Synthesis and characterization of bionanoconjugates from nanodiamonds and recombinant spike protein of porcine epidemic diarrhea virus for biomedical applications

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - THESIS TITLE: SYNTHESIS AND CHARACTERIZATION OF BIONANOCONJUGATES FROM NANODIAMONDS AND RECOMBINANT SPIKE PROTEIN OF PORCINE EPIDEMIC DIARRHEA VIRUS FOR BIOMEDICAL APPLICATIONS HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - THESIS TITLE: SYNTHESIS AND CHARACTERIZATION OF BIONANOCONJUGATES FROM NANODIAMONDS AND RECOMBINANT SPIKE PROTEIN OF PORCINE EPIDEMIC DIARRHEA VIRUS FOR BIOMEDICAL APPLICATIONS Student name : NGO NHAT QUANG Student code : 637429 Class : K63CNSHE Faculty : BIOTECHNOLOGY Supervisor : Dr PHAM DINH MINH : Dr BUI THI THU HUONG DECLARATION I hereby declare that the graduate thesis work is mine All research results have been results during the implementation of the topic The results, the data are completely true, never appeared in any scientific report I also guarantee that the references and useful information for the topic are clearly cited, and all help is appreciated Hanoi, December 27th, 2022 Student Ngo Nhat Quang ii ACKNOWLEDGEMENTS During the process of implementing my graduation project, I have received a lot of attention and help from individuals and groups First of all, I would like to express my respect and deep gratitude to Dr Pham Dinh Minh and Dr Bui Thi Thu Huong for giving me the opportunity to carry out this work, and their huge efforts, enthusiasm, and support throughout the duration of the undergraduate thesis Secondly, I would like to thank the teachers in the Faculty of Biotechnology have helped and taught me during my training at the university Especially the teachers of the Biology department who gave me advice during carrying out Finally, I would like to sincerely thank my family members and friends who always trust, support, and encourage me to complete this report Sincerely thank! Hanoi, December 27th, 2022 Student Ngo Nhat Quang iii CONTENTS LIST OF TABLES vi LIST OF FIGURES vii LIST OF ABBREVIATIONS ix ABSTRACT PART I INTRODUCTION 1.1 Preface 1.2 Objective and Requirements 1.2.1 Objective 1.2.2 Requirements PART II LITERATURE REVIEW 2.1 Pig (Sus domesticus) .4 2.1.1 Introduction of pig 2.1.2 The importance of Pigs in Vietnam .4 2.2 Porcine Epidemic Diarrhea 2.2.1 Introduction of Porcine Epidemic Diarrhea 2.2.2 Distribution of Porcine Epidemic Diarrhea 2.3 Porcine epidemic diarrhea virus (PEDV) .8 2.3.1 Scientific classification 2.3.2 PEDV structure 2.3.3 Genome organization 10 2.3.4 Transmission of PEDV 12 2.4 Prevention and control of PEDV 13 2.4.1 Detection and symptomatic treatment 13 2.4.2 Prevention of PEDV .14 2.5 Nanotechnology and it's potential in vaccine research .15 2.5.1 Introduction of Nanotechnology 15 2.5.2 Types of nanoparticles 16 2.5.3 The immunogenicity of nanoparticles 18 2.6 Nanodiamonds and Fluorescent nanodiamonds 20 PART III MATERIALS AND METHODS 23 3.1 Location and time for research 23 3.1.1 Research location 23 3.1.2 Time for research 23 iv 3.2 Materials, chemicals, and equipment 23 3.2.1 Materials .23 3.2.2 Chemicals, machines, and equipment 23 3.3 Experimental Methods 24 3.3.1 Synthesis of S1:ND, S1:FND conjugates using sonication 24 3.3.2 Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) 25 3.3.3 Western blot 25 3.3.4 Dynamic Light Scattering (DLS) 25 3.3.5 Electrophoretic Light Scattering (ELS) .26 3.3.6 Mouse experiment 27 3.3.7 Indirect Enzyme-linked immunosorbent assay (ELISA) .28 PART IV RESULTS AND DISCUSSION 29 4.1 Optimization of the synthesis conditions of bionanoconjugates (S1:ND and S1:FND) .29 4.1.1 Buffer Optimization for S1:ND conjugates 29 4.1.2 Ratio optimization between S1-protein and ND/ FND 31 4.2 Physical-chemical-biological characterization 33 4.2.1 Biological characterization of S1:ND conjugates 33 4.2.2 Size characterization 35 4.2.3 Zeta potential characterization .37 4.3 Initial evaluation of the immunogenicity of S1:FND in mice model 39 PART V CONCLUSION .41 5.1 Conclusion 41 5.2 Proposal for further work 41 REFERENCES 42 v LIST OF TABLES Table 2.1: Scientific classification of PEDV (NCBI:txid28295) .8 Table 3.1: Materials of research 23 Table 