WO2007120840A2 - Procedes et compositions destines a l'impression de composites de nanotubes biologiquement compatibles - Google Patents

Procedes et compositions destines a l'impression de composites de nanotubes biologiquement compatibles Download PDF

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Publication number
WO2007120840A2
WO2007120840A2 PCT/US2007/009161 US2007009161W WO2007120840A2 WO 2007120840 A2 WO2007120840 A2 WO 2007120840A2 US 2007009161 W US2007009161 W US 2007009161W WO 2007120840 A2 WO2007120840 A2 WO 2007120840A2
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WO
WIPO (PCT)
Prior art keywords
nanoparticles
scaffold
composition
cells
ink
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PCT/US2007/009161
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English (en)
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WO2007120840A3 (fr
Inventor
Nicole Levi
Faith Coldren
David Carroll
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Wake Forest University Health Sciences
Wake Forest University
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Application filed by Wake Forest University Health Sciences, Wake Forest University filed Critical Wake Forest University Health Sciences
Publication of WO2007120840A2 publication Critical patent/WO2007120840A2/fr
Publication of WO2007120840A3 publication Critical patent/WO2007120840A3/fr
Priority to US12/250,820 priority Critical patent/US20090117087A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/10—Mineral substrates

Definitions

  • the present invention concerns methods and compositions useful for the production of three-dimensional constructs of viable cells.
  • Each method has the ability to produce stable porous scaffolds for infiltration of cells.
  • this composition comprises, consists of or consists essentially of a host material (sometimes referred to as a physiologically acceptable polymer) such as; collagen, alginates, fibronectin, elastin, poly(lactide), poly(glycolide), etc., and mixtures or co-polymers, thereof, in some embodiments a bi-phasic dispersant agent such as PEG, and finally a nanophase dispersant.
  • a host material sometimes referred to as a physiologically acceptable polymer
  • collagen alginates, fibronectin, elastin, poly(lactide), poly(glycolide), etc.
  • a bi-phasic dispersant agent such as PEG
  • nanophase dispersant a bi-phasic dispersant agent
  • the function of the host is to provide a scaffolding surface for the growth of tissues, the dispersant can be used to mediate solvent drying, or to aid in the dispersion of the nanophase.
  • the nanophase is used to impart functionalities to the
  • a first aspect of the invention is, accordingly, a method for forming an array of viable cells by depositing, spraying, or printing a cellular composition of the cells on a substrate (e.g., under conditions in which at least a portion of the cells remain viable.
  • the substrate employed is a scaffold that comprises, in combination, nanoparticles and a polymer.
  • a second aspect of the invention is an array (e.g., a tissue scaffold) comprising, in combination,
  • a scaffold comprising nanoparticles and a polymer
  • viable cells deposited (e.g., by printing or ink-jet printing) on the scaffold.
  • a further aspect of the invention is a liquid composition useful for forming a scaffold for viable cells, comprising (a) nanoparticles; (b) polymer; and (c) solvent.
  • a further aspect of the present invention is the use of a liquid composition as described herein for carrying out a method as described herein.
  • Figure 1 Transmission electron micrograph of fibronectin/ SWNT composite printed directly onto formvar coated copper grid. Scalebars are l ⁇ m and 0.5 ⁇ m.
  • Figure 2 Sodium alginate (A) without and (B) with SWNT.
  • Figure 3 Collagen (A) in a water solution, (B,C) PEG solution, both without
  • SWNT (D,E) Collagen in a PEG solution with SWNT; fibrous formations present in this sample.
  • Figure 4 A. AFM of PLGA printed with SWNT suspended in tetraglycol. B. SEM of the same sample which shows the fiber formation in the center of the printed drop.
  • Figure 5 AFM height morphology profiles of (A)decellularized blood vessel material, (B) collagen printed with PEG and SWNT, and (C) PLGA printed with tetraglycol and SWNT.
