Encapsulation of Enzymes within Polymer Spheres To Create Optical

Encapsulation of Enzymes within Polymer Spheres To Create Optical Nanosensors for Oxidative Stress. Se-Hwa Kim ... Publication Date (Web): October 5, ...
0 downloads 0 Views 739KB Size
Download PDF
Anal. Chem. 2005, 77, 6828-6833

Encapsulation of Enzymes within Polymer Spheres To Create Optical Nanosensors for Oxidative Stress Se-Hwa Kim,† Bumsang Kim,‡ Vamsi K. Yadavalli,§ and Michael V. Pishko*,⊥

Huck Institute for the Life Sciences, The Pennsylvania State University, University Park, Pennsylvania 16802-4400, Department of Chemical Engineering, Hongik University, Seoul, South Korea, and Departments of Chemical Engineering, Chemistry, and Materials Science and Engineering, the Pennsylvania State University, University Park, Pennsylvania 16802

We describe the fabrication and characterization of poly(ethylene glycol) (PEG) hydrogel spheres containing the enzyme horseradish peroxidase (HRP) for application as optical nanosensors for hydrogen peroxide. HRP was encapsulated in PEG hydrogel spheres by reverse emulsion photopolymerization, yielding spheres with a size range from 250 to 350 nm. Encapsulated HRP activity and sensitivity to hydrogen peroxide were investigated by the Amplex Red assay based on the fluorescence response as a function of H2O2. These HRP-loaded spheres were then introduced to murine macrophages with Amplex Red in the culture media. After phagocytosis, the biocompatibility of spheres was determined by live cell staining using calcein AM (5 µM). The HRP-loaded PEG hydrogel spheres were activated (i.e., fluorescent oxidized Amplex Red produced within the spheres) by oxidative stresses such as exogenous H2O2 (100 µM) and lipopolysaccharide (1 µg/mL), which induced the production of endogenous peroxide inside macrophages. The results presented here indicate that after polymerization, the enzyme activity of HRP was still maintained and that using these HRP-containing nanospheres, peroxide production could be sensed locally within cells.

In biological studies, real-time measurement of chemical and physical parameters at the single cell level has been of great interest to researchers.1 The use of fluorescent nanoparticles as sensors was first demonstrated by Sasaki and colleagues.2 Optical nanosphere sensors consist of an inert, biocompatible matrix in which a sensing material, an optical reporter, or both are entrapped. Currently, the transduction method used for an optical nanosensor is fluorescence because of its high sensitivity and * To whom correspondence should be addressed. 204 Fenske Laboratory, Pennsylvania State University, University Park, PA 16802. Phone: 814-863-4810. Fax: 814-865-7846. E-mail: [email protected]. † Huck Institute for the Life Sciences, The Pennsylvania State University. ‡ Department of Chemical Engineering, Hongik University, Seoul, 121-791, Korea. § Department of Chemical Engineering, The Pennsylvania State University. ⊥ Departments of Chemical Engineering, Chemistry, and Materials Science and Engineering. (1) Lu, J.; Rosenzweig, Z. Fresenius’ J. Anal. Chem. 2000, 366, 569-575. (2) Sasaki, K.; Shi, Z.-Y.; Kopelman, R.; H, M. Chem. Lett. 1996, 2, 141-142.

