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Generation of reactive oxygen species from porous silicon microparticles in cell culture medium Suet Peng Low,1 Keryn A. Williams,2 Leigh T. Canham,3 Nicolas H. Voelcker1 1 Faculty of Science and Engineering, School of Chemistry, Physics and Earth Sciences, Flinders University, Adelaide, South Australia 2 Department of Ophthalmology, Flinders University, Adelaide, South Australia 3 pSiMedica, Malvern, Worcestershire, United Kingdom Received 30 March 2009; revised 27 June 2009; accepted 29 June 2009 Published online 18 September 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.a.32610 Abstract: Nanostructured (porous) silicon is a promising biodegradable biomaterial, which is being intensively researched as a tissue engineering scaffold and drugdelivery vehicle. Here, we tested the biocompatibility of non-treated and thermally-oxidized porous silicon particles using an indirect cell viability assay. Initial direct cell culture on porous silicon determined that human lens epithelial cells only poorly adhered to non-treated porous silicon. Using an indirect cell culture assay, we found that non-treated microparticles caused complete cell death, indicating that these particles generated a toxic product in cell culture medium. In contrast, thermally-oxidized microparticles did not reduce cell viabil-

INTRODUCTION Porous silicon is formed through anodic etching of silicon wafers in hydrofluoric acid. The pore size, porosity, and thickness of the porous silicon layer can all be controlled by the current, concentration of hydrofluoric acid, and the etching time.1 The resultant porous silicon degrades into non-toxic silicic acid in aqueous solutions. The material’s degradability coupled with the widely acknowledged biocompatibility2–5 have led to research on its use as a biodegradable cell scaffold.6 Control over the rate of degradation is readily achieved by surface modification, such as oxidation or silanisation.5 A variety of mammalian cells have been successfully cultured on porous silicon.7,8 The tunability of the pore size and its biodegradability also make porous silicon an appealing Correspondence to: N. H. Voelcker; e-mail: nico.voelcker@ flinders.edu.au Contract grant sponsors: Australian Research Council, National Health and Medical Research Council, pSiMedica

Ó 2009 Wiley Periodicals, Inc.

ity significantly. We found evidence for the generation of reactive oxygen species (ROS) by means of the fluorescent probe 20 ,70 -dichlorofluorescin. Our results suggest that non-treated porous silicon microparticles produced ROS, which interacted with the components of the cell culture medium, leading to the formation of cytotoxic species. Oxidation of porous silicon microparticles not only mitigated, but also abolished the toxic effects. Ó 2009 Wiley Periodicals, Inc. J Biomed Mater Res 93A: 1124–1131, 2010 Key words: porous silicon; microparticles; biodegradable materials; reactive oxygen species

material for drug-delivery applications.9 Current investigations have focused upon the use of porous silicon microparticles. Microparticles have been used to deliver insulin across intestinal tissue10 and recently, particles loaded with the isotope 32P were used to target and kill tumor cells.11 Another well known property of porous silicon is its photoluminescence,12 and investigations into the causes and decay of porous silicon luminescence have led to the discovery that this surface is capable of generating singlet oxygen (O2 2 ) molecules under 13 certain conditions. To the best of our knowledge, it has not been investigated if the singlet oxygen molecules generated by porous silicon have an effect upon cell viability, or whether singlet oxygen is generated under low or no light conditions. Singlet oxygen is one of many different reactive oxygen species (ROS). ROS play important physiological roles, which are being unraveled.14 They act as signaling molecules and regulate cell proliferation, differentiation, and apoptosis,15 although, at high concentration, they are known to have cytotoxic effects.16,17 In cellular systems, oxygen radicals can be converted into hydrogen peroxide for example via the enzyme superoxide dismutase.18 Hydrogen