  • Nanoparticles for carrying out the present invention may be in any shape and include rods, ellipsoids, spheroids, tubes (single walled and multi-walled), and complex or combined shapes ⁇ e.g., as demonstrated by S. Chen, Z.L. Wang, J. Ballato, S. Foulger, and D.L. Carroll, “Monopod, Bipod, and Tetrapod Gold Nanocrystals", Journal of the American Chemical Society jaO38927. DEC (2003)).
  • the nanoparticles may be composed of- any suitable material including carbon (doped and undoped) metals such as Ag and Au, ceramic (silicon, silica, alumina, calcite, hydroxyapatite, etc.) organic polymers (including stable polymers and bioabsorbable polymers), and composites and mixtures thereof. See, e.g., US Patents Nos. 6,942,897; 6,929,675; 6,913,825; 6,899,947; 6,888,862; 6,878,445; 6,838,486; 6,294,401 ; etc.
  • the nanoparticles may be conductive, semiconductive, or nonconductive (insulating).
  • Carbon nanoparticles include nanotubes (including both single-wall and multi-wall nanotubes), buckyballs, fiillerenes of other configuration (e.g., ellipsoid), and combinations or mixtures thereof.
  • the nanoparticles may be coupled to (e.g., covalently coupled to) other agents (e.g., proteins, peptides, antibodies) or ligands (e.g., to cell-surface proteins or peptides on the cells being delivered) depending upon the particular application thereof.
  • Diameters of the nanoparticles can be from about 0.1 or 4 nanometers to about 1 micron. Lengths of the nanoparticles can be from 0.8 nm to 100, 200, or 500 microns or more.
  • “Viable cells” as used herein include prokaryotic and eukaryotic cells such as gram negative and gram positive bacterial cells, yeast cells, plant cells, and animal cells (e.g., reptile, amphibian, avian, mammalian, etc.). Mammalian cells (e.g., human, mouse, rat, monkey, dog, cat, etc.) are in some embodiments preferred. Cells may be of any type, including precursor, progenitor, or “stem” cells, or may be of any suitable tissue (e.g., liver, pancreas, muscle (e.g., smooth muscle), skin, bone (e.g., osteoblast), cartilage (e.g., chondrocytes), tendon, nerve, etc.). In some embodiments the cells are cancer cells (e.g., colon, lung, breast, prostate, brain, liver, or ovarian cancer cells, etc.).
  • mammalian cells e.g., human, mouse, rat, monkey, dog, cat,
  • Polymers that are used to carry out the present invention may be natural or synthetic and may be bioabsorbable or stable. In general the polymers are preferably physiologically acceptable or biocompatible. Suitable examples include but are not limited to alginate, collagen, fibronectin, polylactide, polyethylene glycol, polycaprolactone, polycolide, polydioxanone, polyacrylates, polysulfones, peptide sequences, proteins and derivatives, oligopeptides, gelatin, elastin, fibrin, laminin, polymethacrylates, polyacetates, polyesters, polyamides, polycarbonates, polyanhydrides, polyamino acids carbohydrates, polysaccharides and modified polysaccharides, and derivatives and copolymers thereof See, e.g., US Patent Nos. 6,991,652 and 6,969,480.
  • solvent as used herein may be any suitable solvent or combination thereof as is known in the art, including but not limited to water, acids such as acetic acid or phosphoric acid, N-methyl-2-pyrrolidone, 2-pyrrolidone, C 2 -C 8 aliphatic alcohol, glycerol, tetraglycol, glycerol formal, 2,2-dimethyl-l,3-dioxolone-4-methanol, ethyl acetate, ethyl lactate, ethyl butyrate, dibutyl malonate, tributyl citrate, tri-n-hexyl acetylcitrate, diethyl succinate, diethyl glutarate, diethyl malonate, triethyl citrate, triacetin, tributyrin, diethyl carbonate, propylene carbonate, acetone, methyl ethyl ketone, dimethylacetamide, caprolact
  • Preferred solvents include, but are not limited to, water, tetraglycol, polyethylene glycol, acetic acid, dimethyl sulfoxide, C 2 -Cs aliphatic alcohol, vegetable oil such as corn oil, isopropyl myristate, 1 -dodecylazacycloheptan-2-one, N-methyl-2-pyrrolidone, and combinations thereof.