6828 Analytical Chemistry, Vol. 77, No. 21, November 1, 2005

relative ease of measurement. These optical nanosensors offer several advantages over direct cell labeling of fluorescent dyes, which is the current method for quantification of intracellular analysis: (i) The hydrogel matrix protects the intracellular environment from any toxic effects of the sensing material; (ii) the matrix also protects the sensing material from potential interferences in the cellular environment, for example, nonspecific binding of proteins; and3 (iii) using multiple sensing materials, nanosensors may be able to measure several analytes at the same time. Most methods to detect intracellular peroxide reported so far have used fluorophores directly as the sensing reporter.4-10 Recently, Chang and colleagues reported synthetic cell-permeable optical probes for hydrogen peroxide (H2O2) in living cells, peroxyfluor-1(PF1).11 They synthesized PF1 using the transformation of arylboronates to phenols. PF1 was verified in live cells with the H2O2 concentration range 10-100 µM; however, the ability to quantify oxidative stress locally may be limited. One other hand, there have been efforts to incorporate biorecognition molecules,12-14 such as enzymes and antibodies, to expand the scope of analytes that can be detected with this technology. Therefore, encapsulation of enzymes and other biomolecules as sensing agents in these nanosensors may yield sensors that can measure reactive oxygen species (ROS) locally within a cell. The enzyme-encapsulated sensors developed in this study are applicable for oxidative stress investigations which have been defined as a disturbance in the oxidant-antioxidant equilibrium in favor of oxidants.15 Oxidative stress is involved in various human (3) Podual, K.; Doyle, F. J.; Peppas, N. A. Biomaterials 2000, 21, 1439-1450. (4) Clark, H. A.; Hoyer, M.; Philbert, M.; Kopelman, R. Anal. Chem. 1999, 71, 4831-4836. (5) Clark, H. A.; Kopelman, R.; Tjalkens, R.; Philbert, M. Anal. Chem. 1999, 71, 4837-4843. (6) Carter, W. O.; Narayanan, P. K.; Robinson, J. P. J. Leukocyte Biol. 1994, 55, 253-258. (7) Ozaki, Y.; Ohashi, T.; Niwa, Y. Inflammation 1986, 10, 119-130. (8) Rajkovic, I. A.; Williams, R. J. Immunol. Methods 1985, 78, 35-47. (9) Rosen, G. M.; Freeman, B. A. Proc. Natl. Acad. Sci. U.S.A. 1984, 81, 72697273. (10) Ueda, N.; Guidet, B.; Shah, S. V. Kidney Int. 1994, 45, 788-793. (11) Aderem, A. Cell 2002, 110, 5-8. (12) Xu, H.; Aylott, J. W.; Kopelman, R. Analyst 2002, 127, 1471-1477. (13) Ai, H.; Jones, S. A.; Lvov, Y. M. Cell Biochem. Biophys. 2003, 39, 23-43. (14) Lvov, Y.; Caruso, F. Anal. Chem 2001, 73, 4212-4217. (15) Favier, A. E.; Neve, J.; Faure, P. Trace Elements and Free Radicals in Oxidative Diseases; American Oil Chemists Society: Champaign, IL, 1994. 10.1021/ac0507340 CCC: $30.25

