Biotin-Doped Porous Polypyrrole Films for Electrically Controlled

ARTICLE pubs.acs.org/Langmuir

Biotin-Doped Porous Polypyrrole Films for Electrically Controlled Nanoparticle Release Youngnam Cho*,† and Richard Ben Borgens†,‡ †

Center for Paralysis Research, Department of Basic Medical Sciences, School of Veterinary Medicine, and ‡Weldon School of Biomedical Engineering, Purdue University, West Lafayette, Indiana 47907, United States

bS Supporting Information ABSTRACT: A novel method for the preparation of biotindoped porous conductive surfaces has been suggested for a variety of applications, especially for an electrically controlled release system. Well-ordered and three-dimensional porous conductive structures have been obtained by the electrochemical deposition of the aqueous biotin
pyrrole monomer mixture into particle arrays, followed by subsequent removal of the colloidal particles. Advantageously, direct incorporation of biotin molecules enhances the versatility by modifying surfaces through site-directed conjugate formation, thus facilitating further reactions. In addition, the porosity of the surfaces provides a significant impact on enhanced immobilization and efficient release of streptavidin-tagged gold nanoparticles. Biotinylated porous polypyrrole (Ppy) films were characterized by several techniques: (1) scanning electron microscopy (SEM) to evaluate surface topography, (2) X-ray photoelectron spectroscopy (XPS) to assess the potential-dependent chemical composition of the films, (3) four-point probe evaluation to measure the conductivity, cyclic voltammetry to observe surface eletroactivity, and contact angle measurement to evaluate the surface wettability, and (4) fluorescence microscopy to image and quantify the adsorption and release of gold nanoparticles. Overall, our results demonstrate that these biotinylated porous Ppy films, combined with electrical stimulation, permit a programmable release of gold nanoparticles by altering the chemical strength of the Ppy
biotin interaction.

’ INTRODUCTION Significant effort has been devoted to developing and constructing a novel methodology using conductive polymer structures in a diverse range of applications, in particular prosthetic devices and tissue engineering scaffolds in the human body.1
7 Because the optimization of the electrode
tissue interface is of great importance in deciding the performance of the implants, a number of approaches have been devoted to inhibiting unpredictable device failure by reducing foreign material response induced by undesirable scar tissue formation.8
11 Recent advances in the performance of these devices have been achieved by combining drug delivery systems, where conducting polymers, such as polypyrrole (Ppy), can be electrochemically deposited on electrodes. These entrap a variety of anions and cations, such as growth factors, anti-inflammatory drugs, adenosine-50 -triphosphate (ATP), glutamate, and protonated dopamine.12
19 Release of biomolecules from these devices is readily initiated by the applied electrical fields. The electrical stimulation induces conformational changes in the polymer structure via repetitive contraction and expansion cycles. In other words, because the diffusion of dopants is voluntarily adjusted to the imposed electrical fields, the unwanted release of the dopant atoms is likely to be avoided. In addition, site-specific drug delivery in the r 2011 American Chemical Society

vicinity of an implant can enhance the performance of drugs while lowering the exposure of the drug to the whole body and, thus, preventing potential toxicity and/or side effects. Conductive polymers as constructive platforms have mostly focused on the development of electrically controlled release of small molecules, such as biomolecules or drugs.2,5,20,21 Indeed, potential functionality of conductive polymers has been clearly demonstrated by several recent in vivo and in vitro studies. The biocompatible nature of these electroactive polymers makes them particularly well-suited to control cellular behaviors in response to external stimuli in various cell types, including osteoblasts, myocytes, bone marrow cells, neurons, endothelial cells, fibroblasts, etc.22
28 Here, we would expand this technology by combining nanoparticles (NPs) within Ppy films to form unique recognition-based tools (Figure 1). Because Ppy serves as the binding site for negatively charged biotin moieties, densely packed biotin molecules readily adsorb streptavidin-tagged gold (SA
Au) NPs. In addition, the introduction of porosity in conductive polymers can significantly augment the surface area Received: January 13, 2011 Revised: March 4, 2011 Published: April 18, 2011 6316

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Figure 1. Schematic illustration shows the electrical voltage applied NP release system. Porous Ppy containing biotin as a dopant anion facilitates the uptake/release of NPs associated with a biotin
streptavidin linkage in the presence of the negatively charged electrical potentials.

