VIET NAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE THE INTERACTION BETWEEN BSA MODIFIED NANODIAMOND WITH SERUM PROTEINS Hanoi, March 2021 VIET NAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY TITLE THE INTERACTION BETWEEN BSA MODIFIED NANODIAMOND WITH SERUM PROTEINS STUDENT: Nguyen Tra My MAJOR: Biotechnology CLASS: K61CNSHE 610743 STUDENT CODE: SUPERVISORS: Phan Huu Ton, Ph D Vietnam National University of Agriculture Pham Dinh Minh, Ph D Vietnam Academy of Science and Technology Hanoi, March 2021 COMMITMENT I assure this is my research All the results are honestly collected and have not been published in any article I accept responsibility for my commitment Hanoi, February 25th, 2021 Nguyen Tra My i ACKNOWLEDGEMENTS The completion of this study could not have been possible without the expertise of Ph D Pham Dinh Minh, Ph D Phan Huu Ton, my thesis adviser I would also like to thanks Ms Quy, Ms Hue, Mrs Loan and Mr Son for supporting me on my thesis A debt of gratitude in also owned to my beloved family and friends for their belief and their help on the spirit Without all of you, none of this would indeed be possible Nguyen Tra My ii TABLE OF CONTENTS COMMITMENT .1 ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF ABBREVIATIONS v SUMMARY viii CHAPTER 1: INTRODUCTION CHAPTER 2: LITERATURE REVIEW .2 2.1 Introduction to Nanodiamonds: Classification, Surface Modification, and Biomedical Applications 2.2 Introduction to Proteomics and Its Application to Protein profiling 2.3 Interaction of Bare-ND and Surface-modified ND with Proteins/ Human Fluids/ Cell culture/ Immune System .6 2.3.1 Interaction of Bare-ND and Surface-modified ND with Proteins 2.3.2 Interaction of Bare-ND and Surface-modified ND with Human fluids 2.3.3 Interaction of Bare-ND and Surface-modified ND with Cell Culture 2.3.4 Interaction of Bare-ND and Surface-modified ND with Immune System: 2.4 Introduction to BSA and previous study about BSA-conjungates NDs: CHAPTER 3: MATERIAL AND METHOD 10 3.1 Materials 10 3.2 Methods .10 3.2.1 Production of Bare-Nanodiamonds 10 3.2.2 Coating Nanodiamonds by Incubating with BSA .10 3.2.3 Interaction of Human Serum Proteins with Bare-ND and BSA-ND 11 3.2.3.1 Effect of Protein Amount and Incubating time 11 3.2.3.2 Effect of Incubating Buffers 12 3.2.3.3 Effect of Washing buffers 12 3.2.3.4 Size and Zeta-potential Measurements 12 3.2.4 BSA-coated Nanodiamonds Interact with Human Serum Proteins for Nano LCMS/MS 13 3.2.5 Liquid Chromatography coupled to Tandem Mass Spectrometry 13 iii 3.2.6 Bioinformatic Analysis 14 CHAPTER 4: RESULTS AND DISCUSSION 15 4.1 Synthesis and Characterization of Oxidative Nanodiamond (bare-NDs) and Protein-modified Bare-NDs (BSA-NDs) 15 4.1.1 Production of Bare-NDs .15 4.1.2 Synthesis and Characterization of BSA modified bare-NDs 16 4.2 Interaction of Human Serum Proteins with Bare-NDs and BSA-NDs .17 4.2.1 Effect of Protein Amount and Incubating time 17 4.2.2 Effect of Incubating Buffers 19 4.2.3 Effect of Washing Buffers 20 4.2.4 Size Distribution and Zeta-potential of Bare-NDs and BSA-NDs Before and After Conjuncgation with Human Serum Proteins 21 4.3 Analysis of Human Serum Proteins Adsorbed onto the Surface of Bare-ND and BSA-ND at Physiological pH 22 4.3.1 SDS-PAGE Analysis 22 4.3.2 Proteomics Analysis 23 4.4 Discussion: 25 CHAPTER 5: CONCLUSION AND RECOMMENDATION .27 REFERENCES 28 iv LIST OF ABBREVIATIONS Abbreviations Definitions ND Nanodiamond BSA Bovine serum albumin BSA-ND Bovine serum albumin conjungates to Nanodiamond HPHT high pressure high temperature CVD chemical vapour deposition DND Detonation MS Mass spectrometry ESI electrospray ionization TDP Top-down proteomics LC-MS/MS Liquid Chromatography Mass Spectrometry cNDs carboxylated nanodiamond RBC red blood cells FND flourescent nanodiamonds FESEM Field emission scanning eelectron microscopy DLS dynamic light scattering SDS-PAGE Sodium dodecyl sulfatepolyacrylamide gel electrophoresis v LIST OF FIGURES Figure Surface modification of nanodiamonds with various functional group (Journal of Material Science & Engineering) Figure 2 The main components of a mass spectrometer (Application review on Merckmilipore.com) Figure Production and physical characterization of bare-NDs (a) The size distribution of nanodiamonds suspension in H2O; (b) TEM image of bareNDs suspension in H2O 15 Figure Synthesis and physical