  • “Support” as used herein may be an article of any suitable shape (flat, curved, formed, etc.) and may be made of any suitable material, including metals, glass, ceramics, organic polymers, and composites thereof.
  • Subjects that may be implanted with constructs or arrays of the present invention include both human subjects and animal subjects (particularly mammalian subjects such as dogs, cats, horses, pigs, sheep, cows, etc.) for veterinary purposes.
  • compositions useful for making scaffolds upon which viable cells may be deposited.
  • the composition comprises: (a) nanoparticles (e.g., from 0.1, 0.5 or 1 percent by weight up to 10,
  • polymer e.g., from 1, 2 or 3 percent by weight up to 40, 50 or 60 percent by weight
  • solvent e.g., from 1 or 5 percent by weight up to 60 or 80 percent by weight, or more
  • live cells as described herein (e.g., 0, or from 0.01 or 0.1 percent by weight up to 50 or 80 percent by weight of live cells).
  • the polymer is preferably physiologically acceptable or biocompatible (that is, suitable for implant in a human or animal subject without unduly excessive adverse reaction).
  • the scaffold is printed separately from the printing or deposition of live cells; in other embodiments the live cells are formulated in and printed with the scaffold ink described herein.
  • the polymer comprises a single polymer; in other embodiments the polymer comprises a combination of different polymers. Where a combination of different polymers is employed, each polymer in the combination — if charged — can be of the same charge or a different charge.
  • the composition is preferably in a form suitable for spraying or ink-jet printing (discussed further below), and hence preferably has a viscosity of from about 1 or 2 centipoise (and in some embodiments at least 20, 30 or 50 centipoise) up to 60, 80, 100, or 200 centipoise or more.
  • the nanoparticles in the composition are stably suspended therein (that is, the composition is stable at room temperature without settling of the nanoparticles for at least two weeks, or more preferably at least one month).
  • compositions described above are applied to a solid support by any suitable means, including spraying or printing. Application may be uniformly or in patterns. In one embodiment, ink-jet printing (e.g., thermal ink-jet printing) is preferred. Thermal ink-jet printing may be carried out with apparatus such as described in US Patent No. 7,051,654 to Boland, but with the scaffold ink compositions described herein, rather than the compositions described therein.
  • the compositions may be applied in a single layer or multiple layers, depending upon the particular end structure or array being produced. Such application forms a "substrate” or "scaffold" on the solid support to which cells may then be applied.
  • the scaffold so formed generally comprises, in combination, nanoparticles (e.g., from 0.01, 0.1, or 1 or 5 to 10, 20 or 50 percent by weight of said scaffold) and a polymer ⁇ e.g., from 99 or 95 to 50, 40 or 20 percent by weight of said scaffold).
  • nanoparticles e.g., from 0.01, 0.1, or 1 or 5 to 10, 20 or 50 percent by weight of said scaffold
  • polymer e.g., from 99 or 95 to 50, 40 or 20 percent by weight of said scaffold.
  • Cells are then applied to the scaffold.
  • the cells may be applied by any suitable means, such as spraying or printing, with ink-jet printing being (in one embodiment) preferred.
  • the cells may be applied as a single application or multiple applications (uniformly or in patterns) to create three dimensional arrays.
  • cells may be sandwiched between multiple layers of nanotube/polymer scaffold layers. Indeed, multiple layers (e.g., 3, 4, 5, 6, 10, 20, 30 or more) of scaffold and cells, in any order or combination, may be carried out to produce the desired structures or arrays such as three-dimensional, contoured, or shaped arrays.
  • the polymers within the scaffold are cross-linked after they are ink-jet printed.