3.2: Chemicals of research 23 Table 3.3: Machines, equipments of research .24 vi LIST OF FIGURES Figure 2.1: Picture of pigs (Sus domesticus) (Beaver & Höglund, 2016) Figure 2.2: Meat consumption per capita in Vietnam from 1990 to 2021 (Kilograms/capita) (Vo, 2022) Figure 2.3: Meat consumption in some countries in 2021 (Kilograms/capita) (Vo, 2022) Figure 2.4: : Photographs of outbreaks of swine viral diarrhea (PEDV) Figure 2.5: Image taken under electron microscope (S H Kim et al., 2015) .9 Figure 2.6: Genome structure of the virus causing acute diarrhea in pigs (Sekhon et al., 2016) 10 Figure 2.7: S-antigen protein structure of PEDV and neutral epitope regions (Okda et al., 2017) 11 Figure 2.8: Several transmission routes of PEDV infection (Zhang et al., 2022) 13 Figure 2.9: A schematic showing where nanotechnology lies among the micro-, nano-, pico-, and femtoranges .16 Figure 2.10: A Diagram of Nanodiamonds B Transmission electron microscopy image of FND (100nm) 21 Figure 2.11: The structure of the NV_ center in nanodiamond (Hsiao et al., 2016) 22 Figure 3.1: Diagram illustrates the principle of Dynamic Light Scattering 26 Figure 3.2: Diagram illustrates the principle of Electrophoretic Light Scattering 26 Figure 3.3: Diagram illustrates the workflow of immunogenicity study in mouse model .27 Figure 4.1: Evaluation of the optimal buffer for synthesis of bionanoconjugates from Recombinant S1-protein (S1) and Nanodiamonds (ND) by Western blot .30 Figure 4.2: Evaluate the optimal ratio for synthesis of bionanoconjugates from Recombinant S1-protein (S1) and Nanodiamonds (ND) by Western blot .31 Figure 4.3: Different ratios (w/w) of S1:FND were analyzed by Western blot The arrow indicates the S1 bands in Western blot paper .32 Figure 4.4: Different ratios (w/w) of S1:FND were analyzed by Western blot 33 vii Figure 4.5: Effect of different temperatures to S1:ND complexes after day of storage by Western blot 34 Figure 4.6: Evaluate the effect of different temperatures to S1:ND complexes after days of storage by Western blot 35 Figure 4.7: Size measurement of FND in DI water and in PBS 36 Figure 4.8: Size measurement of S1-protein, bare-FND, and S1:FND at 1:48 (w/w) ratio 36 Figure 4.9: : Size measurement of S1:FND with different ratios 37 Figure 4.10: Zeta potential measurement of S1-protein, bare-FND, and S1:FND (1:48, w/w) 38 Figure 4.11: Zeta potential measurement of S1:FND with different ratios in PBS and S1-protein free .38 Figure 4.12: Determination of the S1-specific-IgG antibody responses in mice via ELISA 39 viii LIST OF ABBREVIATIONS o C Celsius DLS Dynamic light scattering ELS Electrophoretic Light Scattering ELISA Enzyme-linked immunosorbent assay FND Fluorescent Nanodiamond kDa Kilodalton µg Microgram µl Microliter Min Minute ND Nanodiamond OD Optical density PBS Phosphate-buffered saline PDV Porcine Epidemic Diarrhea PEDV Porcine Epidemic Diarrhea Virus pH Potential of hydrogen S1-protein Recombinant spike protein of Porcine Epidemic Diarrhea Virus Revolutions per minute Rpm SDS-PAGE TEMED Sodium Dodecyl Sulfate–Polyacrylamide Electrophoresis Tetramethylethylenediamine TEM Transmission electron microscopy TFA Trifluoroacetic w/w Weight/weight Gel ix no difference in the optimal ratio for the synthesis of S1:ND and S1:FND complexes The optimal ratio for the synthesis of S1:FND complexes was also 1:48 (w/w) Figure 4.3: Different ratios (w/w) of S1:FND were analyzed by Western blot The arrow indicates the S1 bands in Western blot paper M: protein marker Then, I continued to increase the amount of FND in the conjugation process from 1:48 ratio to 1:60 and 1:72 ratios (w/w) The results in figure 4.4 showed that when adding more FNDs that correspond to the S1:FND ratio of 1:60, and 1:72 (w/w), the binding capacity of S1-protein on the surface of FNDs was not increased This may be explained that the binding capacity of S1-protein on the surface of FNDs was saturated 32 Figure 4.4: Different ratios (w/w) of S1:FND were analyzed by Western blot The arrow indicates the S1 bands in Western blot paper M: protein marker marker 4.2 Physical-chemical-biological characterization 4.2.1 Biological characterization of S1:ND conjugates To characterize the stability of S1:ND