میانکنش نانوذرات با بیومولکول‌ها

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Biomolecules- Nanoparticles: Interaction in Nanoscale وت توس ‎Sajjad Babaei‏ PhD student of Nanobiotechnoogy. Razi University. Kermanshah. Iran 

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Introduction > Nanomaterials that measure 1-1,000 nm allow unique interaction with biological systems at the molecular level. > As nanoparticles and biomolecules are of similar length scale, it seems logical that the combination of biomacromolecules to nanomaterials can provide interesting tool for mimicking the biomolecules which are present at cellular systems, probing the mechanisms of biological processes, as well as developing chemical means by handling and manipulating biological components. 

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> The interactions of biomolecules and _ metal nanoparticles arise that determine the size of nanoparticles, modify the surface of nanoparticles to enhance solubility/biocompatibility/biorecognition, and detoxification of toxic metals. >» the macromolecule and nanoparticles interaction helped in ultra-trace detection, imaging, biomolecules detection, drug and DNA/RNA delivery, cancer therapy, and photodynamic therapy. 

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‎NUCLEIC ACIDS PROTEINS‏ أمععع11ل ‏ 3 رها ‎groups 0‏ ‎macromolecule‏ ‎s that interact ۱۳۷ NANOPARTICLES ‎nanoparticles ‎—_ ‎ 

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> The interaction of nanoparticles with nucleic acids and proteins are very high due to the presence of active functional groups on the surface; while that of carbohydrates and lipids is comparatively lower. 

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Nucleic Acid-Nanoparticles Interaction > Among the various nanoparticles, gold followed by silver and platinum are highly explored for use in various molecular level applications due to their high affinity with nucleic acids. > nanoparticles are highly sensitive spectroscopic reporters for the base-pairing of DNA. 1 5 Aptamer : Aptamers are oligonucleotide or peptide molecules that bind to a specific target molecule. Aptamers are usually created by selecting them from a large random sequence pool. 

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5 molecule Unbound Amplification Elution nN DIN ۷ رصم DIS PRINS DIS RIN DARN DIS RIS 2 5 = 2 1 5 a Next cycle Random nucleic acid library 

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Au @.0@ 0. @ 0 @ @ @ A B The ‘sandwich’ type aptamer-based colorimetric bioassays with gold (A) [56] and silver (B) [57] amplification systems. 

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Protein-Nanoparticles ‏لوا 0 يتنا‎ >» Nanoparticles are capable of strong and important interaction with other molecules. > Gold nanoparticle as metal based beads, have specific importance due to their attractive physical and chemical properties, biocompatibility, and facile surface modification. > In general, nanoparticles have the ability to interact with whole physiological surrounding once when they enter human body. In most of the cases, first molecule they interact with are proteins, which are the main constituens of human body and the driving force of most of the biological processes. 

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> Size of nanoparticles makes them able to enter in almost all parts of human body, including cells and organelles, while flat surfaces can only affect biological processes via cell surface receptors such as integrins. >» When nanoparticles enter biological fluid, the first molecule that will react with nanoparticles, are proteins in more than 95% of all cases. > The result of protein coating on nanoparticles surface is protein corona. > Protein corona may influence cellular uptake, inflammation, accumulation, degradation and clearance of nanoparticles 

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> Proteins of protein corona can change their native conformation, influencing the downstream regulation of protein-protein interactions, cellular signal transduction and transcription of DNA. For a better understanding of interactions between nanoparticles and proteins, we acquire information on binding affinities and stoichioetries for different combinations of proteins and nanoparticles. > Adsorption of protein on nanoparticle surface is aided by hydrogen bonds, solvation forces, Van der Waals interactions, etc. 

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Since different nanoparticles have distinct properties, the ۳ composition of protein corona is unique to each kind of nanoparticles and depends on many parameters Nanoparticle-protein cor complexes ‘Nanoparticle-protein corona 

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وت رن ‎monolayer 4:‏ NP - protein aggregate protein ‏بذ‎ ‎multilayer 

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> The proteins corona can be hard and soft. > It is thought that hard corona proteins interact directly with nanoparticle surface with high affinity, while soft corona consists of loosely bound proteins that interact with hard corona via weak-protein interactions. > It was shown that typical lifetime of hard corona can be even eight hours which indicates that hard corona defines the biological identity of the particle. >» Hard corona is formed in seconds, while the time of forming soft corona varies from seconds to hours. > In contrast to hard corona with lifetime of around eight hours, soft corona will probably be desorbed in ten minutes 

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> protein corona is not a fix layer and the composition of protein corona can be determined by kinetic rate of adsorption and desorption of each protein. Dynamic of protein corona as ,,biological identity” might lead to more clear classification system of nano-safety and could be used for engineering of nanomedical products. 