© 2005 American Chemical Society Published on Web 10/05/2005

inflammatory diseases, such as cancer,16 vascular diseases,17 and neuronal degeneration,18 through biochemical and immune responses on cells, tissues, and organs. The detection of oxidative stress can be potentially useful diagnostic markers; however, there are few established methods for detection of oxidative stress.19,20 Phagocytosis was used for insertion of nano- or microparticles into cells, even though phagocytosis is a delivery method limited to special phagocytic cells, such as dendritic cells, neutrophils, and macrophages. These phagocytic cells are involved in a number of biological events, including the immune system host defense against pathogens. Additionally, macrophages can internalize particles even larger than themselves by an actin-dependent mechanism. Phagocytosis occurs in several steps. First, phagocytic receptors recognize the particles, then these stimulated receptors trigger intracellular signals for polymerization and rearrangement of actin and coordinate the tractional forces that internalize particles. Finally, macrophages make the phagosome containing particles.21 It is in these phagosomes that the HRP-containing nanoparticles described here reside. Here we describe, as a first step toward the development of optical nanosphere sensors containing biorecognition molecules for intracellular analysis, the enzyme horseradish peroxidase (HRP) encapsulated in poly(ethylene glycol) (PEG) hydrogel nanospheres produced via reverse emulsion photopolymerization. PEG hydrogels are hydrophilic and highly water-swollen and have been used for a wide range of biomedical and pharmaceutical applications because of their general biocompatibility.22-25 To determine the potential use of HRP-loaded PEG hydrogel spheres as optical micro- and nanosensors, the size and morphology of the polymer spheres as well as the HRP-catalyzed reaction between H2O2 and 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red) was characterized, in addition to enzyme leaching from the spheres. We also investigated the in vitro applications of HRPloaded PEG hydrogel spheres for the measurement of intracellular peroxide. Biocompatibility of HRP-loaded spheres with macrophages was studied by live-cell staining before and after internalization. Furthermore, the ability of exogenous and endogenous oxidative stressors to activate HRP-loaded PEG hydrogel spheres inside vacuoles of macrophages was examined after sphere internalization by phagocytosis. EXPERIMENTAL SECTION Materials. Sorbital monolaurate, silicone oil, Bradford reagent, and HRP were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Poly(ethylene glycol) (n)-monomethacrylate (n ) 400, (16) Klaunig, J.; Kamendulis, L. Annu. Rev. Pharmacol. Toxicol. 2004, 44, 239267. (17) Jeremy, J.; Shukla, N.; Muzaffar, S.; Handley, A.; Angelini, G. Curr. Vasc. Pharmacol. 2004, 2, 229-236. (18) Butterfield, D. A. Brain Res. 2004, 1000, 1-7. (19) Vendemiale, G.; Grattagliano, I.; Altomare, E. Int. J. Clin. Lab. Res. 1999, 29, 49-55. (20) Beissenhirtz, M.; Scheller, F.; Lisdat, F. Anal. Chem. 2004, 76, 4665-4671. (21) Chang, M. C.; Pralle, A.; Isacoff, E. Y.; Chang, C. J. J. Am. Chem. Soc. 2004, 126, 15392-15393. (22) Peppas, N. A.; Keys, K. B.; Torres-Lugo, M.; Lowman, A. M. J. Controlled Release 1999, 62, 81-87. (23) Harris, J., Ed. Poly(ethylene glycol) Chemistry: Biotechnical and Biomedical Applications; Plenum Press: New York, 1992. (24) Kim, B.; Peppas, N. Biomed. Microdevices 2003, 5, 333-341. (25) Kim, B.; Peppas, N. Int. J. Pharm. 2003, 266, 29-37.

PEGMA) and tetraethylene glycol dimethacrylate (TEGDMA) were obtained from Polysciences Inc. (Warrington, PA). NHydroxysuccinimide-PEG-acrylate (PEG-NHS, MW 3400) was purchased from Nektar Therapeutics (San Carlos, CA). 2-Hydroxy2-methyl-1-phenyl-1-propanone (Darocur 1173) was obtained from Ciba Specialty Chemicals (Tarrytown, NY). Hydrogen peroxide was obtained from VWR (Bridgeport, NJ). Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) was purchased from Molecular Probes, Inc. (Eugene, OR). Phosphate buffered saline (PBS, 0.1 M, pH 7.4) consisted of 1.1 mM potassium phosphate monobasic, 3 mM sodium phosphate dibasic heptahydrate, and 0.15 M NaCl. Milli-Q Ultrapure (Millipore, Billerica, MA) deionized water (18 MΩ cm) was used to prepare aqueous solutions. Preparation of Polymer Hydrogel Spheres Containing the Enzyme. PEG hydrogel spheres containing HRP and control spheres without HRP were synthesized via reverse emulsion photopolymerization of an aqueous monomer mixture in a continuous phase of silicone oil. Before the polymerization, HRP was coupled to PEG-NHS to prepare an acrylate-modified HRP conjugate. A 5-mg portion of HRP was dissolved in 1 mL of PBS, and 5 mg of PEG-NHS was added to the HRP solution. The solution of PEG-NHS and HRP was incubated for 1 h at room temperature. During the incubation, the NHS ester moieties of PEG-NHS react with lysine residues on the surface of HRP, resulting in a HRP molecule with an acrylate functionality. Thus, upon free radical polymerization in a PEG-acrylate system, HRP was covalently tethered to the polymer backbone. Acrylated HRP was stored at 4 °C prior to use. The aqueous monomer mixture was prepared by mixing 1.0 g of PEGMA, 35 µL of TEGDMA as a cross-linking agent, 0.2 mg of acrylated HRP, 500 µL of deionized water, and 50 µL of Darocur 1173 as a UV initiator. The monomer mixture was purged with nitrogen for 10 min and then added to the 100 mL of silicone oil, to which 0.01 g of sorbital monolaurate was added. Sorbital monolaurate was used for the stabilization of the emulsion. The mixture of oil and monomer mixture was then purged with nitrogen and stirred at 14 000 rpm for 5 min at room temperature for the formation of the emulsion. The emulsion solution was irradiated with UV light (EFOS Lite, Mississauga, Ontario) for 30 s. The spheres were separated from the oil by repeated dilution with deionized water, centrifugation and sonication. The spheres were washed with deionized water three times for oil separation and, finally, sonicated with 5 mL of PBS. This suspension of the spheres is composed of nanoscale HRP-loaded PEG hydrogel spheres. The suspension of spheres was filtered using a 0.45-µm-pore filter before size measurement to remove coarse aggregates. Characterization of Polymer Hydrogel Nanospheres Containing HRP. To determine the enzyme activity after encapsulation in the polymer hydrogel spheres and their potential use as a peroxide sensor, various concentrations of H2O2 were introduced to the HRP-loaded PEG hydrogel spheres in the presence of Amplex Red in vitro. The resulting fluorescence spectra were recorded in an L-format configuration using a QM-2000 SE spectrofluorometer (Photon Technologies Intl., Monmouth, NJ). Particle size analysis of the spheres was performed using a ZetaSizer Nano ZS instrument (Malvern Instruments, Inc., Southbrough, MA). The morphology of the spheres was imaged using scanning electron microscope (SEM) (S-3500N, Hitachi, PleasAnalytical Chemistry, Vol. 77, No. 21, November 1, 2005