that is particularly relevant to the amount of incorporated and released molecular cargos. It is apparent that the porous nature of these films allows for efficient transport of large molecules when compared to flat surfaces, thereby imparting additional potential advantages.29,30 Our observations clearly provide proof of concept for the controlled release of NPs from conductive porous Ppy in an electrical potential-dependent way. Given that gold nanospheres play an important role in the development of more efficient technology, including imaging, cancer therapy, and biomedical diagnostics, biotin-loaded porous Ppy could be a tunable platform useful in therapeutic delivery of drug-laden NPs, which are readily conjugated to these surfaces.31,32 With this approach, it is possible to overcome some significant limitations in current methodologies, which include low levels of encapsulation and restricted release of drugs associated with the limited surface area of their carrier.

’ EXPERIMENTAL SECTION Construction of Polystyrene (PS) Colloids. The aqueous suspension of PS spheres with a diameter of 1 μm (Spherotech, Inc., Lake Forest, IL) was adjusted to 5% (w/v) by adding an equal volume of ethanol solution before use. The three-dimensional PS particle arrays were obtained on a clean indium tin oxide (ITO) surface (Delta Technologies) by dipping ITO surfaces in the PS solution. Such process facilitates the self-assembly of PS particles by confining them in layered arrays. The colloidal crystals with a uniform deposition of PS NPs were obtained at room temperature over ∼5 days by slow ethanol evaporation. Finally, the template was air-dried for at least 1 day and kept in a vacuum desiccator until use. Electrochemical Deposition of Biotinylated Porous Ppy on Colloidal Crystal Construction. Ppy was electrochemically prepared on prepared colloidal crystals using a 604 model potentiostat (CH Instruments). A template, platinum gauze, and saturated calomel

electrode were employed as working, counter, and reference electrodes, respectively. For the preparation of biotin-loaded Ppy, the electrochemical deposition was conducted on top of colloidal crystals in an aqueous solution of a mixture of 0.1 M pyrrole (Py) monomer, 9 mM biotin (Molecular Probes), and 0.01 M sodium dodecylbenzenesulfonate (NaDBS, Aldrich) by applying a suitable cathodic potential of 0.7 V according to George et al.33 These films were immediately rinsed with deionized water and dried under nitrogen to avoid any further deposition. For the porous Ppy films, the biotinylated Ppy template was incubated in the mixture of 5% water with 95% tetrahydrofuran (THF) for 24 h, where the PS core was dissolved leaving hollow composites. The resulting porous film was air-dried and stored in a vacuum desiccator until use. The biotin-doped porous Ppy surfaces were further interacted with streptavidin-coated gold (Au) NPs (Nanocs) with a diameter of 1.4 or 5 nm at a concentration of 0.1 mg/mL. For the fluorescent measurement, the resulting surfaces were incubated in the solution of fluorescent-tagged biotin for 2 h. Characterization of Biotinylated Porous Ppy Surfaces. The surface morphology of porous Ppy films were performed on a FEI NOVA nanoSEM (FEI Company) using a 5 kV acceleration voltage. Prior to these measurements, all samples were sputtered with gold
palladium. Survey and high-resolution X-ray photoelectron spectroscopy (XPS) scans were collected with a Kratos Axis ULTRA X-ray photoelectron spectrometer. The instrument was equipped with a monochromatic Al KR X-ray source, small area extraction optics, spherical capacitor electron energy analyzer, and dual-channel plate position-sensitive detector. Survey spectra were taken from 0 to 1100 eV with pass energy of 160 eV. High-resolution scans were collected with pass energy of 40 eV. Data analysis was performed using a commercial software package, Casa 2310, through fitting by Gaussian and Lorenzian functions. The high-resolution data for C 1s, N 1s, S 2p, Au 4f, and O 1s were deconvoluted on the basis of the peak area analysis, and then percent assignments were made for the components under each element. The measurement of surface conductivity was achieved using the four-probe technique (model 6000 of Micromanipulator) at room 6317

