2024 AQA Proteins and Materials Past Paper PDF

327-1714-00L Biocompatible Materials Proteins and Materials HS2024 25.09.2024 Prof. Dr. Katharina Maniura, Empa, Biointerfaces Dr. Markus Rottmar, Empa, Biointerfaces Prof. Dr. Marcy Zenobi-Wong, ETHZ, D-HEST, Tissue Engineering & Fabrication K...

327-1714-00L Biocompatible Materials Proteins and Materials HS2024 25.09.2024 Prof. Dr. Katharina Maniura, Empa, Biointerfaces Dr. Markus Rottmar, Empa, Biointerfaces Prof. Dr. Marcy Zenobi-Wong, ETHZ, D-HEST, Tissue Engineering & Fabrication Katharina Maniura 1  Important facts - motivation ▪ all biomaterials adsorb proteins/biomolecules upon contact to blood or other body fluids, depending on the surface properties of the biomaterial and the composition of the fluid. https://www.composites.polito.it/biomaterials/2021/04/13/protein.html ▪ cells never see the bare biomaterial surface but the adsorbed protein layer. Proteins can change conformation, and thus function, upon adsorption. September 25, 2024 Katharina Maniura 2 Why is it relevant to characterize proteins at surfaces? Slide: courtesy E. Reimhult, Boku September 25, 2024 Katharina Maniura 3 Drastic differences possible Example dental implant surface storage condtitions… Blood1 Blood + bone cells in vivo hydrophobic hydro SLA phobic hydrophobic hydrophilic SLActive The role of nanostructures and hydrophilicity in osseointegration: In-vitro protein-adsorption and blood-interaction studies. Kopf BS, Ruch S, Berner S, Spencer ND, Maniura-Weber K. J Biomed Mater Res A. 2015 Aug;103(8):2661-72. Enhanced differentiation of human osteoblasts on Ti surfaces pre-treated with human whole blood. Kopf BS, Schipanski A, Rottmar M, Berner S, Maniura-Weber K. Acta Biomater. 2015 Jun;19:180-90. September 25, 2024 Katharina Maniura 4 Protein adsorption in biomaterials design Foreign Body Response-Encapsulation Explanted Explanted biosensor: biosensor: peritoneal peritoneal implant implant after after 70 70 days days Clark et al. 1988 Photo: courtesy K. Ward Goals: Is it possible to prevent or minimize protein adsorption? Can we control protein adsorption to trigger specific cellular responses? September 25, 2024 Katharina Maniura 5  Teaching objectives ▪ A consequence of protein adsorption: the foreign body reaction ▪ Introduction into the mechanisms underlying protein adsorption to (bio-)material surfaces and the implications for biomaterials ▪ What is the Vroman effect? ▪ Understand the basic concept of the DLVO theory and why it cannot fully explain protein adsorption to surfaces ▪ What are the basic components of blood? ▪ What analysis techniques are available to study protein adsorption? ▪ Understand benefits and possible drawbacks of the different analysis techniques recommended book series: “Comprehensive Biomaterials”, Elsevier, 2011, ed. Ducheyne, P.; Healy, K.; Hutmacher, D.W.; Grainger, D.W.; Kirkpatrick, C.J. (-> available online via ETH library) Tengvall, P. Protein Interactions with Biomaterials. Comprehensive Biomaterials (2011) section 4.406 September 25, 2024 Katharina Maniura 6 What often follows protein adsorption......the foreign body reaction Definition: The foreign body reaction is mediated by macrophages and foreign body giant cells reflect the end-stage response of the inflammatory and wound healing responses following implantation of a medical device, prosthesis, or biomaterial. Anderson et al., Seminars in Immunology 20 (2008) 86-100. September 25, 2024 Katharina Maniura 7 Foreign body reaction - key characteristics fibrosis Slide courtesy of Dave Grainger, University of Utah wounding acute inflammation Fibrous Capsule September 25, 2024 Katharina Maniura 8 Foreign body response maybe wanted... ….versus complication! September 25, 2024 Katharina Maniura The foreign body reaction performed by macrophages Normal functions of macrophages: long lived phagocytic cells extend long filopodia (8 m) that can attach to polysaccharides on the bacterial surface bacteria are invaginated into a vacuole that fuses with a lysosome. lysosome: two ways of killing bacteria ➔ generation of toxic reactive oxygen species (H 2O2, NO) ➔ lysosomal proteolytic enzymes (lysozyme) some macrophages patrol through the body, some are stationary within a tissue: lymph nodes, lung (alveolar macrophages), liver (Kupffer cells) etc.