6829

anton, CA) and a transmission electron microscope (TEM) (JEM1200 EXII, JEOL USA, Peabody, MA). Application of HRP-Loaded PEG Hydrogel Spheres in Vitro. To study the application of HRP-loaded PEG hydrogel spheres for the study of oxidative stress, the spheres were applied to cells in a monolayer culture. Macrophages (RAW 264.7) were purchased from American Type Culture Collection and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (FBS), 4500 mg of glucose/L, glutamine, NaHCO3, and antibiotics at 5% CO2, 37 °C. Confluent macrophages in a polystyrene tissue culture flask were subcultured every 2 or 3 days. The cells used in this study were between passages 4 and 8. After obtaining macrophages from the culture flasks, the cells were prepared on sterile cover glasses. Twenty-four hours later, prepared HRP-loaded PEG hydrogel spheres in PBS were added to the cell culture medium and were incubated for an additional 24 h to allow the hydrogel nanospheres to undergo phagocytosis. The phagocytic cells were treated with H2O2 (100 µM) or lipopolysaccharide (1 µM) (Sigma, St. Louis, MO) to induce oxidative stress, resulting in fluorescence of the HRP-containing nanospheres. The viability of the macrophages after phagocytosis was demonstrated by a live cell assay with calcein acetoxymethyl (calcein AM) (Molecular Probes, Eugene, OR). Live cells have intracellular esterases that convert nonfluorescent, cell-permeable calcein AM to the green fluorescent calcein. Cleaved calcein is retained within the cells.26 In addition, the activity of the inserted HRP-loaded spheres was also verified using an Amplex Red assay, as specified by the manufacturer’s protocol.27 After both live-cell staining and the Amplex Red assay, the cells were fixed with cold methanol and imaged by confocal microscopy (Olympus Fluoview 300 Confocal Laser Scanning Microscopes, Olympus, Melville, NY). RESULTS AND DISCUSSION Fabrication of HRP-Loaded Polymer Nanospheres. Here, we describe a novel method of reverse emulsion photopolymerization to encapsulate bioactive molecules, such as enzymes and antibodies, within polymer spheres while maintaining their activity. In general, polymer micro- or nanospheres may be prepared by techniques such as emulsion polymerization, dispersion polymerization, and precipitation polymerization.28-31 However, few studies have been performed to encapsulate bioactive molecules within hydrogel nanospheres,7 since several problems exist, such as degradation of the bioactive molecules during the polymerization process and low conversion of polymer product due to the relatively large size of the bioactive molecules. To form the micro- and nanospheres described here, the aqueous precursor solution containing the enzyme, a PEG macromer, and photoinitiator were emulsified in silicone oil. When (26) Knight, M.; Roberts, S.; Lee, D.; Bader, D. Am. J. Physiol. 2003, 284, C10831089. (27) Haugland, R. P. Handbook of Fluorescent Probes and Research Products, 9th ed.; Molecular Probes: Eugene, OR, 2002. (28) Behan, N.; Birkinshaw, C.; Clarke, N. Biomaterials 2001, 22, 1335-1344. (29) Leobandung, W.; Ichikawa, H.; Fukumori, Y.; Peppas, N. J. Appl. Polym. Sci. 2003, 87, 1678-1684. (30) Kamijo, Y.; Fujimoto, K.; Kawaguchi, H.; Yuguchi, Y.; Urakawa, H.; Kajiwara, K. Polym. J. 1996, 28, 309-316. (31) Robinson, D.; Peppas, N. Macromolecules 2002, 35, 3668-3674.

