Download pdf - Biomolecules immobilization

Immobilization of

biomolecules on the

surface of biomaterials

By: Mohsen Norouzi

MSc Student of Tissue Engineering

Islamic Azad University of Najafabad (IAUN)1

Biomaterials must possess bulk properties that permit its function in the bio‐environment, 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.

Preface2

Preface

The 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 engineering

biosensors and biochips for diagnosis (DNA‐chips, 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 self‐cleaning

character like the lotus leaf )

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Preface4

Approaches to improve biointerfaces:

reduction of unspecific protein adsorption

enhanced adsorption of specific proteins

material modification by immobilization of cell

recognition motives to obtain controlled

interaction between cells and synthetic substrate

• Using methods like selfassembly (SAMs),

surface modification, photochemical

immobilization or polymer chemistry,

complex surfaces with immobilized

peptides and proteins can be prepared

Preface5

Biomolecules used in precision immobilization

strategies include proteins, lipids, polypeptides,

polynucleotides and polysaccharides

Immobilization techniques range from relatively

low to extremely high specificity.

characteristics of successful precision

engineered biorecognition surfaces:

presence of one ligand site and the receptor‐ligand

affinity

an appropriate surface density of those sites

spatial distribution of the ligands

Preface6

The 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.

Preface7

Immobilization

Molecules may be immobilized either passively through;

o Hydrophobic

o Ionic interactions

o Covalently 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.

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Immobilization

Immobilization 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.

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Immobilization

The 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-OH

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Immobilization

Surface 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.

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1) unhindered binding. 2) inaccessible binding site. 3) hindered

binding site when adjacent site is occupied. 4) restricted access

binding site.

Immobilization12

Immobilization

Correct 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.

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Proteins 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.

Immobilization14

Preparation of Surface for

Biomolecule Immobilization

Modification of the surface to create

functional groups.

Modification of biomolecules for

covalent attachment to the surface.

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General Route for Immobilization16



Surface Chemistry

Cross-linking Strategies for Protein Immobilization

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Surface Chemistry

Cross-linking Strategies for DNA Immobilization

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Surface engineered scaffolds

Collagen:

major structural component forming the natural ECM of

connective tissues and organs

one of the most established methods for endowing cell

adhesive properties to the scaffolds

Examples:

PLA and PLGA scaffolds chemically grafted with collagen by

plasma treatment have shown enhanced adhesion and

spreading of fibroblasts

Collagen 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

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Gelatin:

a good alternative for collagen because of its

absence of antigenicity and ease of handling

at high concentrations

Example:

Gelatin immobilized onto porous scaffolds by

physical entrapment and chemical crosslinking

showed greatly enhanced surface properties on

attachment, proliferation, and ECM deposition of

osteoblasts

Surface engineered scaffolds22

Cell 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 strategy

Advantages of The surface immobilization of short peptides:

higher stability against conformational change

easy controllability of surface density,

orientation more favorable for ligand–receptor interaction and

cell adhesion

minimizing immune responses and infection

Surface engineered scaffolds23

peptide sequences involved in cellular

interactions by receptor binding:

RGD, IKVAV, and YIGSR

RGD sequence: one of the best known foruse in tissue

engineering applications

Examples:

Immobilization of RGD onto 3-D matrices to improve cell

adhesive properties was previously demonstrated in collagen

gels, showing enhanced adherence of murine melanoma cells

RGD, 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 differentiation

osteoblasts seeded onto the RGD immobilized scaffolds greatly

enhanced mineralization and formation of bone-like tissues

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Hyaluronic acid:

a non-sulfated glycosaminoglycan (GAG), is

a major substance of the gel-like component

in the extracellular matrix of connective

tissues

capable of specific cell interaction via the

CD44 receptor which promotes wound

healing and induces chondrogenesis

Examples:

Chitosan–gelatin composite scaffolds modified with

HA have been shown to increase the adhesion of

fibroblasts

PLGA scaffolds modified with HA supported the

growth of chondrocytes with maintenance of its

original phenotype, showing great potential for cartilage tissue engineering

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Galactose:

utilized in scaffolds for liver tissue engineering

recognized by mammalian hepatocytes through

the asialoglyco protein receptor leading to

regulation of a degradative pathway I

glycoprotein homeostasis

Examples:

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

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Heparin:

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

factors

Examples:

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 factors

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Examples from Literature (1)30

Strategies for design and preparation of

anti-fouling, bioactive (AFB) surfaces

1- Surfaces based on PEG:

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


anti-fouling, bioactive (AFB) surfaces32

p-nitrophenyl chloroformate (NPC), Disuccinimidyl carbonate (DSC), 1,10-Carbonyldiimidazole (CDI), succinic anhydride (SA)





3- Surfaces based on co-polymers:





Examples from Literature (2)37

Basement Material (Substrate):

Synthetic polymer substrates, polystyrene (PS) and poly(lactic-co-glycolic acid) (PLGA), polydimethylsiloxane (PDMS), silica (Si) and titanium (Ti).

