Patent Application
20110311490
This invention relates generally to recombinant bacteriophages for use in tissue engineering.
There are currently more than 2.5 million people who live with spinal cord injuries worldwide, and an estimated 130,000 new cases are reported every year. Aside from rehabilitation therapy for lower severity cord lesions, there is currently no complete cure leading to neural regeneration. Immediately after the cord is transected and axons are severed, glial astrocyte cells, differentiated at the time of injury, invade the site to restore the blood brain barrier and secrete a thick extracellular matrix of chondroitin sulfate proteoglycans that inhibits neuronal growth. This glial scar provides both a physical barrier and a molecularly inhibitive environment for axon growth. In light of the fact that even a healthy spinal cord provides an inhibitive environment for neuronal growth, the absence of spinal cord self-regeneration is understandable. However, axons of the spinal cord neurons can extend through a transected spinal cord in vivo if given a more permissive environment, and multipotent progenitor cells identified in the spinal cord can differentiate into neurons in vitro and in vivo when transplanted into a neurogenic site such as the hippocampus. Tissue engineering for spinal cord regeneration has, to date, focused on providing a permissive and stimulating environment for neuronal regeneration. High density signaling molecule displays, nanostructured materials, and aligned fiber materials have shown promising results for promoting neuronal cell specification and growth.
Tissue regeneration is one of the most exciting and challenging research fields and requires a coordinated integration of various disciplines, including chemistry, molecular biology, materials science, and medicine. In vivo, cells are organized within extracellular matrices (ECM), filamentous protein structures that surround the cells. The ECM provides cells with mechanical support, controls the directional alignment of the cells, and directs cellular behavior by displaying bioactive peptide groups for cell attachment, growth, and differentiation. A number of previous works have demonstrated that desired tissues can be regenerated by (1) directly injecting cells into a tissue site to replenish lost cells, (2) developing three-dimensional scaffolds on which cells can grow into tissues with defined shapes, or (3) a combination of these two methods. The most common approach is to fabricate tissue scaffolds using synthetic biodegradable (or resorbable) materials such as poly(L-lactic acid) and poly(glycolic acid) or natural materials such as collagen, fibrin, and alginate. These scaffolds are seeded with the desired cells, which proliferate or differentiate to form organized cellular matrices (tissues). During the formation of the desired tissues, the scaffolding materials are degraded or resorbed. Recently, biodegradable electrospun nanofibers and self-assembling peptide amphiphiles were shown to have the ability to sustain cellular activity. However, major challenges remain for both approaches. For polymeric materials, it is difficult to ensure the high density and the proper spatial arrangement of the surface-conjugated functional groups, and self-assembling peptide amphiphiles require a laborious and expensive synthesis process for the creation of individual peptide units. Moreover, highly ordered tissue engineering scaffolds cannot be created using self-assembling peptides due to insufficient control of the self-assembling processes.
Currently there lacks a tissue engineering material that can influence cell growth chemically through display of signaling or therapeutic peptide groups, and physically by providing a directionally aligned fibrous support. Current approaches to tissue scaffold fabrication utilize synthetic biodegradable (or resorbable) materials such as poly(L-lactic acid) and poly(glycolic acid) or natural materials such as collagen, fibrin, and alginate. These scaffolds are seeded with the desired cells, which proliferate or differentiate to form organized cellular matrices (tissues). During the formation of the desired tissues, the scaffolding materials are degraded or resorbed. Recently, biodegradable electrospun nanofibers and self-assembling peptide amphiphiles were shown to have the ability to sustain cellular activity. However, major challenges remain for both approaches. For polymeric materials, it is difficult to ensure the high density and the proper spatial arrangement of the surface-conjugated functional groups, and self-assembling peptide amphiphiles require a laborious and expensive synthesis process for the creation of individual peptide units. Moreover, highly ordered tissue engineering scaffolds cannot be created using self-assembling peptides due to insufficient control of the self-assembling processes.
The invention provides for a composition comprising a genetically engineered bacteriophage capable of guiding cell growth and polarization via signaling peptides and directionally aligned structures. The invention provides for modified bacteriophage and its uses thereof The present invention also provides for genetically engineered phage capable of guiding cell growth, migration and/or alignment, providing essential biological effects including proliferation and/or differentiation, which can be performed by expressing specific biological motifs, such as the amino acid sequences RGD, IKVAV (SEQ ID NO:1), DGEA (SEQ ID NO:2) and HPQ, on their coat proteins, on which functional DNA, proteins and cells can be conjugated and/or fixed thereon.
The invention provides for a method of modifying a bacteriophage via genetic engineering and/or chemical conjugation such that the modified phage presents a signaling peptide, therapeutic molecules, or other functional entities. The invention also provides for the use of such a bacteriophage as a material building block for the construction of structures capable of directionally or chemically guiding cell behavior in two and three dimensions.
The invention provides for a recombinant M13 bacteriophage comprising one or more recombinant phage coat protein comprising a signal peptide capable of promoting or causing a desired biological effect.
The invention provides a method of modifying a M13 bacteriophage coat with a biomolecule of interest.
This invention provides a method of conjugating a biomolecule, such as a DNA or protein, or a cell, specific to the signaling peptide via biological and/or chemical complementary interactions to coat proteins.
The invention provides for a recombinant nucleic acid encoding a recombinant M13 bacteriophage genome capable of replicating the recombinant M13 bacteriophage of the present invention.
The invention provides for a phage matrix comprising a plurality of the recombinant M13 bacteriophages of the present invention that are directionally aligned to each other.
The invention provides for making a phage matrix of the present invention, comprising: providing a plurality of recombinant M13 phages of the present invention, and allowing for self-alignment or applying an external force to align the plurality of recombinant M13 phages.
The invention provides a tissue matrix comprising a phage matrix of the present invention further comprising viable cells in contact with said phage matrix.
The invention provides for making a tissue matrix of the present invention, comprising: providing a phage matrix of the present invention, adding one or more viable cells of interest to the phage matrix to form a tissue matrix, and optionally culturing the tissue matrix for a sufficient period of time such that the viable cells grow on the phage matrix.
The invention provides for a method of treating a subject in need of such treatment comprising: making a tissue matrix of the present invention comprising cells of the subject, and implanting the tissue matrix into the subject.
The foregoing aspects and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.
), azimuthal rotation (φ) and diameter (D) of phage bundle unit structure). (I, J) Proposed model of the chiral smectic O* nanofilament structures composed of (+) and (−) rotational smectic C* nanofilaments.
Before the present invention is described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a peptide” includes a plurality of such peptides, and so forth.
These and other objects, advantages, and features of the invention will become apparent to those persons skilled in the art upon reading the details of the invention as more fully described below.
