Patent Application
20110021653
The present invention relates to two-photon initiation systems in general, and, more particularly, to a two-photon initiation system using chromophores to initiate two-photon polymerization of hydrogels for 3D construction of structures for promoting cell growth.
Two-photon absorption has been long known as the simultaneous absorption of two photons of identical or different frequencies in order to excite a molecule from one state (usually the ground state) to a higher energy electronic state via a virtual state transition. Two-photon absorption may be used to precisely fabricate three dimensional hydrogel structures in a process called two-photon lithography (TPL). Hydrogel structures have gained importance because they are useful for a number of biomedical applications such as construction of biosensors, tailoring materials for drug delivery, and creating biocompatible scaffolds that interact with living cells.
Key aspects to consider in making patterned structures include the resolution of TPL, the ease of the technique and the freedom to make arbitrary structures. TPL holds promise for accomplishing these key aspects. For example, TPL-fabricated structures include cantilevers and gratings and for creating defects in photonic crystals. Structures can be fabricated from commercially available monomers, of almost any shape with resolution less than 100 nm. However, while TPL has been used to fabricate a wide variety of arbitrarily shaped structures, it has limitations due to the lack of efficient initiators compatible with hydrogels.
One of the major limitations of TPL used in hydrogel fabrication has been the inability to use effective hydrophobic chromophores in aqueous media. This characteristic has limited the use of TPL for biological applications. Unfortunately, the use of cytotoxic organic solvents such as toluene to solubilize the hydrophobic chromophores is very undesirable because changes from organic solvents used in fabrication to then immersing the resultant structure into an aqueous environment can lead to significant structural distortions (See Chemistry of Materials 2009, 21(10), 2003).
As indicated above, known systems are missing a highly efficient two-photon initiator compatible with two-photon initiated hydrogel polymerization. Because currently known initiators are not efficient, high powered and expensive femto-second laser devices must be used to initiate the two-photon polymerization. See, for example, Campagnola, P. J., J. Biomed. Mater. Res., Part A 2004, 71A, 359 and Shear, J. B., Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 16104. Another approach requires the use of a hydrophobic two-photon chromophore and initiator system to form hydrogel structures.
While other 3D fabrication attempts have been tried, they lack the simplicity of a direct fabrication approach such as TPL. Examples of such alternate technologies include pseudo-3D techniques such as using conventional layer-by-layer 2D approaches. A number of varying approaches are reviewed in Peltola, et al., “A review of rapid prototyping techniques for tissue engineering purposes,” Annals of Medicine (2008) Vol. 40: 268-280. Such approaches result in overly complicated fabrication requiring precision alignment and produce structures having interfaces with weak bonding properties. Known approaches are also typically limited in the ability to produce fine features in structures.
As a result there is a need in the art, satisfied for the first time by the methods and compositions disclosed herein, to provide a true 3D fabrication of a hydrogel material with resulting advantages including a simplified fabrication process and improved bonding properties. As a further advantage, an improved TPL as disclosed hereinbelow provides high resolution in creating structures having features of submicron to nanometer resolution over conventional approaches having features of micron to tens of micron resolution. As a result, fine 3D structures may be fabricated, such as, for example, hydrogel cell scaffolding. Further, high efficiency of novel two-photon initiators disclosed for the first time hereinbelow allow fabrication at faster speeds and/or use of a low peak power laser.
A compound or composition is disclosed which includes:
at least one chromophore initiator compatible with a hydrogel oligomer or polymer, wherein the chromophore has a simultaneous two-photon absorptivity; and
at least one monomer in close proximity to said chromophore, wherein the at least one monomer includes a hydrogel oligomer or polymer.
In one embodiment the chromophore initiator includes a constitutional unit derived from the formula
wherein PEG consists of polyethylene glycol.
In another embodiment a method for creating 3D structures using two-photon direct writing is disclosed. The method includes mixing a resin having a monomer structure with a solvent to create a resin-solvent mixture. A chromophore initiator is added to the resin-solvent mixture to create a second mixture, where the chromophore initiator is comprised of a first constitutional unit derived from a compound having the formula
Two-photon lithography is applied to the second mixture to produce a 3D structure.
