Surface Engineering Using Peptide Amphiphiles

4.415.

Surface Engineering Using Peptide Amphiphiles

W H Suh, University of California, Berkeley, CA, USA; University of California, Santa Barbara, CA, USA M Tirrell, University of California, Berkeley, CA, USA ã 2011 Elsevier Ltd. All rights reserved.

4.415.1. 4.415.2. 4.415.3. 4.415.3.1. 4.415.3.2. 4.415.3.3. 4.415.4. 4.415.4.1. 4.415.4.2. 4.415.4.3. 4.415.5. 4.415.6. 4.415.6.1. 4.415.6.2. 4.415.6.3. 4.415.6.4. 4.415.6.5. 4.415.6.6. 4.415.6.7. 4.415.6.8. 4.415.7. References

Introduction The Amphiphilic Nature of Life’s Events PAs: Synthesis, Physicochemical Characterization, and Self-Assembly Synthesis and Characterization of PAs Double-Tailed (Dialkylated) PAs Single-Tailed (Monoalkylated) PAs The Multifunctional (Soft) Nanoparticle Concept Targeting-Enabled PA Spherical Micelles Single-Tailed PAs for Cancer Cell Biology Control In Vitro Binding Affinity Control via Native Chemical Ligation Protein-Like Structures to 3D Hierarchical Nanostructures Applications in Biomedical Sciences: Tissue and Stem Cell Engineering Helical Structure and Antibacterial PAs Neural Stem Cell Engineering Bone Marrow Mononuclear Cells Ameoblast Cell Engineering for Tooth Regeneration Heparin-Binding PAs for Regenerative Medicine Human Mesenchymal Stem Cell Engineering Dental Stem Cells Bioactivation of Metallic Surfaces Summary and Future Directions

Glossary Angiogenesis Growth of new blood vessels from preexisting vessels. ApoE-null mouse ApoE stands for plasma protein apolipoprotein E, binding to a specific receptor present in liver cells and peripheral cells. It is an essential protein for the normal catabolism of triglyceride-rich lipoprotein constituents. Mice that lack the ApoE gene are either called the ApoE-null or ApoE-knockouts (KO). Apoptosis The process of programmed cell death involving characteristic changes related to cell morphologies such as blebbing, loss of cell membrane asymmetry, and cell shrinkage. Astrogliosis An abnormal increase in the number of astrocytes due to the destruction of neurons. Atherosclerotic plaques Fatty materials such as cholesterol buildup in the arterial wall. Axon A long, slender projection of a nerve cell (i.e., neuron) that conducts electrical impulses away from the neuron’s cell body or the soma. Ball-and-stick model A molecular model of a chemical structure that aims to display both the 3D positions of the atoms and the bonds. bFGF Basic fibroblast growth factor is present in the basement membranes and in the subendothelial extracellular matrix of blood vessels. bFGF is involved in the

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angiogenesis process and, in research, is a critical component in stem cell cultures. Bioactive A material property based on its interacting ability with cellular organisms and tissues in vitro and in vivo. Blastocyst A core–shell structure formed during the early embryogenesis of mammals. The shell is comprised of tropoblasts and the core contains the inner cell mass (ICM or embryoblast) and fluid-filled cavity (blastocoele). Blastocyst, in humans, begin formation 5 days after fertilization. (Cell) Nucleus A membrane-enclosed cell organelle found in eukaryotic cells. The chromosome inside the nucleus is a complex structure made up of DNA and histone molecules. Gene expression control is done here in the nucleus. Cell membrane A biological membrane comprised of a phospholipid bilayer with incorporated proteins. Cell or cellular adhesion The binding of a cell to a surface (i.e., extracellular matrix) or a neighboring cell via adhesion molecules such as integrins, cadherins, and selectins. Cell signaling A complex system of association that governs cellular activities and coordinates cell actions. The breakdown of the cell signaling pathways can lead to disease formation. Traditionally, focus has been given to exploring individual parts within a cell signaling pathway but, in recent times, systems biology approaches have allowed researchers to investigate multiple pathways and link them together to study a network of genes and proteins.

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Surface Engineering

Centriole A barrel-shaped cell structure found in eukaryotic cells. Microtubules comprise the walls and are arranged perpendicular to the pericentriolar material – this associated pair is called the centrosome. Circular dichroism (CD) CD refers to the differential absorption of left and right circularly polarized light and is exhibited by optically active chiral molecules such as proteins. Collagen A family of fibrous proteins rich in glycine and contains hydroxyproline and hydroxylysine. It is found throughout vertebrates and is the most abundant protein in mammals. This protein is a major element in skin, bone, cartilage, teeth, blood vessels, etc. COMPASS forcefield An ab initio forcefield optimized for condensed-phase applications. COMPASS stands for Condensed-phase Optimized Molecular Potentials for Atomistic Simulation Studies. CREKA A pentapeptide with a sequence of CysteineArginine-Glutamic acid-Lysine-Alanine that preferentially homes to tumor tissue. It was identified via in vivo phage display. Cytoskeleton Internal proteinaceous framework of a eukaryotic cell composed of actin microfilaments, intermediate filaments, and microtubules and giving shape to a cell, and providing support for cell extensions such as villi and axons of nerve cells. Cytosol The cytoplasm other than the various membranebound organelles. DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene, commonly used in organic synthesis as a nonnucleophilic base. Dentin A calcified tissue constituting the interior hard part of vertebrate teeth between the enamel and the pulp gum, also written as dentine. Differentiation (cellular) The cellular process of a less specialized cell transforming into a more specialized (thus differentiated) cell type. DMEM Dulbecco-modified Eagle’s medium utilized to maintain cells. It contains varying amounts of inorganic salts, amino acids, glucose, and vitamins. The most concentrated salt is sodium chloride. Specific media formulations are available from vendors. DPC Dodecylphosphocholine, a single-C12-tailed phospholipid. CAS No. 29557-51-5. DPPC 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, a double-C16-tailed phospholipid. CAS No. 63-89-8. DPPE 1,2-Dipalmitoyl-sn-glycero-3phosphoethanolamine, a double-C16-tailed phospholipid. CAS No. 923-61-5. DPPG 1,2-Dipalmitoyl-sn-glycero-3-phospho-(1’-racglycerol) (sodium salt), a double-C16-tailed phospholipid. CAS No. 200880-41-7. DSPE 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, a double-C18-tailed phospholipid. CAS No. 1069-79-0. Dynamic light scattering (DLS) A light-based physics technique used to determine the size distribution of small particles in liquid solutions undergoing Brownian motion. A monochromatic light source (e.g., fixed wavelength laser) is often utilized and the time-dependent fluctuation of the scattering intensity is monitored.

Endocytosis A membrane-based process for eukaryotic cells taking up foreign materials. Endoplasmic reticulum (ER) Extensive, convoluted internal membrane in eukaryotic cells, continuous with the outer nuclear membrane and enclosing a continuous internal space (lumen); involved in the synthesis and transport of membrane proteins and lipids, and of proteins destined for secretion from the cell. Extracellular matrix Macromolecular ground substance of connective tissue, secreted by fibroblasts and other connective tissue cells, generally comprised of proteins, polysaccharides, and proteoglycans. FAM Carboxyfluorescein, a fluorescent dye molecule often utilized to tag proteins and incorporated into micelles and liposomes. Excitation/emission occurs at 492/517 nm. Fluorescence Emission of light by a substance that has absorbed electromagnetic radiation often at a lower wavelength. It involves ground-state excitation via absorption, radiationless decay, and then a radiative transition back to the lower electronically excited state and ceases as soon as the source illumination is removed; it is different from phosphorescence. Fmoc-chemistry 9-Fluorenylmethoxycarbonyl-based synthesis technique. The Fmoc-protecting group attaches to the amine-terminus of an amino acid and is selectively removed by bases such as piperidine and DBU. Glial scar A reactive cellular process involving astrogliosis that occurs after injury to the CNS (central nervous system) and is the body’s mechanism to protect and begin the healing process. Globular protein A class of proteins that have metastablefolded spherical 3D structures and forms colloidal solutions after aqueous medium dissolution. The other class of proteins are fibrous proteins. Glycolipid Lipids containing carbohydrate head groups and serving as cellular recognition sites. Glycoprotein Proteins incorporating carbohydrate chains, ubiquitous in cell membranes and cellular secretions. If they become integral membrane proteins, they are involved in cell–cell interactions. An example is mucin. Golgi apparatus Stacks of flattened membrane sacs present in eukaryotic cells and are involved in directing membrane lipids and proteins and secretory proteins to their correct destination in the cell. Growth factor (GF) A general term for specific peptides and proteins required by particular cells for division and/or differentiation. Examples are epidermal growth factor, basic fibroblast growth factor, and nerve growth factor. Heparin A highly sulfated glycosaminoglycan (polysaccharide) found in mast cells that has anticoagulant activity. This molecule will bind and activate the enzyme inhibitor antithrombin III which then inactivates thrombin and other proteases such as factor Xa. Hirulogs A group of synthetic peptides designed to inhibit thrombin, leading to anticoagulation. HOX Homeobox, a DNA sequence found within genes that are involved in the regulation of patterns of anatomical development (morphogenesis) in animals, fungi, and plants.

Surface Engineering Using Peptide Amphiphiles

Hydrophobicity The physical property of molecular species (often nonpolar) being repelled from a mass of water, or forcing the structuring of water as a solvent around this molecule. The tendency to positively interact with or be dissolved by water will be referred to as hydrophilicity. IKVAV A pentapeptide with a sequence of isoleucine-lysinevaline-alanine-valine that promotes cell adhesion, derived from the extracellular matrix protein laminin. Inflammation or inflammatory response A complex host-initiated biological process following infection or tissue damage or after a particular immune reaction. Integral protein A protein assembly that is permanently associated to the biological membrane and is difficult to isolate without the use of detergents or nonpolar solvents. Integrin receptor (or Integrins) A class of cell receptor molecules that mediate the association between a cell and the surrounding environment (e.g., extracellular matrix, other cells). Integrins are involved in cell signaling processes that often define cell mechanics and regulate the cell cycle. Ion channel A type of pore-forming integral membrane protein that allows selective flow of ions across the plasma membrane of the cell, thus controlling the electrochemical gradient formed inside and outside the cell. Iron oxide Inorganic oxides composed of varying degrees of iron and oxygen. The most common iron oxide is Fe2O3, rust, and Fe3O4, paramagnetic magnetite. IV-H1 A 15-mer peptide with a sequence of Gly-Val-Lys-GlyAsp-Lys-Gly-Asn-Pro-Gly-Trp-Pro-Gly-Ala-Pro derived from the extracellular matrix protein collagen known to promote cancer cell adhesion and spreading. Lipid bilayer A thin membrane composed of two layers of lipid. The cellular membrane is a phospholipid bilayer that incorporated multiple proteins, glycolipids, etc. Lipidation A covalent addition process of a hydrophobic molecule (e.g., palmitic acid) onto a biomolecule (e.g., protein). LyP-1 A cyclic nine-amino acid peptide, CGNKRTRGC, that can preferentially bind to tumors. Lysosome A cellular organelle that contains acid hydrolases capable of breaking up cellular debris (e.g., worn-out organelles, food particles) and waste materials (e.g., engulfed viruses and bacteria). The organelle’s pH is below 5. Magnetic resonance imaging (MRI) A medical imaging technique utilizing an electromagnetic field that allows the visualization of soft tissue based on varying degrees of proton relaxation in different tissues and organs. Mass spectrometry An analytical technique that measures the mass-to-charge ratio of ionized (thus charged) particles. Metabolite The intermediates and products of metabolism and is often restricted to small molecules such as ethanol and glutamic acid. Metallic alloy A partial or complete solid solution of one or more elements in a metallic matrix. Metallic foam A porous (cellular) structure consisting of a solid metal that incorporates a large volume fraction of gas-filled pores (typically 70–95% porosity).