… September 25, 2024 Katharina Maniura 10 Macrophage differentiation and activation on a materials surface Sheikh et al., Materials 2015, 8(9), 5671-5701 September 25, 2024 Katharina Maniura 11 Host response to implant surfaces Grainger; Nat Biotechnol, 2013, (31(6)), 507-509 September 25, 2024 Katharina Maniura 12 Repetition: proteins – basic facts ▪ enormous diversity in structure and function ▪ cell surface recognition proteins ▪ structure proteins (e.g cytoskeleton; extracellular matrix/ ECM) ▪ DNA binding proteins ▪ enzymes ▪ make up about 50% of dry mass of cell ▪ determine shape, structure and function of cells ▪ by specific, reversible interactions with other proteins / substrates, proteins can be activated and perform very distinct tasks examples: enzyme – substrate complexes antibody – antigen interactions cell surface receptors – ligand on substrate / material ▪ interaction is regulated by i) quantity of both interaction partners ii) strength and duration of the interaction September 25, 2024 Katharina Maniura 13 Amino acids – protein building blocks 20 different natural amino acids ▪ 20 natural amino acids ▪ identity is defined by the genetic code ▪ polypeptide chain forms by peptide bonds between the amino group and the carboxyl group of adjacent amino acids https://en.wikipedia.org/wiki/Peptide_bond September 25, 2024 Katharina Maniura 14 Amino acid sequence determines shape and function of proteins ▪ the chemical and physical properties of the amino acid side chains and primary protein structure contribute to the spontaneous folding pattern ▪ interaction of polymer chain with the solvent important to drive polymer folding ▪ polar side chains are exposed on the protein surface facing the solvent residue hydropathy ▪ hydrophobic side chains tend to be buried within soluble proteins Ile 4,5 Val 4,2 ▪ protein folds are stabilized by hydrogen bonds Leu 3,8 Phe 2,8 Cys 2,5 Met 1,9 Ala 1,8 Gly -0,4 Thr -0,7 Ser -0,8 Trp -0,9 Tyr -1,3 Pro -1,6 His -3,2 Asp -3,5 Glu -3,5 Gln -3,5 Asn -3,5 Lys -3,9 Arg -4,5 September 25, 2024 Katharina Maniura 15 Summary protein structure / folding ▪ proteins are made from long, non-branched polypeptide chains ▪ primary structure ▪ folded into -helices and -sheets that are stabilized by H-bonds ▪ secondary structure ▪ assembly of secondary structures to domains (approx. 50 – 300 aa) is mediated by the sum of all local interactions ▪ tertiary structure ▪ quaternary structure September 25, 2024 Katharina Maniura 16 Why do proteins adsorb to surfaces? Amino acids have many different properties September 25, 2024 Katharina Maniura 17 Proteins of the human body and their relevance for biomaterials ▪ host proteins (e.g. from body fluids) readily adsorb to foreign materials cells never “see” material surface, only adsorbed protein layer ▪ occurs within the first seconds to minutes after contact September 25, 2024 Katharina Maniura 18 Blood composition - I Tengvall, P. Protein Interactions with Biomaterials. Comprehensive Biomaterials (2011) section 4.406 September 25, 2024 Katharina Maniura 19 Blood composition - II ▪ total protein concentration in native blood approx. 70 mg/ml ▪ major components (“the big twelve”; > 1mg/ml) include: albumin, IgG, low and high density lipoprotein (LDL/HDL), -macroglobulin, fibrinogen, transferrin, -antitrypsin, haptoglobulins, C3, IgA, IgM Plasma Monomer Protein type concentration Molecular weight (mg/ml) (daltons) Pre-albumin 10 - 40 54,900 Albumin 35 - 45 66,500 IgG 6 - 17 150,000 Fibrinogen* 2.0 - 4.0 340,000 Fibronectin* 0.26-0.38 250,000 (but dimeric) September 25, 2024 Katharina Maniura 20  Surface-protein interactions - I Despite the fact that the displacement of water from the surface of a hydrophilic material represents a large energy barrier to adsorption by proteins, the processes of charge interactions and changes in protein conformation provide adequate favorable energetic changes to drive adsorption. ▪ adsorption process is exothermic (G < 0; release of heat) ▪ in most cases, a change in system entropy (solvent, protein, surface) drives protein adsorption with: 1) increase in the amount of free water liberated from between the adsorbent and adsorbing proteins 2) increase in internal mobility of adsorbed proteins ▪ increased exposure of hydrophobic protein residues and internal molecular entropy suggested to be main driving force of protein adsorption in addition to attractive coulomb and v.d.Waals forces (enthalpy) September 25, 2024 Katharina Maniura 21 Surface-protein interactions - II strength ▪ hydrophobic interactions (“lack of interaction with water”) ▪ electrostatic / coulomb interactions (proteins and surfaces are charged; 1/r 2) ▪ van der Waals interactions (dipole interactions; 1/r 6) ▪ π-π bonding (between rings) ▪ ion bridging (via divalent metals, e.g. Ca 2+, Zn2+) Tengvall, P. Protein Interactions