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Figure 2. (a and b) Typical SEM of templates composed of PS beads with a mean diameter of 1 μm. (c and d) Porous Ppy films doped with biotin were prepared by applying a constant potential of 0.7 V and subsequently immersing the films in aqueous THF solution. temperature. Prior to taking measurements, Ppy films were isolated from ITO surfaces using double-sided tape. When a constant current was applied across the surface, the voltage was measured and further correlated to determine resistance. The sheet conductivity of films was correlated and deduced through the Van der Pauw equation. Measurements were performed at five different locations per Ppy surface. The thickness of the biotin-doped porous Ppy film and porous Ppy film immobilized with gold NPs was assessed using an Alpha Step 500 stylus profilometer, where several measurements were averaged in different directions from the center of the film. The biotinylated porous Ppy film had a thickness of 2.7
4.4 μm, whereas the porous Ppy film immobilized with gold NPs had a thickness of 3.2
5.1 μm. Both the diameter of PS beads and the number of deposition layers might be the key factor deciding the film thickness. Cyclic voltammetry (CV) experiments were carried out using a 604 model potentiostat (CH Instruments) at room temperature. In a three-electrode cell, platinum gauze, saturated silver/ silver chloride, and functionalized Ppy films were used as counter, reference, and working electrodes, respectively, to observe the electrochemical properties of the surface. Cyclic voltammograms were recorded within the potential range from
1000 to 1000 mV in 0.1 M phosphate-buffered saline (PBS) containing 5.0 mM Fe(CN)64
/3
at a scan rate of 50 mV/s. The effects of dopants and the porosity of Ppy on the wettability were studied in the static contact angles using a contact angle analyzer (Ahtech LTS Co.). All samples were imaged at 20 magnification using fluorescent microscopy. The concentration of fluorescent-functionalized biotin on a porous Ppy surface was determined by measuring the intensity of Fourier transform ion cyclotron (FITC) with a fluorescence microscope using excitation/barrier wavelengths of 490/520 nm. The fluorescent intensity of each sample was statistically quantified using “Image J” software by capturing and averaging the FITC labeling.

Gold NPs Release Studies from Biotinylated Porous Ppy. To observe the release behavior of Au NPs from porous Ppy composite, biotindoped porous Ppy samples were placed in borosilicate coverglass chambers consisting of three electrodes, a reference electrode (Ag/AgCl), a counter electrode (Pt), and a working electrode (Ppy film), and stimulated from
2 to þ2 V for different time intervals. All experiments were performed in triplicate.

ARTICLE

’ RESULTS AND DISCUSSION The well-ordered three-dimensional structures assembled from 1.0 μm PS beads were documented by scanning electron microscopy (SEM) images (panels a and b of Figure 2). The organization of colloid crystals into close-packed arrangements was achieved using a convective self-assembly technique driven by capillary forces. As a well-established phenomenon, the evaporation pressure in the dispersion solution was a dominant factor determining the surface quality of a colloidal template.34,35 Notably, rapid evaporation of ethanol containing PS particles leads to non-uniform colloidal dispersion, as well as low surface coverage. On the other hand, slow ethanol evaporation tends to favor more ordered organization with regular and periodic particle arrays as a result of their spontaneous assembly process of the spheres into large ordered domains. Subsequently, conductive Ppy films were potentiostatically electrodeposited on the three-dimensional PS arrays. In recent studies of ours, the optimization in film-forming conditions has been discussed.36 Indeed, the film-growth rate, efficiency, and structural stability are closely correlated to the extent of infiltration into a colloidal crystal template (e.g., the interstitial channels) of such monomers to achieve uniform conductive films. When this is taken into account, it seems obvious that electrochemical deposition is an effective method for controlling film growth, although such process often causes inhomogeneous filling of the precursor polymer that results in the defects or problems of the conductive films. The Ppy films were electrochemically grown by copolymerization of pyrrole and biotin at a constant potential of þ0.7 V versus Ag/AgCl reference and subsequently treated with THF to remove the PS template. Panels c and d of Figure 2 show well-defined porous morphology with highly ordered and interconnected spherical voids, closely resembling the original particle geometry. Biotin-loaded porous Ppy surfaces were further incubated with SA
Au NPs with a diameter of 1.4 or 5 nm. No discernible differences were observed in the morphology because of the small size of Au NPs (data not shown). Meanwhile, because the porosity of the surface has attributed to significant changes in the morphology, electrical conductivity, and surface reactivity, it is thus necessary to explore the surface properties of resulting surfaces. Table 1 shows more detail with regard to electrical conductance and water contact angle. Initially, we assessed the electrical conductivity of Ppy surfaces. The electrical conductivity of flat biotin-doped Ppy films was 7.62 S/cm, whereas porous biotin-loaded Ppy films corresponded to 3.02 S/cm. Although the biotin-modified Ppy still retained its electrical properties, a porous structure with a large effective surface area does apparently result in the decrease in charge transport, thereby lowering the conductivity compared to those in bulk films. However, the electrical conductivity after the immobilization of SA
Au NPs was significantly enhanced to 47.62 S/cm because the inclusion of biotin leads to a proportional increase in the concentration of the Au NPs in the film through strong biotin
streptavidin interactions. Interestingly, the conductivity of the surfaces was again decreased when exposed to the electrical field at a constraint potential of
1.0 V for 20 s. This is direct evidence of the charge transfer coupled to the reduction
oxidation behavior of Ppy species. As a “proof of concept”, these results imply unique features of biotin-doped porous design that can induce the efficient transport of streptavidin-tagged Au NPs coordinated through electrically responsive processes. This trend is consistent with the change in surface 6318