The invention allows the use of a recombinant M13 phage for tissue engineering applications. The invention allows a display of a cell signaling peptide on all 2,700 copies of major coat protein. The invention allows designed cell interaction between phage and the cell, based on engineered peptide displayed on the phage and on the structural alignment of phage matrix. The invention provides a way to display high density array of single, or multiple signaling or therapeutic peptide motifs on a directionally aligned matrix.
The invention provides for a recombinant M13 bacteriophage comprising one or more recombinant phage coat protein comprising a signal peptide capable of promoting or causing a desired biological effect, such as promoting growth of a eukaryotic cell. The recombinant phage coat protein is a recombinant pIIII, pIX, or pVIII. The signal peptide is a peptide of any suitable length that does not interfere with the self-assembly of the M13 phage. The desired biological effect includes, but is not limited to, transport, promote cell interaction, and regulate a cellular behavior, such as promoting growth of a eukaryotic cell. The signal peptide can also be any adhesion peptide sequence, enzymatic substrate peptide sequence, or heparin binding peptide sequence.
The recombinant phages are useful in having one or more the following properties: (1) natural monodisperse nanofibers which can self-assemble nanostructures, (2) high density signaling peptides or motifs can be engineered onto the major coat protein (1.5×1013 motif/cm2), (3) have multi-functionality on pVIII, pIII and pIX, (4) replication through bacterial host cells (such as E. coli), (5) response for external forces, (6) can identification of unknown functional motifs through phage display, and (7) ease removal from the body through lysosomal degradation.
The recombinant phages can be made in by the methods taught herein (e.g. Examples 1 and 7), with the appropriate peptide sequence as selected by one skilled in the art.
The invention provides for a genetically engineered M13 bacteriophage can be utilized to construct a novel tissue engineering material that is able to both support and influence cell growth. High density signaling molecule displays, nanostructured materials, as well as aligned fiber materials, demonstrate the phage promotes neuronal cell specification and growth. M13 phage material combines all of these qualities by providing a high-density array of peptide-based signaling molecules and therapeutic materials, achieved through genetic engineering of its coat proteins, and a directionally ordered nanofibrous liquid crystalline structure which is made possible by its nanoscale dimensions, long-rod shape and monodispersity.
In some embodiments of the invention, the M13 phage display various peptides that promote cell interaction (such as IKVAV (SEQ ID NO:1) or RGD) on all 2,700 copies of its major coat proteins. Through viability assays it is verified that these viruses are biocompatible with neuronal cells. Via microscopy studies and immunostaining it is shown that neural progenitor cells can both proliferate and differentiate when grown on viral surfaces and that there is a preference of cell interaction with the genetically engineered over wild-type phage. Utilizing SEM and bright-field microscopy it is demonstrated that such engineered phage can self assemble into directionally organized structures, which in turn can dictate the alignment and direction of cell growth. Engineered virus-based materials can be used as promising novel substrates for neural cell growth. The modified phages can further be engineered to have greater control over cell behavior at the molecular level, regeneration of various tissues, and help in the research leading to a cure of challenging conditions such as spinal cord injuries.
We have developed radically novel tissue engineering materials to control and guide cell behavior using genetically engineered M13 bacteriophage (viruses). Filamentous M13 phage has several qualities that make them attractive candidates for use as building blocks in tissue engineering scaffolds. The M13 phage has a monodisperse, long-rod shape that enables its self-assembly into directionally ordered liquid crystalline structures. Through genetic engineering, a high-density array of peptide-based signaling molecules and therapeutic materials can simultaneously be displayed on its major and minor coat proteins. We have engineered M13 bacteriophage to display various peptides that promote cell interaction (IKVAV (SEQ ID NO:1), RGD) on all 2700 copies of major coat proteins. We have verified that these viruses are biocompatible to neuronal cells using viability assays. We have shown that neural progenitor cells can both proliferate and differentiate when grown on viral surfaces and that there is a preference of specific cell interaction with the RGD- and IKVAV (SEQ ID NO:1)-peptide engineered phages over wild-type phage. Utilizing SEM and fluorescent-immunostaining microscopy we have demonstrated that such engineered phage can self assemble into directionally organized structures, which in turn dictate the alignment and direction of cell growth in 2D and 3D tissue engineering matrices. These smart and novel engineered virus-based materials can be used as promising novel substrates for neural cell growth. The success of these radically novel materials will enable to manipulate cell behavior at the molecular level and regenerating various tissues, and possibly lead to the discovery of cures for challenging diseases such as spinal cord injuries.
The M13 phage is a bacterial virus composed of a single-stranded DNA encapsulated by various major and minor coat proteins. It has a long-rod filament shape that is approximately 880 nm long and 6.6 nm wide. Through genetic modification, short peptide signaling molecules (<8 residues) can be displayed on all 2700 copies of the pVIII major coat protein, which covers most of the phage surface (>98%).
The M13 phage have several properties that make them ideal for use as a tissue engineering material. In contrast to lytic phages (T4 and lambda phage), which break the host cell wall during phage reproduction, M13 phage is non-lytic, producing little cell debris during amplification and simplifying the amplification and purification processes. Therefore, mass amplification of the virus can be easily realized through its infection of E. coli cells, resulting in a monodisperse population of the phage. Due to their monodispersity and long-rod shape, phage have the ability to self-assemble and have been extensively studied as highly organized liquid crystalline systems. The concentration of the viral suspension, ionic strength of the solution, and externally applied force fields are used to modulate viral organization in these systems and have previously been optimized for the construction of one-, two-, and three-dimensional phage-based materials. In addition, through the insertion of random gene sequences into the phage genome, a large combinatorial library can be displayed on the phage major and minor coat proteins. It has been demonstrated that semiconductor-specific phage can be isolated from the directed evolutionary screening process of phage display. These phage can synthesize various semiconductor and metallic nanocrystals and can also be self-assembled into periodically ordered films and fibril structures (
The large surface area of the recombinant M13 phage of the present invention and their ability to present ligands in high densities make them useful as tissue engineering materials. The number of receptors that are engaged on a progenitor cell plays a pivotal role in its differentiation. The density of chemical signaling, the availability of the stimulating ligand, and the physical signaling present (e.g., surface area available for focal adhesions on nanotextured substrates) have been shown to be critical parameters in regulating cell differentiation. Aside from being a nanofibrous filament, an engineered M13 phage has the potential for presenting a very high ligand density of ˜1.5×1013 epitopes/cm2 (3.3 nm radius, 880 nm length, 2700 pVIII units/phage).