While the novel features of the invention are set forth with particularity in the appended claims, the invention, both as to organization and content, will be better understood and appreciated, along with other objects and features thereof, from the following detailed description taken in conjunction with the drawings, in which:
In the drawings, identical reference numbers identify similar elements or components. The sizes and relative positions of elements in the drawings are not necessarily drawn to scale. For example, the shapes of various elements and angles are not drawn to scale, and some of these elements are arbitrarily enlarged and positioned to improve drawing legibility. Further, the particular shapes of the elements as drawn, are not intended to convey any information regarding the actual shape of the particular elements, and have been solely selected for ease of recognition in the drawings.
The following disclosure describes several embodiments and systems for a two-photon polymerization system using chromophores to initiate two-photon polymerization of hydrogels for 3D construction of structures for promoting cell growth. Several features of methods and systems in accordance with example embodiments are set forth and described in the Figures. It will be appreciated that methods and systems in accordance with other example embodiments can include additional procedures or features different than those shown in the Figures. Example embodiments are described herein with respect to biological cells. However, it will be understood that these examples are for the purpose of illustrating the principles, and that the invention is not so limited.
Additionally, methods and systems in accordance with several example embodiments may not include all of the features shown in these Figures. Throughout the Figures, like reference numbers refer to similar or identical components or procedures.
Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to.”
Reference throughout this specification to “one example” or “an example embodiment,” “one embodiment,” “an embodiment” or various combinations of these terms means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
In one example embodiment this disclosure provides a method of creating 3D cell scaffolds using two-photon direct writing using highly efficient two-photon initiators that are compatible with hydrogel medium. Using the methods and compositions taught herein solves problems present in other techniques while allowing the use of low-cost femtosecond fiber laser, made possible due to the high initiation efficiency of the new initiators.
Referring now to
To a solution of fluorine (10 g, 60 mmol), 1-bromo-2-(2-methoxyethoxy)ethane (24.2 g, 132 mmol), tetrabutylammonium bromide (2.5 g) in degassed DMSO (200 mL), is added NaOH aq (50 wt %, 50 mL). After mechanically stirring at room temperature for overnight, the solution is quenched with water (100 mL), and solvents are distilled under vacuum. The residue solid is dissolved in CH2Cl2, and washed with HCl aq. (1 M, 100 mL×3). The organic layer is dried over MgSO4, and purified by column chromatography to give a viscous liquid.
A two-necked flask containing a mixture of M1 (10.0 g, 25.9 mmol) and paraformaldehyde (7.68 g, 128 mmol) is placed in an ice bath. A 30% HBr solution (35 mL) in acetic acid is then added carefully to this flask, and the mixture is heated to 70° C. and stirred for 24 h under nitrogen until the HBr is consumed. The hot reaction mixture is cooled to room temperature before it is poured into 200 mL of cold water. The resulting mixture is then extracted with methylene chloride, and the combined organic layer is collected and washed with brine. After drying over anhydrous MgSO4, evaporation in vacuo afforded the crude product, which is subjected to purification by column chromatography on silica yield a slight yellow transparent liquid. The yield liquid above is heated in excess triethylphosphite (3 eqv.) at 125° C. for 4 hr, and then the excess triethylphosphite is distilled off to M2.
A flask is charged with bromophenol (1.16 g, 9.6 mmol), methoxypolyethylene glycol (Mn:2000) (19.2 g, 9.6 mmol), triphenylphosphine (PPh3) (6.1 g, 9.6 mmol), and 150 mL of tetrahydrofuran. The flask is immersed in an ice bath, and diethyl azodicarboxylate (1.6 mL, 9.8 mmol) is added dropwise at a rate such that the temperature of the reaction mixture is maintained below 10° C. Upon completion of the addition, the flask is removed from the ice bath and the solution is allowed to stir at room temperature overnight and subsequently at 40° C. for 3 hr. The reaction mixture is cooled to room temperature, diluted with 150 mL of ether, and washed twice with 100 mL portions of saturated aqueous sodium bicarbonate solution. The aqueous layers are combined and back-extracted with 100 mL of ether. The combined organic layers are dried over sodium sulfate. Excess solvent and other volatile reaction components are completely removed under reduced pressure initially on a rotary evaporator and then under high vacuum. The resulting solid is purified under a flash chromatography column.