conjugates under different temperature conditions and storage time, the S1:ND conjugates were stored at room temperature, 4oC, and -20oC for day and days after conjugated The S1:ND conjugate was conjugated with 1:48 ratio (w/w) and PBS buffer • Effect of different temperatures on S1:ND conjugates after day of storage The results in Figure 4.5 showed that the S1-protein bands in the room temperature lane were very faint, while the bands in 4oC and -20oC lanes were as dark as the not stored lane These results suggested that the minimum temperature required for storage of the S1:ND complex was 4oC At room temperature, S133 protein could be degraded so that only small amounts of protein could be shown on the Western blot paper Figure 4.5: Effect of different temperatures to S1:ND complexes after day of storage by Western blot The arrow indicates the S1-protein bands in Western blot paper M: protein marker • Effect of different temperatures on S1:ND conjugates after days of storage In actual use, biomedical products (e.g vaccines) may need to be stored for a period of time (e.g a week) and under different temperature conditions before being used This study evaluated the stability of the S1:ND complex when stored in different temperatures (room temperature, 4oC, and -20oC) for a period of days Similar to the above experiment, the S1:ND ratio and buffer for the conjugation process were 1:48 (w/w) and PBS respectively The results in figure 4.6 also showed that the conjugated S1-protein on ND particle could be degraded at room temperature, therefore no band appeared on Western blot paper The amount of S1-protein in 4oC and -20oC lanes were similar 34 to not stored lanes, suggesting that the S1:ND complex could be stored at 4oC or -20oC for days without being degraded Figure 4.6: Evaluate the effect of different temperatures to S1:ND complexes after days of storage by Western blot The arrow indicates the S1 bands in Western blot paper M: protein marker 4.2.2 Size characterization • Size characterization of FND particles Before measuring the S1:FND complex, the size of FND was measured to compare the change of size before and after conjugation At first, the FND was diluted into Deionized (DI) water and PBS to determine the aggregation of FND in water and a salt solution (e.g., PBS) It can be seen that the size of FND diluted in PBS was 7.8-fold bigger (1194 nm) compared to those diluted in DI water (152.3nm), suggesting that the FND particles might be aggregated in PBS (figure 4.7) This experiment is similar to other researches about the aggregation of FND particles, in that the FND particles are easy to aggregate in salt solution but not in DI water (Hemelaar et al., 2017; Pham et al., 2017; Ho et al., 2021) 35 1400 1194 Diameter (nm) 1200 1000 800 600 400 152.3 200 FND in DI water FND in PBS Figure 4.7: Size measurement of FND in DI water and in PBS • Size characterization of S1:FND complexes The results in figure 4.8 illustrated the change in the size distribution of S1protein and FND before and after coating with S1-protein at 1:48 (w/w) ratio The average sizes of S1-protein, bare-FND, and S1:FND were 68.91nm, 152.3nm, and 285.9nm respectively It can be clearly seen from figure 4.6 that FND particles increased in size to approximately 130 nm in diameter after protein coating This 30 Percentage (%) 25 20 15 Protein S1 10 FND S1:FND (1:48) 0 -5 100 200 300 400 500 600 Diameter (nm) Figure 4.8: Size measurement of S1-protein, bare-FND, and S1:FND at 1:48 (w/w) ratio 36 result indicated that several S1-protein were conjugated onto the surface of FND particles Figure 4.9: illustrated the size of S1:FND complexes when conjugated with different S1-protein and FND ratios (1:3, 1:6, 1:12, 1:48) (w/w) It can be seen that the S1:FND at 1:3 ratio was significantly bigger than other ratios, suggesting the S1:FND could be aggregated at this ratio In other ratios, the sizes are similar to each other Diameter (nm) 600 518.6 500 400 345.3 355.2 300 292.9 285.9 S1:FND (1:24) S1:FND (1:48) 200 100 S1:FND (1:3) S1:FND (1:6) S1:FND (1:12) Figure 4.9: Size measurement of S1:FND with different ratios 4.2.3 Zeta potential characterization To analyze the change in surface chemistry of FND after