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wettability resulting from the contact angle measurements. The water contact angle was examined to define the hydrophilicity after each modification. Biotin-doped porous Ppy film showed remarkable hydrophobicity of 88°, which is relevant to the hydrophobic nature of biotin, indicating that biotin is successfully incorporated as dopant into PPy. Subsequently, the conjugation with streptavidintagged Au NPs resulted in a significant decrease around 27° because of the exposure of hydrophilic moieties from streptavidin.37 On the other hand, electrical stimulation is likely to bring back the hydrophobicity of porous Ppy films to 54°. This is because such porous Ppy surfaces release biotin-linked NP conjugates from the surface. We have conducted a number of studies to establish the beneficial effect of such electrically responsive porous structures on the controlled release of NPs. Initially, we examined the chemical composition of functionalized porous Ppy surfaces before and after electrical stimulation using XPS. High-resolution spectra were recorded for the main core-level peaks of S 2p and Au 4f because the chemical structure of these species would provide critical insights into this issue. As expected, conjugation with 1.4 nm SA
Au NPs exhibited an obvious Au 4f signal, Table 1. Comparison of Conductance and Water Contact Angle of a Porous Ppy Film Doped with Biotin (Ppy
Biotin), Porous Ppy
Biotin Film Immobilized with SA
Au NPs (Ppy
Biotin/SA
Au NPs) without Electrical Stimulation, and Ppy
Biotin/SA
Au NPs with Electrical Stimulation at
1.0 V for 20 s conductance (S/cm) contact angle (deg) 3.02 ( 1.52

88.0 ( 5.0

porous Ppy
biotin/SA
Au NPs

47.62 ( 3.43

27.3 ( 3.6

porous Ppy
biotin/SA
Au NPs

11.36 ( 1.48

54.7 ( 6.8

porous Ppy
biotin

with electrical stimulation

indicating the presence of Au NPs in the porous Ppy structure (Figure 3a). The high affinity between biotin and streptavidin proteins is likely to yield the immobilization of SA
Au NPs to the biotin-doped porous Ppy. In agreement with the Au 4f, the sulfur S 2p signal in Figure 3b shows two strong components at approximately 161.0 and 166.0 eV, respectively, and two weak peaks at 162.5 and 167 eV prior to electrical stimulation. The oxidized sulfur species at 166 eV is most likely to correlate with the use of sodium dodecylbenzenesulfonate (NaDBS) in the preparation of Ppy film. Moreover, the peak at 161.0 eV was associated with the presence of biotin
strepatavidin coordination via their sulfur atoms. Taken together, these observations clearly suggested the presence of SA
Au NPs in the biotinylated porous Ppy. To investigate the influence of the electrical stimulation, experiments were carried out by applying the constant potential of
1.0 V for 60 s. In contrast to the previous results, no XPS signals in Au 4f were observed directly, whereas weak S 2p peaks were observed as shown in panels c and d of Figure 3. Additionally, we considered the effect of porosity on the immobilization of NPs by comparing to flat biotin-doped Ppy and porous biotin-doped Ppy (see Supplemental Figure 1 in the Supporting Information). According to the analysis of XPS, a marked difference was observed in the absorption behavior of SA
Au NPs. In the Au 4f peak region, slight peaks were detected in flat Ppy, while an intense gold signal were observed in porous Ppy. Indeed, porous surfaces provide beneficial binding sites or “channels” through which NPs may adsorb and diffuse very easily. We speculatively attribute these results to high surface area and pore volume of porous Ppy that would lead to the enhanced adsorption capacity for SA
Au NPs. In the initial experiment, we focused on the induced effect of electrical stimulation on the release behavior. We expanded on this experiment to analyze the voltage-dependent release behavior of SA
Au NPs through the studies of the S 2p, C 1s, and N 1s peaks from XPS. As a