To a solution of tris(dibenzylideneacetone)dipalladium [Pd2(dba)3] (0.0274 g, 0.15 mol % relative to the aniline) and bis(diphenylphosphino)ferrocene (DPPF) (0.025 g, 0.225 mol % relative to the aniline) in dry THF (50 mL) under nitrogen is added D1 (4.31 g, 2 mmol) at room temperature, and the resultant mixture is stirred at that temperature for 10 min. Sodiumtert-butoxide (0.25 g, 2.6 mmol) and D3 (0.298 g, 2 mmol) are added to this solution and stirred at 90° C. for 4 h. The reaction mixture is cooled to room temperature and poured into water. The mixture is extracted by toluene (3×100 mL), and the fractions of organic layers are collected together and concentrated in vacuo to give the crude reaction mixture. Purification of the reaction mixture is done by flash column chromatography.
To a stirred solution of D2 (4.61 g, 2 mmol) and B2 (0.639 g, 1 mmol) in THF, is added dropwise a solution of t-BuOK (1 mL, 1M in methanol) at 0° C. After stirring for 2 hr at this temperature, the mixture is quenched with water. THF is removed under reduced pressure, and then the solid is dissolved in methylene chloride, washed with water and dried over Na2SO4. Excess solvent is also removed under reduced pressure, and the residue is purified by column chromatography to give the chromomer (C).
One embodiment of the chromomer includes chromophore initiators comprised of a first constitutional unit derived from a compound having the formula
where PEG represents polyethylene glycol.
For the purposes of two-photon polymerization, the chromophore derived from formula (1) is combinable with a resin compound. In one embodiment the resin comprises a compound having the general structure:
D is selected from the group consisting of N, O, S,
At least one of Rc and Rd consists of a hydrogel structure (oligomer or polymer), including collagen, collagen-GAG(alginate) copolymers, albumin, hyaluronic acid, fibrinogen-fibrin, chitosan, matrigel, alginate, polyhydroxyalkanoates, starch, poly(lactic acid), poly(d-lactic acid), poly(l-lactic acid), poly(d,l-lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid), poly(e-caprolactone), poly(hydroxyalkanoate), poly(3 or 4-hydroxybutyrate), poly(3-hydroxyoctanoate), poly(3-hydroxyvalerate), poly (p-dioxanone), poly(propylene fumarate), poly (1,3-trimethylene carbonate), poly(glycerol-sebacate), poly(ester urethane), polyethylene glycol and hydroxyethyl methacrylate (HEMA) and combinations thereof.
One of Rc and Rd is not present when B is O or S. Note that Matrigel™ Basement Membrane Matrix is manufactured by BD BioSciences, NJ and according to their literature is a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in ECM proteins.
As shown in the examples below, a chromophore according to formula (1) provides highly efficient photoinitiation for a two-photon polymerization process.
A typical resin having a monomer structure as shown below in formula (11) is mixed with solvent. An initiator, Irgacure® 651, is added at 0.5% (w/w), based on the monomers. A chromophore according to formula (1) is added having a concentration in a range of 0.5-5 wt %, based on the monomers. All the components are stirred using a stir bar for about 2 hours to form a hydrogel medium. The hydrogel medium is then ready for two-photon lithography.
As published by the manufacturer, Ciba Specialty Chemicals, Inc., Basel, Switzerland, Irgacure® 651 is a general-purpose, highly efficient solid, free-radical photoinitiator used for the curing of unsaturated pre-polymers in the chemical class of Benzyldimethyl-ketal having a chemical formula Alpha, alpha-dimethoxy-alpha-phenylacetophenone. It will be apparent to those skilled in the art having the benefit of this disclosure that the co-photoinitiator is not limited to Irgacure® 651, but other equivalent photoinitiators may be used such as, for example, Irgacure® 819 and known equivalents.
A typical resin having a monomer structure as shown above is mixed with solvent. A chromophore according to formula (1) is added having a concentration in a range of 0.5-5 wt % based on the monomers. All the components are stirred using a stir bar for about 2 hours to form a hydrolgel medium. The hydrogel medium is then used for two-photon lithography.
