delivery system refers to the use of nano-carriers to transport drugs to specific tissues or cells under the guidance of targeting ligands,and ultimately achieve the purpose of improving the effect of drug treatment.In this study,anti-NNV drugs,nanocarriers and NNV-specific targeting ligands were optimized for screening in order to obtain the optimal combination to construct a nano targeted drug delivery system.The targeting and anti-NNV activity of the system against NNV were evaluated both in vitro and in vivo.The results obtained in this study are as follows:1. Screening of anti-NNV drugsSSN-1 cells were used to isolate NNV from the brain tissue of diseased grouper.Based on the RNA2 gene sequence,the NNV was identified as a member of the red spotted grouper nervous necrosis virus?RGNNV?genotype.Twelve drugs with broad-spectrum antiviral activity were selected for in vitro and in vivo screening of anti-NNV drugs.In vitro,ribavirin showed the best anti-NNV activity,with a 90%inhibitory concentration(IC₉₀)of NNV of 7.18 mg/L,followed by amantadine.In addition,we explored the effects of amantadine and ribavirin on different stages of NNV infection.Results showed that the therapeutic effect of amantadine on NNV infection is better than ribavirin.Ribavirin mainly plays a preventive role on NNV infection.Unlike in vitro anti-NNV activity,amantadine showed the best anti-NNV activity in vivo,followed by ribavirin.The IC₉₀ for amantadine and ribavirin were 27.91 mg/L and 73.25mg/L,respectively.Following amantadine?40 mg/L?and ribavirin?100 mg/L?treatment for 7 days,the survival rates of diseased grouper were 44%and 39%,respectively.For the control groups,the grouper's survival rates were less than 10%.Based on the anti-NNV effects and the characteristics of the drugs,we finally selected amantadine as an anti-NNV drug to build the targeted drug delivery system.2. Screening of nanocarriersFe₂O₃-NPs,Fe₃O₄-NPs,single-walled carbon nanotubes?SWCNTs?,multi-walled carbon nanotubes?MWCNTs?and graphene oxide?GO?were selected as the research objects.Saccharomyces cerevisiae and Artemia salina were used as model organisms to check the biocompatibility,uptake,distribution and metabolism of the selected nanocarriers.The reproduction rate and cell viability of yeast cells were used as evaluation indicators to explore the cytotoxicity of the selected nanocarriers.The results showed that the order of toxicity of nanocarriers to yeast was:Fe₂O₃-NPs>Fe₃O₄-NPs>GO>SWCNTs>MWCNTs.SWCNTs and MWCNTs can enter into yeast cells via direct penetration and endocytosis,and then distributed in various organelles such as cytoplasm,vacuole,lysosome and mitochondria.In addition,SWCNTs can enter the nucleus of yeast cells,while MWCNTs only distributed around the nuclear membrane.Metabolic data showed that SWCNTs have a better metabolic stability than MWCNTs.The toxicity order of the selected nanocarriers to Artemia salina was:Fe₂O₃-NPs>GO>Fe₃O₄-NPs>SWCNTs>MWCNTs.For the larvae,SWCNTs and MWCNTs can be distributed in macrophages,lipid vesicles and intestines.After exposure for 48 h,the SWCNTs and MWCNTs contents in Artemia salina larvae reached the maximum values.Subsequently,MWCNTs were rapidly discharged by Artemia salina,while the process of SWCNTs discharge is relatively slow and stable.Based on the experimental results and the selection criteria of nanocarriers with good biocompatibility,strong penetrability and stable metabolism,we finally selected SWCNTs as the nanocarrier for targeted drug delivery systems construction.3. Screening of NNV specific nanobodiesThe purified NNV was used as an antigen to immunize alpaca,and the variable region gene fragment of the heavy chain antibody was amplified to construct an anti-NNV nanobody phage library.After identification,the actual library capacity was 1.27×10⁸ cfu and the diversity was good,which was enough for the subsequent screening test.Three rounds of affinity panning were performed on the phage library using Gly-HCl elution using NNV as the target.After panning,phages that specifically bind to NNV were effectively