characterization of BSA-ND The nanodiamonds suspension before coating (ND) and after coating with BSA (BSA-NDs) (a) In H2O (b) Zeta-potential of BSA-ND and bare -ND (c) The SDS-PAGE image shows the result of coating bare-ND with BSA The bare-ND protein band pattern is blank, showing the purity of ND containing no contaminants The BSA protein band pattern is shown as the main band of over 66.2 kDa 16 Figure The SDS-PAGE image shows the result of the interaction between bare-ND and human serum proteins at different incubating times and different amounts of human serum proteins .18 Figure 4 The SDS-PAGE image shows the resullt of the interaction between BSA-ND and human serum proteins at different incubating times and different amounts of human serum proteins .18 Figure The SDS-PAGE image shows the result of the interaction of bare-ND and BSA-ND with human serum proteins in different pH conditions .19 Figure The SDS-PAGE image shows the result of the interaction of bare-ND and BSA-ND with human serum proteins in different washing buffers .20 Figure Synthesis and physical charaterization of ND and BSA-ND before and after interacting with serum proteins a) Size distribution in H2O; b) Zetapotential 21 Figure The SDS-PAGE image shows the result of interaction in human serum with bare-ND and BSA-ND The bare-ND protein band pattern is blank, vi showing the purity of ND containing no contaminants The BSA protein band pattern is shown as the the main band of 66.2 kDa 22 Figure The bar chart show the most abundant serum proten groups found in the sample of bare-ND interaction with serum Abundancy was calculated based on MS/MS intensity from MaxQuant 23 Figure 10 The bar chart shows the 10 most abundant serum protein groups found in the sample of BSA-ND interaction with serum Abundancy was calculated based on MS/MS intensity from MaxQuant 24 vii SUMMARY Nanodiamonds (NDs) or nanocrystalline diamond is an immense term that described a continuum of materials It is recently considered as a promising material in proteomics, in cell labeling and tracking, in drug delivery, and especially in vaccine development Numerous studies have shown the linking or conjugating of protein on ND particles but there has not been work on how it interacts in human serum, which is important for predicting their behavior in the human body In this study, we have examined the effect of surface modifications on the interaction of NDs and human serum proteins The adsorbed ability of proteins on the surface of NDs was investigated and confirmed by the change in size before and after modification The interaction between bare-NDs and BSA-NDs with serum proteins is pH dependent and depends on different proteins which are linked with That leads to different behaviors in the human body Keywords: Nanodiamonds; Surface-modified Nanodiamonds; Nanomedicine; BSA viii Nanodiamonds; Carboxylated CHAPTER 4: RESULTS AND DISCUSSION 4.1 Synthesis and Characterization of Oxidative Nanodiamond (bare-NDs) and Protein-modified Bare-NDs (BSA-NDs) 4.1.1 Production of Bare-NDs Size distribution of re-suspended nanodiamonds in H2 O Number Percentage (%) ND 25 20 15 10 0 200 400 600 800 -5 Diameter (nm) Figure Production and physical characterization of bare-NDs (a) The size distribution of nanodiamonds suspension in H2O; (b) TEM image of bare-NDs suspension in H2O Following previous research, in aqueous solution, the surface of bare-NDs are oxidized in strong acid solution (the H2SO4: HNO3 ratio is 3:1 (v/v)) and the hightemperature condition (about 100ºC) (Pham et al, 2013) As shown in Fig 4.1a, the original bare-NDs with a variety of sizes distribute unequally in solution In the range of size from ~50nm to ~400nm, they count different percentages but reach the peak at 100nm Therefore, bare-NDs particles can be aggregate in a deionized water solvent to form differently sized clusters Furthermore, the TEM images showed that the morphology of bare-NDs is not round (Fig 4.1b) 15 4.1.2 Synthesis and Characterization of BSA modified bare-NDs Size distribution of re-suspended nanodiamonds compared to BSANDs in H2O Number percentage (%) ND Zeta Potential of NDs and Protein-coated NDs ND BSAND -15 -10 BSA-NDs 25 20 15 10 -5 200 400 600 -25 800 Diameter (nm) -20 -5 Average Zeta Potential (mV) Figure Synthesis and physical characterization of BSA-ND The nanodiamond suspension before