Therapeutic Micro-Nano Technology BioMEMs - Tejlal Desai & Sangeeta Bhatia

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3.1. INTRODUCTION

Manufacture of new molecular scale biomaterials has become increasingly important for next generation of nanobiomaterials and nanobiotechnology, namely, the design, synthesis and fabrication of nano-devices at the molecular scale from bottom up. Nature has already produced numerous and diverse building units through billions of years molecular selection and evolution [1, 8]. Basic engineering principles for microand nano-fabrication can now be learned through understanding of the molecular self-assembly and programmed assembly phenomena. Selfand programmed assembly phenomena are ubiquitous in nature. The key elements in molecular self-assembly are chemical complementarity and structural compatibility through noncovalent weak interactions.

Several self-assembling peptide systems have been developed ranging from models for studying protein folding and protein conformational diseases, to molecular materials to produce peptide nanofibers, peptide scaffolds, peptide surfactants and peptide ink [20, 21]. These self-assembling peptide systems are simple, versatile, affordable and easy to produce in large scale to spur a new industry. These self-assembly peptide systems represent a significant advance in molecular engineering for diverse nanobiomaterial innovations. We here only focus research activities from our laboratory from the last decade. Those who are interested in a broad range of biomaterials are referred to elsewhere in this encyclopedia.

Molecular self-assembly is ubiquitous in nature and has recently emerged as a new approach in chemical synthesis, nanotechnology, polymer science, materials and engineering. Molecular self-assembly systems lie at the interface between molecular biology, protein science, biochemistry, polymer science, materials science and engineering [13, 14]. Many self-assembling systems have been developed. Molecular self-assembly systems represent a significant advance in the molecular engineering of simple molecular building blocks useful for a wide range of applications. This field is growing at an accelerating pace riding on the tide of nanobiotechnology.

3.2. PEPTIDE AS BIOLOGICAL MATERIAL CONSTRUCTION UNITS

Similar to the construction of a house, doors, windows, and many other parts of house can be prefabricated and programmed assembled according to architectural plans. If we shrink the construction units many orders of magnitude into nanoscale, we can apply similar principles to construct molecular materials and devices, through molecular self-assembly and programmed molecular assembly. Given the growing interest but limited space, only three self-assembling construction units are summarized here. They include: “Lego peptide” that form well-ordered nanofiber scaffolds for 3-D cell culture and for regenerative medicine; “surfactant/detergent peptides” for drug, protein and gene deliveries as well as for solubilizing and stabilizing membrane proteins; “peptide ink” for surface biological engineering. These designed construction peptide units are structurally simple and versatile for a wide spectrum of applications as nanobiomaterials and beyond.

Three distinct classes of self-assembling peptide construction units are described. The first class belongs to amphiphilic peptides that form well-ordered nanofibers. These peptides have two distinctive sides, one hydrophobic and the other hydrophilic. The hydrophobic

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FIGURE 3.1. Two distinct classes of self-assembling peptides construction units. The first class “Lego peptides” belongs to amphiphilic peptides that form well-ordered nanofibers. These peptides have two sides, one hydrophobic (green) and another hydrophilic (red and blue). The hydrophobic side forms a double sheet inside of the fiber and hydrophilic side forms the outside of the nanofibers that interact with water molecules that they can form extremely high water content hydrogel, containing as high as 99.9% water. At least three types of molecules can be made, with −, +, −/+ on the hydrophilic side. The second class of the self-assembling peptide belongs to a surfactant-like molecule. These peptides have a hydrophilic head and a hydrophobic tail, much like lipids or detergents. They sequester their hydrophobic tail inside of micelle, vesicles or nanotube structures and their hydrophilic heads expose to water. At least three kinds molecules can be made, with −, +, −/+ heads.

side forms a double sheet inside of the fiber and hydrophilic side forms the outside of the nanofibers that interact with water molecules that they can form extremely high water content hydrogel, containing as high as 99.9% water. At least three types of molecules can be made, with −, +, −/+ on the hydrophilic side.

The second class of the self-assembling peptide belongs to a surfactant-like molecule. These peptides have a hydrophilic head and a hydrophobic tail, much like lipids or detergents. They sequester their hydrophobic tail inside of micelle, vesicles or nanotube structures and their hydrophilic heads expose to water. At least three kinds molecules can be made, with −, +, −/+ heads. The third class of the self-assembling peptide belongs to molecular ink, which has 3 distinct segments, the ligand, the linker and the anchor. These molecular ink peptides have been used as ink to directly print arbitrary patterns on surfaces.