Figure 3. Records of high-resolution XPS for surface deconvolution analysis of (a and c) Au 4f and (b and d) S 2p spectra of porous Ppy
biotin/ SA
Au NPs (a and b) without electrical stimulation and (c and d) with electrical stimulation at
1.0 V for 60 s. 6319

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Figure 4. Records of high-resolution XPS for surface deconvolution analysis of (a) S 2p, (b) C 1s, and (c) N 1s spectra of porous Ppy
biotin/SA
Au NPs with varying applied voltages.

consequence of these changes, more evident effects were observed in all spectra. Figure 4a shows the S 2p region of the XPS spectra as a function of varying voltage. Along with the steady increase toward positive potentials, it is clear that peak shape as well as the total intensity in the S 2p region becomes apparently identical to those of controls (unstimulated surface, 0 V). With increasing negative bias, such obvious peaks related to C
S
Au at 161.6
162.0 eV, CdS and C
S
C
at 162.0
164.7 eV, and
SO3H
at 165.0
169.0 eV almost disappeared. In parallel to the sulfur signals, C 1s photoelectron peaks were considerably increased and broadened with positive potentials. These series of peaks could be deconvoluted and attributed to
C
C species at 283.0 eV, C
O species at 284.3 eV, and amide
C species at 286.2 eV, apparently confirming the binding of streptavidin to the biotinylated surface. Moreover, the examination of the high-resolution N 1s core level spectra of non-stimulated biotinylated porous Ppy surfaces (0 V) further confirmed two distinct peaks at 399.0 and 400.7 eV, corresponding to amine-like (N
H) and imine-like nitrogen (dN
), respectively, shown in Figure 4c. Taken together, the response was very unique to certain potentials, clearly supporting that progressive release of metal NPs can be manipulated by alternation of the applied voltage. Indeed, such a potential-dependent mechanism may be able to induce the desorption of molecules and further direct an effective delivery system for drug-laden NPs by tuning the release kinetics. We further explored the electrochemical behavior of biotinylated porous Ppy films before and after the electrical stimulation. CV has been used to monitor the electron-transfer reactions through the chemical reactivity of the electroactive species. Because CV of thin polymer films identifies the presence of electrochemical reactions by the presence of reversible oxidation and reduction waves, redox peak currents and the positions

Figure 5. Cyclic voltammogram of 5 mM Fe(CN)63
/4
at porous Ppy
biotin/SA
Au NPs without electrical stimulation (red) and with electrical stimulation (blue) at a scan rate of 50 mV s
1.

would be varied linearly with the nature of the electrolyte solution. Figure 5 shows the effect of the CV curve on the electrical stimulation conducted in ferricyanide probe solutions as an indicator. I
V hysteresis between the adsorption and desorption branch exhibited an obvious magnitude of the oxidation and reduction current before electrical stimulation. Similar to the observations conducted by Gao et al., the improvement in the charge transport of these porous films was demonstrated by incorporation of metal particles.38 In our study, the existence of SA
Au NPs would be the main contributor for charge-transfer 6320

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not as sensitive relative to those from negatively stimulated Ppy surfaces. Interestingly, such observations are in good agreement with our XPS data as well as several other reports related to the drug release patterns from Ppy. Panels b and c of Figure 6 show representative plots of total fluorescence intensity as a function of both the correlation potential and the decay time by quantitative analyses of the fluorescence images. When the surfaces were subjected to negative electric potentials, a much higher release of SA
Au NPs occurred. The release pattern and the variation in the electrical potential seem to be fairly similar to the results from the study by Luo et al.39 This was likely the production of excess hydrogen bubbles originating from the electrode as a result of the positive potential pulse, and this could result in the failure of the release system.39,40 These studies provide the promising possibility that fine control of NPs release from porous Ppy films when electrical stimulation applied is linked with the porous morphology of the films as well as chemical affinities.