  • Such cross-linking can be carried out by any suitable technique, such as separately applying (e.g., by ink-jet printing through a different orifice) a cross-linking agent (e.g., a carbodiimide, an aldose sugar, D-I- glyceraldehyde, genipin, etc.) onto the scaffold, by utilizing polymers that are cross- linked upon exposure to light (e.g., UV light) or heat, etc.
  • a cross-linking agent e.g., a carbodiimide, an aldose sugar, D-I- glyceraldehyde, genipin, etc.
  • An advantage of cross- linking is, in some embodiments, to maintain or enhance the physical integrity of the scaffold.
  • the arrays or constructs may be cultured further in vitro in accordance with known techniques to grow the cells (e.g., for subsequent implantation as a prosthesis or the like in a subject, or for the commercial production of a desired compound such as naturally occurring or transgenic protein or peptide from the cells in a fermentation process).
  • the growth or proliferation of the viable cells can be enhanced while they are growing in vitro by subjecting the viable cells to an electric field or current sufficient to enhance the proliferation thereof of said viable cells.
  • the electrical field or current may be achieved by any suitable means, such as by connecting the scaffold (directly or indirectly) to a power supply, and/or connecting culture media in which the cells are cultured to a power supply.
  • the present invention has a number of applications. Particular applications include, but are not limited to, the following:
  • A. Electrically conductive scaffolds By including electrically conductive nanoparticles, the scaffolds can be operatively associated with a current source (such as a battery or voltage regulator) and used to electrically stimulate cells thereon (e.g., muscle cells, nerve cells, skin cells, or any other cell type for which electrical stimulation stimulates growth or enhances proliferation thereof).
  • a current source such as a battery or voltage regulator
  • electrically stimulate cells thereon e.g., muscle cells, nerve cells, skin cells, or any other cell type for which electrical stimulation stimulates growth or enhances proliferation thereof.
  • Particular electrically conductive nanoparticles include, but are not limited to, metal and carbon nanoparticles and nanotubes, including nanowires.
  • Such scaffolds can also be used for applying heat to the scaffolding.
  • the elastic modulus of the scaffold can be increased by at least 20 or 50 percent, up to 200 or 500 percent or more, as compared to a scaffold of the same configuration and composition without nanoparticles.
  • A. Patterned scaffolds By including nanoparticles in the scaffold in an appropriate amount (e.g., from 0.001 or 0.01 percent by weight, up to 10 or 20 percent by weight of the ink composition), cell scaffolds with improved definition of topographical features (such as lines, ridges, wells, vias, composite shapes, etc.) are obtained.
  • aspect ratios (A/B) of topographical features on the printed scaffold are in some embodiments preferably at least 1, 2, or 3 (where A is the heighth (or depth) and B is the width of the topographical feature, when the topographical feature is measured in cross-section.
  • D. Contrast agents are in some embodiments preferably at least 1, 2, or 3 (where A is the heighth (or depth) and B is the width of the topographical feature, when the topographical feature is measured in cross-section.
  • Nanoparticles used to carry out the present invention can comprise or contain a contrast or imaging agent to provide detectability of the scaffold in an imaging system such as NMR, X-ray, or the like.
  • contrast or imaging agents can comprise Gd complexes, metals such as Fe, and Fe3 ⁇ j, encapsulated contrast agents such as fullerene and encapsulated Gd complexes. See, e.g., US Patent No. 6,797,380.
  • Nanoparticles used to carry out the present invention can comprise or contain an antimicrobial ⁇ e.g., antibacterial) agent, such as when the scaffolds are used as a tissue implant scaffold to grow cells for tissue implantation.
  • Antimicrobial metal (including metal alloy) particles can comprise any suitable metal materials ⁇ e.g., silver) or bi-, tri- or multicomponent or alloyed metals, typically of a size of from 2 ran to 1000 nm).
  • Nanopartic.es can be formed of a polymer such as a biodegradable polymer ⁇ e.g., PLGA) that contain an active agent to be released into the scaffold.