3.2.1. Lego Peptide

Molecular-designed “Lego Peptide”, at the nanometer scale, resembles the Lego bricks that have both pegs and holes in a precisely determined manner and can be programmed to assemble in well-formed structures. This class of “Lego peptide” can spontaneously assemble into well-formed nanofibers [16]. The first member of the Lego peptide was serendipitously discovered from a segment in a left-handed Z-DNA binding protein in yeast, Zuotin (Zuo means Left in Chinese, tin means protein in biology) [15].

These peptides form beta-sheet structures in aqueous solution thus they form two distinct surfaces, one hydrophilic, the other hydrophobic, like the pegs and holes in Lego bricks. The hydrophobic sides shield themselves from water thus facilitate them to selfassemble in water, similar to that as seen in the case of protein folding. The unique structural feature of these “peptide Lego” is that they form complementary ionic bonds with regular repeats on the hydrophilic surface (Fig. 3.2). The complementary ionic sides have been classified into several moduli, i.e. modulus I, II, III, IV, etc., and mixed moduli. This

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FIGURE 3.2. Molecular models of several self-assembling peptides, RAD16-I, RAD16-II, EAK16-I and EAK16II. Each molecule is 5nm in length with 8 alanines on one side and 4 negative and 4 positive charge amino acids in an alternating arrangement on the other side.

classification is based on the hydrophilic surface of the molecules that have alternating + and − charged amino acid residues, either alternating by 1, 2, 3, 4 and so on. For example, charge arrangements are for modulus I, − + − + − + −+; modulus II, − − + + − − ++; modulus III, − − − + ++; and modulus IV, − − − − + + ++. The charge orientation can also be designed in the reverse orientation that can yield entirely different molecules (Fig. 3.2). These well-defined Lego peptides undergo ordered self-assembly, resembling some polymer assemblies.

The Lego peptide molecules can undergo self-assembly in aqueous solutions to form well-ordered nanofibers that further associate to form nanofiber scaffolds. One of them, RADA16-I, is called PuraMatrix, because of its purity as a designed biological scaffold in contrast to other biologically derived scaffolds from animal collagen and Matrigel which contain unspecified components in addition to known materials.

Since these nanofiber scaffolds contain 5–200 nm pores and have an extremely high water content (>99.5% or 1–5 mg/ml), they have been used as three-dimensional (3-D) cellculture media. The scaffolds closely mimic the porosity and gross structure of extracellular matrices, allowing cells to reside and migrate in a 3-D environment and molecules, such as growth factors and nutrients, to diffuse in and out very slowly. These peptide scaffolds have been used for 3-D cell culture, controlled cell differentiation, tissue engineering and regenerative medicine applications.

3.2.2. Surfactant/detergent Peptides

We designed a second class of surfactant/detergent peptides with hydrophobic tails and hydrophilic heads, taking advantage of their self-assembly properties in water [10– 12]. Several surfactant peptides have been designed using nature’s lipid as a guide. These peptides have a hydrophobic tail with various degrees of hydrophobicity and a hydrophilic

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FIGURE 3.3. Molecular models of peptide surfactants/detergents. Left upper panel. A6D, V6D, V6D2 and L6D2. KKL6, KKV6. D (Aspartic acid) bears negative charges and A (alanine), V (valine) and L (leucine) constitute the hydrophobic tails with increasing hydrophobicity. Each peptide is about 2–3 nm in length, similar to biological phospholipids. K (lysine). Left lower panel. Molecular structures of individual glycine tail-based surfactant peptides. G4D2, G6D2, G8D2 and G10D2. The tail length of glycines varies depending on the number of glycine residues. The lengths of these molecules in the extended conformation range from 2.4 nm of G4D2 to 4.7 nm of G10D2. Color code: carbon, green; hydrogen, white; red, oxygen; and blue, nitrogen.

head, either negatively charged aspartic and glutamic acids or positively charged lysine or histidine (Fig. 3.3). These peptide monomers contain 7 to 8 amino acid residues and have a hydrophilic head composed of aspartic acid and a tail of hydrophobic amino acids such as alanine, valine or leucine. The length of each peptide is approximately 2 nm, similar to that of biological phospholipids [10–12]. The length can also be varied by adding more amino acids, one at a time to a desired length as shown in figure 3.3.

Although individually these peptide surfactants/detergents have completely different composition and sequences, these peptides share a common feature: the hydrophilic heads have 1–2 charged amino acids and the hydrophobic tails have four or more consecutive hydrophobic amino acids. For example, A6D (AAAAAAD), V6D (VVVVVVD) peptide has six hydrophobic alanine or valine residues from the N-terminus followed by a negatively charged aspartic acid residue, thus having two negative charges, one from the side chain and the other from the C terminus; likewise, G8DD (GGGGGGGGDD), has eight glycines following by two asparatic acids with three negative charges. In contrast, K2V6 (KKVVVVVV) has two positively charged lysines as the hydrophilic head, following by six valines as the hydrophobic tail [10–12].

