Biomolecules immobilization

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Immobilization of biomolecules on the surface of biomaterials

Immobilization of biomolecules on the surface of biomaterialsBy: Mohsen NorouziMSc Student of Tissue EngineeringIslamic Azad University of Najafabad (IAUN)1Biomaterials must possess bulk properties that permit its function in the bioenvironment, but also the best surface properties. It is difficult to design materials that fulfill both needs.A common approach is to fabricate with adequate bulk properties followed by a special treatment to enhance the surface properties.Preface2PrefaceThe broad interdisciplinary area where properties and processes at this interface are investigated and biofunctional surfaces are fabricated is called Biological Surface Science.Examples:medical implants in the human body (dental implants, artificial hip and knee joints, artificial blood vessels and heart valves, etc.)tissue engineeringbiosensors and biochips for diagnosis (DNAchips, etc.)(clinical diagnostics, environmental control, food production)Bioelectronics (systems to get information storage and processing ) and artificial photosynthesis (clean energy)biomimetic materials (mimic the functional properties of biological materials/components in order to achieve new and better materials; ow friction from the sharkskin or selfcleaning character like the lotus leaf )


Preface4Approaches to improve biointerfaces:reduction of unspecific protein adsorptionenhanced adsorption of specific proteinsmaterial modification by immobilization of cell recognition motives to obtain controlled interaction between cells and synthetic substrateUsing methods like selfassembly (SAMs), surface modification, photochemical immobilization or polymer chemistry, complex surfaces with immobilized peptides and proteins can be preparedPreface5Biomolecules used in precision immobilization strategies include proteins, lipids, polypeptides, polynucleotides and polysaccharidesImmobilization techniques range from relatively low to extremely high specificity. characteristics of successful precision engineered biorecognition surfaces:presence of one ligand site and the receptorligand affinityan appropriate surface density of those sitesspatial distribution of the ligandsPreface6The use of short peptides for surface biorecognition has proved to be advantageous over the use of the long chain native ECM proteins, since the latter tend to be randomly folded upon adsorption, being the receptor binding domains not always sterically available.

Preface7ImmobilizationMolecules may be immobilized either passively through;HydrophobicIonic interactionsCovalently by attachment to activated surface groups.Non-covalent surfaces are effective for many applications; however, passive adsorption fails in many cases.Covalent immobilization is often necessary for binding of molecules that do not adsorb, adsorb very weakly, or adsorb with improper orientation and conformation to non-covalent surfaces.Covalent immobilization may result in better biomolecule activity, reduced nonspecific adsorption, and greater stability.8ImmobilizationImmobilization reaction should have several characteristics;Firstly, the reaction should occur rapidly and therefore allow the use of low concentrations of reagents for immobilization.The chemistry should require little, if any, post-synthetic modification of ligands before immobilization to maximize the number of compounds that can be generated by solution or solid-phase synthesis and minimize the cost of these reagents.Immobilized ligands must be in an oriented and homogeneous manner.9ImmobilizationThe immobilization process should occur selectively in the presence of common functional groups, including amines, thiols, carboxylic acids, and alcohols.Amino-NH2,Carboxy-COOH,Aldehyde-CHO,Thiol-SH,Hydroxyl-OH10ImmobilizationSurface density of the ligand should be optimized.Low density surface coverage will yield a correspondingly low frequency.High surface densities may result steric interference between the covalently immobilized ligand molecules, impending access to the target molecules.111) unhindered binding. 2) inaccessible binding site. 3) hindered binding site when adjacent site is occupied. 4) restricted access binding site.

Immobilization12ImmobilizationCorrect orientation of the ligand molecules on the surface, and using a spacer arm are important and critical and makes the ligand available for the target.