The invention provides for a recombinant nucleic acid encoding a recombinant M13 bacteriophage genome capable of replicating the recombinant M13 bacteriophage of the present invention. The recombinant nucleic acid can also be a replicon in a suitable microorganism, such as bacterial host cell, such as E. coli. Nucleic acid sequences and methods of maintaining and replicating such replicons are well known to one skilled in the art.
The recombinant phages comprise a short peptide motif of amino acids that is displayed on a coat protein of M13 phage. The coat protein can be pIII, pVIII, and/or pIX. In some embodiments, the peptide is 3-8 amino acids long. One skilled in the art can create partial libraries that contained randomized framing amino acids around the sequence of interest, before could successfully display the desired sequences, that also accommodated phage requirements for replication and packaging by bacteria.
Peptide sequences that promotes cell interaction include, but are not limited to, IKVAV (SEQ ID NO:1), GRGD (SEQ ID NO:3), DGEA (SEQ ID NO:2), YIGSR (SEQ ID NO:4), and RGD. IKVAV (SEQ ID NO:1) promotes neurite growth, cell differentiation and cell migration via interaction with neuronal cell receptors (LBP 110 and nucleolina). GRGD (SEQ ID NO:3) promotes cell attachment through integrin interaction. DGEA (SEQ ID NO:2) promotes cell migration and neurite growth by B1 integrin interaction. YIGSR (SEQ ID NO:4) promotes cell attachment and migration through 67 kDa laminin receptor.
Transport can include, but is not limited to, transport to the nucleus, transport to the endoplasmic reticulum, retention to the endoplasmic reticulum, transport to the mitochondrial matrix, and transport to the peroxisome. A peptide sequence capable of transport to the nucleus is -Pro-Pro-Lys-Lys-Lys-Arg-Lys-Val- (NLS) (SEQ ID NO:5). A peptide sequence capable of transport to the endoplasmic reticulum is H2N-Met-Met-Ser-Phe-Val-Ser-Leu-Leu-Leu-Val-Gly-Ile-Leu-Phe-Trp-Ala-Thr-Glu-Ala-Glu-Gln-Leu-Thr-Lys-Cys-Glu-Val-Phe-Gln- (SEQ ID NO:6). A peptide sequence capable of retention to the endoplasmic reticulum is -Lys-Asp-Glu-Leu-COOH (SEQ ID NO:7). A peptide sequence capable of transport to the mitochondrial matrix is H2N-Met-Leu-Ser-Leu-Arg-Gln-Ser-Ile-Arg-Phe-Phe-Lys-Pro-Ala-Thr-Arg-Thr-Leu-Cys-Ser-Ser-Arg-Tyr-Leu-Leu- (SEQ ID NO:8). Peptide sequences capable of transport to the peroxisome include, but are not limited to, -Ser-Lys-Leu-COOH (PTS1) and H2N-Arg-Leu-X5-His-Leu- (PTS2) (SEQ ID NO:9).
Useful adhesion peptide sequences include: RGD, DVDVPDGRGDLAYG (SEQ ID NO:10), CGGNGEPRGDTYRAY (SEQ ID NO:11), FHRRIKA (SEQ ID NO:12), TMKIIPFNRLTIGG (SEQ ID NO:13), KQAGDV (SEQ ID NO:14), PHSRN (SEQ ID NO:15), REDV (SEQ ID NO:16), LDV, DGEA (SEQ ID NO:2), GFOGER (SEQ ID NO:17), IKVAV (SEQ ID NO:1), YIGSR (SEQ ID NO:5), RNIAEIIKDI (SEQ ID NO:18), VAPG (SEQ ID NO:19), KHIFSDDSSE (SEQ ID NO:20), and HAV.
Useful enzymatic substrate peptide sequences include: GPQGIWGQ (SEQ ID NO:21), GPQGIAGQ (SEQ ID NO:22), QPQGLAK (SEQ ID NO:23), LGPA (SEQ ID NO:24), APGL (SEQ ID NO:25), YKNR (SEQ ID NO:26), NNRDNT (SEQ ID NO:27), YNRVSED (SEQ ID NO:28), LIKMKP (SEQ ID NO:29), VRN, AAAAAAAA (SEQ ID NO:30), NQEQVSP (SEQ ID NO:31), and GLVPRG (SEQ ID NO:32).
Useful heparin binding peptide sequences include: BBXB (SEQ ID NO:33), XBBXBX (SEQ ID NO:34), KRSR (SEQ ID NO:35), PRRARV (SEQ ID NO:36), FAKLAARLYRKA (SEQ ID NO:37), FHRRIKA (SEQ ID NO:38), RHRHRK (SEQ ID NO:39), LRKKLGKA (SEQ ID NO:40), KHKGRDVILKKDV (SEQ ID NO:41), and YKKIIKKL (SEQ ID NO:42) (where X=any hydrophilic amino acid and B=any basic amino acid).
In some embodiments, the recombinant M13 bacteriophage of the present invention comprises a signal peptide is derived from a signal motif and/or neurotrophic factor that promotes the growth and/or maturation of neuronal cells. Such pages are useful for regenerating neural tissue. In some embodiments, the recombinant M13 bacteriophage is capable of promoting the growth and/or maturation of neuronal cells, cell adhesion, the migration of cells and/or axon extension, the formation of bundles that facilitate further extension of individual axons, or the like. Such cells include, but are not limited to, neural progenitor cells (NPCs). The NPCs can also be astrocytes and oligodendrocytes.
Neurogenesis, or the birth of functional neurons, is most active during prenatal development. During this period, the growth and maturation of neuronal cells is guided by chemically attractive and repellent signals and directional contact guidance. Signaling comes from soluble diffusible molecules, proteins bound on extracellular matrix components such as laminin, and cell adhesion molecules. Contact guidance influences both the migration of cells and axon extension. Cell migration is directed along radial glial cell extensions, and axon growth occurs along previously formed axon fiber paths, resulting in the formation of bundles that facilitate further extension of the individual axons. Neural progenitor cells (NPCs) have been identified in several areas of the adult central nervous system, including the subventricular zone (SVZ), the dentate gyms of the subgranular zone of the hippocampus, and even the spinal cord. Whereas the hippocampus and the SVZ have demonstrated neurogenesis activity, spinal cord NPCs have only been shown to generate astrocyte and oligodendrocytes in vivo. However the ability of these cells to differentiate into neurons has been demonstrated in vitro as well as in vivo by transplanting them to a more neurogenic zone of the hippocampus.
Signaling biomolecules play a critical role in controlling cellular behavior. For example, neuronal cells receive guidance as their receptors interact with extracellular matrix molecules, such as laminin, and cell adhesion molecules, such as L1 and CHL1. Various short signaling peptide motifs (RGD and IKVAV (SEQ ID NO:1)) have been shown to influence their proliferation, differentiation, migration, and axon extension. In addition, neurotrophic factors [such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins (NT-3, NT-4/5)] and their concentration gradients in extracellular environments have been shown to enhance neuronal survival, proliferation, migration, differentiation, axon growth, and synaptic plasticity.