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FIGURE 3.4. Design of various peptide materials. (a) Lego Peptide, also called ionic self-complementary peptide has 16 amino acids, 5 nm in size, with an alternating polar and nonpolar pattern. They form stable -strand and -sheet structures, thus the side chains partition into two sides, one polar and the other nonpolar. They undergo self-assembly to form nanofibers with the nonpolar residues inside (green) and + (blue) and − (red) charged residues form complementary ionic interactions, like a checkerboard. These nanofibers form interwoven matrices that further form a scaffold hydrogel with very high water content, >99.5% water (images courtesy of Hidenori Yokoi). (b) Surfactant/detergent peptides, 2 nm in size, which has a distinct head group, either positively charged or negatively charged, and a hydrophobic tail consisting of six hydrophobic amino acids. They can self-assemble into nanotube and nanovesicles with a diameter of 30–50 nm (image courtesy Steve Santoso and Sylvain Vauthey). These nanotubes go on to form an inter-connected network, which has similar been observed in other nanotubes. (c) Ink peptide, This type of peptide has three distinct segments: a functional segment where it interacts with other proteins and cells; a linker segment that can not only be flexible or stiff, but also sets the distance from the surface, and an anchor for covalent attachment to the surface. These peptides can be used as ink for an inkjet printer to directly print on a surface, instantly creating any arbitrary pattern, as shown here. Neural cells from rat hippocampal tissue form defined patterns. (Images courtesy S. Fuller & N. Sanjana).

These peptides undergo self-assembly in water to form nanotubes and nanovesicles having an average diameter of 30–50 nm [10–12]. The tails consisting of alanines and valines produce more homogeneous and stable structures than those of glycines, isoleucine and leucine. This property may be due to their hydrophobic and hydrophilic ratios. These monomer surfactant peptides were used for molecular modeling. The negatively charged aspartic acid is modeled as red and positively lysine is blue with the green as the hydrophobic tails. They form tubular and vesicle structures.

Quick-freeze/deep-etch sample preparation where the sample was flash-frozen at −190◦C instantly produced a 3-D structure with minimal structural disturbance. It revealed a network of open-ended nanotubes with a helical twist using transmission electron microscopy (Fig. 3.5) [10–12]. Likewise, A6K cationic peptide also exhibited similar nanotube structures with the opening ends clearly visible.

It is interesting that these simple surfactant peptides can produce remarkable complex and dynamic structures. This is another example of building biological materials from the bottom up.

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FIGURE 3.5. Quick-freeze/deep-etch TEM image of V6D dissolved in water (4.3 mM at pH 7) at high-resolution and AFM image of A6K. A) The images show the dimensions, 30–50 nm in diameters with openings of nanotube ends. Note opening ends of the peptide nanotube may be cut vertically. The strong contrast shadow of the platinum coat also suggests the hollow tubular structure. There are openings at the ends with the other ends possibly buried. The diameter of the V6D nanobutes is 30–50 nm. B) The nanotubes of A6K peptide surfactant/detergent. The openings are clearly visible in the AFM image. It should be pointed out that V6D and other anionic peptide surfactants cannot be imaged on negatively charged mica surface with AFM because they do not adhere to mica surface well.

How could these simple surfactant peptides form such well-ordered nanostructures? There are molecular and chemical similarities between lipids and the peptides since both have a hydrophilic head and a hydrophobic tail. The packing between lipids and peptides is however likely to be quite different. In lipids, the hydrophobic tails pack tightly against each other to completely displace water, precluding the formation of hydrogen bonds. On the other hand, in addition to hydrophobic tail packing between the amino acid side chains, surfactant peptides also interact through intermolecular hydrogen bonds along the backbone.

These peptide surfactants/detergents have been found to be excellent materials for solubilizing, stabilizing and crystallizing several diverse membrane proteins. These simply designed peptide detergents may now open a new avenue to overcome one of the biggest challenges in biology–to obtain high resolution structures of membrane proteins. Study of membrane proteins will not only enrich and deepen our knowledge of how cell communicate with their surroundings, thus all living system response to their environments, but these membrane proteins can also be used to fabricate the most advanced molecular devices, from energy harness devices, extremely sensitive sensors to medical detection devices, we cannot now even imagine.

