Direct Three-Dimensional Microfabrication of Hydrogels via Two-Photon Lithography in Aqueous...

Direct three-dimensional microfabrication of hydrogels via two-photon lithography in aqueous solution

Shalin J. Jhaveri, Jesse D. McMullen, Rint Sijbesma, Loon-Seng Tan, Warren Zipfel, andChristopher K. OberDepartment of Materials Science and Engineering, Cornell University, Ithaca, NY

Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY

Department of Applied Engineering and Physics, Cornell University, Ithaca, NY

Department of Chemical Engineering and Chemistry, Eindhoven University of Technology,Eindhoven, Netherlands

Nanostructured & Biological Materials Branch, United States Air Force Research Laboratory,Dayton, Ohio,

The ability to precisely microfabricate hydrogels in three-dimensions has gained tremendousimportance as it may be useful for a number of biomedical applications such as constructionof biosensors, tailoring materials for drug-delivery and creating soft scaffolds that interact withliving cells.1–3 The two most important aspects to consider while making patterned structuresare the ease of the technique and the freedom to make arbitrary structures. Two-photonlithography (TPL) stands out when both of these aspects are considered. Using a confocal laserscanning microscope equipped with a femtosecond mode-locked near IR laser, one canmicrofabricate structures, from commercially available monomers, of any shape and havingresolution less than 100 nm.4–6 TPL has been used to microfabricate a wide variety ofarbitrarily shaped structures including cantilevers,7 gratings,8 photonic crystals,9 and forcreating defects in photonic crystals.10,11

One of the major current limitations of multiphoton lithography used in hydrogel fabricationhas been the inability to use effective hydrophobic chromophores in aqueous media. Thischaracteristic has limited the use of multiphoton lithography for biological applications,because the use of cytotoxic organic solvents such as toluene to solubilize the hydrophobicchromophores is very undesireable.12 In addition, the direct net shape fabrication of hydrogelsin water without the need to exchange solvents can offer advantages during the hydrogelproduction step. This is particularly useful since changes from organic solvents used infabrciation to water in use can lead to significant structure distortions. Our objective for thiswork has been to fabricate hydrogels capable of releasing growth factors from very precise 3Dstructures to be placed on neural electrodes.13

Chemical reactions in a two-photon process are tightly confined at the focal point, generallyto the order of cube of laser wavelength.14 Since the cross-sections are very small, a very intensesource of light is needed which often results in unwanted damage to the structures if insensitivechromophores are used. Hence, molecules having excellent two-photon sensitivity are neededwhich enable lower laser powers for two-photon excitation.15

Correspondence to: Christopher K. Ober.Supporting Information. Experimental procedures. This material is available free of charge via the Internet at http://pubs.acs.org.

NIH Public AccessAuthor ManuscriptChem Mater. Author manuscript; available in PMC 2010 April 27.

Published in final edited form as:Chem Mater. 2009 April 27; 21(10): 2003–2006. doi:10.1021/cm803174e.

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http://pubs.acs.org

Researchers interested in biological imaging have previously studied a variety of methods tosolubilize hydrophobic chromophores in water including synthesis of complex water solublechromophores using multi-step reactions, used water soluble quantum dots for multiphoton invivo biological imaging, or through use of amphiphilic block copolymer complexes.16–18 Incommon with this prior work we are interested in applying oil soluble chromophores for usein aqueous solution. We have instead elected to solubilize hydrophobic chromophores withlarge two-photon absorption cross-section using a non-ionic surfactant (Pluronic F127®

(PF127)) and applying the resulting complex with an oil soluble photoradical initiator tofabricate hydrogel microstructures which could be useful for biomedical applications. Thehydrophobic chromophores were used for free-radical photopolymerization of 2-hydroxyethylmethacrylate (HEMA) and poly(ethylene glycol) diacrylate (PEGDA) in aqueous medium.

A simple one-step method is described to produce synthetic microfabricated hydrogelstructures via TPL. To the best of our knowledge, this is the first report in which TPL has beenperformed using hydrophobic chromophores with large two-photon absorption (TPA) cross-section (δ) directly in aqueous solution. Previously, Campagnola et al. and Shear et al. havecrosslinked proteins in aqueous solution via multiphoton excitation. They utilizedphotosensitizers having low two-photon sensitivity which require extraordinarily highthreshold laser powers for microfabrication.19,20 Their work is very promising for potentialtissue scaffold fabrication, however, crosslinking of the proteins requires very long exposuretimes and high energies. The resulting protein aggregates may cause immunogenic responsein vivo.21 This communication reports the direct net shape fabrication of biocompatible acrylateand methacrylate hydrogels which require much lower exposure times for free-radicalcrosslinking thereby reducing the fabrication time.

