Environmentally Responsive Hydrogel Nanocomposites for Biomedical Applications Synthesized Via PCμCP Hariharasudhan D. Chirra, J. Zach Hilt Department of Chemical and Materials Engineering, University of Kentucky, Lexington, KY RESEARCH OBJECTIVES RESULTS AND DISCUSSION CONCLUSIONS EXPERIMENTAL METHODS  Precise XY control at the microscale was achieved using microcontact printing (μCP).  Nanoscale control over thickness of hydrogels has been achieved using atom transfer radical polymerization.  Crosslinker parameters (e.g., crosslinker MW and amounts) were used to control the response behavior of patterned hydrogels.  Ultrathin hydrogels grown via ATRP respond instantaneously and so can be used for point of care applications.  Gold nanoparticles were loaded into hydrogel microstructures using in-situ precipitation.  Environmentally responsive tunable smart hydrogels synthesized via PC μ CP can potentially be used for “instantaneous” biomedical applications at the micro- and nanoscale. Devices that are spatially functionalized with smart hydrogels, especially at the micro- and nanoscale, are of high interest in the diagnostic and therapeutic fields. Using nanocomposites, hydrogels can be tailored to exhibit synergistic effects of the component inside the crosslinked matrix along with the hydrogels response property, thereby expanding their potential applications in the biomedical field. The goal of this project was to develop novel methods to fabricate precise 3-D patterned smart hydrogels to devices (e.g., silicon, gold) followed by the integration of nanoparticles to the hydrogels matrix. Specifically, polymerization controlled by micro contact printing (PCμCP) was applied to synthesize a controlled array of environmentally responsive hydrogels over surfaces followed by in-situ precipitation of particles 1,2. These methods enable for the fabrication of device platforms harnessing the unique abilities of hydrogels, such as diagnostic and therapeutic devices, microarrays, and clinic-on-a-chip devices. INTRODUCTION  Controlled synthesis of smart hydrogels over gold  XY control using microcontact printing (μCP)  Z/thickness control using polymerization techniques – UV, atom transfer radical polymerization (ATRP)  Characterization of responsive nanocomposite patterns  Loading of gold nanoparticles to the hydrogel matrix  Responsive studies to external stimuli  Environmentally responsive studies for biomedical applications μCP – 1-Octadecanethiol (ODT; 1 mmol) UV photopolymerization – Methacrylic acid, MAA (80 mol), TEGDMA (20 mol), 2,2-dimethoxy-2-phenylacetophenone (DMPA; 1 wt%), UV light of intensity 17.5 mW/cm 2 Initiator – Bromoisobutyrate terminated undecyl disulfide (3 mmol) for 24 hours ATRP – NIPAAm:crosslinker (100-x:x mol), Bipyridyl (3 mmol), copper bromide (1 mmol), copper powder (0.1 wt%), nitrogen atmosphere N-Isopropylacrylamide (NIPAAm) Ethylene glycol dimethacrylate; 1 EG unit Poly (ethylene glycol) n dimethacrylate; Varying EG units NHNH O O O n O O Patterned Hydrogels Synthesized via µCP and ATRP  Hydrogels of varying crosslinking density (NIPAAm: Crosslinker – 95:5, 90:10, 85:15, 80:20, 70:30) and crosslinker molecular weight (EGDMA, TEGDMA, PEGnDMA, n = 200/400/600) studied.  The effectiveness of ATRP for controlled growth was studied by varying the polymerization reaction time from 4 to 48 hours.  The dry state thickness was measured using AFM imaging at different reaction times.  Precise hydrogel patterns of square shapes are formed from initiator molecules.  A very well defined XY controlled pattern is obtained using μCP. Normalized AFM images of patterned hydrogels showing spatial XY patterns and Z controlled hydrogel growth Controlled Hydrogel Growth via ATRP Controlled growth of 10% crosslinked NIPAAm hydrogel patterns in nanometer scale with increasing polymerization time Δ T Δ pH Poster Background: Non contact AFM image of an array of 10 µm 95:5 mol% PNIPAAm-co-EGDMA hydrogel squares grown over gold via ATRP. ATRP Environmentally Responsive Hydrogels Response profile of different PNIPAAm hydrogels (squares) at increasing temperature showing broad transition. Inset: Surface response of 30% PEG600DMA crosslinked PNIPAAm hydrogel showing sharp transition  Liquid cell set up of AFM with an auto tunable T controller. Equilibration was allowed for 15 minute after every temperature stimuli.  Swelling behavior of hydrogels can be tailored by varying crosslinking density.  