S1-protein coating, the zeta potential of S1-protein, bare-FND particles, and S1:FND complexes were also measured The measurements showed that all types of particles have negatively charged surfaces After coating S1-protein onto the FND’s surface, the zeta potential changed from -34.1 mV to -12.19 mV (figure 4.10) This implied that the S1:FND complex was unstable and tends to clump together and agglomerate The change in the zeta 37 potential of the S1:FND complex could be explained by the fact that the S1protein has a zeta potential close to zero (-11.36 mV), suggesting that the surface of FND was covered by the S1-protein after conjugation process -11.36 -12.19 -34.1 -40 -35 -30 S1 Protein -25 -20 -15 Zeta potential (mV) -10 -5 S1-FND (1:48) Bare-FND Figure 4.10: Zeta potential measurement of S1-protein, bare-FND, and S1:FND (1:48, w/w) S1 Protein -11.36 S1-FND (1:48) -12.19 S1-FND (1:24) -10.3 S1-FND (1:12) -12.09 S1-FND (1:6) -13.3 S1-FND (1:3) -10.66 -15 -10 -5 Zeta potential (mV) Figure 4.11: Zeta potential measurement of S1:FND with different ratios in PBS and S1-protein free 38 Figure 4.11 illustrated the zeta potential of S1:FND complexes when conjugated with different S1-protein and FND (1:3, 1:6, 1:12, 1:48) (w/w) All the ratios had similar zeta potential and were similar to the figure of S1-protein This suggests that FND particles at all the ratios were covered by S1-protein after conjugation 4.3 Initial evaluation of the immunogenicity of S1:FND in mice model To test the immunogenicity of the S1-protein conjugate with negatively charged NDs compared to the free S1-protein, the optimized mixture of S1:FND conjugates were immunized in a mouse model (in vivo experiments) S1-specificIgG antibody responses in mice vaccinated with PBS (group 1), free S1 (group 2), and S1:FND (1:48) (group 3) were evaluated via ELISA The sera of each group were mixed and diluted 1:1000 The ELISA results (figure 4.12) indicated that a stronger S1-specific-IgG response was induced in mice vaccinated with S1:FND conjugate (group 3) compared to the free S1-protein (group 2) However, the Absorbance (OD450 nm) amount of S1-specific-IgG induced in mice vaccinated with S1:FND complex was 3.5 2.5 1.5 0.5 3.1191 3.4123 0.4409 PBS S1 Protein S1:FND Figure 4.12: Determination of the S1-specific-IgG antibody responses in mice via ELISA 39 only 1.09-fold higher than those in mice vaccinated with S1-protein This implied that the immunogenicity of the S1:FND conjugate is not significantly stronger than S1-protein Compared to other research, the immunogenicity of bionanoconjugate between ND and H7N9-protein showed that after conjugated with ND, the immunogenicity in mice was increased significantly compared to free H7N9protein (Pham et al., 2017) Another research using bionanoconjugates between ND and A/H5N1-protein also showed a significant increase in immunogenicity of mice vaccinated with bionanoconjugate compared to those vaccinated with free A/H5N1-protein (Ho et al., 2021) 40 PART V CONCLUSION 5.1 Conclusion The nanoconjugates were synthesized most effectively in PBS buffer and S1:ND/FND ratio of 1:48 (w/w) The minimum temperature required for storage of the S1:ND conjugate was 4oC After being coated by S1-protein, the size of FND increased by 130 nm, and the zeta potential changed from -34.1 mV to -12.19 mV The immunogenicity in mice vaccinated with S1:FND conjugate was 1.09-fold higher than those vaccinated with S1-protein 5.2 Proposal for further work Due to the limited time in the research process, the report has some limitations Because of this, I would like to give some recommendations: Further evaluate the physical-chemical-biochemical properties of the bionanoconjugates including luminescence, safety Studying the interaction of the S1:FND conjugates with in vitro cultured animal cells Measurement of the immunogenicity of S1:FND conjugates in experimental pigs 41 REFERENCES Agnihotri, S A., Mallikarjuna, N N., & Aminabhavi, T M (2004) Recent advances on chitosan-based micro- and nanoparticles in drug delivery Journal of Controlled Release : Official Journal of the Controlled Release Society, 100(1), 5–28 Allen, T M., Hansen, C B., & 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