6830 Analytical Chemistry, Vol. 77, No. 21, November 1, 2005

Figure 1. Particle size analysis of HRP-loaded PEG hydrogel spheres produced via reverse emulsion polymerization. Size distribution of 300 ( 50 nm ) 69.12 (%) of HRP spheres.

Figure 2. (a) TEM and (b) SEM images of HRP-loaded PEG hydrogel spheres.

the emulsion was exposed to UV light, the initiator produced free radicals that attacked the acrylate and methacrylate groups of the macromer. The precursor mixture was thus gelled, resulting in the formation of solid spheres with a loading of ∼0.2 mg HRP/g of spheres. Sphere sizes from 250 to 350 nm were readily produced, with 69% of the total yield found to be within these size

Figure 3. (a) Fluorescence spectra of oxidized Amplex Red produced by HRP-loaded PEG hydrogel sphere suspension in various conditions: sphere suspension after adding Amplex Red (‚‚‚) and sphere suspension after adding H2O2 (90 nM) and Amplex Red (s). (b) Change of absorbance of the HRP-loaded PEG hydrogel spheres as a function of hydrogen peroxide concentration. Absorbance was measured by a plate reader after Amplex Red assay. Error bars represent standard deviation.

limits. Figure 1 shows a particle size distribution of the spheres synthesized via reverse emulsion polymerization that produced an average sphere size of ∼302.5 nm in diameter. Verification of Encapsulated HRP Activity. To verify that the enzyme, HRP, was encapsulated within the PEG hydrogel spheres, the solution of the spheres was centrifuged, and both the spheres and the supernatant were examined using a Bradford total protein assay, which colorimetrically indicates the presence of protein. The supernatant did not show any protein, whereas the spheres exhibited a change in absorbance maximum at 595 nm. This demonstrated that the enzyme was, indeed, encapsulated within the spheres and that there was no leaching into the surrounding media after encapsulation (as quantified using HRP enzyme activity and Bradford protein assays). The morphology of the spheres was studied by SEM and TEM. (Figure 2a,b) As shown in Figure 2a and b, spheres were uniform, under 500 nm, and not aggregated. Amplex Red was used to verify that the HRP encapsulated in PEG hydrogel nanospheres was active after the fabrication procedure and consequently may be used to detect for H2O2 in solution. Amplex Red has been earlier reported as an excellent reagent for the detection of H2O2 as a fluorogenic substrate for peroxidase.32,33 There are several qualities that make Amplex Red a good substrate for analysis, including very little background fluorescence or absorbance. Moreover, this substrate is resistant to photobleaching. In the presence of HRP, Amplex Red reacts with H2O2 in a 1:1 stoichiometry to produce the red fluorescent oxidation product, resorufin. Resorufin has excitation and emission maximums of ∼563 and 585 nm, respectively.27 The intensity change of the spheres resulting from the enzymatic reaction was determined quantitatively using both a (32) Mohanty, J.; Jaffe, J.; Schulman, E.; Raible, D. J. Immunol. Methods 1997, 202, 133-141. (33) Zhou, M.; Diwu, Z.; Panchuk Voloshina, N.; Haugland, R. Anal. Biochem. 1997, 253, 1162-168.