Referring now to
Also located in the beam path will be a computer controlled high speed shutter 3 (for example a shutter, beam slicer or pulse picker); a beam expander 28, a turning (ultrafast) mirror 4, an (optional) turning (ultrafast) mirror 5, (optional) computer controlled steering (ultrafast) mirrors 6,7, steering lens 8, a tube lens 9, a laser line dichroic mirror 10, an objective lens 51, an LED 11, a focuser (dark field and bright field) 12, a fluorescence spot 30, an (optional) polarizer for anisotropy measurement, an (optional) dichroic mirror 14; an IR filter 15, a fiber collector lens 17 and an (optional) multimode confocalization fiber 32. A Personal Computer (PC) 36 will operate to send a control signal to an Avalanche Photodiode (APD) 34 and a synchronization signal to the femtosecond laser 20. A multi-axis nanopositioning stage 50 is adapted to holds and manipulate a substrate for fabrication by two photon polymerization. A CCD camera 42, or equivalent imaging device will be optically located to receive images from the stage.
The laser 20 may include, for example, a femtosecond fiber laser, a Ti:Sapphire femtosecond laser, other conventional ultrafast lasers and equivalents. Those skilled in the art having the benefit of this disclosure will recognize that various other configurations may also be used to accomplish two-photon lithography using the novel principles disclosed herein. Such alternatives may use a dual laser configuration, a de-scanned laser configuration, an inverted beam-path, a commercial microscope system, may have no need for imaging, and may employ only one of either stage-scanned or beam-scanned options. The two-photon cross-section for the chromophore is expected to be ca. 1000 GM. The operation wavelength is expected to be about 780 nm which is a typical fiber laser fixed wavelength. The fabrication speed, NA of the lens, laser peak power and frequency are all related. At 100 um/s fabrication speed, the average laser power (not peak power) of about 5 mW is required with a NA for the lens of about 1.4. Commercially available fiber lasers having parameters of about 100 fs pulse and 100 MHz frequency can provide a laser power up to 65 mW.
Having described one example of a design for two-photon fabrication, it is believed that a discussion of example operational modes will enhance the understanding of this disclosure. Typically, two-photon manufacturing is accomplished when an ultrafast pulsed laser is focused into controlled locations in a reactive monomer (herein referred to as the “material”) to induce a photo-crosslinking polymerization reaction. The wavelength of the laser needs to be such that the material will exhibit very little single-photon absorption and a high amount of two-photon absorption. This is necessary to ensure that a photo-crosslinking reaction will occur tightly confined to the focal spot, deep within the material and not just at the first surface of the material. A shutter controls when the material is exposed to the laser and either a 3D stage, a 2D scanned mirror system (a scan head), beam shaping optics or any combination therein, can be used to control where the material is exposed to the laser light. For ease of language, the 3D stage or scan head are herein referred to as the “intensity distributor”.
The pulse duration, repetition rate, average laser power, numerical aperture of the focusing lens, scan speed of the focal spot in the monomer, two-photon cross section of the absorber, polymerization efficiency, intensity distributor resolution and repeatability, and system losses are all relevant parameters to the manufacturing process. Sufficient enough peak intensity (peak power/area) must be present to induce the threshold limited two-photon photo-crosslinking reaction. With the advent of our super efficient (high two-photon cross section and high polymerization efficiency) materials, as disclosed herein for example, it is now possible to induce the photo-polymerization reaction with greatly reduced peak intensities (less average power, less tightly focusing optics, and longer pulse durations) and thus induce photo-crosslinking over larger areas and in less time than ever before.
The generalized processes by which two-photon fabrication is accomplished is described below with reference to
Referring now to
A structure is generated 130 by moving the sample in three dimensions relative to the focusing lens to irradiate said chromophore to cause a simultaneous two-photon or two-photon absorption in said chromophore to produce two photon polymerization of the hydrogel medium. In example embodiments the hydrogel medium is composed of compounds as described hereinabove.
The invention has been described herein in considerable detail in order to comply with the Patent Statutes and to provide those skilled in the art with the information needed to apply the novel principles of the present invention, and to construct and use such exemplary and specialized components as are required. However, it is to be understood that the invention may be carried out by specifically different equipment, and devices, and that various modifications, both as to the equipment details and operating procedures, may be accomplished without departing from the true spirit and scope of the present invention.
