Electroconductive Gels for Controlled Electrorelease of Bioactive

Chapter 15

Electroconductive Gels for Controlled Electrorelease of Bioactive Peptides

Downloaded by KTH ROYAL INST OF TECHNOLOGY on August 29, 2015 | http://pubs.acs.org Publication Date: September 24, 1998 | doi: 10.1021/bk-1998-0709.ch015

Anthony Guiseppi-Elie, Ann M . Wilson, and Andrew S. Sujdak Research and Development Department, ABTECH Scientific, Inc., P.O. Box 376, Yardley, PA 19067-8376

We have synthesized highly hydrophilic, chemically responsive materials we call electroconductive gels. They are formed as an interpenetrating network of an electroconductive polymer and a hydrophilic hydrogel. Polymers were formulated form UV polymerizable hydrophilic monomer such as hydroxyethyl methacrylate (HEMA), N-[tris(hydroxymethyl)methyl] acrylamide (HMMA), and tetraethylene glycol diacrylate (TEGDA), and oxidatively polymerizable electroactive monomer such as aniline and dianiline. The hydrogel network was formed by UV-photoinitiated polymerization and the electroconductive polymer subsequently formed by chemical oxidative coupling induced by immersion in aqueous FeCl3 solutions, by electropolymerization, or by a combination of these methods. Interaction between both networks was accorded by the difunctional 3-sulfopropylmethacrylate (SPM). These materials retain more electroactivity and display fast cation transport with K diffusivity (D =5.31xl0- cm S ) that are an order of magnitude larger than that of electropolymerized polyaniline (D =3.12xl0- cm S ). The electroconductive polyaniline gels are shown to be more stable under ambient conditions and to efficiently imbibe and release Ca under electrostimulation. +

7

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-1

o

8

2

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++

Hydrogels have been established as controlled release materials suitable for the passive delivery of bioactive peptides (1). Electroconductive hydrogels are now being developed as controlled release materials for the programmable delivery of bioactive peptides to cells and tissues grown in culture. Our goal is to address the challenge of maintaining control over cell metabolism, differentiation, and proliferation by providing for the precisely controlled (quantity, timing, and duration) delivery of bioactive agents and peptides.

©1998 American Chemical Society

In Tailored Polymeric Materials for Controlled Delivery Systems; McCulloch, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

185


186 These materials have been integrated into single well and 24-well controlled electrorelease cell culture vessels (2). These devices are suitable for the study of the potentiation of insulin secretion by HIT-T15 cells under the influence of electroreleased bradykinin (3). Electroconductive polymers have emerged as field-responsive materials suitable for controlled electrorelease (4), as transducer-active elements in gas, chemical and biological sensors (5), as responsive films in solid state devices, and as mechanical actuators (6). Indeed, electroconductive membranes have been shown to release bioactive molecules such as adenosines-triphosphate (ATP) (7) and the 11 amino acid peptide, insulin, as well as organic molecules such as 2,6-anthraquinone disulfonate (8). Electrorelease applications are based on chemically or electrochemically induced changes in oxidation state of the polymer, concomitant changes in the charge density of the material, and consequent diffusive egress of charge-neutralizing "dopants" previously held in association with the electroactive polymer. Both anionic and cationic molecules may be electroreleased (9). The preparation of water-containing electroconductive polymers has been generally achieved by the electropolymerization of the electroactive monomer form a solution that contained suitable anionic polyelectrolytes (10,11). Other types of hydrogels have been found useful in the controlled release of bioactive molecules (12). By formulating and/or synthesizing an electroconductive hydrogel, we believe that such a composite material will facilitate diffusion, provide a biocompatible environment for the retained bioactivity of amino acid, peptide, or protein agents, accelerate redox switching times, and offer increased loading capacity for the bioactive molecules of interest. The electroconductive polymers of this study are interpenetrating networks of inherently conductive polymers, such as polyaniline or polypyrrole, formed within water-swellable, electrode-supported or freestanding, HEMAbased hydrogels. We conceptualize these smart materials as precursors to artificial endocrine organs, capable of delivering function-regulating peptides to cells and tissues grown in culture and hypothesize that precise quantities of bradykinin will be electroreleased into cell culture media after programmed electric pulses are delivered to the electroconductive polymer film. Experimental Materials. The hydrogel membranes were formulated from hydrophilic, U V polymerizable monomer and electroactive aniline monomer. The hydrophilic and UV-polymerizable components consisted of ophthalmic grade 2hydroxyethyl methacrylate (HEMA) (Polysciences), N[tris(hydroxymethyl)methyl] acrylamide (HMMA) (Aldrich), poly(ethylene glycol)(200) monomethacrylate (PEG200MMA) (Polysciences), potassium salt of 3-sulfopropylmethacrylate (SPM) (Sigma), and poly-(2-hydroxyethyl methacrylate) (pHEMA) (MW = 300,000) (Polysciences). The formulation was also made to contain tetraethylene glycol diacrylate (TEGDA) which served as In Tailored Polymeric Materials for Controlled Delivery Systems; McCulloch, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