Figure 6. (a) Fluorescence micrographs showing electrically responsive release of gold NPs (5 nm in diameter) as a function of applied potentials and times. Ppy
biotin/SA
Au NPs were further incubated with florescence-labeled biotin. (b) Fluorescent intensity at different applied potentials for Ppy
biotin/SA
Au NPs. The y axis denotes mean measured fluorescence intensity. (c) Fluorescent intensity for Ppy
biotin/SA
Au NPs as a function of time and applied potentials. The y axis shows the normalized fluorescence intensity based on the value at 0 s.

enhancement. The CV curve after electrical stimulation showed a dramatic decrease in the magnitude of the redox current. It seems likely that the desorption of Au NPs from the surfaces would be responsible for lowering the electrical activity between the ferricyanide species in solution and the porous Ppycoated electrode, consequently inhibiting efficient electron transfer. Subsequently, in addition to the evidence supporting the electrically triggered response, we observed the effect of SA
Au NP release in response to various applied potentials and at various times by fluorescence microscopy. After deposition of SA
Au NPs on porous Ppy, FITC-labeled biotin was incubated for 2 h. The immobilization of FITC-labeled biotin via a streptavidin bridge was visually detected by the fluorescent intensity. Figure 6a shows the fluorescence images of FITC/ biotin-captured porous Ppy before and after electrical stimulation. Representative fluorescence images of controls or unstimulated surface (e.g., 0 V) revealed spatially homogeneous dispersion of FITC/biotin throughout. On the other hand, the areas where negative potentials were applied resulted in a marked decrease in fluorescence at 60 s, apparently supporting the interpretation of a diffusion of fluorescently tagged SA
Au NPs. On the contrary, the level of the fluorescence trace slightly decreased by applying þ1 V, whereas the areas upon the exposure of þ2 V remained remarkably constant. That is, the reduction in fluorescence was observed but

’ CONCLUSIONS We developed an electrically programmable NP release system from porous conductive films by taking advantage of the high affinity of the biotin
streptavidin interaction. The addition of biotin during electrochemical deposition of pyrrole yields broad flexibility because it opens the possibilities for the facile incorporation of new derivatives. Notably, these engineered surfaces offer the substantial likelyhood for improved adsorption/desorption of NPs by controlling the porosity of the films. With this approach, we offer a new strategy for electrical responsive NP release mechanisms. This novel system prompted research focus toward the development of implantable drug carriers for hyperthermal therapeutic applications. ’ ASSOCIATED CONTENT

bS

Supporting Information. High-resolution XPS Au 4f spectra of (a) flat Ppy
biotin/SA
Au NPs and (b) porous Ppy
biotin/SA
Au NPs (Supplemental Figure 1). This material is available free of charge via the Internet at http://pubs.acs. org.

’ AUTHOR INFORMATION Corresponding Author

*Telephone: 765-494-7600. [email protected].

Fax:

765-494-7605.

E-mail:

’ ACKNOWLEDGMENT We appreciate the excellent illustrations and graphics by Michel Schweinsberg and the assistance with manuscript preparation by Jennifer Danaher. We also acknowledge financial support from the General Funds of the Center for Paralysis Research, the state of Indiana, and a generous endowment from Mari Hulman George. ’ REFERENCES (1) Abidian, M.; Kim, D.; Martin, D. Adv. Mater. 2006, 18, 405. (2) Cui, X.; Wiler, J.; Dzaman, M.; Altschuler, R.; Martin, D. Biomaterials 2003, 24, 777. (3) Kim, D.; Martin, D. Biomaterials 2006, 27, 3031. (4) Thompson, B.; Moulton, S.; Ding, J.; Richardson, R.; Cameron, A.; O’Leary, S.; Wallace, G.; Clark, G. J. Controlled Release 2006, 116, 285. 6321

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