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> Parameters affecting protein corona : 1. Surface charge of nanoparticle : Acting as important parameter in protein interaction, surface charge of nanoparticle can also denaturate the adsorbed proteins. It was found that proteins can denaturate when they interact with positively or negatively charged ligands, whereas neutral ligands can keep the native structure of proteins. 

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2. Surface functionalization and Coating : Since nanoparticles are travelling through different environment of an organism, they may get pre-coated with different proteins and that pre-coatingcan determine which new proteins will bind to nanoparticle- protein complexes. 3. Hydrophobicity and Hydrophilicity : Nanoparticles withcharged orhydrophobic surfaces, tend to adsorb and denaturate more proteins than neutral and hydrophilic surfaces. due to clustering of hydrophobic polymer chain that forms distinct protein-binding sites. 

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4. Nanoparticle size : Same nanoparticles with different sizes have unlike compositions of protein corona. where nanoparticles have different sizes, from 70-700nmand that the amount of bound plasma proteins increased with increasing available surface area at constant particle weight. Nanoparticles surface area, available for protein binding, increases with decreasing of particle size. 5. Biological environment : Many in vitro studies have been done to examine nanoparticle-protein interaction, but it was still hard to predict the behavior of nanoparticles in living biological environment. two different cellular media are used, Dulbecco's Modified Eagle's Media (DMEM) and Roswell Park Memorial Institute medium (RPMI) with differently sized citrate coated gold nanoparticles. 

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> NP-protein interactions are changing all the time, even within the same environment. Different factors can affect the kinetics of protein adsorption on the NP surface. One of the factors that influence the nanoparticle protein corona (NP-PC) composition is amount of proteins that may interact with NP surface. >» Apart from the amount of proteins, affinity of the protein toward the NP surface also affects the ‏.0م3050‎ > Different proteins can arrange themselves differently ‏توت رت وتو واه‎ 

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immune ecomnition B 6 Alteredprotein or Protein un-folding function ‎the‏ تور ‎NP Surtace ‎ 

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S-layer and NPs ۳ > The paracrystalline proteinaceous surface layers (S- layers) are one of the most common § surface structures present in all major phylogenetic groups of bacteria and in almost all archaea. >» They are composed of protein or glycoprotein monomers of a molecular weight between 40 and 200 kDa with the ability to selfassembling. >» S-layers have been used as templates for the fabrication of different inorganic nanocrystal arrays 

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Lipids-Nanoparticles Interaction > Liposomes are self-assembled lipid structures. > liposomes encapsulated with nanoparticles have found enormous scopes in various biomedical fields such as drug design, transport, imaging, targeted delivery and therapy. > The encapsulation of nanoparticles in liposomes provides a biologically inspired route in designing therapeutic agents and as a means of reducing nanoparticle toxicity. >» The hybrid lipid/nanoparticle conjugates have diverse biomedical applications including imaging of cancer ‏رتیه‎ drug/gene 061۱۳۷, ‏تا‎ therapy, immunoassay, cell/protein separation, biosensing etc. 

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> Currently, little is known about the influence of nanoparticles on physicochemical properties of lipid vesicles such as stability, elasticity, membrane fluidity and bilayer phase behavior. > In lipid vesicles, nanoparticle encapsulation can be achieved by trapping the particles within the aqueous core or in the hydrophobic bilayer. > To be embedded in the lipid bilayers, the nanoparticles must possess two important features : علاقائط أمأمنا 3 ‎Mala‏ ات رات لابامطد لإعط1 .1 2. and should have a hydrophobic surface (by coating with appropriate agents such as sterylamine) 

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> When the nanoparticles are entrapped within bilayers, it can lead to changes in lipid packing and may disrupt lipid-lipid interactions amongst the head groups and/or ‏الاعج‎ 35. > Disruption of such interlipid interactions can result in changes in lipid bilayer phase behavior, which is related to the degree of lipid ordering and bilayer viscosity. >» When some charged nanoparticles are adsorbed onto cell surface, the membrane undergoes deformation and lipids in the constituent bilayers will be reorganized due to electrostatic interaction between the lipids and nanoparticles/proteins. 