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Micelle An aggregate of surfactant molecules dispersed in a liquid solution. This higher-order structure forms above the critical micelle concentration (CMC), often in the mM range. Mitochondrion A membrane-enclosed organelle found in most eukaryotic cells and the main source of ATP (adenosine triphosphate), used as a chemical energy source. It also contains nucleic acids. MSC (mesenchymal stem cell) Bone marrow stromal cells. A population of multipotent adult stem cell cultures discovered from the mesenchyme and the marrow that has been shown to differentiate into osteoblasts, chondrocytes, myocytes, and adipocytes. It is now also identified in nonmarrow tissue such as muscle and the dental pulp. Nanoparticles Particles that are submicron, often 100 nm or below. Nanostructure Structure exhibiting significant submicron features. Native chemical ligation (NCL) A technique that reacts a C-terminal thioester with another peptide (or protein) containing an N-terminal cysteine residue. Neural progenitor cell (NPC) Progenitor cells have a tendency to differentiate into a specific cell type with a limited amount of division possible. The term is sometimes used in place of neural stem cells, but (neural) stem cells can replicate indefinitely. Neural stem cell (NSC) Self-renewing, multipotent adult stem cells able to generate the main phenotypes of the nervous system – neurons, astrocytes, and oligodendrocytes. NiTi (nickel–titanium) A metallic alloy made up of nickel and titanium. Nuclear magnetic resonance (NMR) A property that magnetic nuclei have in a magnetic field and applied electromagnetic (EM) pulse(s), causing the nuclei to absorb energy from the EM pulse and radiate this energy back out. The energy-radiated back out is at a specific resonance frequency that depends on the strength of the magnetic field and other factors. Nuclear overhauser effect (NOE) The transfer of spin polarization from one spin population to another via cross-relaxation in NMR spectroscopy. NOE differs from spin coupling in the respect that it is a through-space observation rather than through bonds. Nuclear transport Macromolecules such as RNA and proteins associate with karyopherins to enter and exit the cell nucleus via the nuclear pore complexes (NPCs), whereas small molecules can be transported without any regulatory process. Osteogenic differentiation Cellular differentiation into fibroblast cells that, in addition, express the bone sialoprotein (or osteopontin) and osteocalcin. PBX Pre-B cell leukemia homeobox refers to a family of transcription factors. They act as cofactors in the transcriptional regulation mediated by homeobox proteins during embryonic development and cellular development. Peptide amphiphile (PA) A molecule that covalently links hydrophobic lipid tails together with hydrophilic peptide head groups. A linker segment (e.g., PEG) can be added in the middle.

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Surface Engineering

Peripheral membrane protein A class of proteins that temporarily associate with the biological membrane either by attaching to integral membrane proteins or penetrating the peripheral region of the lipid bilayer. Phospholipid A class of lipids comprising fatty acid chains (i.e., diglyceride), a phosphate group, and a small organic molecule such as choline. Photocurable Polymerization occurring while exposed to a light source. Posttranslational modification (PTM) The chemical modification of a protein after the translational process. Examples include phosphorylation, ubiquitination, palmitoylation, and acetylation. Quantum dot A size- and shape-defined semiconductor whose excitons are confined in all three dimensions, often a few nanometers in size. RGD A tripeptide with a sequence of Arginine-GlycineAspartic acid and is derived from the extracellular matrix protein, fibronectin. Peptide, proteins, and materials incorporating the RGD sequence have been shown to promote cellular adhesion. Ribosome A component within the cell that associates with mRNAs and tRNAs in the cytosol to produce peptides and proteins enzymatically (the translation process). SC4 A 12-mer bactericidal peptide that forms helical structures that have a sequence of KLFKRHLKWKII. Self-assembly A term utilized to describe the processes in which a disordered system of preexisting components forms an organized structure or pattern as a consequence of specific, local, and cooperative interaction. If the components are molecules, the process is termed molecular self-assembly. Self-renewal A cellular process that allows an unlimited number of cell divisions and replications. Silanize or silanization Surface functionalization via silane molecules such as (3-aminopropyl)-triethoxysilane, (3-mercaptopropyl)-trimethoxysilane, and octadecyltrimethoxysilane.

Spinal cord injury (SCI) An injury to the spinal cord that causes myelopathy (loss of nerve function) or damage to nerve roots or myelinated fiber tracts that mediate signals to and from the brain. Stem cells Cells that are able to self-renew and differentiate into multiple cell types. Mammalian stem cells are broadly categorized into either embryonic stem cells or adult stem cells. Theranostic application An application that enables both medical diagnostics and treatment. Transcription The process of single-stranded DNA being copied into the complementary RNA. Translation Part of the protein biosynthesis process that involves the production of polypeptides via the association among mRNA, tRNA, and the ribosome. Transplantation Instillation of an organ from one body to another for the purpose of replacing the recipient’s damaged or absent organ. It can also involve the use of cell therapy sources such as stem cells and bone marrow cultures. Vacuole A membrane-bound cavity in the cytoplasm of eukaryotic cells that contains air, water, cell sap, and digested food. It is present especially in plant and fungal cells while found to a lesser extent in animal and bacterial cells. Vascular endothelial growth factor (VEGF) VEGF is a signal protein involved in the stimulation of new blood vessel production. Vesicle A supramolecular assembly made up of lipid bilayers created by double-tailed lipid molecules such as phospholipids. Basically, it is a membrane-enclosed sack that has a hydrophilic core. Artificially prepared vesicles are referred to as liposomes. Viscosity A measure of the resistance of a fluid being deformed by either shear stress or tensile stress. W3K A 17-mer peptide with a sequence of WAAAAKAAAAKAAAAKA that is alanine-rich but watersoluble and forms worm-like micelles upon lipidation. Wound healing An intricate and dynamic process in which tissues and organs repair themselves after sustaining injury.

Abbreviations

SEM TEM TFA

DMF PEG SDS

4.415.1.

Dimethyl formamide, a common solvent used in peptide synthesis. Poly(ethylene glycol) Sodium dodecylsulfate, a common anionic C12-alkylated surfactant molecule.

Introduction

Biomaterials1–20 can be divided into two different categories: one that exists in nature21–25 or one that (favorably) mimics nature.26–32 One facet of applications involving biomaterials is in the field of tissue and bio(medical) engineering. Bioengineers, on a regular basis, prepare biomaterials to influence

Scanning electron microscopy Transmission electron microscopy Trifluoroacetic acid

biological events where genetic codes are transcribed and then translated within a dynamic environment in the cell. When taking into account the complexity involving biological systems, it is critical that surface properties are well understood all the way down to a single molecule or even an element. Multiple surface engineering techniques are available depending on the mode and degree of action, meaning the change of

Surface Engineering Using Peptide Amphiphiles

physicochemical properties posttreatment (engineering). Surface cleaning, etching, and depth profiles are often done using radiation (beam) (i.e., plasma treatment33–35; electron beam lithography36,37; UV38–41; ultrasound16,42) (Mode 1). Synthetic chemistry-based techniques43 are used when specific molecules need attachment (i.e., covalent bond formation; selfassembled monolayer) or a passivation of polymeric-networked materials (i.e., layer-by-layer technique44–47; core–shell material synthesis15,48; polymer coating on glass via photocuring49,50) is desired (Mode 2). Spatial positioning of molecules and nanoparticles on extended surfaces with nanometer-level precision can be achieved via printing methods (i.e., microcontact stamping51–58; dip-pen lithography printing59,60; ink jet printing61–66 (Mode 3). Intermolecular associative and repulsive forces (interfacial phenomena)67,68-induced structuring can be useful as well to achieve well-defined nanostructured surfaces, and this is where self-assembly or cooperative assembly of molecules and (nano)materials69–72 plays a critical role in the outcome of the final complex structure (Mode 4). (Figure 1) In this chapter, we cover surface engineering efforts performed using peptide amphiphiles (PAs) (see Chapter 2.205, Self-Assembling Biomaterials). PAs provide a platform of bioactive nanostructured materials that provide biomaterials with a controlled display of functional and biologically responsive moieties on the periphery of the resulting biomaterial construct; PAs have the potential to mimic nature. The hierarchical two- and three-dimensional (3D) structures formed by PAs via self-assembly processes are spherical vesicles and micelles, lipid layers, and fibrous high-aspect-ratio structures (Figure 2).17,18,74–80 The usual characterization methods for such higher-order structures involve dynamic light scattering (DLS), circular dichroism (CD), fluorescence spectroscopy, and microscopy. One of the key features of a lipidated peptide is that sequences incorporating a distinct secondary structure (i.e., a-helix) will often experience stabilization of the structure over a time domain compatible enough to elicit a cell (signaling) response (i.e., adhesion). We highlight work by several different groups over the past two decades reporting PAs0 (nano)structural features as well as biological responses in cellular and animal models. We view ‘surfaces’ as critical components having unique spatial and temporal domains; they exist over multiple length scales and time domains often bioactive enough to interact with living systems undergoing dynamic changes with varied magnitudes and frequency depending on the type of biosystem. PAs can form surfaces that present bioactive subdomains and physicochemically unique properties that may architecturally and biochemically reproduce materials present in nature (i.e., the cell membrane, subcellular machinery, the extracellular matrix) (see Chapter 4.406, Protein Interactions with Biomaterials and Chapter 2.207, Extracellular Matrix: Inspired Biomaterials).

4.415.2.

The Amphiphilic Nature of Life’s Events

The cell membrane (shown in Figure 3) comprises multiple amphiphilic and hydrophobicity-controlled structures: lipids, proteins, polysaccharides, and nucleic acids.15 Among the aforementioned biological entities, proteins interact with other biomolecules in a complex but intricate manner; most

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importantly, the role and the fate of proteins within a specific cell biology subsystem (i.e., receptor-mediated signaling pathway, cellular adhesion process, cytoskeleton rearrangement, endocytosis, inflammatory response, translation, transcription, ion channeling, posttranslational modification, apoptosis, nuclear transport, differentiation, self-renewal, etc.) will depend strongly on the dynamics associated with the protein’s primary and secondary structures – for example, the hydrophobicity and the a-helical propensities will vary in accordance with the incorporated amino acid residue characteristics. We consider a cell membrane as a 2D soft surface that can be mimicked on flat surfaces. In living systems, however, the cell membrane exterior and interior are not interfaced to a hard surface (except in bone) and is, in fact, three-dimensionally responsive through multiple cellular and extracellular signaling pathways that play critical roles in the overall function of cells. In this respect, we can define self-assembly processes occurring in multiple biological systems constituting 3D structural cues and niches – meaning that coassembly of subcomponents are important to produce functionality (see Chapter 4.401, The Concept of Biocompatibility).

4.415.3. PAs: Synthesis, Physicochemical Characterization, and Self-Assembly PAs (or lipopeptides; Figure 2) are a class of small molecules that link peptides (as the hydrophilic head group) together with hydrocarbons (hydrophobic tail). Unlike naturally occurring lipids, this platform of materials is synthetically prepared and usually has a molecular weight of 1000–3000. The use of PAs to address biomaterials and bioengineering questions has been developed since the mid-1990’s. A highly efficient solid-phase synthesis method was employed by Tirrell and coworkers where dialkyl hydrocarbon chains were covalently bonded to bioactive peptide sequences such as Gly-Val-Lys-Gly-Asp-Lys-Gly-Asn-ProGly-Trp-Pro-Gly-Ala-Pro (denoted as [IV-H1] from here on) derived from the extracellular matrix protein collagen, as shown in Figure 4.80–85 The chemistry behind such PA synthesis is Fmoc-chemistry.86–89 The motivation to utilize bioactive peptides displaying amphiphilic construct is to mimic aspects of native designs – for instance, the stem cell niche or the tumor microenvironment. The [IV-H1] sequence is a 15-mer biologically proved to promote cancer cell adhesion and spreading while physicochemically observed (via CD, NMR, etc.) to form a triple-helical structure, often the secondary protein structure naturally occurring in collagen (co)assembly.80,83

4.415.3.1. Synthesis and Characterization of PAs The synthesis protocol outline for synthesizing a single-tailed PA is given below (Figure 5). Characterization generally involves mass spectrometry, CD, and NMR. Protocols developed by Tirrell and coworkers80–85 can be easily applied to both dialkylated and monoalkylated PA synthesis.