The spinal cord contains a large number of neurons with long directionally aligned projections. Migration and neurite extension are key components in neuron differentiation and growth. Both processes are, in part, governed by contact guidance of the growth cones and the focal adhesions they make on the surrounding surfaces. Similar to the physiological alignment of axons that has been observed along other axon fibers and glial cell extensions, cell neurites have been shown to preferentially extend along the direction of the aligned texture (fibers, grooves, ridges) of their given synthetic substrates.
The invention provides for a phage matrix comprising a plurality of the recombinant M13 bacteriophages of the present invention are directionally aligned to each other. In some embodiments, each recombinant M13 phage is in contact with at least one or more other recombinant M13 phage. The recombinant phage of the phage matrix can be directionally aligned or oriented to each other. The phage matrix can be a 2-D phage film, or any other 2-D structure or 3-D structure (see
The invention provides for making a phage matrix of the present invention, comprising: providing a plurality of recombinant M13 phages of the present invention, and allowing for self-alignment or applying an external force to align the plurality of recombinant M13 phages. The external force can be a shear force.
In some embodiments, the recombinant M13 bacteriophages of the present invention are capable of forming a self-aligned structure that promotes the directional guidance of neuronal cells in vivo.
In some embodiments, the self-aligned structure comprises an alternating nematic-cholesteric structure. The phage concentration affects spacing, width, and height of ridges within alternating nematic-cholesteric periodic structures.
In some embodiments, the self-aligned structure comprises a cholesteric helix ribbon structure. In some embodiments, the cholesteric helix ribbon structure comprises an induced left-handed helicity or an induced right-handed helicity. In some embodiments, the self-aligned structure comprises a cholesteric helix ribbon structure comprising first group of ribbons with a left-handed helicity and a second group of ribbons having a right-handed helicity, wherein there is a grain boundary structure wherein the two groups meet. In some embodiments, the self-aligned structure comprises a cholesteric plywood morphology.
In some embodiments, the self-aligned structure comprises a chiral smectic O* nanofilament structures. In some embodiments, the self-aligned structure comprises an iridescent phage films. In some embodiments, the self-aligned structure comprises a film exhibiting opposite polarized optical responses between the left and right sides of the film. In some embodiments, the self-aligned structure comprises a smectic O zig-zag morphology and is composed of stacked nanofilaments. In some embodiments, the self-aligned structure comprises of nanofilaments comprising smectic C bundle structures. In some embodiments, the self-aligned structure comprises a film exhibiting inversion center symmetry depending on the areas illuminated. In some embodiments, the film comprises patterns from the left (L) and right (R) sides which exhibited mirror symmetry, wherein the middle (M) areas exhibits the L and R diffraction patterns joined together in a V shape. In some embodiments, the self-aligned structure comprises a chiral smectic C nanofilament structure. In some embodiments, the self-aligned structure comprises chiral smectic O* nanofilament structures comprising (+) and/or (−) rotational smectic C* nanofilaments.
In some embodiments, the matrix comprises a self-aligned structure, such as a phage-based optical film, and a soft or hard tissue regenerating material. In some embodiments, the self-aligned structure comprises a structure shown in
The invention provides a tissue matrix comprising a phage matrix of the present invention further comprising viable cells. The viable cells include, but are not limited to eukaryotic cells. The eukaryotic cells are animal cells, such as human cells. The viable cells can be cells of any tissue. The viable cells can be osteoblasts, chondroblasts, hepatocytes, enterocytes, urothelials, neural cells, fibroblasts. The tissue matrix may be made in vitro.
The invention provides for making a tissue matrix of the present invention, comprising: providing a phage matrix of the present invention, adding one or more of a viable cell of interest to the phage matrix to form a tissue matrix, and optionally culturing the tissue matrix for a sufficient period of time such that the viable cells grow on the phage matrix. Growing can be an increase of number cells, an increase of the cell size, and/or an adaption of the viable cell to the phage matrix, such as adhering to the phage matrix.
Removal of the Engineered Phage from the Animal Body
The recombinant M13 phage of the present invention can be removed from a body of a recipient animal by any one of two means with little side effect after the regeneration of the desired tissues. One major requirement for tissue engineering materials is that they need to be removed from the body or degraded after the regeneration of the desired tissues. Commonly used synthetic (e.g., polylactic acids and polyglycolic acids) and natural materials (e.g., collagen, fibrin, and polysaccharide materials like chitosan and glycosaminoglycans) are degradable through natural metabolic processes. Previous studies have shown that phage injected into animals are removed in two ways. The phage can be internalized through endocytosis and degraded by lysosomes. Specific ligands, such as integrin binding sequences (RGD), can increase the efficiency of internalization. Injected phage are also quickly localized to the liver and spleen and degraded by macrophages.
Any non-native material in the body may cause unexpected immune responses. The biological nature of the M13 phage may also cause cytotoxicity or an immune response in surrounding cells. We expect that immune response problems encountered in the use of the engineered phage will be minor compared to those seen with conventional tissue engineering materials (polymeric and peptide amphiphile materials).
The present invention provides for a method of constructing regenerative tissue engineering materials from genetically engineered phage has many advantages over conventional methods and materials. Such advantages are:
The invention provides for a method of treating a subject in need of such treatment comprising: making a tissue matrix of the present invention comprising cells of the subject, and implanting the tissue matrix into the subject. The subject is an animal, such as a mammalian animal, such as a human. The subject can be a human patient suffering from tissue damage, such as spinal cord damage. The method can further comprise removing cells from the subject wherein the cells are the type of cells in which the subject is in need of replacing or regeneratings. The cells removed from the subject are added to a phage matrix to produce the tissue matrix. In some embodiments, the cells are neurons or neural cells, and the tissue damage is a damaged nerve or spinal cord.
The invention provides for a method of delivering a drug to a subject, comprising administering a therapeutically effective amount of a recombinant M13 phage to the subject in need of such treatment. The recombinant M13 phage comprises a signal peptide that directs the uptake or endocytosis of the phage by a particular cell and/or directs the phage to a specific part of the cell. For example, signal peptide sequences useful for this method includes those which promote transport.
The invention can be used in a medical device manufacturing industry. Additionally the platform of phage materials could be used for further academic research focused on the influence of displayed signaling and therapeutic peptide motifs on cell behavior. The engineered phage can be internalized into cells easily without any toxic effect. Therefore, the engineered phage can also be used as drug delivery vehicles.