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> Since the membrane is negatively charged, positively charged nanoparticles are attracted more towards the surface of cell-membrane and show higher levels of internalization when compared to uncharged and negatively charged particles. >» Hence, depending on their size and surface chemistry, embedded nanoparticles may influence the stability and function of hybrid vesicles, domain formation, phase separation etc 

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Schematic diagram for the synthesis of SPIO@Liposome-ICG-RGD. A. SPIO nanoparticles was coated with liposome (SPI0@Liposome). B. ICG molecules were loaded into the lipid layer of magnetic liposomes (SPIO@Liposome+IC6). C. RGDs were conjugated to obtain the SPIO@Liposome-ICG-RGD probes 

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Carbohydrates-Nanoparticles 0 Interaction > Although 56۷6۲۵۱ ‏تلاوت تا‎ worked 00 nanomaterials functionalized with proteins, peptides, DNA and RNA during the last decade, very few of them reported on nanoparticles covered with carbohydrates. > But the results of a research carried out by Chun- Cheng Lin (et. Al) are as follows: they have demonstrated that mannose-encapsulated gold nanoparticles (m-AUNP) can be used as a probe to target specific proteins (Con A) in living bacteria. 

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>» They found that the binding of m-AuNP to Con A ۳ exhibited a strong multivalent effect and that the binding specificity of Con A for the multivalent carbohydrate-encapsulated gold nanoparticles (carbohydrate-AUNP) was similar to that of the monovalent counterparts. > they also show that the affinity of m-AuNP for Con A can be adjusted by altering the nanoparticle size or sugar moiety. > Our results demonstrate that nanoparticles can be excellent multivalent carbohydrate carriers for lectins and that carbohydrate-AuNP has great potential as 2 inhibitors of protein-carbohydrate interactions in biological system. 

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31 Schematic illustration of the interactions of carbohydrate-AuNP and Con A on the biosensor chip used .in the competition assays 

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Interactions of Macromolecules and a Nanoparticles ‏ا ع ايلا‎ 1. Biotemplates and Biomimetics : The study of biosynthesis of nanomaterials offers a valuable contribution into materials chemistry. The ability of some microorganisms such as bacteria and fungi to control the synthesis of metallic nanoparticles offers a viable approach as an alternative to chemical and physical ones. Recently, Sadowski et al. (2008) have reported the biosynthesis of silver nanoparticles using Penicillium fungi. 

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>» Various organic molecules and polymers such as amino acids, citric acid, vitamins, cyclodextrin, chitosan, starch, etc. can be employed as biotemplates for the synthesis of metal nanoparticles. > Biomimetic processes: Titanium is a well-known bone repairing material widely used in orthopedics and dentistry. It has a high fracture resistance, ductility, and weight to strength ratio. Unfortunately, it suffers from the lack of bioactivity, as it does not support cell adhesion and growth well. 

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> Ma et al. (2003) have employed a biomimetic process, to form a nanocrystallite apatite coating on metal. A thin bone-like apatite layer was coated onto titanium (Ti) metals via an alkali pretreatment. Their work has shown that the apatite layer grown in this way exhibits nanostructure and has similar stoichiometry to that of natural bone. > It was also observed that the thickness of the apatite layer increases as the immersion period increases. 

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2. Drug and gene Delivery : Nanoparticle-based drug delivery systems are increasingly being used for treatment of certain types of cancer, as opposed to chemotherapy or radiation therapy. The application of magnetic nanoparticles (MNPs) as carriers for drug delivery overcomes this major disadvantage of nonspecificity. The objectives are two fold: (1) to reduce the amount of systemic distribution of the cytotoxic drug, thus reducing the associated side-effects; and (2) to reduce the dosage required by more efficient, localized targeting of the drug. In magnetically targeted therapy, a cytotoxic drug is attached to a biocompatible magnetic nanoparticle carrier such as superparamagnetic iron oxide nanoparticles. 