coating (ND) and after coating with BSA (BSANDs) (a) In H2O (b) Zeta-potential of BSA-ND and bare -ND (c) The SDS-PAGE image shows the result of coating bare-ND with BSA The bare-ND protein band pattern is blank, showing the purity of ND containing no contaminants The BSA protein band pattern is shown as the main band of over 66.2 kDa The BSA-ND complexes were synthesized by simply mixing BSA protein with ND solution, then sonicated this mixture for 30 minutes Based on the previous study, 16 at the protein: ND ratios (w/w) is 1:12, the bare-NDs presented the highest ability to bind trimeric HA protein (Pham et al., 2017) Therefore, in this study, the amount of BSA and ND was used to ensure the ratio of 1:12 (w/w) The changes in size distribution in deionized water of bare-NDs before and after coating with BSA protein are shown in Fig.4.2a After coating with BSA, the size of bare-ND particles increases by about 20nm in diameter It means there was a considerable amount of BSA protein that is bound to the surface of bare -NDs and also demonstrated the ability of bareNDs in protein capture This feature was also shown in Fig 2c The bare-ND protein band pattern is blank, reflecting the purity of ND containing no contaminants For further investigation of the surface properties of bare-NDs and BSA-NDs, we measured the Zeta-potential of both particles The results showed the both of them have negatively charged surfaces (Fig 4.2b) It can be seen that all values are less than -10mV, which means particles have good dispersion in the deionized water solvent For more detail, the average zeta potential values of bare-NDs and BSA-NDs are 19.2±3.85 and -16.1±3.88 4.2 Interaction of Human Serum Proteins with Bare-NDs and BSA-NDs 4.2.1Effect of Protein Amount and Incubating time 17 Figure The SDS-PAGE image shows the result of the interaction between bare-ND and human serum proteins at different incubating times and different amounts of human serum proteins Figure 4 The SDS-PAGE image shows the resullt of the interaction between BSA-ND and human serum proteins at different incubating times and different amounts of human serum proteins The interaction of bare-ND and BSA-ND with serum proteins was examined at different times and different amounts of serum proteins (Fig 4.3 and 4.4) At 60 18 μgproteins, after minutes of incubating, the lane appeared protein bands which can easily see by eyes, showing the interaction ability between bare-ND, BSA-ND, with serum in a short period time Moreover, with 60 μg, in 30 minutes and 120 minutes, the protein band is bolder, saying that there are more proteins interact with ND particles Compared to 300 μg and 600 μg, the pattern of the band has not many changes, but the number of proteins interact with not fold as expected That can be explained by the instability of particles in solution, there may be aggregation On another side, despite the amount of albumin in the original serum is the largest but with nanoparticles, we can easily see that not appeared on the protein band Therefore, the interaction of proteins does not rely on the ratio amount of proteins in serum To mimic the interaction between ND particles and serum protein, even when the amount of protein increase the pattern remains the same, in this study, we decided to choose the amount of serum protein (300 μg) 4.2.2 Effect of Incubating Buffers Figure The SDS-PAGE image shows the result of the interaction of bare-ND and BSA-ND with human serum proteins in different pH conditions 19 The effect of pH condition on the interaction of bare-ND and BSA-ND with serum proteins was shown in Fig 4.5 The SDS-PAGE results show that these interactions are pH-dependent In both ND-S and BSA-ND-S, in acidic buffers, protein lanes are bold, meaning interaction between nanoparticles with serum is most stable in an acidic environment (0.1% TFA) However, the basic pH lanes have the lightest shade The pattern of two particles in the same environments still keeps the same but the condensation is different that states the effect of BSA after coating and change the surface of nanoparticles 4.2.3 Effect of Washing Buffers Figure The SDS-PAGE image shows the result of the interaction of bare-ND and BSA-ND with human serum proteins in different washing buffers The effect of the washing buffer on the interaction of bare-ND and BSA-ND with serum proteins was shown