  • a polymer such as a biodegradable polymer ⁇ e.g., PLGA
  • Nanoparticles comprised, consisting of, or consisting essentially of a free-radical scavenger can be utilized to produce a scaffold that scavenges such free radicals and reduces their deleterious effects on cells grown thereon. Examples include, but are not limited to, fullerene and transition metal oxides. H. Others. Other applications of the present invention include quantum dot nanoparticles ⁇ e.g., CdSe QD from Evident Technologies) for tracking of targeted or tagged agents within the scaffold, transition metal oxides for catalytic crosslinking. etc.
  • PLGA polylactic co-glycolic acid
  • Collagen I and fibronectin are natural biopolymers found in vivo and alginates have been shown to act as viable artificial replacements similar to glycoaminoglycosans which naturally occur in the body.
  • PLGA is a material used in sutures and as additional material in tissue scaffolds, which hydrolyses into glycolic and lactic acids which are reabsorbed by the body.
  • Collagen and other extracellular matrix proteins are typically reincorporated into the tissues following implantation.
  • a variety of cell types are known to have increased proliferation on nanofibrous materials such as collagen fibrils or carbon nanotubes.
  • SWNT Single-wall carbon nanotubes
  • Table 1 Viscosities for biopolymer/ carbon nanotube composites using a cone on late viscometer. 1
  • InkJet printing offers a viable alternative for polymer scaffold development in tissue engineering as well as for other device manufacturing needs. We have shown that not only can carbon nanotubes be printed in polymeric systems, but they generate the formation of fibers within the matrix which could be valuable in allowing cellular penetration and fluid flow into the designed scaffold.
  • the fibrous structures that form using the inkjet printing system are similar to the surface features of real tissue. Techniques like inkjet printing allow placement of cells directly into the scaffolds to form a complete material. Our technique allows fibrous structures to form directly from the printed material without the need for added materials or coatings onto the waiting substrates, which decreases the need to manipulate the 5 printed system.
  • Supplementation to the properties of the scaffold by carbon nanotubes include increased strength and compressibility as shown in non-printed polymeric systems and further offer the advantage to employ the conductive nature of the SWNT for electrical stimulation of the seeded cells.
  • We have developed new materials for use in an inkjet printing system which incorporate carbon nanotubes 0 for their beneficial properties while also adjusting the polymer morphology toward a more preferred cell substrate.
  • Print cartridges are prepared by first removing residual ink, sonicating the entire cartridge
  • a 10,000MW polyethylene glycol (PEG) solution consisting of Ig PEG, lmg HiPCo carbon single-wall nanotubes (Carbon Nanotechnologies Inc.) in 10ml water was horn sonicated (Branson) on 20%duty cycle at 40% power for ten minutes.
  • ImI of this solution was suspended in a 3000 MW PEG solution prepared by adding 100mg/ml PEG in water and sonicating in a water bath for 10 minutes to obtain a uniform solution. This dispersion of nanotubes was uniform and printed repeatedly without any clogging. We refer to this solution as nanotube stock A.
  • nanotube stock B A stock of O.lmg/ml HiPCo tubes in tetraglycol was sonicated with a horn sonicator on duty cycle 40% and power of 20% for ten minutes and a uniform solution was obtained. We refer to this solution as nanotube stock B.
  • Biopolymer/ nanotube solutions were prepared using nanotube stock A with sodium alginate and collagen I.
  • Nanotube stock B was used with PLGA and fibronectin stocks.
  • To prepare the solutions equal amounts of the above-described biopolymer and nanotube stocks were pipetted together and immediately printed. All solutions retained a uniform dispersion of nanotubes following mixing of the polymer and tubes. Printing of the solutions followed immediately and all solutions were : printed onto clean glass slides, or copper grids for electron microscopy observation.

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Abstract

La présente invention concerne un procédé permettant de former un échafaudage cellulaire à l'aide d'une composition d'encre qui contient, de manière associée, un solvant, des nanoparticules et un polymère physiologiquement acceptable ; puis d'imprimer par jet d'encre ladite composition sur un support solide afin de former ledit échafaudage cellulaire à partir de celle-ci. Par la suite, des cellules viables peuvent être déposées sur l'échafaudage par impression à jet d'encre par exemple.