13Proteins are much more sensitive to their physiological environments and can easily be degraded or denaturated by physical or chemical effects. Protein`s 3-D confirmation must not change during immobilization procedure.DNA molecules are much more stable then proteins.It is easier to immobilize DNA molecules.Immobilization14Preparation of Surface for Biomolecule ImmobilizationModification of the surface to create functional groups.Modification of biomolecules for covalent attachment to the surface.15General Route for Immobilization

16General Route for Immobilization


General Route for Immobilization18Surface ChemistryCross-linking Strategies for Protein Immobilization

19Surface ChemistryCross-linking Strategies for DNA Immobilization

20Surface engineered scaffoldsCollagen:major structural component forming the natural ECM of connective tissues and organsone of the most established methods for endowing cell adhesive properties to the scaffoldsExamples: PLA and PLGA scaffolds chemically grafted with collagen by plasma treatment have shown enhanced adhesion and spreading of fibroblastsCollagen modification by conjugation reactions onto PLA scaffolds grafted with polymethacrylic acid also has improved cell spreading and growth for use in cartilage tissue engineering.its immunogenicity has limited its applications

21Gelatin:a good alternative for collagen because of its absence of antigenicity and ease of handling at high concentrationsExample:Gelatin immobilized onto porous scaffolds by physical entrapment and chemical crosslinking showed greatly enhanced surface properties on attachment, proliferation, and ECM deposition of osteoblastsSurface engineered scaffolds22Cell adhesive peptides:Rather than immobilizing the whole protein, chemical conjugation of short chain peptide moieties derived from the cell adhesive proteins onto the polymer surface can be a much more effective strategyAdvantages of The surface immobilization of short peptides: higher stability against conformational changeeasy controllability of surface density,orientation more favorable for ligandreceptor interaction and cell adhesionminimizing immune responses and infectionSurface engineered scaffolds23peptide sequences involved in cellular interactions by receptor binding:RGD, IKVAV, and YIGSRRGD sequence: one of the best known foruse in tissue engineering applicationsExamples: Immobilization of RGD onto 3-D matrices to improve cell adhesive properties was previously demonstrated in collagen gels, showing enhanced adherence of murine melanoma cellsRGD, along with other short peptide sequences such as IKVAV, YIGSR, RNAIAEIIKDI from laminin, and HAV from N-cadherin, was also used for engineering of neural tissue.PLA scaffolds modified with RGD by plasma treatment not only resulted in improved adhesion of the osteoblast-like cells, but also supported its growth and differentiationosteoblasts seeded onto the RGD immobilized scaffolds greatly enhanced mineralization and formation of bone-like tissues24

25Hyaluronic acid:a non-sulfated glycosaminoglycan (GAG), is a major substance of the gel-like component in the extracellular matrix of connective tissuescapable of specific cell interaction via the CD44 receptor which promotes wound healing and induces chondrogenesisExamples: Chitosangelatin composite scaffolds modified with HA have been shown to increase the adhesion of fibroblastsPLGA scaffolds modified with HA supported the growth of chondrocytes with maintenance of its original phenotype, showing great potential for cartilage tissue engineering

2626Galactose:utilized in scaffolds for liver tissue engineeringrecognized by mammalian hepatocytes through the asialoglyco protein receptor leading to regulation of a degradative pathway I glycoprotein homeostasisExamples: Porous scaffolds immobilized with galactose have been fabricated to improve hepatocyte attachment, viability, and metabolic functions. Gelatin sponges modified with galactose were shown to support hepatocyte adhesion and function such as release of lactate dehydrogenase (LDH), albumin secretion, and urea synthesis. Perfusion culture of hepatocytes with galactose-derivatized PLGA scaffolds further improved viability and functional activity of the cells

27Heparin:intensively studied for growth factor releasing matrices in tissue engineering.a highly sulfated GAG constituting the extracellular matrix, and is known for its specific interactions with various angiogenic growth factorsExamples:Heparin binding has been shown to preserve the stability and biological activity of the growth factors. A wide variety of scaffold matrices, including nanofibers, prepared from collagen, fibrin, chitosan, alginate, PLA and PLGA, have been incorporated or immobilized with heparin to achieve sustained release of growth factors28

29Examples from Literature (1)

30Strategies for design and preparation of anti-fouling, bioactive (AFB) surfaces1- Surfaces based on PEG:

312- Surfaces based on anti-fouling comb-like polymers

Strategies for design and preparation of anti-fouling, bioactive (AFB) surfaces32

p-nitrophenyl chloroformate (NPC), Disuccinimidyl carbonate (DSC), 1,10-Carbonyldiimidazole (CDI), succinic anhydride (SA) Strategies for design and preparation of anti-fouling, bioactive (AFB) surfaces33

Strategies for design and preparation of anti-fouling, bioactive (AFB) surfaces343- Surfaces based on co-polymer