Linkage Material:

Polydopamine

Chemical/Physical Method:

Dipcoating a biomimetic polymer (PD) thin film onto the polymer surface followed by conjugation of adhesion peptides and neurotrophic growth factors to the biomimetic polymer film. Because amine and thiol groups can be covalently conjugated to a PD layer via the quinone group, PD coating exhibits latent reactivity to various nucleophiles with those functional groups

Immobilized Material:

ECM protein-derived adhesion peptides, fibronectin [Arg-Gly-Asp (RGD)] and laminin [Try-Ile-Gly-Ser-Arg (YIGSR)], and neurotrophicfactors, NGF and GDNF

Goal:

Modification of tissue engineering scaffolds for improving the efficacy of stem cell therapy by generating physicochemical stimulation promoting proliferation and differentiation of stem cellssurface modification for efficient and reliable manipulation of human neural stem cell (NSC) differentiation and proliferation

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Result/Effectiveness:

highly efficient, simple immobilization of neuro trophic growth factors

and adhesion peptides onto polymer substrates.

greatly enhance differentiation and proliferation of human NSCs

(human fetal brain derived NSCs and human induced pluripotent stem

cell derived NSCs) at a level comparable or greater than currently

available animal derived coating materials (Matrigel) with safety issues.

versatile platform technology for developing chemically defined, safe,

functional substrates and scaffolds for therapeutic applications of

human NSCs.

efficient surface immobilization of proteins and peptides to a diverse

range of materials, including polymer scaffolds, ceramic substrates, and

metal devices, for stem cell culture and transplantation.

versatile platform technology for efficient development of biomimetic

substrates and scaffolds that induce desirable stem cell behavior and

enhance stem cell function

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Examples from Literature (3)

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ZrO2, TiZr and Ti with its naturally occurring oxide layer TiO2

Linkage Material:

Specific adsorbing peptides (Pep5 (SHKHGGHKHGGH KHGSSGKG)) are

used as anchor molecules to immobilize oligodesoxynucleotides (ODNs)

on the implant surface (anchor strand, AS)


The BAM is conjugated to a complementary ODN strand (CS) which is

able to hybridize to the AS on the implant surface to immobilize the

BAM. The ODN double strand allows for a controlled release of the BAM

adjustable by the ODN sequence and length.


biologically active molecules (BAMs), e.g. antibiotics or growth

factors immobilize the parathyroid hormone (PTH) fragment 1-34

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Successful immobilization of biologically active PTH (1-34)

The high potential of the established surfaces to achieve an increased

osseointegration of variable implants, especially for patients with risk

factors. the development of bioinductive implant surfaces might

increase the healing capacity in the bone, especially for patients with

risk factors such as osteoporosis, where the healing of bone fractures is

disturbed.

The ability of PTH (1-34) to induce the differentiation of osteoblast

precursor cells C2C12 was detected by the quantification of the ALP

activity.

The conjugation of PTH with CS only slightly decreased the Alkaline

phosphatase(ALP) activity, indicating that the biological activity was

almost completely maintained. The application of the immobilization

system on the three materials allows for the modification of the surfaces

with PTH (1-34) as the ALP activity could be increased while

unspecifically bound PTH (1-34) itself showed no effect.

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Examples from Literature (4)

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gold, platinum, glass and titanium

Linkage Material:

Peptide motifs


We synthesized bifunctional quartz-binding peptide QBP1–RGD and titanium-binding peptide TiBP1–RGD peptides via solid phase peptide synthesis and immobilizes these peptide conjugates on the surface through

directed assembly in a single step


poly(ethylene glycol) anti-fouling polymer and the integrin-binding

RGD sequence

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We successfully imparted cell-resistant properties to gold and platinum surfaces using gold- and platinum-binding peptides, respectively, in conjunction with PEG.

several-fold increase in the number and spreading of fibroblast cells on glass and titanium surfaces using quartz and titanium-binding peptides in conjunction with the integrin ligand RGD.

Control over the extent of cell–material interactions by relatively simple and biocompatible surface modification procedures using inorganic binding peptides as linker molecules.

Targeted assembly proved to be an efficient way of immobilizing large molecules (i.e. PEG) through, first, coating the inorganic binding peptides and then performing the conjugation reaction.

Directed assembly, on the other hand, is preferred for the immobilization of small molecules by synthesizing a single chimeric molecule with bi functional domains.

Control over the extent of cell–material interactions can be achieved by relatively simple and biocompatible surface modification procedures using GEPIs as linker molecules.

QBP1 and the TiBP1 facilitate the immobilization of RGD on both surfaces while preserving its functionality as a recognition site for cells.

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References:

Prof. Marco Mascini, Immobilization of Biomollecules, Grenoble, 2004.

Laia Francesch de Castro , Surface modification of polymers by plasma polymerization techniques for tissue engineering, doctorate thesis, Universitat liull, Barcelona .

Hyun Jung Chung, Tae Gwan Park, Surface engineered and drug releasing pre-fabricated scaffolds for tissue engineering, Advanced Drug Delivery Reviews 59 (2007) 249–262.

Tina Micksch et al, A modular peptide-based immobilization system for ZrO2, TiZr and TiO2 surfaces, Acta Biomaterialia (2014).

Qian Yu et al, Anti-fouling bioactive surfaces, Acta Biomaterialia 7 (2011) 1550–1557.

Dmitriy Khatayevich et al, Biofunctionalization of materials for implants using engineered peptides, Acta Biomaterialia 6 (2010) 4634–4641.

Kisuk Yang et al, Polydopamine-mediated surface modification of scaffold materials for human neural stem cell engineering, Biomaterials 33 (2012) 6952e6964.

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Thanks for your attentionGood luck

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