PCT/US2007/009161 2006-04-14 2007-04-13 Procedes et compositions destines a l'impression de composites de nanotubes biologiquement compatibles WO2007120840A2 (fr)

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US12/250,820 US20090117087A1 (en) 2007-04-13 2008-10-14 Methods and compositions for printing biologically compatible nanotube composites of autologous tissue

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US74485506P 2006-04-14 2006-04-14
US60/744,855 2006-04-14

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US7520951B1 (en) 2008-04-17 2009-04-21 International Business Machines (Ibm) Corporation Method of transferring nanoparticles to a surface
WO2009102484A2 (fr) * 2008-02-14 2009-08-20 Wake Forest University Health Sciences Impression par jet d’encre de tissus et de cellules
EP2203129A1 (fr) * 2007-10-15 2010-07-07 Wake Forest University Health Sciences Procédés et compositions pour l'impression de composites nanotubes de tissu autologue biologiquement compatibles
CN104399119A (zh) * 2014-12-02 2015-03-11 淮安皓运生物科技有限公司 基于3d生物打印制备高力学性能软骨的方法
WO2015048355A1 (fr) * 2013-09-26 2015-04-02 Northwestern University Réticulation de poly(éthylène glycol) de matériaux mous pour l'adaptation des propriétés viscoélastiques pour la bio-impression
US9238090B1 (en) 2014-12-24 2016-01-19 Fettech, Llc Tissue-based compositions
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EP2203129A1 (fr) * 2007-10-15 2010-07-07 Wake Forest University Health Sciences Procédés et compositions pour l'impression de composites nanotubes de tissu autologue biologiquement compatibles
EP2203129A4 (fr) * 2007-10-15 2011-11-23 Univ Wake Forest Health Sciences Procédés et compositions pour l'impression de composites nanotubes de tissu autologue biologiquement compatibles
US9005972B2 (en) 2008-02-14 2015-04-14 Wake Forest University Health Sciences Inkjet printing of tissues and cells
WO2009102484A2 (fr) * 2008-02-14 2009-08-20 Wake Forest University Health Sciences Impression par jet d’encre de tissus et de cellules
US8691274B2 (en) 2008-02-14 2014-04-08 Wake Forest University Health Sciences Inkjet printing of tissues and cells
WO2009102484A3 (fr) * 2008-02-14 2009-12-03 Wake Forest University Health Sciences Impression par jet d’encre de tissus et de cellules
US9301925B2 (en) 2008-02-14 2016-04-05 Wake Forest University Health Sciences Inkjet printing of tissues and cells
US7520951B1 (en) 2008-04-17 2009-04-21 International Business Machines (Ibm) Corporation Method of transferring nanoparticles to a surface
US10731046B2 (en) 2012-11-27 2020-08-04 Tufts University Biopolymer-based inks and use thereof
EP2925822A4 (fr) * 2012-11-27 2016-10-12 Univ Tufts Encres à base de biopolymères et leur utilisation
US10035920B2 (en) 2012-11-27 2018-07-31 Tufts University Biopolymer-based inks and use thereof
US10173357B2 (en) 2013-09-26 2019-01-08 Northwestern University Poly(ethylene glycol) cross-linking of soft materials to tailor viscoelastic properties for bioprinting
WO2015048355A1 (fr) * 2013-09-26 2015-04-02 Northwestern University Réticulation de poly(éthylène glycol) de matériaux mous pour l'adaptation des propriétés viscoélastiques pour la bio-impression
CN104399119A (zh) * 2014-12-02 2015-03-11 淮安皓运生物科技有限公司 基于3d生物打印制备高力学性能软骨的方法
US9238090B1 (en) 2014-12-24 2016-01-19 Fettech, Llc Tissue-based compositions
US11938246B2 (en) 2014-12-24 2024-03-26 Fettech, Llc Tissue-based compositions and methods of use thereof

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