It was found that only when both the initiator 2,2-dimethoxy-2-phenyl acetophenone (Irgacure651) and the 2-photon sensitive chromophore (AF240) were present in the polymerizablemixture could hydrogel microstructures be formed. While the exact mechanism for theobserved photopolymerization is presently unknown, there are at least two possibilities: (a) thechromophore behaves as a photosensitizer, which emits an up-converted fluorescent light inthe UV-visible regime on simultaneous absorption of two near IR photons. Subsequently, theinitiator (Irgacure 651) on absorbing the fluorescent light starts free-radical polymerizationand (b) an intermolecular electron transfer occurs from the two-photon-excited AF240 toIrgacure 651. It has been reported by several groups that the apparent α-cleavage quantumefficiency (Φα) of Irgacure 651 (leading to generation of benzoyl and substituted radicals) inthe wavelength region from 313 to 365 nm is close to 100%.22,23 Therefore, it is notunreasonable to assume that Φα would be also significant in the longer wavelength region (i.e.365–400 nm) where the emission tail on the “blue side” of AF240 can overlap as the blueemission tail of AF240 extends below 410 nm.24 In the confined, hydrophobic environmentprovided by the PF127 energy transfer would be expected to be highly efficienct.

The structures of the chemicals used for TPL are shown in Scheme 1. PF127 was chosen as asurfactant for dispersion of the hydrophobic chromophores because it is also FDA approvedand has been previously used with no cytotoxic effects, when used in appropriateconcentrations.25,26

The TPA chromophore, designated AF240, has a linear D-π-A motif where the donor (D) isdiphenylamine and the acceptor (A) is benzothiazole. It has a fluorene ring which is thermallyand photochemically stable and by locking the biphenyl unit into the fluorenyl ring, greaterelectron delocalization through increased π molecular orbital overlap is provided.27, 28 Thisplanar feature enhances molecular polarizability and TPA sensivity.29 The monomers, 2-hydroxyethyl methacrylate and poly(ethylene glycol) diacrylate and the initiator, Irgacure 651,are commonly used in synthesizing highly biocompatible hydrogels.30–32 The presence of

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water in the prepolymer solution helps in controlling important properties of the resultingstructure such as modulus and the pore size which can be fine tuned for its intended application.It also enables precise net shape fabrication which is not possible when an organic solvent isused in the fabrication step only to be replaced with water.

Photophysical studies of the resins (prepolymer solution) with chromophores dissolved indifferent solvents are shown in Figure 1. The chromophores are prone to aggregate andsubsequently precipitate, when dispersed in water alone, resulting in reduced concentration ofthe solvated dye and a decrease in the action cross-section value, a product of fluorescencequantum yield (ΦF) and two-photon absorption cross-section (δ; GM=1 × 10−50 cm4 secphoton−1 molecule−1), as compared to when they are dissolved in toluene. When the solventis water with added PF127, an increase and greater consistency in the δΦF values is seen. Asimilar effect was seen when Tian et al. solubilized different hydrophobic chromophores inwater using the block copolymer poly(methacrylic acid)-block-polystyrene.18 This effect isbelieved to be due to micelle formation which leads to a dilution effect and reduced aggregationof the chromophores. The large error bars (Fig. 1) when the solvent is water with no PF127may be caused by reduced solubility of the chromophore. In this case the monomers themselveshelp in solvating the chromophores in water. There was no significant difference when thechromophore:PF127 molar ratio was changed from 1:10 to 1:20.

Two parameters, namely required TPL threshold power (Pth) and the polymerization windowrange available to make hydrogel microstructures, were chosen to compare the efficiency ofdifferent monomer mixtures. The TPL threshold power is defined as the local absorbed energydensity below which no polymerization occurs.33 The polymerization window range wascalculated using the formula Pmax/Pth, where Pmax was the maximum power with which thestructures could be microfabricated, beyond which damage to the structures would occur. Whentwo-photon initiated polymerization was performed with the chromophore dissolved indifferent solvents, the best performance, as expected, was when the solvent was toluene.Approximately, 12 times the concentration of AF240 was needed to achieve similar Pth valueswhen the chromophore was dispersed in water in the presence of PF127 (1:10) (Table 1). Whenthe chromophore was solvated in water alone, in the absence of PF127, only single-layerstructures could be fabricated, since any multi-layer microstructures were damaged becauseof the very high threshold power required. The highly efficient AF240 dye was used in thesestudies to demonstrate a proof-of-principle that PF127 can be used to directly microfabricatehydrogels in water. We believe that the use of dyes having even higher TPA cross-sectionswill eliminate the need of high concentrations of dye required for microfabrications, therebyreducing any potential toxicity issues.