4.415.3.2. Double-Tailed (Dialkylated) PAs Double lipidation of cationic peptides often leads to potent antibiotic agents. In the context of surface engineering, double-tailed (low molecular weight) PAs displaying bioactive

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Mode 2 – On/off

Energy source

Mode 3

hv

Xab+

RO

R

SH

R

Print

O

O

O

Si O

O

O

Si O

O O O

Liposomes

Cast

H2O

hv Si

Si O

Polar head Hydrophobic tail sed ces Pro

H2O

Print

OR

NH2

O

H2O -assembly

Scan

Si

RO

H2O H2O

Chemical reaction

HO

Nonnative surface

Biomolecules

Self

X

e–

Polymers Polyelectrolytes

Peel off

R = hydrocarbon X = NH2, SH, hydrocarbon, etc.

Mode 4

Evaporation

H

Drying

Si

Si

Native surface

Surface cleaning, etching, depth profiling

Chemistry-based surface passivation of layers and networks

Spatial placement of physicochemically defined patches of functional domains

Intermolecular forces based nanostructuring

Patches

Removed nonnative surface Removed native surface

Coating

Surface engineered native surface

Self-assembled surface coating X

NH

2

SH R

R

R

Transferred molecular components Nanostructured topological features

Si Si Si Si OO O OO O O O O O O O

Si

Si

Si

(x,y)

Surface engineering result

(m,n)

(a,b)

Chemically redefined surfaces Distinct spatial addresses

Component X

Component M

Component A

Molecular level resolution

Figure 1 Modes of surface engineering. See main text for details. This review will cover mode 4, in depth. Various combinations of all four modes, however, can lead to unique surfaces with hierarchically, physicochemically, and biologically distinctive properties.

Surface Engineering

Mode 1

Surface Engineering Using Peptide Amphiphiles

O

H N

O

N

H O

O

O

N

O

H H N

N

O

O

NH2

HN

Peptide amphiphile chemical structure

H

225

O

O OH H

O

N

O

N

H O

N OH

OH O

O O

Hydrophobic tail

Spacer

Hydrophilic head

Self-assembly

Vesicle Ball stick model

Micelle

Self-assembled structures

Figure 2 Self-assembled structures of peptide amphiphiles. Based on the degree of lipidation, peptide amphiphiles will form vesicles or micelles. The chemical structure represents the double-tailed peptide amphiphile (C16)2-Glu-C2-PEG-GRGDSP which is synthesized from covalent linkages among glutamate dialkyl ester 1’,3’-dihexadecyl L-glutamate (two hexadecanols attached to glutamic acid; the hydrophobic tail), 3,6,9-trioxaundecanedioic acid (polyethylene oxide spacer), and a 6-mer peptide head group Glu-Arg-Glu-Asp-Ser-Pro (the hydrophilic head).73 The ball-and-stick model represents the single-tailed peptide amphiphile C16-WA4KA4KA4KARGD,74 created using Accelrys DS Visualization software.

Secreted proteins Cell membrane n+

Surface proteins

DNA

M

RNA

RNA

Localized proteins (i.e., enzymes)

RNA

Nn

+

Ion channel Nucleus ABC Cytosol

A+BC metabolites

Cytoskeleton 10 μm (a)

Blood cell

Epithelial cell

Multifunctional sub-10 nm (soft) nanoparticle Bioactive subdomain 1 (i.e., imaging) Ligand–substrate interaction Glycoprotein

Blastocyst

Bioactive subdomain 2 (i.e., targeting) Matrix micelle or vesicle Globular Glycolipid protein

1–2 nm

(b)

Muscle cell

Nerve cell

Surface Protein Integral (c) protein channel protein

Cytoskeleton Peripheral membrane protein

Figure 3 A cell as a multifunctional entity. (a) The breakdown of cellular machinery shows that a mammalian cell is essentially a multifunctional micron-sized particle comprised of internal and external components that function in coordination. The external components are (1) the cell membrane, (2) surface proteins/ion channels, and (3) the cytoskeleton, while the internal components are (4) the cytosolic organelles (i.e., ribosome, endoplasmic reticulum, golgi apparatus, mitochondrion, vacuole, lysosome, and centriole) and (5) the nucleus (which contains nucleic acids). Another major component in a living cell consists of the active biological processes that occur in a hierarchical and consistent fashion. These so-called biological pathways involve proteins, metal ions, nucleic acids, amino acids, external stimuli, and other biomolecules to work in concert with one another. The represented scanning electron microscopic (SEM) image shows two murine BV-2 microglia cells attached to a surface. (b) Multiple cell types exist differing in their function, shape, and size. (c) The cell membrane consists of many functional biomolecules, including multiple forms of functional proteins, displayed carbohydrates, and the lipid bilayer. A bioactive and nanometer-sized material (i.e., multifunctional micelles, liposomes, and inorganic nanoparticles) can directly interact with such biomolecules present on or inside a cell as shown in the illustration. It is important to precisely define the nature of such interactions in order to elicit a biologically definable process through certain types of surface engineering presented in Figure 1. Adapted from Suh, W. H.; Suh, Y. H.; Stucky, G. D. Nano Today 2009, 4, 27–36.

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Surface Engineering

Fmoc

Protected peptide

O

Lipidation

Deprotection H2N

O

H N

O

Protected peptide

O O

N H

H N O

Protected peptide

O

Cleavage

Protection groups (i.e., Boc, tBu)

+

Product

+ Beads

Figure 4 Synthetic outline of peptide amphiphile based on Fmoc-chemistry. A protected peptide is first prepared on a solid polymer bead using a standard peptide synthesizer. The amine-protecting Fmoc group is removed using base reagents (i.e., piperidine, piperidine/DBU, or 4-methylpiperidine in DMF), followed by lipid tail (or linker) addition. After the hydrocarbon tail addition process is complete, the peptide amphiphile and the protecting groups are cleaved under a mild acid condition (i.e., 95% TFA). Adapted from Berndt, P.; Fields, G. B.; Tirrell, M. J. Am. Chem. Soc. 1995, 117, 9515–9522.

sequences can be embedded into lipid bilayers created by phospholipids via vesicle fusion. This feature allows facile screening of functional molecules that play an intricate role in the cell adhesion process or to probe biomolecular forces in a controlled environment that may provide information crucial for our increased understanding of biological systems such as membrane protein trafficking and protein–biomolecule interactions. Dialkylated hydrophilic moieties including PAs will form vesicles in liquid media (Figure 6).91 Depending on the number of lipid bilayers formed and size of the soft spheres, such vesicles (often termed liposomes) can be categorized into small unilamellar vesicles (SUVs, < 100 nm diameter), larger unilamellar vesicles (LUVs, 100–1000 nm), multilamellar vesicles (MLVs, 100 nm to 20 mm), and multivesicular vesicles (MVVs, 100 nm to 20 mm). Generally, when PA-incorporated films are rehydrated and liposomes have formed, it is further purified via extrusion to predominately populate the working solution with SUVs before lipid bilayer experiments are conducted to minimize the heterogeneity of the surface being created (Figure 7) (see Chapter 1.124, Polymer Films Using LbL SelfAssembly).92 Peptide amphiphile (C16)2-Glu-C2-(GPO)4-GVKGDKGNP GWPGAP (herein (C16)2-Glu-C2-(GPO)4-[IV-H1], where O is hydroxyproline) was constructed by Tirrrell and coworkers and compared to poly(ethylene glycol) (PEG) lipids for its ability to promote melanoma cell adhesion, based on ligand accessibility as a means to control cell responses on a bioactive lipid bilayer.93 The head group lengths for the studied lipids (neutron reflective data) were 8.8 nm for (C16)2-Glu-C2-(GPO)4-IV-H1, while for DSPE-PEG-120, -750, -2000, and -5000, the lengths were 1.6, 3.5, 9.0, and 16.8, respectively. When the particular PA was mixed 50:50 with the PEG lipids and spread out as lipid bilayers, melanoma cells M14 clone #5 adhered to only 50% DSPE-PEG120, -750, and -2000. For DSPE-PEG-5000, the head group length of the PEG lipid (16.8 nm) exceeds the PA head group length (8.8 nm) so the bioactive sequence IV-H1 – a known sequence to promote melanoma cell adhesion – was not accessible and PEG molecules are known antifouling agents,94–97 hence no cell attachment and proliferation (see Chapter 3.320, Nanostructured Polymeric Films for Cell Biology).

4.415.3.3. Single-Tailed (Monoalkylated) PAs Monoalkylated amphiphilic constructs self-assemble into spherical micelles with a hydrophobic core and hydrophilic head groups at the periphery of the soft nanoparticle (Figure 8). This is a contrasting feature compared to doubletailed (low molecular weight) amphiphile construct, as liposomes have a hydrophilic core surrounded by a lipid bilayer as mentioned earlier. Tirrell and coworkers prepared an alanine-rich 17-mer peptide sequence containing single-tailed PA and profiled its conversion from spherical micelles into worm-like fibrous micelles based on modulation of physicochemical properties (i.e., shear induction).74 The particular PA is C16-WA4KA4KA4KA (C16W3K herein); above its critical micelle concentration (CMC), the PA molecules form spherical micelles or fibrous structures depending on shear, time, concentration, and temperature, and C16-W3K switches into fibrous structures. Multiple examples of monoalkylated PAs exist and we further examine their physicochemical and biological properties in the later sections of this chapter.

4.415.4. Concept

The Multifunctional (Soft) Nanoparticle

Multiple chemically distinctive PAs can comprise different higher-order structures such as liposomes and spherical micelles (Figure 9) with nanometer-scale precision (see Chapter 3.319, Characterization of Nanoparticles in Biological Environments; Chapter 3.319, Characterization of Nanoparticles in Biological Environments; and Chapter 4.429, Ordered Mesoporous Silica Materials). We describe some recent examples of molecular delivery and theranostic applications using multicomponent liposomes and spherical micelles.

4.415.4.1. Targeting-Enabled PA Spherical Micelles Ruoslahti, Tirrell, and coworkers utilized a double-tailed PEG construct appended with a homing (targeting) peptide sequence (i.e., CREKA, LyP-1) for targeting atherosclerotic plaques or

Surface Engineering Using Peptide Amphiphiles

227

Peptide amphiphile synthesis protocol 0. Calculations (using excel is helpful) and preparation a. Obtain a clean synthesis vessel. b. Weigh peptide conjugated beads and calculate the mmol g-1. c. Calculate how much 4 equivalent carboxylic acid (lipid tail), 4 eq. HOBt (1hydroxybenzotriazole), 4 equiv. HBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3tetramethyluronium hexafluorophosphate), 8 equiv. DIEA (N,N-diisopropylethylamine) are needed. Steps 1 through 12 should be done inside a chemical hood. 1. Swelling

Beads with protected peptides

a. Add the pre-weighed beads to the clean synthesis vessel. b. Add 10–15 ml of DCM (dichloromethane). c. Shake slowly for 30 min. 2. Washing a. Stop shaking and empty DCM. b. Add 10–15 ml of DMF (dimethylformamide) and shake to wash for 5 min. c. Stop shaking and empty DMF. d. Repeat step 2(b) and 2(c) twice more.