with Biomaterials. Comprehensive Biomaterials (2011) section 4.406 September 25, 2024 Katharina Maniura 22 Colloidal theory fails to explain protein adsorption DLVO theory Vtotal = VA +VR v.d.Waals electrostatic attraction repulsion https://doi.org/10.1016/j.ijpx.2018.100002 BUT: proteins are not colloidal particles as they can undergo conformational changes September 25, 2024 Katharina Maniura 23 Surface and protein properties that determine adsorption ▪ protein adsorption and desorption depends on bulk and surface protein concentration, molecular weight of protein, temperature and activation energy / surface energy ▪ follows Langmuir-like behavior Tengvall, P. Protein Interactions with Biomaterials. Comprehensive Biomaterials (2011) section 4.406 September 25, 2024 Katharina Maniura 24 Protein adsorption kinetics The Langmuir model ▪ “Langmuir” model assumptions: ▪ no multilayer formation ▪ uniform surface binding sites ▪ binding to one site does not depend on the neighbors / microenvironment September 25, 2024 Katharina Maniura 25  Vroman effect - I ▪ proteins with highest mobility (low molecular weight) that are structurally flexible and exert no columbic repulsion generally “arrive” first on the surface ▪ small proteins like albumin readily adsorb from plasma short after initial blood-material contact ▪ the adsorbed proteins modify the surface properties ▪ replacement by large, adhesive proteins with time that have a higher surface affinity but lower bulk concentration - albumin – non adhesive for cells - adhesion proteins (i.e. fibronectin) display integrin binding sites (RGD motif) and enable cell adhesion Holden, M. A. & Cremer, P. S. Microfluidic tools for studying the specific adsorption and displacement of proteins at interfa ces. Annu Rev Phys Chem 56, 369–387 (2005). Horbett, T. A. Principles underlying the role of adsorbed plasma-proteins in blood interactions with foreign materials. Cardiovasc Pathol 2, S137–S148 (1993). Vroman et al, L. (1980). Interaction of high molecular weight kininogen, factor XII, and fibrinogen in plasma at interfaces. Blood (55): 156. September 25, 2024 Katharina Maniura 26 Vroman effect - II ▪ HSA - human serum albumin ▪ IgG - Immunoglobulin G / antibodies ▪ FN - fibronectin ▪ Fib - fibrinogen ▪ HMWK – high mol. weight kininogen Tengvall, P. Protein Interactions with Biomaterials. Comprehensive Biomaterials (2011) section 4.406 September 25, 2024 Katharina Maniura 27 Protein corona (on (nano) particles) / conditioning film - I Nel, Mädler, Velegol, Xia, Hoek, Somasundaran, Klaessig, Castranova, Thompson; Nat Mater, 2009, (8(7)), 543-557 September 25, 2024 Katharina Maniura 28 Protein corona (on (nano) particles) / conditioning film - II Nel, Mädler, Velegol, Xia, Hoek, Somasundaran, Klaessig, Castranova, Thompson; Nat Mater, 2009, (8(7)), 543-557 September 25, 2024 Katharina Maniura 29 Consequences of protein adsorption – I ▪ upon protein adsorption – lowered system energy / release of heat ▪ proteins tend to adsorb slightly more to hydrophobic surfaces ▪ higher protein concentration in solution yields thicker protein film - from single protein solution: 1-5 nm (70-350 ng/cm2) - from full serum / plasma: 3-20 nm (210-1400 ng/cm2) ▪ thickening of the protein layer occurs with time ▪ both, intact and proteolytically fragmented proteins may bind to surfaces and reside in their close vicinity ▪ increased residence time leads to an increase in conformational changes with more denaturation on hydrophobic surfaces ▪ degree of denaturation increases with surface residence time September 25, 2024 Katharina Maniura 30 Consequences of protein adsorption – II ▪ firm but not completely irreversible binding – protein exchange process (Vroman effect) occurs more frequently on hydrophilic surfaces ▪ surfaces become opsonized (antibody decoration) and thus recognized as foreign bodies by the cells and the immune system ▪ adsorbed proteins participate in surface-initiated coagulation ▪ both, cells and bacteria may specifically or unspecifically bind onto adsorbed protein, which in turn can co-regulate cell signaling ▪ surface-bound and protein fragments interact extensively with surrounding cells and tissues which in turn regulate homeostasis (recruitment of specific cells, cell differentiation, wound healing) September 25, 2024 Katharina Maniura 31 Protein adsorption analysis methods ▪ spectroscopy ▪ electron spectroscopy (XPS, AES); ion spectroscopy (SIMS) ▪ mass spectroscopy (MALDI-TOF) ▪ infrared spectroscopy, fluorescence spectroscopy, UV-Vis ▪ evanescent field methods ▪ SPR, OWLS ▪ acoustic