spectrophotometer and an absorbance plate reader. The emission of the fluorescent product was monitored at λem ) 585 nm with λex ) 565 nm. Resorufin emission at 585 nm was also recorded by an absorbance plate reader. Figure 3a shows the spectra of HRP-loaded nanospheres in the absence and presence of hydrogen peroxide. Spectra were recorded within 1 min and after an incubation time of 10 min. The fluorescence intensity of the spheres containing HRP increased significantly due to resorufin produced by the HRP-catalyzed reaction between Amplex Red and H2O2. Figure 3b shows the spectra of Amplex Red within HRPloaded spheres in a broad range of H2O2 concentrations. The change of absorbance intensity was clearly measurable from 500 nM to over 250 µM. Above this concentration, the spectra were observed to plateau. Above 1 mM, absorbance decreased with concentration, consistent with substrate inhibition inherent in the enzyme. Thus, encapsulated HRP activity was verified using Amplex Red assay using both fluorescence and absorbance measurements. To quantify the response time of HRP-loaded spheres to changes in peroxide concentration, a kinetic analysis of oxidized Amplex Red production was performed. The Amplex Red assay was performed with HRP-loaded PEG hydrogel nanospheres and H2O2 (100 nM), and the intensities of the fluorescence were measured kinetically (Figure 4). In 10 min, the fluorescence was increased by nearly 300% over the baseline intensity. After 30 min, there was no further change in the fluorescence intensity of the HRP-loaded spheres. Application of HRP-Loaded PEG Hydrogel Spheres in Vitro. To demonstrate the ability of these nanospheres to detect intracellular peroxide, we applied HRP-loaded PEG hydrogel spheres to the macrophages. Macrophages are capable of phagocytosis, a process by which the cell engulfs extracellular particles, creating vacuoles. By simply adding sterile HRP-loaded PEG spheres to a macrophage culture, the cells were observed to Analytical Chemistry, Vol. 77, No. 21, November 1, 2005

6831

Figure 4. Kinetics of HRP-loaded PEG hydrogel spheres. Fluorescence intensities at 585 nm were measured after Amplex Red assay.

uptake the spheres spontaneously. The macrophages were exposed to the PEG nanospheres for 24 h, and after this period, the vacuoles were observed inside the macrophages using confocal microscopy. Figure 5 shows the viability of cells before and after phagocytosis through live-cell staining with calcein AM. Macrophages that had taken up nanoparticles were viable when compared to nonphagocytotic cells. We postulated that the HRP-loaded PEG hydrogel spheres inside macrophages could be used to detect local peroxide production by compounds such as LPS. The macrophages that had intracellular HRP-loaded PEG hydrogel spheres in phagosomes were prepared as described above. H2O2 (100 µM) was then introduced into the cell culture media. Exogenous H2O2 activates cellular signaling pathways, such as extracellular signalregulated kinase 1 (ERK1) and ERK2 in many cell types, and increases intracellular production of ROS. Thus, the HRP nanospheres inside vacuoles could be activated (i.e., oxidized Amplex Red and became fluorescent) by exogenous oxidative stress, such as H2O2 through ROS production inside vacuoles. Confocal images on the left column in Figure 6 indicate the HRP-loaded PEG hydrogel spheres were activated by H2O2 through Amplex Red assay. Although the spheres were within phagosomes, the nanospheres responded to oxidative stress, H2O2 (Figure 6). This result suggested that the spheres may be developed to biosensors to detect intracellular peroxide generation with individual subcellular compartments. Additionally, we exposed nanospheres-containing macrophages to an endogenous source of oxidative stress, LPS. One would anticipate the concentration of peroxide within a phagosome would be in the hundreds of micromolar, although peroxide rapidly reacts with free iron via the Fenton reaction to create hydroxyl radicals.34 Macrophages containing HRP-loaded PEG hydrogel nanospheres were treated with LPS (1 µg/mL), an endotoxin which mediates proinflammatory responses.35 In response to LPS, macrophages, which play a central role in the immune system, (34) Zarley, J. H.; Britigan, B. E.; Wilson, M. E. J. Clin. Invest. 1991, 88, 15111521. (35) Kim, S. H.; Lessner, S. M.; Sakurai, Y.; Galis, Z. S. Am. J. Pathol. 2004, 164, 1567-1574.