187 the UV-polymerizable cross-linker and 2,2-dimethoxy-2-phenylacetophenone (DMPA) which served as the photoinitiator. Aniline (An), dianiline (DAn), 3aminopropyltrimethoxysilane (y-APS), 3-methacryloxypropyltrimethoxysilane (y-MPS), and dodecyltrichlorosilane were obtained form Aldrich (Milwaukee, WI). Prior to formulation, H E M A , H M M A , and TEGDA were each passed over inhibitor remover columns (Aldrich) to remove the polymerization inhibitor, hydroquinone monomethyl ether (MEHQ), purged with dry argon, and stored in dark bottles at 4°C. All other reagents were used as received. Table I shows the formulae for a typical electroconductive gel dope. To each 5 g batch of the foregoing formulation was added 20% by weight of DI water (1 g) and ethylene glycol (1 g) as mixed solvent. Table I. Formulation of an electroconductive gel dope based on polyaniline. Compounds in Formulation 2-Hydroxyethyl methacrylate (HEMA) N-[Tris(hydroxymethyl)methyl] acrylamide (HMMA) Poly(ethyleneglycol)(200)monomethacrylate (PEG200MMA) 3-sulfopropylmethacrylate (SPM) Tetraethylene glycol diacrylate (TEGDA) (cross-linker) Poly-(2-hydroxyethyl methacrylate) (pHEMA) 300,000 2,2-Dimethoxy-2-phenylacetophenone(DMPA) (photoinitiator) Aniline (An) Dianiline (DAn) Total of Reagents Water (solvent) Ethylene glycol (solvent)

Mole % 57.85 10 5

g% 50.44 11.74 8.78

5 3 2 2

8.25 6.077 1.74 3.43

15 0.15 100

9.36 0.19 100 20 20

Electrodes. Electrode-supported hydrogel membranes (10 |xm - 30 \im thick) were cast onto 1.00 cm x 1.75 cm x 0.05 cm planar metal gold electrodes (PME Au-118, ABTECH Scientific, Yardley, PA), microlithographically fabricated Interdigitated Microsensor Electrode (IME) arrays (IME 1050-M-Au-P, ABTECH) or onto 7 mm x 50 mm x 0.5 mm ITO-coated (s. Ag°/AgCl, 3 M CI") form deaerated 1.0 M An, 0.01 M DAn, and 2.0 M HC1 held at 20 °C. Oxidatively polymerized polyaniline hydrogels were prepared by exposure (1 hr) of the aniline-containing hydrogel to 0.10 M FeCb at 20 °C. The interdigit spaces of the IME devices were chemically modified with y-APS or y-MPS to improve adhesion prior to hydrogel casting. The film was allowed to grow on each electrode and also between the digits of the pair of electrodes such that it formed a fully contiguous membrane. Results and Discussion Electroconductive Gel Synthesis. The synthesis of electroconductive hydrogels proceeds form two separate polymerization reactions. The first reaction is the UV-initiated polymerization of the hydrophilic polymer network formed from acrylate and methacrylate monomers in the formulation. The second reaction is the oxidative polymerization of In Tailored Polymeric Materials for Controlled Delivery Systems; McCulloch, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.