Cap

3. Deprotection (Fmoc to free amine)

Vessel compartment

a. Mix 2% piperidine, 2% DBU, and 96% DMF (recommended final volume 20–30 ml). b. Add the deprotection solution to the emptied synthesis vessel from 2. c. Shake moderately (beads are well dispersed during) for 1 h. d. Stop reaction, empty the Fmoc deprotection solution; proceed to the wash step. *Fmoc = 9H-fluoren-9-ylmethoxycarbonyl 4. Washing

5. (Optional) Amine test

Frit Three-way knob

a. Wash 3 DMF (10–20 ml volumes; 5 min/ea). b. Empty the final washing solution and proceed to either step 5 or 6. 6. Coupling

*Hose connection

24/40 joint

a. Coupling reagents from 0(b) are dissolved in 20 ml DMF, 2 ml DCM. b. The coupling solution is added to the dried bead containing synthesis vessel from 4. c. Shake moderately for, at least, 2–4 h but overnight reaction is also acceptable. d. Stop reaction, empty the remaining coupling solution; proceed to the wash step. 7. Washing

Synthesis vessel

a. Wash in the following order, first: 2 DMF, 2 DCM, 1 MeOH (10–20 ml volumes). b. Do the final washing step: 1 DCM, 2 MeOH (also 10-20 ml volumes). c. Dry under vacuum at room temperature. 8. Cleavage a. Prepare a clean glassware (100 ml Erlenmeyer flask recommended) to receive the as-prepared peptide amphiphile solution. b. Mix 2.5% water, 2.5% TIS (triisopropylsilane), and 95% TFA (trifluoroacetic acid) (final vol. 10–20 ml) c. Add the cleaving solution to the dried bead containing synthesis vessel from 7. d. Shake slowly for approximately 4 h. e. Stop reaction and collect the crude PA solution into the precleaned glassware from 8(a) via vaccum filtration or nitrogen gas purging. 9. Evaporation a. Transfer the collected crude PA in TFA (approximately 10–30 ml) to a 50 ml plastic tube. b. Evaporate TFA via nitrogen gas purging. c. After evaporation is complete (usually after overnight purging), the crude PA oil can be stored below -20 C or undergo the final precipitation step. 10. Precipitation a. Prepare a cold solution of ether (i.e., diethyl ether). b. Add 20–30 ml of cold ether into the 50 ml plastic tube containing the PA oil. c. Hand shake, vortex, and/or sonicate the PA-ether dispersion for approximately 5–10 min until white/ivory colored precipitates are visible. d. Centrifuge at 1500 rpm for 5 min. e. Empty the ether supernatant solution. f. Repeat steps (a) through (e) twice more. Discard ether after product identification. 11. Drying a. After the final precipitation (/wash) from 10, the collected crude PA pellet/particles are dried under nitrogen gas purging until the ether has visibly evaporated. b. The crude PA solid is further dried under vacuum for 1–2 h (room temperature). c. The crude PA is ready for HPLC separation. Store below -20 C when appropriate. 12. Separation 13. Lyophilization

Figure 5 Peptide Amphiphile Synthesis Protocol. A macrophotograph of the synthesis vessel and the beads with protected peptides are shown on the far left. The optional amine test (Step 5) is usually done using the Kaiser test90 in which the beads will turn blue if amine groups are exposed.

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Surface Engineering

Liquid Self-assembly

SUV

Amphiphilic constructs

LUV

nano-sized

MLV

MVV

Micron-sized

Figure 6 Lipid vesicles. Adapted from Jones, M. N. Adv. Coll. Interfac. Sci. 1995, 54, 93–128.

Amphiphilies

Self-assembly SUV Diffusion

Adsorption and deformation

Rupture

Spreading

tumors (see Chapter 3.312, Cell Culture Systems for studying Biomaterial Interactions with Biological Barriers).76,98 The resulting spherical micelles have a diameter of 25–35 nm and their in vivo circulation half-life time was 2–3 h. The added feature of the PA was the covalent linkage of either a bioimaging probe or a therapeutic agent; this makes them a multifunctional nanoparticle (type V MFNPS) (Figure 9). Synthesis-wise, the anticoagulant peptide hirulog99 (CF*PRPGGGGNGDFEEIPEEYL where F* is D-Phenylalanine and a cysteine was added to the N-terminus of hirulog for functionalization), CREKA,100 or LyP-1 (cyclic nine-amino acid peptide CGNKRTRGC)101,102 were linked to DSPEPEG2000-maleimide via Fmoc-chemistry methods (Figure 10). DSPE-PEG2000-CREKA spherical micelles, in particular, are able to home to the aortic tree of atherosclerotic mice within 3 h of injection (Figure 10). Thus, when CREKA-targeted hirulog micelles were injected into ApoE-null mice, the antithrombin activity was significantly higher compared to the wild-type mice or when nontargeted micelles were administered.

Rupture propagation

4.415.4.2. Single-Tailed PAs for Cancer Cell Biology Control Lateral diffusion

Lipid bilayer Hydrophilic surface Figure 7 Vesicle fusion mechanism (hypothesis) on a hydrophilic surface. Adapted from Stroumpoulis, D.; Parra, A.; Tirrell, M. AIChE J. 2006, 52, 2931–2937.

(a)

(b)

Hartgerink and coworkers103 prepared C16-WYPWMKKHH, C16-RQIKIWFQNRRMKWKK, and C16-WYPWMKKHHRQI KIWFQNRRMKWKK to study their effects on cancer cell proliferation and inhibition of growth. The sequence WYPWMKKHH disrupts HOX/PBX/DNA complex formation, while the RQIKIWFQNRRMKWKK sequence allows cargo delivery into cells. The C16 hydrocarbon chain allowed the self-assembly process to occur; the resulting stable population of spherical micelles incorporated stable secondary structures (i.e., a-helix) when embedded in a micelle that predominantly comprised anionic surfactant molecules (i.e., SDS) (Figure 11).

(c)

(d)

Figure 8 TEM of C16-W3K Peptide Amphiphile. (a) Spherical versus (b, c) fibrous micelles. C16-W3K initially forms spherical micelles (a). However, fibrous micelles are observed after 1 day (b) and highly cross-networked nanofibrous micelles dominate after 3 days (c). (d) Molecular mechanics method optimized W3K structure via the COMPASS Force Field produced by Accelrys Software Inc. The image shows that three lysine residues are evenly distributed along the peptide backbone, presenting no angular and longitudinal asymmetry around the a-helix. Adapted from Shimada, T.; Lee, S.; Bates, F. S.; Hotta, A.; Tirrell, M. J. Phys. Chem. B 2009, 113, 13711–13714.

Surface Engineering Using Peptide Amphiphiles

229

Hard nanoparticles Polymer matrix

Metal oxide matrix

Matrix metal oxide Imaging probe

Imaging probe Fluorescent probe

Fluorescent dye

Bioactive component

Chemical functionality Pore

Targeting agent

Type II MFNPS 50–200 nm in overall size

Type I MFNPS 50–200 nm in overall size

Metal oxide coating

Bioactive functionality

Imaging probe Imaging probe Optical probe

Fluorescent probe

Bioactive functionality

Bioactive functionality

Type III MFNPS <20 nm in overall size

Cargo space Matrix Imaging probe Targeting agent

Type IV MFNPS

Soft nanoparticles Micelle

Vesicle Hydrophilic surface

Hydrophilic core

Fluorescent probe

Hydrophobic core

Bioactive component Hydrophobic cargo

Hydrophilic cargo

Type V MFNPS Figure 9 Categorization of multifunctional nanoparticles. Five distinctive multifunctional nanoparticle systems (MFNPS) can be conceptualized based on soft/hardness of the constituting material, core–shell structure, functional subdomains, and size. Adapted and expanded from Suh, W. H.; Suh, Y. H.; Stucky, G. D. Nano Today 2009, 4, 27–36.

PEG2000 spacer O

X

(OCH2CH2)45

(a)

N H

DSPE lipid tail O O− O P O O H O O O

(c) Fluorescence intensity  1000 (au)

300

(d) (b)

250 200 150 100 50 0 FAMFAMlabeled, CREKA nontargeted micelles micelles

FAMFAMCREKA + CREKA + unlabeled unlabeled, CREKA nontargeted micelles micelles

Figure 10 Schematic representation of a modular DSPE-PEG PA-based multifunctional micelle (a, b) and ex vivo imaging of the aortic tree of atherosclerotic mice (c, d). Adapted from Peters, D.; Kastantin, M.; Kotamraju, V. R.; et al. Proc. Natl. Acad. Sci. USA 2009, 106, 9815–9819.

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20

(b)

(c) Molar ellipticity (deg cm2 dmol-res−1  1000−1)

(a)

200 nm

Peptide 2 PO4 Peptide 2 Tris Peptide 2 PO4 / SDS Peptide 2 Tris / SDS

15 10 5 0 −5 −10 −15 190

200 nm

(d)

200

210

220

230

240

250

Wavelength (nm)

Figure 11 Structural analysis of peptide amphiphile C16-WYPWMKKHH-RQIKIWFQNRRMKWKK via TEM at (a) low concentration (2 mg ml1, spherical micelles), (b) high concentration (12 mg ml1, fibrous micelles), and via (c) CD; (d) schematic representation of the a-helix-incorporating spherical micelle of the analyzed PA. Adapted from Aulisa, L.; Forraz, N.; McGuckin, C.; Hartgerink, J. D. Acta Biomater. 2009, 5, 842–853.103

HS

+ NH3 O

O

+ H3N

+

NH3 O − S O O

S O

SH

HS + +

NH3

O

EYFP-MESNA

HS

O HS

+

NH3 SH

+

NH3

HS

O

n=1

Cys-PEG-DSPE containing liposomes + NH3 O

H N

O

+ n O

SR

H N O SH

O

O

O HS

Cys-PEG-DSPE micelle

O HS

CNA35-MESNA

NH+ 3

+

(a)

NH3

SH

(b)

O

EYFP-liposomes

n=9

Figure 12 Preparation of multivalent CNA35 (a) vesicles and (b) micelles. Adapted from Reulen, S. W. A.; Brusselaars, W. W. T.; Langereis, S.; Mulder, W. J. M.; Breurken, M.; Merkx, M. Bioconj. Chem. 2007, 18, 590–596; Reulen, S. W. A.; Dankers, P. Y. W.; Bomans, P. H. H.; Meijer, E. W.; Merkx, M. J. Am. Chem. Soc. 2009, 131, 7304–7312.

4.415.4.3. In Vitro Binding Affinity Control via Native Chemical Ligation

4.415.5. Protein-Like Structures to 3D Hierarchical Nanostructures

Micelles and vesicles were prepared with a collagen binding protein CNA35 – 35 kDa collagen binding domain present in an adhesion protein from Staphylococcus aureus – displayed on the exterior of DSPE-PEG2000 that had moderate collagen binding affinities (Kmicelle 0.5 mM, Kliposome 3  1 nM) and allowed facile detection via fluorescence spectroscopy.104,105 The native chemical ligation (NCL) method106,107 was employed successfully to create covalent linkages between recombinant proteins and the PEG-conjugated vesicles and micelles; NCL exclusively occurs at the protein C-terminus – often not involved in any binding activities with high specificity. These examples (Figure 12) demonstrate a highly effective instillation of multiple functionalities (i.e., multiple collagen binding sites) into soft nanoparticles that may be necessary for targeting sites with multivalent architectures. The NCL method should be easily expanded to other nanoparticle systems (i.e., iron oxides and quantum dots) coated with peptide moieties.