The invention having been described, the following examples are offered to illustrate the subject invention by way of illustration, not by way of limitation.
We constructed several engineered phage (
IKVAV- (SEQ ID NO:1), RGD-, and wild-type M13 phage were amplified to 12-liter volumes via E. coli infection (
To construct and characterize phage-based tissue scaffold structures: One-dimensional phage-fibers were fabricated using electrospinning and conventional wet-spinning (Lee, S.-W. and A. M. Belcher, Virus-Based Fabrication of Micro- and Nanofibers Using Electrospinning. Nano Letters, 2004. 4(3): p. 387-390; hereby incorporated by reference). Long-range ordered smectic liquid crystalline films were fabricated through meniscus flowing force-assisted cast film processes (Lee, S. W., et al., Ordering of quantum dots using genetically engineered viruses. Science, 2002. 296(5569): p. 892-895; Lee, S.-W., S. K. Lee, and A. M. Belcher, Virus-based alignment of inorganic, organic, and biological nanosized materials. Advanced Materials (Weinheim, Germany), 2003. 15(9): p. 689-692; hereby incorporated by reference). Using these well-established techniques, we constructed one-dimensional fibers and two-dimensional cast films as described below.
Construction of liquid crystalline solutions using genetically engineered phage. We constructed liquid crystalline solutions using pVIII-engineered phage (
Fabrication of one-dimensional fibers. We fabricated one-dimensional fibers using mild spinning conditions by spinning the liquid crystalline suspensions (20-100 mg/ml) into an ethanol fixative (70% aqueous ethanol).
Fabrication of liquid crystalline films. We fabricated two-dimensional ordered liquid crystalline phage films (
In order to investigate the cytotoxic effect of the phage materials, we tested the individual phage as additives to the cell culture media and then tested drop cast films of the wild-type phage and phage displaying IKVAV- (SEQ ID NO:1) and RGD-motifs.
Neural progenitor cell cultures: We investigated the cytotoxicity of the engineered phage on neural progenitor cells. The neural progenitor cells used in these experiments were isolated from the hippocampi of adult female Fischer 344 rats, as previously described (Saha, K., et al., Biomimetic interfacial interpenetrating polymer networks control neural stem cell behavior. J Biomed Mater Res A, 2007. 81(1): p. 240-9; hereby incorporated by reference). The cells were seeded at a density of 3x103 cells/cm2 into 96-well plates with clear bottoms (Greiner Bio-One, Monroe, N.C.) precoated with polyornithine and laminin. They were grown in 100 μL serum-free DMEM/Hams F-12 medium with N-2 supplement (Invitrogen, Carlsbad, Calif.) and 20 ng/ml bFGF (Peprotech, Rocky Hill, N.J.) at 37° C. and 5% CO2.
Phage as a media additive: Different concentrations of constructed phage (displaying IKVAV (SEQ ID NO:1) and RGD) and wild-type phage were added to the media to approximate various potential dissolution conditions at the cell material interface. Estimating the footprint of the phage to be a 6.6×880 nm rectangle, we calculated that the cell growth area in each well (0.34 cm2) could be completely covered by approximately 5×109 phage. This quantity of phage was used to set a median phage concentration, and phage concentrations that were 10× greater and 10× less were tested to see if the phage produced any significant trends in cell behavior. To keep the concentration of the phage and nutrients in the media constant, 50 μL of the phage/media were changed daily.
CyQUANT Assays: The proliferation and metabolic activity of the cells were measured after 1, 3, and 5 days of growth in both the presence and absence of phage. The high throughput [12] CyQUANT assay (Molecular Probes, Eugene, Oreg.) was used per manufacturer's instructions. A Safire™ microplate reader with Xflour™ Software (Tecan Systems, Inc, San Jose, Calif.) was used to take the measurements. To obtain the data, 4-6 samples were tested at each given condition. CyQUANT fluorescent dye binds to nucleic acid material in the cell, thus allowing the amount of DNA in the culture to be quantified and indicating the number of cells present at the time of the test. There was no significant difference in the proliferation rate between the control population of cells in plain media and the cells grown in the presence of phage (
WST-1 Assays: We also used a WST-1 assay (Roche, Indianapolis, Ind.) to evaluate cell viability. The assay is based on the activity of mitochondrial dehydrogenase (NADH), which cleaves water soluble tetrazolium salt to formazan to produce a detectable color change. Mitochondrial activity can also be correlated to cell number and cell activity (with higher activity indicating a higher potential for proliferation and differentiation).
Cell Differentiation and Maintenance: To further assess the cellular responses to the phage, we performed immunostaining to visualize cell state specific markers. As in the above experiments, the cells were plated on polyornithine- and laminin-coated surfaces. The cells were grown in the presence (5×1010 phage particles) and absence of IKVAV-phage in either progenitor media supplemented with bFGF, as described above, or in neural differentiation media [serum-free DMEM/Hams F-12 medium with N-2 supplement, 1 μM retinoic Acid, and 5 μM forskolin (BioMol, Plymouth Meeting, Pa.)]. After 3-6 days in culture, the cells were fixed by formalin (Ted Pella Inc., Redding, Calif.) and their nuclei stained with DAPI (4′,6-diamidino-2-phenylindole). Mouse anti-nestin antibody (1:1000, BD Biosciences, San Jose, Calif.) was used to indicate the presence of nestin, a neural progenitor specific protein, and anti-β-tubulin III IgG was used to indicate the presence of β-tubulin III (1:400, Sigma-Aldirich), a neuron-specific neurofilament protein.
Through immunofluorescent staining, we confirmed that neural progenitor cells can adhere to the phage-films and stay viable while growing on the substrate. In addition, the cells maintained their progenitor state and differentiated when subjected to defined media conditions. In order to prepare drop-cast phage films, 304 droplets of 1012 phage/mL of either wild-type or genetically modified phage were deposited on vinyl cover slips (VWR) and allowed to incubate overnight at room temperature. Neural progenitor cells were seeded on the top of the phage films. After remaining in culture for 5 days, the cells were formalin-fixed, and the cover slips were double stained with anti-fd phage antibody (1:500) and anti-nestin antibody.
We studied the effects of the phage films displaying peptide-signaling motifs on growth and differentiation of the neural progenitor cells. We constructed the 2D films using RGD-, IKVAV- (SEQ ID NO:1), and wild-type (no insert) phages on (100) silicon substrates by casting the phage suspensions as previously reported (Lee, S. W., et al., Ordering of quantum dots using genetically engineered viruses. Science, 2002. 296(5569): p. 892-895; and Lee, S.-W., S. K. Lee, and A. M. Belcher, Virus-based alignment of inorganic, organic, and biological nanosized materials. Advanced Materials (Weinheim, Germany), 2003. 15(9): p. 689-692; hereby incorporated by reference). After the film was dried onto the silicon substrates, the film surface was rinsed two times with PBS, and neural progenitor cells were seeded on top. The cells were cultured in neural differentiation media with media changes every two days. After two and eight days of maintaining culture conditions, the silicon substrates were removed from culture media and fixed in 2% gluteraldahyde. The samples were then processed for Scanning Electron Microscopy (SEM) imaging.