189 electroactive monomers that are trapped within the pre-polymerized hydrophilic network. Figure 1 shows the structure of the various monomers used in this work. The first reaction produces a clear, optically transparent membrane of controlled thickness supported on the chosen conductive or insulating substrate. Membranes containing aniline and dianiline monomer of molar concentrations used in this study were visibly indistinguishable from blank membranes. That is, they were visibly colorless and optically transparent. The formation of the electroconductive network by oxidative polymerization was done by three separate schemes. The first scheme (I) was via chemically based, oxidative polymerization of aniline that was formulated and trapped within the pre-polymerized hydrophilic hydrogel membrane. Contact of the 20 - 35 \im thick and 0.142cm area gel membrane with 25 |xl of aq. 0.1 M FeCb at a p H of 1.4 produced a clear, emerald green colored solution and a more intense emerald green membrane. The color developed on the time-scale of minutes, reaching an unchanging intensity after about 10 min. Other oxidizing agents used included acidified hydrogen peroxide (pH 2, ca. 30 % H2O2), ammonium peroxydisulphate, and dilute acidified potassium permanganate. In all cases there was visible evidence for diffusion of some aniline and/or dianiline out of the gel membrane into the aqueous bathing solution, as both hydrogel swelling and aniline oxidative coupling occurred. This synthesis scheme produces a somewhat un-controlled reaction as several competing forces are simultaneously at work. Contact of the aqueous FeCk solution with the membrane causes its immediate swelling with concomitant influx of water, transport of ions into and out of the gel, and transport of unreacted monomer and glycerol out of the gel. Ferric ions react with aniline and dianiline monomers to produce cation-radicals that couple within the gel matrix and also diffuse out of the gel. The latter possibly accounting for the development of the pale green color in the bathing solution. For aniline oligomers of increasing molecular weight, diffusion out of the gel becomes restricted and future molecular weight increases occurs within the gel, favoring larger molecular weight chains of polyaniline within the gel. The result is polyaniline (likely the emeraldine salt) formed within the matrix of the hydrophilic hydrogel network. The second scheme (II) was via electropolymerization of aniline within the electrode-supported, pre-polymerized hydrogel membrane. In this scheme, the electrode-supported membrane was placed in an electrochemical cell containing deaerated 1.0 M An, 0.01 M DAn, and 2.0 M HC1 held at 20 °C. The aniline was then potentiostaticly (0.70 V vs. Ag°/AgCl, 3 M CI") electropolymerized until the passage of a total of 200 mC cm- of charge. Of course, such a reaction may be performed on a blank electrode leading to the familiar potentiostaticly electropolymerized polyaniline, on a blank hydrogel membrane i.e. one containing no aniline or dianiline monomer within the gel, on a membrane containing electroactive monomer within the gel, or on a membrane containing electroactive monomer that had been exposed to FeCb 2

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H,C-) as a function of scan rate reveals the diffusivity for K that is 5.81 x 10 cm sin polyaniline hydrogels (III) compared to 3.12 x 10 cm s- in electropolymerized polyaniline. 1

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Figure 3. Cyclic voltammograms of electropolymerized PAn and PAn hydrogels in deaerated 0.1M KC1 at 20 °C. a) First scan obtained at 5 mV s- . b) Second scan obtained at 10 mV s . 1

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Material Under Test

Figure 4. Anodic charge density of electropolymerized PAn and PAn hydrogels calculated from the 1 C V scan obtained at 5 mV s- in deaerated 0.1M KC1 at 20 °C over the range 0.00 V to 0.90 V vs. g/AgCl, 3M c r . st