Furthermore, several cationic peptide surfactants have been tested for their ability to encapsulate DNA and deliver DNA into cells [12].

3.2.3. Molecular Ink Peptides

Whitesides and collaborators developed a microcontact printing technology that combines semi-conducting industry fabrication, chemistry, and polymer science to produce

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FIGURE 3.6. Molecular models of peptide surfactant/detergent nanobutes. Color code: hydrophobic tail, green; red, negatively charged head (V6D); and blue, positively charged head (V6K, or KV6).

defined features on a surface down to the micrometer or nanometer scale [2, 7, 13]. Following microcontact printing, a surface can be functionalized with different molecules using a variety of methods, such as covalent coupling, surface adhesion, coordination chemistry. Surfaces have now been modified with a variety of chemical compounds. Furthermore peptides and proteins as inks have also been printed onto surfaces. This development has spurred new research into the control of molecular and cellular patterning, cell morphology, and cellular interactions and fueled new technology development.

Molecular surface assembly can be targeted to alter the chemical and physical properties of a material’s surface. Surface coatings instantly alter a material’s texture, color, compatibility with, and responsiveness to the environment.

Molecular ink peptides have been directly printed on surfaces and to allow adhesion molecules to interact with cells and adhere to the surface (Fig. 3.7). These peptides have three general regions along their lengths: a ligand for specific cell recognition and attachment, a linker for physical separation from the surface, and an anchor for covalent attachment to the surface [9, 18]. The ligands may be of the RGD (arginine-glycine-aspartic acid) motif that is known to promote cell adhesion, or other sequences for specific molecular recognition, or specific cell interactions. The linker is usually a string of hydrophobic amino acids such as alanine or valine. The anchor can be a cysteine residue for gold surfaces, asparatic acid linking on amine surface, or lysine linking on carboxylic surface.

This approach may facilitate research into cell–cell communication. Recently, we have moved one step further: using proteins or peptide as ink, we have directly microprinted specific features onto the non-adhesive surface of polyethylene glycol to write any arbitrary patterns rapidly without preparing the mask or stamps (Fig. 3.7). The process is similar to using an ink pen for writing—here, the printing device is the pen and the biological substances are the inks [9].

This simple and rapid printing technology permitted us to design arbitrary patterns to address questions in neurobiology that could not have been possible.

Since understanding of correct complex neuronal connections is absolutely central for comprehension of our own consciousness, human beings are always interested in finding ways to probes the question deeper. However, the neuronal connections are exceedingly complex, we must dissect the complex neuronal connections into smaller and manageable units to study them in a well-controlled manner through systematic biomedical engineering approaches. Therefore, nerve fiber guidance and connections can now be studied on special engineered pattern surfaces that are printed with protein and peptide materials. The surface

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FIGURE 3.7. Molecular structure of molecular ink peptide. A) This class of peptide ink has three general regions along their lengths: a ligand for specific cell recognition and attachment, a linker for physical separation from the surface, and an anchor for covalent attachment to the surface. Color code: carbon, green; hydrogen, white; red, oxygen; blue, nitrogen; yellow, thiol group. B) Cells adhere to printed patterns. The protein was printed onto a uniform PEG inhibitory background. Cells adhered to patterns after 8–10 days in culture spelling MIT. (Cell images courtesy Sawyer Fuller & Neville Sanjana).

substrate can be either from purified native proteins or from self-assembling peptide containing cell adhesion motifs, prepared through systematic molecular engineering of amino acids. Previous studies have shown that nerve fibers attach and outgrow extensively on the self-assembling peptide matrices [3]. We are interested in studying in detail of the nerve fiber navigation on designed pattern surfaces. Studies of nerve fiber navigation and nerve cell connections will undoubtedly enhance our general understanding of the fundamental aspects of neuronal activities in the human brain and brain-body connections. It will also likely have applications in screening neuropeptides and drugs that stimulate or inhibit nerve fiber navigation and nerve cell connections.

3.3. PEPTIDE NANOFIBER SCAFFOLD FOR 3-D CELL CULTURE, TISSUE ENGINEERING AND REGENERATIVE MEDICINE

3.3.1. Ideal Synthetic Biological Scaffolds

The ideal biological scaffold [22] and its building blocks should meet several important criteria: 1). derived from chemically-defined, synthetic sources which are present in

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native tissue; 2). amenable to design and modification to customize specific functional requirements; 3). allow cell attachment, migration, cell-cell, cell-substrate interactions, and recovery of cells from the scaffold; 4). exhibit no cytotoxicity or biocompatibility problems while chemically compatible with aqueous solutions, cell culture and physiological conditions; 5). compatible with microscopy, molecular and cell biology analyses, flow cytometry; 6). sterile and stable for long shelf life at room temperature, transportation, bioproduction and closed system cell therapy culture; 7). economically viable and scaleable material production, purification and processing; 8). exhibit a controlled rate of material biodegradation in vivo with non-detectable immune responses and inflammation; 9). foster cell migration and angiogenesis to rapidly integrate with tissues in the body. 10). injectable along with cells, compatible with cell delivery and surgical tools.