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Another approach to cancer treatment is hyperthermia where the tumor region is heated locally to the intended temperature without damaging normal tissue. The procedure involves dispersing magnetic particles throughout the target tissue and applying an AC magnetic field with sufficient strength and frequency to cause the particles to heat. The generated heat conducts into the immediately surrounding diseased tissue, and the cancer is destroyed. 

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Application Drug/Gene delivery Drug delivery Photodynamics Drug delivery Drug/gene delivery Imaging (MRI) Imaging In vitro diagnostics Gene delivery Materials Chitosan Dextrane Gelatine Alginates Liposomes Starch Branched polymers Carbon based carriers Polylactic acid Poly(cyano)acrylates Polyethyleinemine Block copolymers Polycaprolactone SPIONS USPIONS Cd/Zn-selenides Silica-nanop articles Mixtures of above Ferrofluids Quantum dots Various 

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Application Optical imaging Drug delivery Hyperemia therapy hyperthermia Encapsulation Drug delivery Drug delivery Drug delivery passive or controlled release roperties wide range of excitation, no photo bleachin| Biocompatible, carry hydrophobic cargo Superparamagnetic, ferromagnetic, paramagnetic Biocompatibility Biocompatibility Biocompatible Less polydispersity, biocompatible Biodegradable CdSe, CdTeete Liposomes, micelles Tron oxide or cobalt based, aggregates in dextran Spheres, rods or shell Spheres, shells Carbon nanotul fullerene, graphene PAMAM ete Chitosan, PLGA 2-10 nm 50-1000 am 3.2-7.5 am 50-100 am 200 1 1-5 0 10-1000 am Quantum dows Lipids ISuperparamagnetic jiron oxide (SPIO) 

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Application Drug/gene delivery Drug delivery/gene delivery Drug/gene delivery Gene delivery Materials Liposomes Chitosan Gelatine Dextrane Starch Alginates Branched polymers Block copolymers Polylactic acid Polycaprolactone Polyethyleinemine Poly(cyano)acrylates Silica-nanoparticles Mixtures of above Particle class Natural materials or derivatives Dendrimers Polymer carriers Various 

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Bioimaging and Magnetic Resonance Imaging .3 > The rapid development of bio-medical sciences demands new advanced techniques and instruments to investigate cells and cellular processes. > In the last years, luminescent nanoparticles (NPs) have attracted growing attention as a versatile and promising tool for bio-imaging. > Bio-imaging involves developing multifunctional nanoparticles with tailored optical and/or magnetic properties for visualizing complex cellular structures (in tissues and organs), receptors, tumor cells, and masses. 

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An interesting and novel application of nanoparticles in biology is their use as intracellular magnetic labels in nuclear magnetic resonance imaging (MRI). The presence of MNPs near a cell results in a much faster rate of magnetic relaxation of protons in the cell. Iron oxide nanoparticles are the most commonly used superparamagnetic contrast agents. Dextran-coated iron oxides are biocompatible and are excreted via the liver after the ‏لاي يفا‎ و > > 

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Limitation Exposure to ionizing radiation and relatively low spatial resolution Relatively high dose of ionizing radiation, limited soft tissue resolution, and exposure to ionizing radiation Relatively low sensitivity Relatively law spatial resolution Limited spatial resolution and unsuitable for examination of digestive organs and bone Advantage Noninvasiveness and high sensitivity Noninvasiveness and high contrast resolution Noninvasiveness and high spatial resolution Noninvasiveness, and no harmful effect by nonionizing radiation Noninvasiveness, real time, low cost, and no harmful effect by nonionizing radiation Nanoparticles Radio-labeled nanoparticles Gold, silver, and iodine nanoparticles Iron oxide nanoparticles, No limit cobalt ferrites, and 603+ labeled nanoparticles Quantum dots Silica, nanobubble Millimeters to centimeters Technique PET/SPECT a Optical Ultrasound 

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4. Sensors and Biosensors > Localized surface plasmon resonance of metal nanoparticles has been exploited in several ways for sensing applications, because this optical characteristic is the basis of various new and highly promising set ups to transduce biorecognitive interactions into visible signals. >» Semiconductor nanoparticles are viable for sensors due tothree main reasons: 1. the sensitivity of surface plasmon band to its immediate environment offers an opportunity to detect attached molecules and environmental changes. 