Close to two decades ago, Yamada et al.108 reported that dialkylated PAs made up of C12 and C16 tails along with 14 residues of L-glutamic acid (Figure 13) self-assembled into helical super structures (nanotubes) comprising single-walled PA bilayers. Transmission electron microscopic (TEM) and CD analysis suggested that the PAs formed tube-like nanostructures and also displayed a-helical secondary structures at 0.1 wt% concentration (Figure 14). During the same period, Kunitake and coworkers also reported identical nanostructures formed from self-assembly of ammonium-based amphiphilic molecules in aqueous media.109 In the 1990s, Tirrell and colleagues80,81,85 realized that controlling the mode of lipidation on PAs instilled with a collagen-model head group enabled the controlled mimicking of protein-like (secondary) structures by conducting an extensive investigation into the relationship between the degree of

231

Surface Engineering Using Peptide Amphiphiles

OH OH OH OH OH O C O C O C O C O C O H2C CH2 CH2 CH2 CH2 O O O CH2 O H CH2 O H CH2 O H H H2C H CH2 H N N N N N N N N N N N N H CH2 O H CH2 O O H H2C O H CH2 O H CH2 O H H 2C CH2 CH2 CH2 CH2 N O C O C O C O C O C O H OH OH OH OH OH

O

H N

N H N H

OH O C H2C H2C O O

O

OH O C O CH2 CH2 O H H N N N N H H C O H CH O 2 2 H2C CH2 C O C O OH OH

OH C CH2 CH2 O N H

OH C CH2 CH2 O N H

OH O C CH2 H CH2 O NH2 N N CH O H CH 2

2

CH2 C O OH

OH OH OH O C O C O O C CH2 CH2 CH2 O O O H CH2 H CH2 H CH2 H N N N N N N N CH2 O H CH2 O H CH2 O H CH2 O CH2 CH2 CH2 CH2 C O C O C O C O OH OH OH OH

dC12-E14

CH2 C O OH

OH C CH2 CH2 O N H

NH2

dC16-E14

CH2 CH2 C O OH

Figure 13 Double-tailed poly(L-glutamic acid) peptide amphiphile chemical structures. Adapted from Yamada, K.; Ihara, H.; Ide, T.; Fukumoto, T.; Hirayama, C. Chem. Lett. 1984, 1713–1716.

(b)

(a)

(d)

(e) 10

B

A

(c)

250

l/nm

−10 2C12Glu14 −20

2C16Glu14 Random coil

−30

C

B

[q]/10−3 deg cm2-unit-dmol−1

200 0

A

−40

Figure 14 TEM of dC12-E14 (a–c) and dC16-E14 at 0.1 wt% (stained). Scale bar in b, c equals 50 nm. (d) dC16-E14 at 0.1 wt% (stained). Scale bar in b,c equals 50 nm. (e) CD spectra of lipidated Glu14 at pH 9.0, 25 C, 5.0x1004 M. Adapted from Yamada, K.; Ihara, H.; Ide, T.; Fukumoto, T.; Hirayama, C. Chem. Lett. 1984, 1713–1716.

alkylation (i.e., chain length, number of chains per molecule) and their secondary structure stabilization. The experiments involved the use of the peptide sequence (GPO)4[IV-H1] (GPO)4 where [IV-H1] is the alpha1(IV)1263-1277 collagen sequence (GVKGDKGNPGWPGAP) and C6, C8, C10, C12, C14, C16, and diC12 tails. Based on multiple CD and NMR analyses, C6-(GPO)4[IV-H1](GPO)4 can form a triple-helix structure below 30  C with the [IV-H1] region within the PA being in a triple-helical environment; the PA melting temperature is increased when the monoalkyl chain length is increased. The denaturation temperature for the C16 PA is comparable to the original (GPO)4[IV-H1](GPO)4 sequence. However, the degree of stabilization differs between mono(Tm ¼ 55.0  C) and dialkylated (Tm ¼ 71.2  C) PAs. All in all, whether a helix-forming peptide sequence is covalently linked to a single hydrocarbon tail or a double tail, synthesized PAs can oligomerize, self-assemble, and form various stable structures that may be crucial in positively (or negatively)

influencing biological systems. More recently, Stupp and coworkers prepared 12 different PAs (Table 1) that self-assembled into fibrous structures made up of spherical micelles having exposed bioactive peptide sequences RGD and IKVAV. TEM was utilized as seen in the figures to unmistakably show amphiphilic constructs forming fibrous structures based on pH-tuning; this change in physical property allowed the formation of gellike macroscopic materials (Figure 15). Table 1 lists single-tailed PAs that form fibrous worm-like 3D structures explored in this work. In Section 4.415.6, we highlight research that utilized mono-lipidated PAs in biomedical applications.

4.415.6. Applications in Biomedical Sciences: Tissue and Stem Cell Engineering Mode 4 of surface engineering, mentioned earlier in this chapter (Figure 1), can be divided into three subcategories when

232

Surface Engineering C18 chains. The particular 12-mer peptide sequence (SC4)114 is a potent antibacterial peptide that displays activity at nanomolar concentrations against Gram-negative bacterial species and micromolar concentrations against Gram-positive bacteria. SC4 also neutralizes endotoxins while displaying no hemolytic activity sub-100 mM. The bactericidal mechanism appears to be coming from the helix-forming nature of the peptide sequence that can permeabilize through the cell membrane. Lipidation of SC4 resulted in a 30-fold increase of biocidal activity against Staphylococcus aureus, Steptococcus pyogene, and Bacillus anthracis (Gram-positive), but there was little or no increase of antibacterial activity against Escherichia coli and Pseudomonas aeruginosa (Gram-negative) strains. In addition, lipidated SC4 PAs neutralized lipopolysaccharides (LPS) more than SC4 alone (3–6 times increased inhibition of LPS activity). Red blood cells were observed to undergo lysis at micromolar PA concentrations (antibacterial activity induction occurs at the sub-1 mM to tens of nanomolar range), whereas endothelial cell toxicity was negligible. The molecular modeling representation based on 2D NMR data (i.e., Nuclear Overhauser Effect) suggests that the monoalkylated SC4 is helical in secondary structure and that has also been validated by CD experiments where the PAs were embedded in liposomes mimicking eukaryotic and bacterial cell membranes (Figure 17). The next study by Tirrell and coworkers112 showed that lauric acid (C12)-modified YGAA(KKAAKAA)2 (termed AKK PA) had increased antibacterial activity, whereas YG(AKAKAAKA)2 (termed KAK PA) did not in comparison to C12-SC4 PA. Here, the importance of helical structure was once again demonstrated; CD results as well as the helical wheel diagram analysis shown in Figure 18 suggest that KAK PA does not share structural homology with SC4 PA. However, AKK PA displays a wider hydrophobic region, with cationic regions grouped on one side (similar to the SC4 PA) when an a-helical structure

bioactive PAs are involved: (1) lipid bilayer embedded bioactive PAs, (2) fibrous PA incorporating bioactive PAs, and (3) 3D gel matrices displaying bioactive PAs and other biomolecules (Figure 16) (see Chapter 4.409, Surfaces and Cell Behavior and Chapter 3.312, Cell Culture Systems for studying Biomaterial Interactions with Biological Barriers).111 In this section, we highlight the use of PAs to influence biological systems; we consider micelles with high-aspect ratios to be ideal for tissue engineering both on 2D surfaces and on 3D matrices.

4.415.6.1. Helical Structure and Antibacterial PAs Tirrell, Mayo, and colleagues112,113 studied single-tailed PAs for their antibacterial ability. In one instance, KLFKRHLKWKIINH2 (amidated C-terminus) was monoalkylated with C12 and Table 1 List of PA molecules that form nanofibers via pH control, divalent ion induction, and concentration. The chemical structure of C16-C4G4S(PO4)RGD (PA 4) is shown in Figure 15. Molecule

N-terminus

Peptide (N to C)

Charge pH 7

1 2 3 4 5 6 7 8 9 10 11 12

H C6H11O C10H19O C16H31O C22H43O C10H19O C16H31O C16H31O C16H31O C16H31O C16H31O C16H31O

CCCCGGGS(PO4)RGD CCCCGGGS(PO4)RGD CCCCGGGS(PO4)RGD CCCCGGGS(PO4)RGD CCCCGGGS(PO4)RGD AAAAGGGS(PO4)RGD AAAAGGGS(PO4)RGD CCCCGGGS(PO4) CCCCGGGS(PO4)KGE CCCCGGGS(PO4)RGDS CCCCGGGSRGD CCCCGGGEIKVAV

–2 –3 –3 –3 –3 –3 –3 –3 –3 –3 –1 –1

Adapted from reference.110

(a)

(b)

(c)

200 nm

150 nm

(d) H N O

O N H SH

SH H N O

O N H SH

SH H N O

O N H

H N O

O HO P OH O O H O N N N H O H

H N O

NH H2N

O OH O OH

NH

Figure 15 TEM images of nanofibers from PAs 6 and 7 from Table 1. Samples were (negatively) stained with phosphotungstic acid. (a) The aspect ratio of the nanofibers is much higher for C10–A4G3S(PO4)RGD (PA 6) compared C16–A4G3S(PO4)RGD (PA 7). (b) PA 7 will form a highly networked gel. Macrophotograph of PA 6 gel is provided on the far right (c). (d) Example chemical structure of PA 4 from Table 1. Adapted from Hartgerink, J. D.; Beniash, E.; Stupp, S. I. Proc. Natl. Acad. Sci. USA 2002, 99, 5133–5138.

Surface Engineering Using Peptide Amphiphiles

is formed (embedded in a bacterial cell membrane mimic). AKK PA also shows reduced hemolytic activity, different from SC4 PA – although the AKK peptide sequence has a strong hydrophobic property, it is less than SC4 peptide. The structure–activity relationship, at least in cytotoxicity, is well demonstrated by Chu-Kung et al.112

233

4.415.6.2. Neural Stem Cell Engineering Palmitic acid (C16) modified A4G3-IKVAV (IKVAV-PA) was utilized in Silva et al. to create an artificial 3D extracellular matrix (Figure 19) ideal for supporting murine neural progenitor cells (NPCs) at physiological conditions (see Chapter 2.209, Materials

Liposomes or micelles

(a)

NSC seeding

-Factors Neural stem cells

Precleaned metal oxide surface (i.e., glass)

Self-assembled supported NSC differentiation Neural stem cell adhesion lipid bilayers w/ peptides potential screening on 2D lipid bilayer matrix e orc e al f sic ral cu y h NSC + P ructu . -Factors t c t s + seeding +e Neural stem cell adhesion on 3D fibrous matrix

3D fibrous matrix w/ peptides

(b)

Differentiated NSCs

NSC differentiation potential screening

(c)

(d)

200 nm

5 μm

200 nm

Figure 16 Surface engineering of surfaces using bioactive peptide amphiphiles for tissue engineering applications (schematic a). Electron microscopy images of a 3D peptide amphiphile gel matrix: SEM (b, c) and TEM (d) images. Adapted from Tirrell, M. WFPC Project 4.4.5: Modular, switchable, synthetic, extracellular matrices for regenerative medicine; In AFIRM Annual Report; Vandre, R., Ed.; 2009; pp II-53–II-56.

DPC micelles

30

SDS micelles

20 10 0 SC4

[q] (103 deg cm2 dmol-1)

SC4 −10

C12-SC4 C18-SC4

C12-SC4 C18-SC4

−20

(b)

(a) DPPC liposomes

30

DPPE/DPPG liposomes C12-SC4

20

C18-SC4 10 SC4

SC4 0 C12-SC4 C18-SC4 −10 −20 190

(a)

(c) 200

210

220

230

240

(d) 250 190 l (nm)

200

210

220

230

240

250

(b)

(c)

(d)

Figure 17 CD spectra of SC4 (unbroken lines), C12-SC4 (dashed lines), and C18-SC4 (dotted lines) in micelle and liposome membrane mimics. In micellar mimics, the SC4 amphiphiles showed spectra consistent with helical conformation in DPC (a-a) and somewhat less helix in SDS (a-b). Liposome membrane mimics showed spectra indicating little SC4 amphiphile structure in DPPC (a-c), the erythrocyte mimic, but a more structured state in bacterial-mimicking DPPE/DPPG liposomes (a-d). The SC4 peptide spectra indicate little structure under any condition. Spectra in water: NOE-derived structures of C12-SC4 in DPC micelles (b) Superposition of the 24 final structures, using residues Lys-1 through Trp-9 for alignment. (c) A ribbon backbone representation of one structure, showing the overall helical fold and a less-ordered C-terminus. Polar residues are shown in blue and apolar residues in gray. The fatty acid tail is shown as a ball-and-stick model. (d) An axial view of the average structure, demonstrating the distribution of charged side chains (arginine and lysine) in an amphipathic helix. The colors are as described in (c) looked similar to those in DPPC liposomes; results at 37 C were similar for all conditions. Adapted from Lockwood, N. A.; Haseman, J. R.; Tirrell, M. V.; Mayo, K. H. Biochem. J. 2004, 378, 93–103.