3.3.2. Peptide Scaffolds

One aspect of molecular self-assembly for production of nanobiomaterials is to fabricate three-dimensional peptide scaffolds by exposing the self-assembling peptide to a salt solution or to physiological media that accelerate the formation of macroscopic structures [3, 16, 17, 22]. Scanning electron microscopy (SEM), transmission EM (TEM), and atomic force microscopy (AFM) [6] reveal that the matrices formed are made of interwoven nanofibers having a diameter of 10 nm and pores of 5–200 nm in size. If the alanines are changed to more hydrophobic residues, such as valine, leucine, isoleucine, phenylalanine or tyrosine, the molecules have a greater tendency to self-assemble and form peptide matrices. These simple, defined and tailor-made self-assembling peptides have provided the first de novo designed scaffolds for three-dimensional cell culture, with potential implications for basic studies of cell growth and applied studies in tissue engineering and ultimately regenerative medicine.

Since these nanofiber scaffolds contain 5–200 nm pores and have an extremely high water content (>99.5% or 1–5 mg/ml), they are of potential utility in threedimensional cell-culture media. The scaffolds closely mimic the porosity and gross structure of extracellular matrices, allowing cells to reside in a three-dimensional environment and molecules, such as growth factors and nutrients, to diffuse in and out very slowly.

We have shown that a variety of tissue cells encapsulated and grown in threedimensional peptide scaffolds exhibit interesting functional cellular behaviors, including proliferation, functional differentiation, active migration and extensive production of their own extracellular matrices (Fig. 3.8). When primary rat neuron cells are allowed to attach to the peptide scaffolds, the neuron cells not only project lengthy axons that follow the contours of the scaffold surface, but also form active and functional synaptic connections (Fig. 3.8a). Furthermore, some neuronal progenitor cells migrate long distances into the three-dimensional peptide scaffold (Fig. 3.8b). Recently, the same peptide scaffold has been used for animal brain lesion repair where the severed brain section could be re-patched (Fig. 3.8c) (Ellis-Behnke, R., personal communication).

Similarly, when young and adult bovine chondrocytes are encapsulated in the peptide scaffold, they not only maintain their differentiated state, but also produce abundant type II and type XI collagen as well as glycosaminoglycans (Fig. 3.8d). Moreover, adult liver

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FIGURE 3.8. Self-assembling peptides form a three-dimensional scaffold woven from nanofibers 10 nm in diameter. The scaffolds have been applied in several three-dimensional cell culture studies and in tissue engineering applications. (a) Rat hippocampal neurons form active nerve connections, each green dot represents a single synapsis. (b) Neural cells from rat hippocampal tissue slide migrate on the three-dimensional peptide scaffold. Cells on the polymer membrane (left) and on the peptide scaffold (right) are shown. Both glia cells (green) and neural progenitors (red) migrate into the three-dimensional peptide scaffold (image courtesy of C. Semino).

(c) Brain damage repair in hamster. The peptide scaffold was injected into the optical nerve area of brain that was first severed with a knife. The cut was sealed by the migrating cells after two days. A great number of neurons form synapses (image courtesy of R. Ellis-Behnke). (d) Chondrocytes from young and adult bovine encapsulated in the peptide scaffold. These cells not only produce a large amount of glycosaminoglycans (purple) and type II collagen (yellow), characteristic materials found in the cartilage, but also a cartilage-like tissue in vitro (images courtesy of J. Kisiday & A. Grodzinsky). (e) Adult rat liver progenitor cells encapsulated in the peptide scaffold. The cells on the two-dimensional dish did not produce cytochrome P450 type enzymes (left-panel). However, cells in three-dimensional scaffolds exhibited cytochrome P450 activity (right panel). (image courtesy of C. Semino).

progenitor cells proliferate and differentiate into cells that express enzyme activities that metabolize toxic substances (Fig. 3.8e).

3.3.3. PuraMatrix in vitro Cell Culture Examples

One of the Lego peptides, RADA16-I now called PuraMatrix for its single component purity, is now commercially available for research (BD Bioscience). PuraMatrix has been used to culture diverse types of tissue cells including stem and progenitor cells, as well as