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2. the reversible aggregation of plasmon resonant particles through specific linkers provides an excellent means for colorimetric assays. 3. The ultra-bright light scattering from each plasmon resonant particle makes the optical detection of a single molecular target possible. Enzymes are also commonly used in biosensors because of their high specificity. Biosensor applications require a highly active immobilized enzyme system that allows the maintenance of an efficient connection between the sensing molecule and the transduction component of the biosensor. 

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Immobilization strategies include : covalent bonding, physical adsorption, cross-linking, encapsulation, or entrapment. A range of biosensors immobilized with specific enzymes are already available that include urease base biosensors for urea, lipase based biosensors for triglycerides, glucose oxidase-based glucose biosensor, acetylcholine esterase biosensors for pesticide detection and many more. 

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Nanostructure Probe moieties ‘Analytes Limit of Substrate Detection _ References ‏عم‎ detection type mechanism NPs Envelope (E) protein of “AngiWest Nile Virus 50 paiml Colloidal SERS Neng et al™ ‘Wert Nile Virus (WN) (WNY) envelope (&) immunoglobulin GNPs Dengue ant-NSI antibody NSI antigen 0.074 jgimt ‘Fiber optic Camara ee al® Gold-enpped sien Raman label engged MS RNA marker of 27 Ghee Pang ee ab nanoparticle array _hairpin-DNA probes HPAI vires: Gold-capped sills Apeamer Bacteria 30 cfu Glass Yoo etal ppanoparticle array Au triangle Detection probe conjugited HBV DNA 50am Siieon Leta rpanearray with Ag nanorice@MGITC@ wafer SiO,, DNA-capture probe HIV-1 noucralizing gp120 ‏تاو مود‎ ITO glass monoclonal antibody ۳۳ Analytes Sensing parameters chitosan-polyar Creatine Amperometric Iginate-pyrrole Glucose Amperometric u nanopatticles/chitosan/TiO,~graphene a-Fetoprotein Amperomettic raphene oxide-chitosan DNA Amperometric chitosan/ionie liquid~graphene composites Bovine serum albumin (BSA) Electrical |Alginate-titanium dioxide nanocomposite Protein Flectrochemical impedance silver/guar gum NH, Optical Frerrite magnetie/chitosan Glucose Potentiometric Jopper nanopatticleichitosan/carbon nanotube ‏جرا‎ Amperometric nCNT Cholesterol Electrical itosan-g-polypyrrole (CHIT--PPy) nanomicelles Urea Optical Choline Blectrochemiluminescent DNA Polymerisation chitosan-Au nanocomposite Dopamine SERS carboxymethylcellulose/gelatin/TiO; ‘Superoxide dismutase “Amperomettic 

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THANK YOU FOR YOUR ATTENTION! 

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: References > M. Rai and N. Duran (eds.), Metal Nanoparticles in Microbiology,DO! 10.1007/978-3-642-18312-6 6, # Springer- Verlag Berlin Heidelberg 2011. > Jasmin Sutkovié, Amina JaSarevié. A review on Nanoparticle and Protein interaction in biomedical applications. PERIODICALS OF ENGINEERING AND NATURAL SCIENCES.Vol. 4 No. 2 (2016). > Saptarshi et al. Journal of Nanobiotechnology 2013, 11:26. >» Romana Parveen, Tooba Naz Shamsi, Sadaf Fatima. Nanoparticles-protein interaction: Role in protein aggregation and clinical implications. International Journal of Biological Macromolecules 94 (2017) 386-395. >» Ying Xu, Guifang Cheng, Pingang He,* Yuzhi Fang. A Review: Electrochemical Aptasensors with Various Detection Strategies. Electroanalysis 2009, 21, No. 11, 1251 - 1259. 

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> PB Santhosh et al 2012 J. Phys.: Conf. Ser. 398 012034. >» Chun-Cheng Lin, et al. Quantitative analysis of multivalent interactions of carbohydrate-encapsulated gold nanoparticles with concanavalin A. CHEM. COMMUN. , 2003, 2920-2921.