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Surface Engineering

as Artificial Stem Cell Microenvironments).115 The working concentration was 0.5 wt% of PA in NPC medium. The NPC experiment confirmed the utility of PA gels that allowed the survival of NPCs over a 3-week period encapsulated (three-dimensionally) – there was no abnormal cell behavior arising from the use of PA gels versus the poly(D-lysine) control. Differentiation profiling revealed that NPC grown inside the soft, nanofibrous PA gel scaffolds produced an increased population of cells identifiable as neurons compared to laminin and poly(D-lysine) controls; the underlying mechanism, however, is not clear. Additional 2D experiments using self-assembled IKVAV-PA coatings on substrates showed that the 2D PA matrix system works equally well compared to the 3D system in promoting the differentiation of NPCs into neurons; these findings suggested that the degree of bioactive sequence IKVAV displayed on the nanofibrous matrices influenced NPC biology rather than the dimensionality of the prepared extracellular matrices. Backof-the-envelope calculations (2D surface approximated) show that the density of bioactive epitope IKVAV in the self-assembled nanostructures (1014–1015 cm2) far exceeds the numbers for naturally occurring laminin (1011–1012 cm2) by more than 1000-fold. It was also noted that injection of IKVAV-PA gels in animals resulted in no detrimental effects on the host. Niece et al.116 expanded on the aforementioned work and prepared 7 PAs with distinct functional subdomains: (1) lipid tail, (2) b-sheet inducer, (3) spacer, and (4) bioactive peptide

sequence were systematically varied (Figure 20). Nanofibrous gels presenting the IKVAV sequence (highlighted PA box) had the highest NPC viability and, in addition, substantially outperforming the other PAs in promoting neuronal differentiation. The gelation of PAs was modulated by controlling the hydrophobicity instilled into the midpeptide region (subdomains 2, 3) – comparing the kinetic and the turbidity data, the viscosity and the speed of PA gelation was PA1>PA2>PA3. Tysseling-Mattiace and coworkers117 utilized the monoalkylated C16-AAAA-GGG-E-IKVAV peptide amphiphile (IKVAVPA) (Figure 21) to study its efficacy and regenerative potential in a murine spinal cord injury (SCI) model (see Chapter 6.630, Biomaterials for Spinal Cord Repair). The injected PA gels biodegraded in vivo in 4 weeks. Injection of PA gels (0.5 wt %) and controls were monitored up to several months for hindlimb functional recovery; the IKVAV-PA gel-injected mice had the best behavioral recovery – significant enough to categorize as a functional improvement. Immunohistochemical and biochemical analyses revealed that astrogliosis (i.e., glial scarring at the SCI site) and programmed cell death (i.e., apoptosis of oligodendroglial cells after SCI) were both attenuated. In addition, it was found that IKVAV-PA nanofibers foster the regeneration of sensory and motor axons, a goal of high clinical relevance (see Chapter 2.220, Extracellular Matrix as Biomimetic Biomaterial: Biological Matrices for Tissue Regeneration).

AKK KAK SC4 C12-AKK C12-KAK C12-SC4

PC: % R-helix

PE/PG: % R-helix

12 15 26 19 18 32

5 8 29 0 11 100

26 19 93 44 -

E. coli D H5R > 65 > 65 19 11 > 65 1

E. coli S. epidermidis ML-35 ATCC 12228 > 65 > 65 > 65 > 65 34 48 30 11 > 65 > 65 4 2

(a) A

K A A

(a)

(c)

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(b)

35 25 15 5 −5 −15 −25 −35 35 25 15 5 −5 −15 −25 −35 195

215

KAK

AKK

SC4

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C12-AKK

C12-SC4

235 195

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235 195

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Figure 18 Physical and biological properties of AKK, KAK, SC4 peptides, and their respective peptide amphiphiles. (a) Helicity based on solvent/vesicle conditions and representative minimum bactericidal concentration (MBC) data based on different strains. PC ¼ vesicles from dilauroylphosphatidylcholine and PE/PG ¼ vesicles from dilauroyl phosphatidylethanolamine and dilauroyl phosphatidylglycerol. (b) CD spectra: Sodium phosphate buffer (triangle), PC (square), and PE/PG vesicle (circle). (c) Helical wheel diagrams: (c-a) KAK, (c-b) AKK, and (c-c) SC4. Charged residues are in black. Adapted from Chu-Kung, A. F.; Bozzelli, K. N.; Lockwood, N. A.; Haseman, J. R.; Mayo, K. H.; Tirrell, M. V. Bioconj. Chem. 2004, 15, 530–535.

V

(a)

K I

A V

Approximately 1014–1015 IKVAV sequences displayed

(b)

(c)

300 mm

1 mm

(d)

3 mm

Figure 19 (a) Molecular graphics representation of C16-A4G3-IKVAV before and after self-assembly. (b) SEM and (c) macrophotograph of nanofibrous gel prepared in cell medium (DMEM). (d) PA with cerebral spinal fluid. Adapted from Silva, G. A.; Czeisler, C.; Niece, K. L.; et al. Science 2004, 303, 1352–1355.

235

Surface Engineering Using Peptide Amphiphiles More recently, Schaffer and coworkers118 demonstrated that RGD-incorporating peptides could be readily prepared into 2D surfaces for stem cell tissue engineering. GGGNGEPRGDTYRAY (bsp-RGD(15)) and GRGDSP were lipidated and embedded into phospholipid bilayers at fixed mol%. Murine neural stem cells (NSCs) adhered to the RGD-functionalized surfaces and, for bsp-RGD(15) PA, the NSCs had a large fraction of single adherent cells, comparable to the laminin control (Figure 22). NSCs cultured on both 2D PA surfaces differentiated into both neurons and astrocytes.

optimum ratio density of the bioactive PA119 that promotes cell adhesion of bone marrow mononuclear cells (BMNCs) (Figure 23). This study reports that 10% (molar) doping of C16-V3A3K3RGDS in C16-V3A3E3 allows BMNCs to adhere effectively on fibrous PA structures in vitro (Figure 24) (see Chapter 4.411, Peptide- and Protein-Modified Surfaces). However, the degree of 2D surface adhesion that can be considered a dramatic increase was not observed, while the increase in proliferation of encapsulated BMNCs was 6 times more efficient compared to other controls.

4.415.6.3. Bone Marrow Mononuclear Cells

4.415.6.4. Ameoblast Cell Engineering for Tooth Regeneration

Webber et al. have demonstrated a dilution-based PA scaffolding approach by mixing bioactive C16-V3A3K3RGDS with C16-V3A3R3, C16-V3A3K3, and C16-V3A3E3 to determine the (a)

Arg-Gly-Asp-incorporating PAs were self-assembled into nanofibers under physiological conditions at 1 wt% concentration;

(b)

(c)

50 nm

(d)

50 nm

50 nm

50 nm NH2

H2N

NH O O N H

H N O

O N H

O

H N

N H

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O N H

O

H N

O

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N H

O O

PA1. C16H31O-AAAAGGGEIKVAV-COOH

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O

N H

OH

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O

H N

O N H

O

H N

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O HO P OH O

PA4. C16H31O-AAAAGGGSPRGD-COOH

N H

O

NH 115

Silva et al. HO O N H

H N O

HO O N H

H N

Niece et al.

O N H

O

H N

O N H

O

H N

PA2. C16H31O-SLSLGGGEIKVAV-COOH

O N H

O O

HO O

116

H N

O H N

O

H N

N H

O N H

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OH O

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N H

H N O

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O

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OO HO P OH O

PA5. C16H31O-SLSLAAASPRGD-COOH

O N H

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O

N H

H N

N H

O

O

N H

OH O

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H N O

HO O N H

H N O

Epitope segment PA7. C16H31O-SLSLAAAEEE-COOH

O N H

H N O

O N H

H N

O

OH

O N H

O OH

OH

O O

HO O

OH

O

OH

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PA6. C16H31O-AAAAGGGEEE-COOH

O

H N

N H

OH OH O

OH

O

O

N H

O

OH NH2

Spacer

H N

O O

H N

N H

O

N H

β-sheet segment

O

OH

H N

O

PA3. C16H31O-SLSLAAAEIKVAV-COOH

Lipophilic segment

H N

NH2

O

O

N H

O

O HO O

HO O

OH OH O

NH2

H2N

NH2

O O

H N

N H

O

O

OH

O

O

H N

OH

O O

OH

Figure 20 TEM images of self-assembled nanofibers from (a) C16-A4G3E-IKVAV, (b) C16-SLSLA3E-IKVAV, (c) C16-A4G3S(PO4)-RGD, and (d) C16-SLSLA3S(PO4)-RGD. The studied PA chemical structures are also given. Adapted from Niece, K. L.; Czeisler, C.; Sahni, V.; et al. Biomaterials 2008, 29, 4501–4509.

(a)

(b)

(c)

Figure 21 Nanofibrous C16-AAAA-GGG-E-IKVAV peptide amphiphile gel matrix. (a) Schematic representation, (b) SEM of bundled nanofibers (scale bar ¼ 200 nm), and (c) TEM of nanofibers (scale bar ¼ 100 nm). Adapted from Tysseling-Mattiace, V. M.; Sahni, V.; Niece, K. L.; et al. J. Neurosci. 2008, 28, 3814–3823.

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ameoblast-like cells (LS8) and primary enamel organ epithelial (EOE) cells were grown inside the 3D PA matrix and injected into the enamel organ epithelia of murine embryonic incisors (in vitro organ culture model) Chapter 6.619, Materials in Dental Implantology. The peptide sequence is a branched

RGD (BRGD-PA, Figure 25) and increased the cell proliferation and Ca2þ accumulation when cultured with LS8 cells and EOE cells. The microinjection of BRGD-PA into the enamel organ epithelia of incisors resulted in cell proliferation at the site of injection, with the eventual level of amelogenin and

(a)

(b)

(c)

(d)

BF

Nestin

Figure 22 Bright field and immunofluorescence images of NSCs after 5 days. NSCs grown on (a) EggPC, (b) 40% GRGDSP PA, (c) 20% bsp-RGD(15) PA, and (d) Laminin treated surfaces. All NSCs expressed Nestin (green). Cells on EggPC and 40% GRGDSP PA surfaces formed loosely attached aggregates, whereas NSCs on laminin, and to a lesser extent, 20% bsp-RGD(15) PA, grew as monolayer adherent cells. Scale bar ¼ 100 mm. Adapted from Ananthanarayanan, B.; Little, L.; Schaffer, D. V.; Healy, K. E.; Tirrell, M. Biomaterials 2010, 31, 8706–8715.

(b)

(a)

Figure 23 Molecular graphics representation of binary PA fibers assembled from 90% C16-V3A3E3 diluent and (a) 10% RGDS PA accented in yellow or (b) 10% DGSR scrambled PA accented in green. Adapted from Webber, M. J.; Tongers, J.; Renault, M.-A.; Roncalli, J. G.; Losordo, D. W.; Stupp, S. I. Acta Biomater. 2010, 6, 3–11.