We observed that the growth patterns of neuronal cells depended on the signaling peptides displayed by the phage films.
We established a method to culture neural progenitor cells within three dimensional liquid crystalline phage matrices. Additionally we have been able to successfully proliferate and differentiate the neural progenitor cells within the phage matrices. Three dimensional phage matrices were constructed using the nematic liquid crystalline suspensions of the RGD-, IKVAV- (SEQ ID NO:1) and wild-type-phages (
When the neural progenitor cells were grown in differentiation media conditions, the cells were able to differentiate and extend dendrites after four days of culture (
We studied the directional control of ordered phage substrate on cell polarization, by observing the growth patterns of 3T3 fibroblast cells. RGD-, IKVAV- (SEQ ID NO:1) and wild-type phage were used to construct the orientationally-ordered 2D phage film substrates using shear forces. 1 μL of the phage solution (10 mg/mL) were placed on the surface of a coverslip and sheared across the surface of the slip using another coverslip (
We successfully constructed pVIII-engineered phage that display short peptide signaling motifs (IKVAV (SEQ ID NO:1), RGD, DGEA (SEQ ID NO:2) and EQS), their control phage, and a random peptide phage library using site specific mutagenesis methods. Our studies on the effects of phage on neural progenitor cell viability using WST-1 and CyQUANT assays showed that the engineered phages have no cytotoxic effects on the neuronal cells compared to the control samples grown in the absence of phage. The differentiation experiment results showed RGD- and IKVAV- (SEQ ID NO:1) phage films showed that neurons can spread on the phage film and grow long dendrites throughout the samples. Wild-type phage substrate showed the least amount of cell spreading, the cells instead preferred to growing together in aggregates. We have demonstrated that such RGD- and IKVAV- (SEQ ID NO:1) peptide engineered phage can self assemble into directionally organized structures, which in turn dictated the alignment and direction of cell growth in 2D and 3D tissue engineering matrices. These smart and novel engineered virus-based materials can be used as novel substrates for neural cell growth and will enable to manipulate cell behavior at the molecular level and regenerating various tissues.
Partial Library Method. Functional signaling peptide motifs of the nervous system extra cellular matrix (RGD and IKVAV (SEQ ID NO:1)) were expressed on pVIII of M13 phage for use as a tissue engineering matrix. The nanofilamentous M13 bacteriophage covered with 2700 copies of the major coat protein pVIII, present a great surface for a controlled dense and uniform peptide display previously shown successful in regulating cell adhesion and differentiation behaviors (K. Saha, E. F. Irwin, J. Kozhukh, D. V. Schaffer, K. E. Healy, J Biomed Mater Res A 81, 240 (2007); J. C. Schense, J. Bloch, P. Aebischer, J. A. Hubbell, Nat Biotechnol 18, 415 (2000); G. A. Silva et al., Science 303, 1352 (2004)). However, as pVIII is involved in many phage replication processes within its Escherichia coli bacteria host (D. Stopar, R. B. Spruijt, C. J. Wolfs, M. A. Hemminga, Biochim Biophys Acta 1611, 5 (2003); M. Russel, Journal of Molecular Biology 231, 689 (1993); hereby incorporated by reference), any genetic modifications or peptide additions to pVIII must be able to accommodate its biological roles in making a viable phage. To circumvent such biological censorship inherent to phage replication, we used a partially constrained insert library. Constraining a short functional motif, by itself unfavorable for expression on pVIII protein (i.e. RGD), among degenerate residues in an 8mer insert, allowed us to display these peptides on every copy of pVIII for tissue engineering. Further we constructed four chemically diverse series (His, Trp, Glu, Lys) of 8mer partial peptide libraries to systematically analyze pVIII insert expression capabilities. By constraining one to six repeated amino acids within the insert, we observed several biochemical and size related compensation mechanisms of the library residues to make the full insert favorable to expression of pVIII. This novel method, to present unfavorable peptide groups on all of the major coat proteins of the M13 bacteriophage can serve to better design and evaluate pVIII inserts and further exploit the phage as functional nanobiomaterial.
Inverse PCR method for M13 phage cloning. To present peptide motifs on every copy of M13 major coat protein an inverse PCR cloning method was adapted (G. Chen, Courey, A. J., BioTechniques 26, 814 (1999); D. Qi, K. B. Scholthof, J Virol Methods 149, 85 (April 2008); hereby incorporated by reference). The insert was positioned between the first and the fifth amino acids of the wild-type pVIII, replacing wildtype M13KE residues 2-4 (Ala-Glu-Gly-Asp-Asp (SEQ ID NO:51) to Ala-(Insert)-Asp). All primers were ordered from IDT DNA technologies (Coralville, Iowa). To allow for re-circularization of the vector following the PCR, a PstI restriction site was created upstream of the insert location using a QuikChange® Site-Directed Mutagenesis Kit by changing position 1372 of M13KE vector from T to A (CTGCÁG (SEQ ID NO:52)), as previously described (V. A. Petrenko, G. P. Smith, X. Gong, T. Quinn, Protein Eng 9, 797 (September 1996); hereby incorporated by reference). The resulting DNA was verified by picking blue plaques resulting from phage transformation, isolating the DNA using common biological methods (J. Sambrook, D. W. Russell, Molecular Cloning: A Laboratory Manual (CSHL Press, ed. 3rd, 2001)), and sequencing at the University of California, Berkeley DNA sequencing facility (Berkeley, Calif.). For the inverse PCR reaction, the forward primer was designed to include a PstI restriction site, an insert sequence, and a segment complimentary to the gVIII 3′-5′ strand. The reverse primer designed to make the M13 plasmid linear, also included the PstI restriction site and was fully complimentary to the gVIII 5′-3′ region. To incorporate the gene sequences, polymerase chain reaction (PCR) was performed using Phusion™ High-Fidelity DNA Polymerase, the two primers, and a PstI mutated M13 vector as the template. The obtained product was purified on an agarose gel, eluted with spin column purification, digested with PstI enzyme, and re-circularized with an overnight ligation at 16° C. with T4 DNA Ligase (New England Biolabs, Ipswich, Mass.). The ligated DNA vector was transformed into XL 10-Gold® Ultracompetent bacteria cells, and the amplified plasmid verified via DNA sequencing.