6832 Analytical Chemistry, Vol. 77, No. 21, November 1, 2005

Figure 5. The viability of phagocytic macrophages. Live cell assay with calcein AM was performed. Macrophages were imaged by confocal microscope (a) before phagocytosis of HRP-loaded PEG hydrogel spheres and (b) after phagocytosis. Green color represents calcein in live cells. The vacuoles are visible in the macrophages after phagocytosis (b). White scale bars in each picture represent 10 µm.

secrete various cytokines36 and ROS such as peroxide and superoxide anions.37 The LPS concentration (1 µg/mL) used in this study is of a sufficient concentration to induce inflammatory responses in a cell culture system. Peroxide produced by LPSstimulated macrophages activated HRP-loaded PEG hydrogel spheres inside macrophages (Figure 6, right). The fluorescence intensities of activated HRP spheres were measured compared to a negative control, which consisted of phagocytic macrophages containing nanospheres without Amplex Red added to the culture medium. The fluorescence of the negative control was negligible (data not shown). Our in vitro application results suggested the HRP-loaded hydrogel spheres could be developed as an oxidative stress biosensor and for medical diagnosis due to the response to LPS. CONCLUSIONS Here, we describe the development of PEG hydrogel nanospheres containing an active enzyme and its potential use as an (36) Fujihara, M.; Muroi, M.; Tanamoto, K.; Suzuki, T.; Azuma, H.; Ikeda, H. Pharmacol. Ther. 2003, 100, 171-194. (37) Victor, V. M.; De la Fuente, M. Physiol. Res. 2003, 52, 789-796.

Figure 6. Application of HRP-loaded PEG hydrogel spheres in vitro. Live cell assay with calcein AM and Amplex Red assay for activation of HRP-loaded PEG hydrogel spheres were performed. The phagocytic macrophages were stimulated by H2O2 and LPS to activate (i.e., oxidize Amplex Red and create fluorescence) the HRP spheres. The macrophages after phagocytosis were imaged by DIC (a) and fluorescence microscopy (b, c). In (a), “N”, represents the nucleus, and the black arrows indicate vacuoles due to phagocytosis. In (b) and (c), green and red colors indicate the live cells and activated HRP-loaded PEG hydrogel spheres by oxidative stress, respectively (white arrows indicate the activated HRP-loaded PEG hydrogel spheres inside macrophages). All white scale bars represent 10 µm.

intracellular optical sensor. Enzyme activity after nanosphere fabrication and enzyme encapsulation was determined using HRP as a model enzyme. HRP was encapsulated in PEG hydrogel spheres by reverse emulsion photopolymerization, and its enzymatic activity was maintained after polymerization. The fluorescence emission response of the HRP-loaded PEG hydrogel spheres changed as a function of H2O2 concentration in the presence of Amplex Red, and no leaching of HRP was observed from the spheres. The HRP-loaded hydrogel spheres were introduced via phagocytosis inside macrophages and were found to respond to both exogenous and endogenous sources of oxidative stress. The results presented here suggest that this method to prepare hydrogel nanospheres containing active HRP may be extended to other enzyme and recognition molecules.

Such hydrogel nanospheres could be potentially used as optical nanosensors for intracellular analysis in the study of oxidative stress injury, drug screening, or in micro total analysis systems. ACKNOWLEDGMENT We gratefully acknowledge the support of NASA (BIOTECH01-0023-0131). S.H.K. acknowledges fellowship support from the Huck Life Sciences Institute of Penn State University through its Integrative Biological Sciences Program.

Received for review April 28, 2005. Accepted August 31, 2005. AC0507340 Analytical Chemistry, Vol. 77, No. 21, November 1, 2005

6833