methods ▪ QCM ▪ optical techniques ▪ AFM, SEM, light / fluorescence microscopy, superresolution microscopy ▪ ellipsometry, contact angle measurement (label free) ▪ radiolabeling techniques ▪ others ▪ ELISA ▪ SDS-PAGE ▪ chromatography September 25, 2024 Katharina Maniura 32 Solution depletion methods ▪ sample with known surface area is placed into protein solution of known concentration ▪ after defined incubation times, solution is separated from solid surface ▪ concentration of the remaining proteins in the supernatant is measured by UV-Vis, circular dichroism, photoluminescence, colorimetric protein assays (BCA, Lowry) ▪ further analysis of supernatant by electrophoresis / ELISA possible September 25, 2024 Katharina Maniura 33 SDS-PAGE steps of protein purification ▪ individual polypeptide chains form a complex with negatively charged sodium dodecyl sulfate (SDS) and migrate through a porous polyacrylamide gel ▪ determination of mol. weight possible September 25, 2024 Katharina Maniura 34 Chromatography ▪ mixture of proteins in solution is passed through a column containing a porous solid matrix ▪ different proteins are retarded to different extends by interaction with the matrix and can be collected separately as they flow out of the bottom of the column ▪ protein separation based on charge, hydrophobicity, size and affinity possible September 25, 2024 Katharina Maniura 35 Mass spectrometry September 25, 2024 Katharina Maniura 36 Proteomics https://microbenotes.com/proteomics/ September 25, 2024 Katharina Maniura 37 Enzyme Linked Immunosorbent Assay (ELISA) ▪ either direct adsorption to surface or specific binding to surface-immobilized primary capture antibody ▪ enzyme conjugated secondary detection antibody binds specifically to primary antibody ▪ after addition of appropriate substrate, enzyme produces detectable signal (UV-Vis, fluorescent, etc.) Phoenix Pharmaceuticals, Inc. September 25, 2024 Katharina Maniura 38 Enzyme Linked Immunosorbent Assay (ELISA) ▪ Advantages ▪ extremely high signal obtained by enzyme amplification ▪ easy to automate (multi-well plates and readers) ▪ multiplexing with immobilized capture antibodies possible ▪ good specificity ▪ Problems ▪ not in real time ▪ steric effects influence the result (orientation of target and proximity of enzyme) ▪ only works if high affinity antibody available September 25, 2024 Katharina Maniura 39 Fluorescence microscopy September 25, 2024 Katharina Maniura 40 Contact angle measurement ▪ surface energy measurements Tengvall, P. Protein Interactions with Biomaterials. Comprehensive Biomaterials (2011) section 4.406 September 25, 2024 Katharina Maniura 41 Ellipsometry ▪ Advantages ▪ label free, in situ technique ▪ imaging possible (2 µm lateral resolution) ▪ 0.1 – 1nm sensitivity (1 ng/µm2) ▪ Problems ▪ requires flat reflecting surfaces ▪ change in phase and amplitude of ▪ special flow through cuvette polarized light provides information needed about thickness and refractive index of adsorbed protein layer ▪ difficult to resolve correct film thickness September 25, 2024 Katharina Maniura 42 Optical Waveguide Lightmode Spectroscopy (OWLS) OWLS measurement of media adsorption on 2% BSA blocked glass surface 600 550 LB media 500 SB media 450 buffer rinse 400 mass [ng/cm ] 2 buffer rinse 350 300 250 200 SB 150 LB 100 2% BSA injection media injection 50 0 0 30 60 90 120 150 180 210 240 time [min] ▪ evanescent field technique ▪ changes in optical path length (effective refractive index change upon protein adsorption) within the evanescent field are captured ▪ label free, 1 ng/µm2 sensitivity, requires optical waveguides that can be easily functionalized with transparent coatings September 25, 2024 Katharina Maniura 43  Quartz crystal microbalance (QCM) ▪ acoustic method; changes in resonance frequency of a quartz oscillator is proportional to the adsorbed mass ▪ entrapped water within the protein layer is measured as well ▪ difficult to determine mass adsorption kinetics September 25, 2024 Katharina Maniura 44 Summary analysis techniques ▪ various techniques available to study protein adsorption ▪ label free vs. label detection methods ▪ in situ vs. ex situ techniques ▪ combinatorial sensing and chemical mapping techniques is required to understand the dynamic arrangement and conformation of proteins on (bio-)material surfaces ▪ in situ sensing of the build-up of hierarchical structures allows to understand the effect of surface engineering for the host response September 25, 2024 Katharina Maniura 45 Thank you for your attention. September 25, 2024 Katharina Maniura 46