Partial Library Cloning Method: All the libraries were cloned into the M13 vector following the above scheme. For the partial libraries the primers were designed to constrain a region of interest (i.e. RGD), and to allow degeneracy within the flanking codons (i.e. XXXRGDXX). As in previous phage libraries, a 32 codon degeneracy was used (X═NNK, N=A/T/C/G, K=G/T) to reduce the bias among presented amino acids, and eliminate two of the three potential stop codons (V. A. Petrenko, G. P. Smith, X. Gong, T. Quinn, Protein Eng 9, 797 (September 1996); G. P. Smith, V. A. Petrenko, Chemical Reviews 97, 391 (March-April 1997); hereby incorporated by reference).
XXDP
(NNK)2 . . . 3′
DP
. . . 3′ (SEQ ID NO: 69)
X
DP
NNK
. . . 3′ (SEQ ID NO: 70)
XX
DP
(NNK)2
. . . 3′ (SEQ ID NO: 71)
XXDP
(NNK)
2 . . . 3′ (SEQ ID NO: 72)
XXXDP
(NNK)3 . . . 3′
XXXXDP
(NNK)4 . . . 3′
XXXDP
(NNK)3 . . . 3′
XXXDP
(NNK)3 . . . 3′
XXDP
2 (NNK)2 . . . 3′
XXDP
(NNK)2 . . . 3′
XDP
3 NNK . . . 3′
§Full primer sequence shown only for the 1st listed, for the rest only the PstI restriction site and the insert portion is included
For a fully unconstrained library, degeneracy was allowed at all of the 8 positions. See Table 1 for primer and corresponding library sequences. To reduce the advantage of wild-type or fast-amplifying phage, the cells were allowed to recover for only 30 minutes after transformation (K. A. Noren, C. J. Noren, Methods 23, 169 (February 2001); hereby incorporated by reference). To analyze the library sequence space the cells were plated in agarose top, and plaques formed after overnight incubation at 37° C., were then picked and sequenced.
Genetic Engineering for Multifunctional Phage. Using inverse PCR and partially constrained library methods described above (A. Merzlyak, S. Indrakanti, S. W. Lee, Nano Lett 9, 846 (2009); hereby incorporated by reference), we were able to display a variety of functional motifs on pVIII and pIII phage proteins. RGD and IKVAV (SEQ ID NO:1) are well known peptides utilized for regulating and promoting cell adhesion and differentiation of neural progenitor cells (A. Merzlyak, S. Indrakanti, S. W. Lee, Nano Lett 9, 846 (2009); K. Saha, E. F. Irwin, J. Kozhukh, D. V. Schaffer, K. E. Healy, J Biomed Mater Res A 81, 240 (2007); J. C. Schense, J. Bloch, P. Aebischer, J. A. Hubbell, Nat Biotechnol 18, 415 (2000); G. A. Silva et al., Science 303, 1352 (2004); all of which are hereby incorporated by reference). The above peptides along with their nonspecific controls RGE, and IQVAV (SEQ ID NO:1) were engineered on pVIII, for a dense signal displayed for tissue engineering applications. HPQ is a biotin-like motif found previously to bind streptavidin molecules. An HPQ conjugated phage can be used in a variety of biotechnological application to deliver streptavidin conjugated therapeutics to the target cells (A. Hajitou et al., Cell 125, 385 (2006); hereby incorporated by reference). HPQ was displayed in a linear form on pVIII as FSHPQNT (SEQ D NO:112) (Kd=125 μM) (J. J. Devlin, L. C. Panganiban, P. E. Devlin, Science 249, 404 (1990); P. C. Weber, M. W. Pantoliano, L. D. Thompson, Biochemistry 31, 9350 (1992); all of which are hereby incorporated by reference), and in a cys-constrained form C-HPQGPP-C (SEQ ID NO:80) (Kd=0.230 μM)(16) on pIII. Although the constrained HPQ motif has been found to have a much higher affinity of binding for streptavidin, there are only 5 copies of pIII protein per phage, and so the potential applications of delivery are different for the differently designed phage, depending on the necessary delivery of the therapeutic molecule. Furthermore to deliver therapeutic molecules to cells, multifunctional phage were designed with pVIII RGD and pIII HPQ and vice versa, along with the non-physiological controls, on wild-type or RGE phage (Table 2).
DP
DP
EDP
DP
DP
DP
DP
SAE
SAE
SAE
DP
SAE
DP
SAE
DP
SAE
DP
AE
DP
DP
DP
DP
DP
DP
DP
DP
DP
DP
DP
DP
DP
DP
IKVAV- (SEQ ID NO:1) and RGD-phage engineered as described above to display peptides at the N-termini of the pVIII major coat proteins, and the wild-type M13 phage were amplified to 12-liter volumes via E. coli infection (
Construction and characterization of phage-based tissue scaffold structures. One-dimensional phage-fibers were fabricated using electrospinning and conventional wet-spinning (Lee, S.-W. and A. M. Belcher, Virus-Based Fabrication of Micro-and Nanofibers Using Electrospinning. Nano Letters, 2004. 4(3): p. 387-390; hereby incorporated by reference). Long-range ordered smectic liquid crystalline films were fabricated through meniscus flowing force-assisted cast film processes (Lee, S. W., et al., Ordering of quantum dots using genetically engineered viruses. Science, 2002, 296(5569): p. 892-895; Lee, S.-W., S. K. Lee, and A. M. Belcher, Virus-based alignment of inorganic, organic, and biological nanosized materials. Advanced Materials (Weinheim, Germany), 2003, 15(9): p. 689-692; hereby incorporated by reference). Using these well-established techniques, we constructed one-dimensional fibers and two-dimensional cast films as described below.
Construction of liquid crystalline solutions using genetically engineered phage. We constructed liquid crystalline solutions using pVIII-engineered phage (
Fabrication of one-dimensional fibers. We fabricated one-dimensional fibers using mild spinning conditions by spinning the liquid crystalline suspensions (20-100 mg/ml) into an ethanol fixative (70% aqueous ethanol).
Fabrication of liquid crystalline films. We fabricated two-dimensional ordered liquid crystalline phage films (
Self-templating is a commonly observed material design theme in nature, whereby preexisting structures determine the organization of subsequent structures. Here, we developed novel bio-inspired “self-templating” hierarchical structures using a chiral colloidal particle, M13 phage. By controlling the interplay between competing interfacial forces and liquid crystalline phase transitions of phage solutions, we created novel chiral structures with controllable long range order on the centimeter scale. The resulting structures were used as selective optical reflectors/filters and grid prisms and exhibited structural colors commensurate with their structures. Using genetically engineered M13 phages, we also developed bio-inspired functional biomaterials for soft and hard tissue regeneration. Our phage-based model system provides insight into understanding the complexities of the hierarchical self-assembly of biomolecules in biological process.