Since the swelling of hydrogels is so important in a number of biomedical applications, theextent of swelling was studied using fluorescence microscopy of the microfabricated structures.The observed fluorescence is due to excitation of the AF240 chromophore which is retainedin the hydrogels immediately after fabrication. It is possible to remove the chromophore fromthe hydrogel microstructures using extended extraction times. The swelling ratio was definedby the change in the width of the structure after immersion in water with respect to the widthof dry gel. The width of the structures was calculated using Metamorph imaging software.Tanaka et al. have explained the lateral swelling of patterned hydrogels which depends on filmthickness and the aspect ratio of the pattern.34–36

As shown in Figure 2, when net shape water microfabricated waffle was made with AF240dissolved in water in the presence of PF127, the swelling ratio was ∼10% (laser power = 21.5mW). The water swollen gel was dried to assess the effect of water on the gel dimension. Thepatterned microstructure has an aspect ratio of 0.5 and a thickness of 100 nm. When the samestructure was made with a higher laser power of 28 mW, the swelling ratio decreased to ∼ 5%.

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When much thicker hydrogel slabs were made in the presence of water having a height of 3µm, the swelling ratio was ∼20%. By varying the thickness, aspect ratio as well as the laserpower used to microfabricate the structures, hydrogels having different swelling ratios couldbe produced. Hence, one can easily create microstructures with customized swelling propertiesusing TPL. The structures remained adhered to the silanized glass coverslip after numerousdeswelling- swelling studies. Delamination of structures from glass substrate was not observedat least up to fabrication of structures having an aspect ratio of 1.5.

In conclusion this is, to the best of our knowledge, the first time synthetic hydrogelmicrostructures have been fabricated via TPL using hydrophobic chromophores having largeTPA cross-section directly in aqueous solution. PF127, a non-ionic surfactant, was used toenhance the dispersion and effectiveness of the chromophore in water. Although, toluene isstill a superior solvent for dissolving the hydrophobic chromophores, the use of water and thesimple methods for using otherwise insoluble chromophores extends this technique fornumerous biological applications.

Supplementary MaterialRefer to Web version on PubMed Central for supplementary material.

AcknowledgmentsWe acknowledge the use of the facilities of the Cornell Center of Materials Research, Cornell NanobiotechnologyCenter, the Microscopy and Imaging facility at Cornell University and its facility manager Carol Bayles. This workwas supported in part by NIH NSR01-044287, Air Force Research Laboratories and by the NBTC, an STC programof the National Science Foundation under Agreement Number ECS-9876771.

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Figure 1.Two-photon excitation (TPE) action cross-section values of the prepolymer solution when thechromophore is dissolved in toluene (○); water with PF127 (1:10 chromophore : PF127 ratio)(●); water with PF127 (1:20 chromophore : PF127 ratio) (■) and water without PF127 (▲).

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Figure 2.Fluorescence image of the microfabricated waffle in (a) swollen and (b) dry condition. Swellingratio of the microstructure is ∼ 10%. The microstructure was obtained by polymerization ofthe monomers via TPL directly in water in the presence of PF127 (1:10). The laser power usedwas 21.5 mW and the aspect ratio of the structure was 0.5.

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Scheme 1.Chemical components used for the microfabrication of hydrogels directly in water. The SEMimage shown is representative of the possible hydrogel microstructures obtained after two-photon lithography. Scale bar = 10 µm.

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Table 1

The laser power (threshold and maximum) and the polymerization window range available to microfabricate thehydrogel structures directly in the different type of solvents.

Solvent AF240(mM)

Pth(mW)

Pmax (mW) Pmax/Pth

Toluene 1.3 23.3 ±3.3

38.9 ± 5.5 1.7

DI Water withPF127 (1:10)

12 24.9 ±3.5

31.1 ± 4.4 1.3

DI Water 12 38.9 ±5.5

38.9 ± 5.5 negligible


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