enriched,and the positive rate was significantly increased.From the third round of panning,120 phage monoclonals were randomly selected for affinity verification,and 22 high-affinity monoclonals were selected for sequence determination.Based on the sequence similarity of nanobodies,eight nanobodies were selected for the prokaryotic expression.After expression,the target protein with a size of about 17 k Da can be detected in the cell supernatant.Ni-NTA resin and anion exchange column were used to purify the expressed nanobodies,and the affinity of the purified nanobodies to NNV was determined.The results showed that NNV-VHH 6 has the strongest affinity with NNV.In addition,yeast two-hybrid and western blot results showed that NNV-VHH 6 binds to the P-domain of NNV both in vivo and in vitro.Based on the above results,we finally selected NNV-VHH 6 as the targeting ligand to construct the targeted drug delivery system.4. Construction of drug delivery system and its targeting ability to NNVAmantadine,SWCNTs and NNV-VHH 6 were selected as anti-NNV drugs,nano-carrier and targeting ligand to construct a nano targeted drug delivery system?SWCNTs-P-A-Nb?.At the same time,a drug delivery system?SWCNTs-P-A?without nanobodies was constructed as the control for subsequent experiments.The morphological characteristics,element types,particle size distribution,Zeta potential and thermal stability of the drug delivery system were characterized to determine the successful construction of the drug delivery system.In vitro,the targeting ability of drug delivery system to NNV was analyzed by fluorescence microscopy,transmission electron microscopy?TEM?,and flow cytometry.Compared with SWCNTs-P-A,more SWCNTs-P-A-Nb can enter into the infected cells,and the specific binding of SWCNTs-P-A-Nb to NNV can be clearly observed by TEM.In vivo,the targeting ability of drug delivery system to NNV was observed using a live imaging system and a fluorescence microscope.The results showed that SWCNTs-P-A mainly existed in the abdomen of the diseased grouper,while SWCNTs-P-A-Nb could enter the brain tissue of the diseased fish.In addition,SWCNTs-P-A-Nb was distributed in various tissues such as muscle,intestine,liver and kidney of fish,but it cannot enter into eyes.In general,the targeted drug delivery system has a good targeting ability to NNV both in vitro and in vivo.5. Anti-NNV activity of the targeted drug delivery systemAt the same drug concentration,SWCNTs-P-A-Nb significantly inhibited the apoptosis of SSN-1 cells than amantadine and SWCNTs-P-A.In addition,SWCNTs-P-A-Nb has a stronger anti-NNV activity by Western blot and RT-q PCR experiments.In vivo,at the same drug concentration,the inhibition rate of SWCNTs-P-A-Nb on NNV is significantly higher than that of amantadine and SWCNTs-P-A.When the amantadine concentration was 40 mg/L,the survival rates of the diseased grouper increased to 27%,39%,and 51%within 10 days after treatment with amantadine,SWCNTs-P-A,and SWCNTs-P-A-Nb.The highest drug contents in the brain tissue of grouper from the amantadine treatment group,SWCNTs-P-A treatment group and SWCNTs-P-A-Nb treatment group were 11.88,19.34 and 34.57 mg/Kg,respectively.After the treated fish was transferred to clean seawater,the amantadine treatment group and the SWCNTs-P-A treatment group were quickly discharged and completely metabolized after 120 h.The SWCNTs-P-A-Nb treatment group showed a slower metabolic trend,and it was not completely metabolized from the brain until 192 h.The above results indicate that SWCNTs-P-A-Nb can increase the anti-NNV activity of amantadine and prolong the action time of the drug.In summary,amantadine,SWCNTs and nanobody NNV-VHH 6 were selected as anti-NNV drugs,nanocarrier and targeting ligand to construct a nano targeted drug delivery system.The system has good targeting ability to NNN in vitro and in vivo,and improves the anti-NNV activity of amantadine.This study will provide new ideas for the prevention and control of VNN,lay the foundations and provide important references for the production of fish without specific pathogens.