Above LCST (40 o C), NIPAAm behaves hydrophobic – dehydrates to a collapsed rough state. Below LCST (25 o C) – relatively less hydrophobic – swells to a smooth gel.  Bulk broad response should be similar to the sharp surface response for ultrathin hydrogels.  Sharp response can be harnessed for the fabrication of stimuli sensitive biosensors, and controlled drug delivery therapeutic devices. Gold Nanoparticle 3-D image courtesy: Nannes et al., J. Leukoc. Biol. 2005, 78, 585 In-situ Precipitated Gold Nanoparticles (GNP) in a Hydrogel Matrix  80:20 mol% of poly(methacrylic acid):TEGDMA hydrogels immersed in cycles of gold salt solution and reducing agent (trisodium citrate dihydrate).  Blank hydrogel shows no absorbance (transparent).  After reduction, the reduced hydrogel (purple colored) showed SPR peaks at 582 nm (cycle 1), 555 nm (cycle 2), and 552 nm (cycle 3) confirming GNP presence.  Increased concentration was observed with each reduction cycle.  Particle loading was done using in-situ precipitation. Absorbance spectra obtained during the various steps of concentrating gold nanoparticles in the hydrogel matrix via in-situ precipitation. Optical images showing (a) applied prepolymer solution over micropatterned gold surface, (b) polymerized microstructures attached to the surface, and (c) swollen hydrogel gold nanocomposites after reduction process. (Scale µm) (a) (b) (c) In-situ Precipitated Responsive Nanocomposites  Surfaces patterned using μCP are treated with hydrophilic prepolymer solution, which is then UV- photopolymerized.  Microstructured hydrogel patterns are then immersed in chloroauric acid solution and subsequently reduced to form GNPs.  Here, there is not a covalent attachment, so responsive swelling causes removal of most of the microstructured hydrogels. pH responsive behavior of GNP precipitated nanocomposites. (a) Swollen nanocomposite in water, (b) collapsed nanocomposite with the acidic stimulus (pH = 2.5; citric acid; t = 8 seconds), and (c) swelling back to original state with basic stimulus (pH = 8.7; sodium phosphate; t = 4 seconds). Scale – 250 μm (a) (b) (c)  In the presence of water, the nanocomposites will swell to an equilibrium state.  Due to the pH responsive nature of the hydrogel, the addition of an acidic buffer collapsed the nanocomposite while a basic buffer caused the nanocomposite to swell.  The supernatant from multiple response cycles analyzed for released GNPs. The UV-Vis spectra did not show the characteristic SPR peak of GNPs, indicating that the GNPs did not leach out of the hydrogels.  The incorporation of GNPs into responsive hydrogel systems have potential applications in various biomedical devices, such as biosensors, microarrays, etc.  The GNPs can act as functionalization points for the attachment of biomolecules, which can create a response due to a specific biochemical stimuli or be used to capture desired analytes.  ATRP grown patterns enable the creation of films with nanoscale precision, which would likely be more attractive for biosensor applications. Above: Schematic representation of the steps involved in the synthesis of thin smart hydrogel nanocomposites Below: ATRP reaction mechanism  Initial reaction is the same since ATRP starts from initiator radicals on the surface.  Further growth is controlled by reaction-diffusion mechanism, i.e. mobility of growing radical chain is restricted by mesh network.  Increase in crosslinker molecular weight (analogous to crosslinker chain length) increases thickness.  This controlled nm Z growth of hydrogels can be used effectively for biomedical diagnostic and therapeutic applications.  Tunable thin film hydrogels can be prepared by varying crosslinking density. Changes in frequency of 12 hour grown hydrogels showing instantaneous T responsive behavior for two cycles. Inset images: Normalized 3-D AFM images showing the temperature sensitive behavior of 10% PEG400DMA crosslinked hydrogel at 25 o C and 45 o C respectively Surface attached hydrogels Response Kinetics of Hydrogels  Saubrey equation: mass α –(Δf); at low T more water content and at high T less water content.  Larger mesh network (PEG600DMA system) system swells more than smaller mesh network (PEG400DMA system).  Instantaneous response of hydrogels to temperature change is observed.  Fast response is highly useful for point of care biomedical diagnostics. Reference [1] Biswal D., Chirra H. D., Hilt J. Z., Biomedical Microdevices, 10 (2008), [2] Chirra H. D., Biswal D., Hilt J. Z., Polymers for Advanced Technologies, in press.