in Fig 4.6 Comparing three types of washing buffer (H2O, PBS 1X, and 2% triton X100), protein lanes which were washed by H2O is bold, while which was washed by 2% Triton X 100 is lightest Some the proteins that have molecular weight are under 45kDa was washed out therefore can not appear Therefore, we can see the stability of nanoparticles and serum is affected by detergents The attachment on NDs of protein is very strong, it can not be completely 20 broken by washing buffers, just in presence of denaturing factors like sodium dodecyl sulfate 4.2.4 Size Distribution and Zeta-potential of Bare-NDs and BSA-NDs Before and After Conjuncgation with Human Serum Proteins Number Percentage (%) Size Distribution of Re-suspended Nanodiamonds in H2O ND 25 BSAND ND-S Zeta Potential of NDs and Protein-coated NDs BSAND-S ND 20 ND-S BSAND BSAND-S 15 10 0 100 200 300 400 500 600 700 -30 -20 -10 Average Zeta Potential (mV) -5 Diameter (nm) a) b) Figure 7Synthesis and physical charaterization of ND and BSA-ND before and after interacting with serum proteins a) Size distribution in H2O; b) Zeta-potential Figure 4.7 a showed the size distribution and Zeta-potential of bare-NDs and BSA-NDs before and after interacting with serum proteins It can be easily seen that all the lines are not in the same position, there must be changes in the diameter of all particles after modified surface compared to bare-ND That also proved the interaction of serum protein with nanoparticles For further investigation of the surface properties of bare-NDs and BSA-NDs before and after conjugation with serum proteins, we compared the Zeta-potential of all ND particles Figure 7b showed that all Zeta-potential value is negatively charged and less than -10mV, which means ND particles have good dispersion in a deionized water solvent 21 4.3 Analysis of Human Serum Proteins Adsorbed onto the Surface of Bare-ND and BSA-ND at Physiological pH 4.3.1 SDS-PAGE Analysis Figure The SDS-PAGE image shows the result of interaction in human serum with bare-ND and BSA-ND The bare-ND protein band pattern is blank, showing the purity of ND containing no contaminants The BSA protein band pattern is shown as the the main band of 66.2 kDa Within the physiological pH of 1X PBS and the amount of serum is 300 μg, the interaction between ND particle and serum is shown in Fig.8 There is no protein bands on ND land, proving the purity of nanoparticles When bare-ND modified by BSA, and interact with serum, compared with bare-ND interact with serum, easily see that many of serum protein interact with nanoparticle It is likely to be similar to the behavior of ND particles in vivo 22 4.3.2 Proteomics Analysis Figure The bar chart show the most abundant serum proten groups found in the sample of bare-ND interaction with serum Abundancy was calculated based on MS/MS intensity from MaxQuant The received raw file data were analyzed by MaxQuant proteomics program, which allowed as to identify 80 protein group in this experiment Serum albumin, protein have biggest amount in serum is not the most abundant protein present in the MS results In the top 10 abundant serum proteins that interact with bare-NDs, majority are proteins that take involve in the immunology process and lipid metabolism In which Apolipoprotein A-I, Apolipoprotein E, Apolipoprotein B-100 belongs to the Apolipoprotein family, a family that has a function in lipid binding, therefore, play in important role in lipid metabolism and transport Complement C3, Inter-alpha-trypsin inhibitory heavy chain H4, complement C4 immunoglobulin gamma-1-chain C region involves in immunity responses by different ways such as participate in inflammatory responses caused by trauma (Inter-alpha-trypsin inhibitory heavy chain H4); complement activation (complement C3 and complement C4); or component of 23 constant region in Immunoglobulin heavy chains (Immunoglobuli gamma-1 chain C region) The most abundant protein that interacts with bare NDs is Gelsolin, that is important in calcium regulation Gelsolin also is reported in actin-binding and regulation, or in cillium biogenesis Albumin has the main function is maintaining homeostasis of blood by managing the moving of ions such as zinc, calcium, magnesium The last one is Hisidine-rich glycoprotein This protein can regulate many biological processes, for example, immune pathogen removal, or chemotaxis, etc (All information provided by UniProt) Figure 10 The bar chart shows the 10 most abundant serum protein groups found in the sample of BSA-ND interaction with serum Abundancy was calculated based on MS/MS intensity from MaxQuant Bovine serum albumin is often used as a coating factor for ND to be more active in vivo, because in such condition ND aggregation hindering its mobility We assess the capture ability of this fully BSA-coated ND, and as expected the BSA-AND still allows robust adsorption of serum proteins The component of proteins varies and not restricted to a specific type of protein, in here we observed the serum proteins with molecular weight ranging from very low (a few kDa) to high (over 116 kDa), 24 accompanied with respective manifold protein sizes Noticeably, the BSA-ND can even enrich several proteins with their corresponding band observed in Figure compared with crude serum samples From this sample, we detected the signal of 79 proteins (excluding keratins) with the relatively similar protein corona component with the ND and HA-ND sample, indicating a certain shared pattern between the three samples, despite some intensity differences Compared with bare-ND, BSA-ND showed different preferences in interacting with serum proteins Not like bare- ND, BSA-ND does not interact strongly with gelsolin-and actin-depolymerizing protein, and its plasma isoform has been recognized as a potential biomarker of inflammatory-associated medical conditions, allowing for the prediction of illness severity, recovery, efficacy of treatment, and clinical outcome BSA-ND interacts most strongly with apolipoprotein A-I, and it has an affinity almost similar to other proteins (complement C3, apolipoprotein E, histidine rich glycoprotein, vitronectin, apoliporotein B, gelsolin, complement C4-B, anti alpha trypsin inhibitor, and kininogen 1) In which, the distinghed differences between bareND and BSA-ND are that kininogen and Ig gamma C chain not present on the list of 10 most abundant proteins that are captured by the two nanoparticles 4.4 Discussion: After coated with proteins, the increase in the size of bare-NDs revealed that protein can conjugate on ND surface to form a new complex The linkage between NDs and protein is strong and can not be broken without the presence of denaturing factors, such as sodium dodecyl sulfate (Chen et al., 2006) Some of the serum proteins can be washed away by 2% Triton X100, which can’t be seen by PBS or H2O (Fig 4.6) Comparing two samples ND-S and BSA-ND-S, it is observed that ND-S can capture more proteins than BSA-ND-S (Fig 4.9 and 4.10) It can be explained that the binding site of bare ND is occupied by most of BSA molecule before interaction with serum, the surface binding site leave for proteins in serum is limited Besides, in serum 25 solution, the interaction of BSA with NDs influencethe ability of other proteins in serum binding to this complex Furthermore, this proved that BSA-NDs have great protein capture ability The identified results for unique proteins in the top 10 serum proteins of bareND consist of Serum Albumin and Ig gamma-1 chain C region, while in BSA-ND are vitronectin and kininogen In the top 10 of the most abundant proteins identified, three types of apolipoprotein were found The last feature that has been discussed here is that after coating BSA with bare-ND, several proteins still can be captured since the band of protein appear clearly.There are two possible theoretical explanations for this scene First, BSA after coating with NDs can also contact other proteins in human serum Second, the long chain of BSA can not fully binding with bare-ND so there are adhesive sites available on the surface of complex BSA-NDs 26 CHAPTER 5: CONCLUSION AND RECOMMENDATION In conclusion, we have demonstrated that there is notably interaction between ND particles and hundreds of types of serum proteins These interactions is pHdependent There is a significant difference between bare NDs and BSA modified NDs in interaction with serum proteins Bare-NDs and BSA modified NDs attract to different types of proteins, that may make them have different behavior in the human body This study provided information about how bare-ND and BSA-ND interact with human serum protein and factors that can affect that interaction However, there 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