(a)

(b)

5 mm

20 mm

(c)

(d)

2 mm

500 nm

Figure 24 BMNCs cultured on surfaces coated with 10% RGDS PA, exhibiting extensive process formation (a–c) in contact with the PA-coated surface at higher magnification. (d) Scale bars are 20 mm (a), 5 mm (b), 2 mm (c), and 500 nm (d). Adapted from Webber, M. J.; Tongers, J.; Renault, M.-A.; Roncalli, J. G.; Losordo, D. W.; Stupp, S. I. Acta Biomater. 2010, 6, 3–11.

Surface Engineering Using Peptide Amphiphiles

ameloblastin protein expression (within the extracellular matrix) increasing, which is an indication that a population of cells is undergoing a differentiation process. In this study, multiple control PAs were utilized that consisted of D-amino acid RGD epitope and a rearranged terminal amino acid sequence (still displaying the same charge density) – the controls did not present significant positive responses via in vitro experiments.

237

HBPA confirmed the potential of HBPA–heparin sulfate nanostructures in vascularization and soft tissue regeneration.121 Two rodent animal models were utilized that showed no harmful effects (i.e., inflammation, toxicity) arising from PA gel injections. These studies demonstrated that fibrous PA gels displaying bioactive subdomains on the nanofiber surface can potentially find usefulness in the area of wound healing and cell (transplantation) therapy.

4.415.6.5. Heparin-Binding PAs for Regenerative Medicine Rajangam et al.120 prepared two heparin-binding PAs (HBPAs) that self-assembled into bundles of nanofibers (Figure 26). The bioactivities of the PA gels were investigated using an in vitro angiogenesis assay using bovine pulmonary arterial endothelial cells sandwiched between two layers of either PA gel. The chemical structure of HBPA is C16-A4G3LRKKLGKA and the scrambled PA (SPA) is C16-A4G3LLGARKKK. Several different protein growth factors (GFs; i.e., angiogenic heparin binding growth factor, bFGF, VEGF) were supplemented to the PA–heparin gels prepared for in vitro assays. HBPA–heparin gel supplemented with GFs outperformed SBA-heparin gel’s ability (with or w/o GFs) to form tubules in three dimensions with continuous lumens; this higher bioactivity of HBPA may be due to the stabilization of interactions among the exposed heparin-binding sequences (on the nanofiber matrix) and heparin via specific and nonelectrostatic interactions (Figure 27). In vivo angiogenesis studies conducted by Ghanaati et al. using

(a)

4.415.6.6. Human Mesenchymal Stem Cell Engineering PAs with photo-cross-linkable diacetylene units bearing a bioactive sequence RGD were utilized as silica surface-coating materials to influence human mesenchymal stem cell biology (Figure 28).122 The studied PA is an extended version of the ones studied by Huang et al.123 mentioned earlier. Peptide sequences KKLLA(K)-(CH2)4-NH2 and RGDSKKLLA(K)(CH2)4-NH2 were linked to HOOC-C8-diacetylene-C12 monoalkyl chain. Soft lithography techniques were utilized to prepare nanofibrous PA (micron-sized) patterns that include channels and holes (see Chapter 4.413, Patterned Biointerfaces). The interesting observation noted in this particular study was that bioactive PA nanofibers that were aligned perpendicular to the micron-sized channels promoted hMSC attachment and subsequent alignment (nanofiber alignment:micron channel alignment ¼ 35%:45%) to an identical degree (Figure 29). This example of surface bioactivity and architectural engineering

(b)

KKGGGAAAK-palmitoyl RGDSW-K NH2 O H2N

NH H2N

N H

H N N H O HO O

HO O N H

H N

O

O NH

O HN

H N

H2N O

H2N

O N H

H N O

O N H

H O N N H O

N H O NH2H N N H O O

H N

E

300 nm

O

Figure 25 (a) Schematic chemical structure of the branched RGD peptide amphiphile (BRGD-PA). (b) TEM image of BRGD-PA injected incisor primordia 5 days after injection. The PAs assembled into nanofibers within the enamel organ epithelia, with cytoplasm extension into the nanofibers. Adapted from Huang, Z.; Sargeant, T. D.; Hulvat, J. F.; et al. J. Bone Miner. Res. 2008, 23, 1995–2006.

HBPA

SPA

NH2 H O H O H O H O H O H O H O H N N N N N N N N N N N N N N N H H H H O H O H O H O O O O O NH2 NH N NH2 H H N NH2 NH H O H O H O H O H O H H O H O N N N N N N N N N N N N N N N H O H O H O H O H O H O H O O NH2

NH2 O NH2

NH2 O NH2 NH2

Figure 26 Molecular structures of heparin binding PAs and a schematic representation of heparin–nucleated HBPA nanofibers interacting with GF and receptors. The HBPA nanofibers (blue) are shown with adsorbed heparin chains on them (red). Heparin is known to bind and activate VEGF (purple), FGF-2 (yellow), and FGF receptor (green). The design proposes that the heparin–FGF-2–FGF receptor complex is further stabilized by their anchoring on HBPA nanofibers. For the control PA, it is possible that the absence of heparin stabilization by hydrophobic interactions and via the consensus format in the SPA nanofibers is responsible for the decreased bioactivity seen in SPA–heparin gels. Adapted from Rajangam, K.; Arnold, M. S.; Rocco, M. A.; Stupp, S. I. Biomaterials 2008, 29, 3298–3305.

238

Surface Engineering

via nanofibrous PAs may thus provide an excellent platform of biomaterials capable of influencing cytoskeleton organization and associated cell signaling pathways, leading to controlling key stem cell biology. Jun and colleagues124 subsequently reported five different C16-tailed PAs all incorporating (cell-mediated) proteolytically cleavable sequence GTAGLIGQ125 which should prove useful in future cell encapsulation studies in a 3D nanofiber gel matrix (Figure 30); an additional 1–4-mer terminal head group dictated each PA’s modular nature mediating and/or promoting integrin-receptor-based cellular responses by hMSCs. Among the PAs, C16-RGDS PA nanofibrous extracellular matrix (coating; 0.1 wt%) most successfully promoted hMSC adhesion and further osteogenic differentiation (i.e., mineral deposition). The same group further demonstrated that multiple different viscosity-controlled 3D PA gel matrices (1 wt%) (Figure 31) can be prepared via doping bioactive PAs into non-bioactive PAs (C16-GTAGLIGQS)126 – an approach identical to the work done by Stupp and coworkers119 to control the cell biology of BMNCs. The terminal 4–5-mer peptide sequences are derived

(a)

from naturally occurring motifs associated with integrinmediated binding architecture (RGDS), elastin (VAPG), collagen type I (DGEA), and laminin (YIGSR).

4.415.6.7. Dental Stem Cells Galler et al.127 prepared a single-tail C16-GTAGLIGQERGDS PA (Figure 32) to create a gel matrix (1 wt%) for dental stem cell (i.e., stem cells from human exfoliated deciduous teeth (SHED), dental pulp stem cells (DPSC)) engineering. Mineral deposition was observed for dental stem cells cultured with osteogenic supplements (i.e., beta-glycerophosphate (betaGP), dexamethasone (dex), KH2PO4 (KPh)) (Figure 33). As it turns out, the SHED-PA system showed promising tissue engineering results that suggest its potential application in soft dental tissue regeneration (i.e., dental pulp), whereas the DPSC-PA system may find utility in mineralized dental tissue engineering (i.e., bone, dentin) – mineral deposition tendency was far greater for DPSC than for SHED (see Chapter 2.208, Artificial Extracellular Matrices to Functionalize Biomaterial Surfaces).

(b)

SPA SPA-heparin

30

q (mdeg)

20 10

0 185 −10

Wavelength (nm) 205

225

245

−20 −30 Figure 27 (a) TEM of SPA–heparin nanofibrous gel (stained) and (b) CD spectra showing predominant b-sheet formation for SPA in the presence of heparin (black) compared to predominantly helix formation without heparin (gray). The SPA–heparin spectra have been corrected for contribution by heparin alone. Adapted from Rajangam, K.; Arnold, M. S.; Rocco, M. A.; Stupp, S. I. Biomaterials 2008, 29, 3298–3305.

(a)

PA molecule 1 KKLLA(K)-(COC8H16)-diacetylene-(C12H25) NH2

(b) O

O H2N

N H O H O H N NH2 N N N N H O H O H O NH2

PA molecule 2 RGDSKKLLA(K)-(COC8H16)-diacetylene-(C12H25) NH2 O OH N O O O O H H H H H N N H2 N N NH2 N N N N N N H H H H H O O O O O OH O NH NH2 H2N NH

200 nm

(c)

O

200 nm

Figure 28 Chemical structure of photocurable and/or bioactive peptide amphiphiles (a) and their SEM images of fibrous nanostructures (randomly oriented, b, and aligned, c). Adapted from Mata, A.; Hsu, L.; Capito, R.; Aparicio, C.; Henrikson, K.; Stupp, S. I. Soft Matter 2009, 5, 1228–1236.

Surface Engineering Using Peptide Amphiphiles

(a)

(b)

(c)

40 mm (d)

239

40 mm

40 mm (f)

(e)

2 mm

2 mm

1 mm

Figure 29 SEM analysis of hMSCs cultured on nanofibrous PA gel surfaces. (a, d) Randomly orientation no micron-sized topology, (b, e) aligned microchannels, and (c, f) 40-mm-sized patterned holes. Adapted from Mata, A.; Hsu, L.; Capito, R.; Aparicio, C.; Henrikson, K.; Stupp, S. I. Soft Matter 2009, 5, 1228–1236.

(b)

(a)

C16-RGDS

C16-DGEA

(e)

(d)

(c)

C16-KRSR

C16-RGES

C16-S

Figure 30 TEM images of nanofibers that form multilayered PA surface coatings of (a) C16-RGDS, (b) C16-DGEA, (c) C16-KRSR, (d) C16-RGES, and (e) C16-S. The PAs were prepared at 0.1 wt% concentrations. Scale bar represents 40 nm. Adapted from Anderson, J. M.; Kushwaha, M.; Tambralli, A.; Bellis, S. L.; Camata, R. P.; Jun, H. W. Biomacromolecules 2009, 10, 2935–2944.

4.415.6.8. Bioactivation of Metallic Surfaces Titanium-based metallic implants are popular choices for orthopedic, dental, and bone replacements possessing great biocompatibility and mechanical stability. Stupp and coworkers demonstrated that self-assembling bioactive PAs are suitable surface modifiers for Ti–6Al–4V metallic foams128,129 and nickel– titanium (NiTi) shape memory metallic alloys (see Chapter 2.201, Bio-inspired Silica Nanomaterials for Biomedical Applications).130 Branched PAs identical to those developed by Guler et al.131,132 were utilized which contained the bioactive sequence RGD (Figure 34). NiTi metal sheets were first polished, cleaned, and silanized (using aminopropyltriethoxysilane (APTES)), then a 0.05 wt% PA solution was drop-cast onto the metallic surface. C16-A3L3K-ERGDS (BRGD-PA2), in particular, was used to achieve the bioactivation of nonbiologically responsive surfaces. Primary bovine pulmonary artery endothelial cells (CPAE) and mouse calvarial preosteoblastic cells (MC3T3-E1) were cultured on bioactivated metal surfaces to evaluate the efficacy of the surface engineering procedures. Proliferation assays indicated that MC3T3-E1 and CPAE cells

grew best on TiNi surfaces silanized and then covalently modified with the branched PA (Figure 35).

4.415.7.