The design of hierarchical structures ordered over lengths ranging from the nano- to macroscale is of great interest in many science and engineering fields including chemistry, physics, biology, material science, and electric engineering. Various bottom-up self-assembly processes have been used to create higher order structures from inorganic and organic nanoparticles, nanowires, and nanosheets (1-4). The resulting structures have exhibited exquisite electrical and optical properties (5, 6) and performed biomedical functions such as drug delivery or tissue regeneration (7-9). Recently, top-down self-assembly processes have created complex structures that can be used to develop sophisticated microelectronics and photonic devices (10-12). Microfabricated nanocolumn structures can be induced to form helical structures and undergo biomimetic actuating motions through the control of interfacial forces (13, 14). Although these examples have demonstrated the potential of using self-assembly processes to develop novel functional materials, the functional and structural complexity of manmade materials does not compare to that of natural systems (e.g. diatoms, seashells, bones) (15-17).
In nature, helical molecules such as DNA, collagen, chitin, cellulose, and many other biological macromolecules play a critical role in life processes and in the formation of structures during the morphogenesis of cells, tissues, and organs (19, 20). In addition, these chiral building block materials are used to create diverse hierarchical structures that are made by selftemplating, in which pre-existing structures in given microenvironments determine the organization of subsequent structures. Synergic interactions between neighboring molecules result in the formation of materials with varied qualities. For example, type I collagen can selfassemble into various hierarchical structures with tunable properties: orthogonally aligned collagen fibers are found in optically transparent corneal tissues (18), whereas the twisted nematic phase of collagen fiber bundles is found in the blue-colored soft tissues of many avian and mammalian skins (19, 20). Diverse, hierarchically organized collagen fibers interact with surrounding proteins and molecules to create tissues that range from soft cartilage to hard, mineralized teeth and bones (15). However, due to limitations in modifying biological building blocks and limited knowledge about their self-assembly, the study of the relationship between hierarchical structures and their diverse functions is challenging. Here, we report a novel approach of using M13 bacteriophage (phage), a monodisperse chiral mesogen, to construct various hierarchical structures. We created self-templating supramolecular structures by controlling the interfacial forces and liquid crystalline phase transitions of phage suspensions deposited on a substrate from the suspension's meniscus. In the deposition process, the helicity of the M13 phage played a critical role in controlling the resulting self-templated structures. The periodic structures exhibited tunable optical properties with transparent to full range color characteristics. In addition, by genetically engineering the phage, we could impart specific biological functions to the resulting ordered structures to guide the growth of soft and hard tissues. Our approach can be expanded to the supramolecular assembly of many other chiral molecules and can be used to construct sophisticated functional chiral structures for the development of optical and photonic materials and tissue regenerating materials. In addition, our approach provides a means for better understanding the self-assembly of chiral molecules and gives insight into hierarchical chiral structure-function relationships in nature.
M13 phage is a filamentous bacterial virus (21). It is covered along its length with 2700 alpha helical protein subunits (pVIII) that exist in a right-handed helical arrangement with fivefold symmetry (
We created diverse self-templating supramolecular structures by modulating the interfacial forces and liquid crystal phase transitions of phage solutions along a meniscus (
We fabricated the alternating nematic striped pattern at a low phage concentration (0.05-2.0 mg/mL), with stripes forming perpendicular to the pulling direction of the substrate (
We also constructed self-templating cholesteric helix ribbon structures by controlling the geometry of the meniscus (
At higher concentrations (4-6 mg/mL) iridescent prism-like optical films were created (
In an effort to mimic various helical structures in nature, we created functional phage films with tunable properties for optical materials and tissue regenerating materials (
Inspired by the fact that hierarchically organized nanofilament structures play a critical role in controlling the morphogenesis and function of desired tissues and organs, we constructed biomimetic tissue scaffolds using self-templating phage structures. We genetically modified the phage to display an RGD-integrin binding peptide or an osteopontin-like highly negative charged peptide motif (tetra-glutamate EEEE (SEQ ID NO:105)) to mimic natural soft and hard tissue templates. When MC-3T3 preosteoblast cells were cultured on top of the directionally organized RGD-phage films, they recognized the aligned microstructures and biochemical cues (RGD motifs) of the films and grew along the direction of the viral film microstructures (
Theoretical modeling of the continuum elastic behavior of chiral bundles has predicted that rod-like molecules with handedness or lack of mirror symmetry prefer to induce mutual torque between neighboring molecules, resulting in twisted helical arrangements (32). Due to the incompatibility between ordering and twisting during the crystallization of the chiral molecules in a confined space, it has been known that various chiral or achiral materials form higher ordered chiral structures including cholesteric (25), chiral smectic C* (27), or helical nanofflament phases (33). Using the M13 system, we demonstrated multiple new findings related to biological structures. First, our self-assembled microstructures exhibited a novel selftemplating ability, in which pre-deposited structures controlled the morphology of subsequently deposited structures. Second, we showed that both left-handed and right-handed macroscopic helical structures could be formed regardless of the intrinsic helicity of the building block molecules. Third, the resulting self-templating liquid crystalline structures exhibited controllable long range order on the centimeter scale, whereas fewer crystalline ordered structures were observed on shorter scales. Finally, genetic modification of the viral particles greatly expanded the functions of the resulting hierarchical structures. The selective display of peptides that bind to synergistic molecules on the viral structures could have several significant applications in tissue regeneration and bone biomineralization.
In summary, we developed novel self-templating helical supramolecular structures using monodisperse viral colloidal nanoparticles. Self-templating was induced mainly by the helical structure of the phage bundles and the interfacial forces in the meniscus areas. The interplay between the competing interfacial forces and liquid crystal transitions played a critical role in producing diverse left- and right-handed helix supramolecular structures. Using various microscopy techniques, optical characterization, and mathematical modeling, we elucidated the structure-function relationship of the supramolecular helix structures. Our phage-based study may provide insight into understanding the complex self-assembly of biomolecules and the selftemplating process that is commonly observed in living systems. The resulting helical structures could be useful in fabricating advanced optical and photonic materials and tissue regenerating materials.
While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.
This application claims priority to PCT International Patent Application No. PCT/US2009/038449, filed Mar. 26, 2009, which claims priority to U.S. Provisional Patent Application Ser. No. 61/039,755, filed Mar. 26, 2008, both of which are hereby incorporated by reference.
The invention described and claimed herein was made in part utilizing funds supplied by the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 61039755 | Mar 2008 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2009/038449 | Mar 2009 | US |
| Child | 12891699 | US |