1


195 Electrochemical Impedance Spectroscopic Characterization. Electrochemical impedance spectroscopy (EIS) was performed on polymer coated IMEs immersed in deaerated 0.10 M KC1 at 20°C EIS interrogation was done over the range 0.10 mHz to 100 kHz using a sine wave voltage pattern of 100 mV peak voltage. The two-electrodes of the interdigitated array served as working and auxiliary/counter electrodes respectively while the reference electrode (Ag/AgCl) was placed in the electrolyte and in close proximity to the hydrogel surface. In this configuration the impedance measured was the inplane impedance of the solvent-cast hydrogel membrane. On un-treated IME devices, immersion in 0.10 M KC1 solution lead to swelling of the hydrogel and often also lead to hydrogel disbondment from the IME device. This we believed to be the result of interfacial shear stress incident to swelling of the gel. Surface modification of the IME device with 3-aminopropyltrimethoxy silane followed by derivatization of the primary amine with methoxyPEG(5000)-epoxide eliminated all evidence of adhesion loss. Hydrogel-coated IME devices, having achieved equilibrium, could be immersed in electrolyte for extended periods. Figure 6 shows a Bode plot of the impedance (magnitude and phase) of pristine electropolymerized PAn and various PAn hydrogel coated IMEs. The polyaniline-free hydrogel was found to give an impedance spectrum similar to that of aqueous 0.1 M KC1 (15). This reflects the "solution like" character of the hydrogel membrane and indicates an ion concentration and ion mobility within the gel that is on the order of the bathing solution (16). There are subtle differences at the extremes of frequency that clearly distinguishes these media but these shall be overlooked for the purpose of this discussion. The influence of the polyaniline formed within the gel by the action of exposure to 0.1 M FeCb is to appreciably reduce the low frequency impedance of the gel but also to increase the high frequency (> 1 Hz) impedance magnitude and produce a significant change in the phase behavior. Consistent with the electrochemical data, the development of color, and the change in gel hydration, this clearly supports the formation of polyaniline within the gel matrix. Electropolymerization within the gel results in a significant further reduction in impedance magnitude and produces frequency independence of both magnitude and phase. The combined action of treatment with FeCU and electropolymerization is to produce still further reduction in the impedance magnitude and frequency independence. The impedance behavior of such a gel (III) is similar to that of pristine polyaniline that is formed by direct electropolymerization. Neither FeCb nor electropolymerization, performed separately on the hydrogel, produces this polymer impedance character. Hydrogel Hydration. The water content of electroconductive hydrogels was studied gravimetrically by casting films of the gel dope into pre-weighted glass weighing boats. The UV-polymerized gel membranes were subsequently reacted for 30 min with 0.1 M FeCb prepared in 10% ethanol-water. The unreacted monomer and the ethylene glycol co-solvent were subsequently In Tailored Polymeric Materials for Controlled Delivery Systems; McCulloch, I., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

196

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Figure 5. Cathodic charge density of electropolymerized PAn and PAn hydrogels calculated form the 2 CV scan as a function of scan rate in deaerated 0.1M KCl at 20 °C over the range -0.5 to 0.9 V vs. Ag/AgCl, 3M CI". nd

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Figure 6. Bode plot (a) magnitude and b) phase angle) of the in-plane electrochemical impedance of electropolymerized PAn and PAn hydrogels in deaerated 0.1M KCl, 20 °C.