Summary and Future Directions

Peptides amphiphiles are ideal molecular building blocks that self-assemble into hierarchical bioactive structures with controlled and defined physicochemical properties.18,133 Two additional promising platforms of amphiphilic biomaterials involving bioactive moieties and nanosized structures described here can be used to explore biomedical applications6 including regenerative medicine,134 theranostics,135 and drug delivery,136,137 exploiting the stability of the final structures. The utilization of polymeric micelles has been well investigated for multiple biomedical applications,138–140 and here, we summarize a few recent excerpts. First, MacKay et al. formulated a recombinant production of 62 kD polypeptide that self-assembles into spherical micelles consisting of a

240

Surface Engineering

TEM

(a)

(b)

(c)

PA-RGDS

PA-VAPG

Moderate gel

Moderate gel

(d)

PA-DGEA

(e)

PA-YIGSR

PA-S

Viscous solution Viscous solution

Strong gel

0% PA-S

Improved gelation after combining with PA-S Doping 50% PA-S

Doping 75% PA-S

2 mm Figure 31 Macrophotographs of viscosity-controlled peptide amphiphile gels (hydrogels). Adapted from Anderson, J. M.; Andukuri, A.; Lim, D. J.; Jun, H. W. ACS Nano 2009, 3, 3447–3454.126

OH CH3 O N H

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CH3

O

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H3C

CH

O

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CH3

O

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CH2

CH2 N H

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CH2

CH2

C O

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CH2

OH

NH2

NH

CH2 N H

OH O

C NH NH2

1

2

3

4

100 nm

Figure 32 Chemical structure of C16-GTAGLIGQERGDS. The numbering refers to function: (1) hydrocarbon chain, (2) enzyme-cleavable site, (3) calcium binding, and (4) cell adhesion promoting sequence. A representative nanofibrous TEM is given. Adapted from Galler, K. M.; Cavender, A.; Yuwono, V.; et al. Tissue Eng. A 2008, 14, 2051–2058.

core–shell structure141; a hydrophobic core incorporating a hydrophobic drug (i.e., doxorubicin) covered with a hydrophilic corona shell made up predominantly of elastin-like protein sequence XGVPG (X ¼ V,A,G) – herein, the PA will be referred to as CP-dox (chimeric polypeptide doxorubicin as coined by the original authors). The main feature of this particular system is that the self-assembled spherical micelles are sub-100 nm and the hydrophobic moiety can be any small molecule with aromatic groups. The second feature is that the attached therapeutic molecule was acid-labile which meant that the anticancer drug, doxorubicin, will release upon cellular uptake and endosomal or lysosomal processing (pH < 6). In vivo studies involving the treatment of malignant tumors with CP-dox revealed 96% tumor volume decrease compared to 50% decrease when only treated with doxorubicin. In addition, animal survival rate increased more than twofold when doxorubicin-only control increased 30%. This CP-dox also decreased the accumulation of doxorubicin in the animal’s heart, important for overcoming any dose-limiting side effects

that may result in cardiomyopathy. Second, Hubbell and coworkers,142 more recently, developed a polymeric micelle system that incorporates carbon monoxide (CO)-releasing ruthenium (Ru) metal centers. The polymeric backbone comprises hydrophilic PEG, Ru(CO)3Cl instilled poly(ornithine acrylamide) and hydrophobic poly(n-butylacrylamide); it is a triblock copolymer system that self-assembles into 30–40 nm spherical micelles. The CO molecules release in a sustained manner in vitro after cysteine introduction (to 80% FBS cell medium) and effectively attenuated the LPS or Toll-like receptor ligand-induced NF-kB (proinflammatory transcription factor) activation of human acute monocytic leukemia cells, THP-1. Third, functional recovery after SCI was achieved via a diblock copolymer monomethoxy poly(ethylene glycol)poly(D,L-lactic acid) self-assembled into 60 nm spherical micelles. The polymeric micelles, in this case, deterred the spreading of damage caused by the primary SCI process involving the neuronal cell membrane breakage and calcium influx. It is interesting to note that Stupp and coworkers reported the use

Surface Engineering Using Peptide Amphiphiles

(a)

(b)

(c)

200 nm

200 nm

200 nm

(d)

(e)

(f)

200 nm

200 nm

200 nm

241

Figure 33 TEM images (cryo-TEM analysis) showing mineral (spherical shapes) deposition for cells treated with osteogenic supplements (4 week culture). (a) SHED control, (b) SHED cultured with betaGP and dex, (c) SHED cultures with KPh and dex, (d) DPSC control, (e) DPSC cultured with betaGP and dex, and (f) DPSC cultured with KPh and dex. Adapted from Galler, K. M.; Cavender, A.; Yuwono, V.; et al. Tissue Eng. A 2008, 14, 2051–2058. O

O N H

N H

O OH NH NH HO O H O H O H O H O H O H O H O N N N N N N N OH N N N N N N H O H O H O H O H O H O H O OH N O OH O

O N H

–E

O

O H O H O H O H O H O H O H O N N N N N N N OH N N N N N N H O H O H O H O H O H O OH O OH

–E

C16-RGDS PA; C16-AAALLLK-ERGDS (a)

Metallic NiTi surface

H O N

O OH HN NH HO

(b)

Silanization

(c)

Pyr-RGDS PA; Pyr-eAhx-AAALLLK-ERGDS

Peptide amphiphile covalent attachment

Figure 34 Titanium surface coated with peptide amphiphiles (chemical structure, molecular representation provided in a and b) and their fibrous schematic (c). Adapted from Sargeant, T. D.; Rao, M. S.; Koh, C. Y.; Stupp, S. I. Biomaterials 2008, 29, 1085–1098.

of a nanofibrous micellar matrix to promote functional recovery after SCI, as highlighted in Section 4.415.6.2.117,143 It will be interesting to see how the SCI rescue process differs between the two micellar systems where one is spherical and the other fibrous. More up-to-date research involving polymeric micelles can be found in reviews by Bae and colleagues,144 Bae and Kataoka,145 Park and coworkers,146 and Hennink and coworkers.147 The second platform involves organic–inorganic nanocomposites or multifunctional nanoparticle systems (MFNPS) (Figure 9).15,148,149 In one example, Ruoslahti and

coworkers100,150 utilized inorganic oxide nanoparticles that were magnetic resonance imaging (MRI)-active and incorporating a targeting feature (i.e., CREKA) to home-in on tumor tissue. The core material is iron oxide, and the surface chemistry is such that different peptides and small molecules can be attached in a facile manner. Second, Labhasetwar and colleagues,151 more recently, demonstrated image-guided drug therapy to treat xenograft breast tumors in mice utilizing iron oxide nanoparticles (10–25 nm magnetite particles) coated with oleic acid, Pluronic F127, and incorporating hydrophobic near-IR dye molecules (overall hydrodynamic diameter

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Figure 35 Atomic Force Microscopy analysis of TiNi surfaces with and without peptide amphiphiles (first row). Cells grown on each surfaceengineered TiNi material: day 1 (2nd two rows), day 3 (3rd two rows, and day 7 only for TiNi surface with covalently attached peptide amphiphile. Adapted from Sargeant, T. D.; Rao, M. S.; Koh, C. Y.; Stupp, S. I. Biomaterials 2008, 29, 1085–1098.

determined via DLS was 240 nm). In vitro toxicity testing using MCF7 breast cancer cells revealed no apparent toxicity from the prepared particle system. Inorganic species-incorporating nanomaterials will be useful for detecting small tumors in the body with high precision and sensitivity,152–157 but the key obstacle will be the body’s inability to process highly dense and essentially non-biodegrading inorganic structures158–161 such as iron oxide, silicon dioxide, and titanium dioxide after prolonged accumulation. Biodistribution studies have shown nanoparticle accumulation in key organs such as the liver, spleen, and kidney162–164 which can be long-term risk factors for patients. Of course, the patient-to-patient treatment response variations and dose-dependent-toxicity over multiple time domains will be difficult to extensively study on humans,149 and so, it will be important and crucial to develop in vitro cell-based assessment models163,165 with these particular platforms of biomaterials. Further examples of successful utilization of hard nanoparticles in biomedical applications are recently reviewed.15,48 The modulated stability of injectable self-assembled structures made up of amphiphilic constructs that include PAs and copolymers will be key for the development of new biomaterials for biomedical applications. Recently, Tirrell and

coworkers166 explored the internalization mechanism of PAs using an in vitro cancer cell model (i.e., SJSA-1). The particular PA consisted of a C16 tail and a peptide sequence derived from p53, a tumor suppressor protein (p5314-29, Pep; LSQETFSDLWKLLPEN). The results point to individual PA monomers (or unimers) being taken up by cells rather than the entire intact self-assembled spherical micelles (soft nanoparticles) being internalized, as often observed in solid inorganic (hard) nanoparticle endocytosis46,48,161,167; inorganic oxide nanoparticles, as mentioned earlier, are condensed solids (density of 2–5 g ml1),168 and cellular organisms will not be able to process or degrade them using preexisting cellular machineries such as the lysosome or the lysozyme protein.169–174 Aforementioned monomer dissociation/uptake mechanism will increase the instability of micelles introduced to the body, as multiple biological entities (i.e., proteins, ions, tissues, cells) undergoing dynamic changes are present in vivo. Park, Cheng, and colleagues175 utilized an 60-nm-sized block copolymer micelle system made from PEG-PDLLA (poly(ethylene glycol)-b-poly(D,L-lactic acid)) to demonstrate that the dynamic instability of polymeric micelles is important for the delivery of hydrophobic small molecules encapsulated inside the micelles to the cell cytosol via membrane-mediated

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Figure 36 TEM images of C16-W3K-RGD and iron oxide nanoparticles. (a) C16-W3K-RGD peptide amphiphiles showing worm-like fibrous morphologies, negatively stained with PTA (phosphotungstic acid), (b) C16-W3K-RGD fixed and negatively stained, showing globular particle-like morphologies, (c) magnetite iron oxide nanoparticles prepared in organic phase, and (d) aggregated magnetite nanoparticles after phase transfer with C16-W3K-RGD peptide amphiphiles.

transportation. The challenge of self-assembled structure (i.e., micelles, vesicles) instability can be overcome by employing chemistry-based techniques such as cross-linking (via polymerization) of the hydrocarbon chains,176,177 as demonstrated by Biesalski et al.,178 or utilizing hard nanoparticles such as iron oxides and metallic particles that are magnetically susceptible153,155,179–184 and surface-engineering the nanoparticles with bioactive functionalities. It will be important, however, to control the degree of polymerization, as extensive cross-linking or fixation will lead to the loss of self-assembling capabilities of PAs that may be crucial for bioactivity control (Figure 36(a,b)); moreover, the application-specific development of hybrid nanoparticles will be crucial as aggregations (Figure 36(c,d)), and oxidation of the core oxide nanoparticles may lead to decreased magnetic susceptibility and injectability for in vivo experiments. The development of PA-based surface engineering of lipid bilayers, spherical micelles, and nanofibrous micelles has advanced in great quality and quantity in the past three decades, with continued increase in interest and work involving PAs (see Chapter 5.531, Peripheral Nerve Regeneration and Chapter 5.532, Nerve Tissue Engineering).81,109,185–188 In particular, many research groups around the world developed multidisciplinary projects linked to translational medicine; regenerative medicine applications such as nerve/bone regeneration, wound healing, stem cell differentiation, and transplantation. In order for such practical applications to further advance the precise understanding of surface engineering principles, stability of assembled structures, biodistribution, and the cell–material interface created by peptide-based materials and living systems will be very important. Two key aspects of PAs’ capabilities to control biological systems will be to control protein and nucleic acid function within and outside the cell103,166,189–194 and to three-dimensionally mimic the naturally occurring extracellular matrices7,11,73,115,118,195–197 – if these two functions are combined and well defined, then some of the undefined components pertaining to biomedical applications, such as stem cell tissue engineering and targeted drug delivery, can incorporate more quantitative subcomponents.

Acknowledgments WHS thanks the Otis Williams Postdoctoral Fellowship in Bioengineering, UCSB (sponsored by the Santa Barbara Foundation). In addition, the authors thank the Armed Forces

of Regenerative Medicine (AFIRM) and the University of California, Berkeley for funding. The magnetite iron oxide nanoparticles experimented with in Figure 36 were a kind gift from Dr. Cafer Yavuz.

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