197 removed by sequential washing, each for 30 min, with 100% ethanol, 75%, 50% and 25% ethanol-water mixtures, and finally, with pure DI water. The resulting fully hydrated gel was scrupulously sapped dry with lint free Kimwipes and then weighed to obtain the hydration weight. Samples were then dried to constant weight by the application of repeated 3-hr drying periods in a Turbo Vac 500 dryer (Zymark, Hopkinton, MA) followed by reweighing after each drying period. The Turbo Vac was set with a temperature differential corresponding to 35 °C in the sample chamber and 10 °C in the condenser. Constant weight was often reached within 4 such drying cycles. Repeated hydration and dehydration cycles revealed the polyaniline-free gel (I) and the electroconductive polyaniline gel (II) to contain 57 ±8% and 80 ±2% hydration respectively. The difference between the dry weight of the gel and weight immediately following UV polymerization yields the monomer extractables. The polyaniline-free gel and the electroconductive polyaniline gel were found to contain 28 % and 33 % extractables respectively. The extractables content calculated from the monomer dope formulae and assuming 100% conversion of acrylate monomer to polymer for these two materials was 30.7 % and 28.6% respectively. Electrical Conductivity Characterization. The electrical conductivity of the various solvent-cast gel membranes was determined directly on the polymercoated IMEs and also on freestanding electroconductive gels. The latter measurements were judged unreliable as the probe tip readily penetrated the gel thereby giving rise to spurious results. The four point technique yielded the resistance values for the various materials measured on the IME device: PAn (EP) = 3.2 S cnr , PAn Gel (FeCl ) = 4.6 x 10 S cm-*, PAn Gel (EP) = 8.4 x 10- S cm- , and PAn Gel (FeCb-EP) = 3.9 x 10- S cm- . These DC resistances correlate well with the above reported impedance measurements. 1

5

3

1

1

1

1

Environmental Stability. The stability of the electroconductive gels was studied by monitoring the four-point resistance over time during exposure to ambient laboratory air. The relative humidity and temperature were simultaneously monitored with each conductivity measurement. No attempts were made to normalize or correct the resistance data for variations in either RH or temperature. Relative humidity varied between 55 % and 68 % over the course of the reported measurement and were typically 66% RH. Temperature varied from 20 to 24 degrees and showed an upward trend into the summer months. Figure 7 shows a plot of the resistance expressed as sheet resistance and normalized resistance (Rt-Ro/Ro) over an approximate 100-day period. The electroconductive gel formed exclusively by electropolymerization shows the most dramatic loss of conductivity. The electropolymerized polyaniline shows excellent well-documented stability but with still some evidence of decay in its conductivity over the 100-day period. The electroconductive gel formed by the process of oxidative chemical polymerization followed by



198

0

20

40 60 80 Time (days)

100

Figure 7. Stability of electropolymerized PAn and PAn hydrogels measured over a 100-day period under laboratory conditions.


199


electropolymerization (III) shows almost imperceptible change in electrical conductivity over the 100 day period. UV/Visible Spectrophotometry. UV-Vis spectra were taken on a Beckman DU-7 High-Speed spectrophotometer equipped with a water-jacketed six-cell changer. The gel was cast onto ITO-coated borosilicate glass plates (0.7 cm x 5 cm x 0.05 cm) that fitted directly into the 1 cm path-length cuvette and were set perpendicular to the beam direction. The cuvette was filled with 0.1 M FeCb solution, equilibrated to 20 °C, and the glass plates introduced at time zero. Spectra were obtained in the rapid scan mode at 1200 nm/min (15 s each) with 1 minute intervals between scans. Figure 8 shows UV-Vis spectra over the range 400 nm to 700 nm of the gel membrane during exposure to 0.1 M FeCb. The reaction was followed in this way for a total of 13 min. The development of polyaniline within the gel membrane could be clearly seen from the change in absorbance over the range 400 - 700 nm. This could be compared with a blank gel studied in the same manner and also presented in Figure 8. Electrorelease. Electrorelease using the electroconductive gel formed by the process of oxidative chemical polymerization followed by electropolymerization (III) was performed using the method described by Schlenoff et al. (17). The gel was cast, UV polymerized, and electropolymerized on gold-coated (1,000A) polystyrene scintillator electrodes. The divinylbenzene (2%) cross-linked polystyrene substrate contains dissolved primary and secondary fluorescent dyes (

Electroconductive Gels for Controlled Electrorelease of Bioactive

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