REACTIVE OXYGEN SPECIES FROM POROUS SILICON MICROPARTICLES

peroxide in turn can be converted into hydroxyl radicals via a Fenton-mediated reaction.16 This study aimed to test the biocompatibility of porous silicon particles with mammalian cells in an indirect assay. Whilst a complementary direct assay would be informative, porous silicon is known to interfere with a variety of colorimetric viability assays.5,19 The results indicated that non-modified or untreated porous silicon particles caused a loss in cell viability. Our investigations led us to believe that the porous silicon particles were generating ROS, thus causing cell death. Generation of ROS was confirmed using the probe 20 ,70 -dichlorofluorescin diacetate. In contrast, thermally-oxidized particles did not exert toxic effects on mammalian cells. METHODS AND MATERIALS Chemicals Dulbecco’s

Modified Eagle’s Medium (DMEM), penicillin, and streptomycin were purchased from Sigma Chemical Company (St Louis). Phenol red-free DMEM was purchased from Invitrogen (Carlsbad). Fetal bovine serum was purchased from Bovogen Biologicals (Essendon, Australia). Alamar Blue was obtained from Biosource International (Camarillo). Phosphate buffered saline (PBS, pH 7.2) consisted of 3.7 mM NaCl, 2.7 mM KCl, 5.3 mM Na2HPO4, and 1.8 mM KH2PO4 in MilliQ water, and was filtered through a 0.2 lm mesh membrane before use. L-glutamine,

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scans were averaged at a resolution of 4 cm21. A background was obtained using a silicon wafer.

Cells Human lens epithelial cells (SRA 01/04) were used with permission from Professor Venkat Reddy from the Kellogg Eye Center, University of Michigan, Ann Arbor. Cells were maintained in DMEM supplemented with 5 mM Lglutamine, 100 IU/mL penicillin, 100 lg/mL streptomycin, and 10% fetal bovine serum. This medium is referred to as complete medium. Cells were maintained in a humidified incubator at 378C with 5% CO2 in air.

Direct cell culture on BioSiliconTM membranes Human lens epithelial cells were seeded onto sterilized porous silicon BioSiliconTM membranes that were nontreated or thermally-oxidized. The membranes were used for optimal viewing of cellular morphology on porous silicon. Before cell seeding, the membranes were cut into 1 cm2 pieces and placed into a 24-well plate. Cells were seeded onto the membrane surfaces at a density of 50,000 cells per well and then incubated at 378C for 24 h. A DiOC5(3) fluorescence stain was employed for visualization of cells. DiOC5(3) was added to the cells at a final concentration of 3 mM and incubated for 2 h at 378C with 5% CO2 in air. After incubation, the membranes were rinsed with PBS and images were captured with a Leitz Laborlux II fluorescence microscope equipped with a Nikon (DS5M) digital camera.

Indirect cell viability assay Porous silicon Porous silicon microparticles and membranes were provided by pSiMedica (Malvern). The microparticles were generated from porous silicon membranes (BioSiliconTM) that were ball-milled and subsequently classified by High Force Research (Bowburn) to particles with dimensions between 20 and 60 lm and having a porosity of 68–70%. Thermally-oxidized microparticles were produced by treatment of particles at 6008C for 1 h. Sterilization of the porous silicon particles and silicon wafers for cell viability studies was done by dry heat treatment at 1608C for 8 h. Silicon wafers (termed bulk silicon wafers in this article) with a resistivity of 3–6 Ohm cm, from Silicon Quest International (Santa Clara), were used as control samples in the cell-based assays.

Transmission FTIR Porous silicon surface chemistry was determined via Fourier-transform infrared (FTIR) spectroscopy. BioSiliconTM membranes were used, since the porous silicon microparticles scattered the infrared beam and a spectrum could not be collected. Membranes were heat-treated at 6008C for 1 h to generate thermally-oxidized porous silicon. Spectra were collected on a Nicolet Avatar 370 spectrometer operating under transmission mode. Thirty-two

To determine if toxic products are released from the porous silicon microparticles into the cell culture medium, an indirect cell viability assay based upon Alamar Blue was utilized. In a 24-well plate, human lens epithelial cells were seeded at a density of 50,000 cells/well and allowed to attach overnight. Before further use, each well was washed twice with sterile PBS. Sixty milligram of non-treated or thermally-oxidized porous silicon microparticles were placed into separate sterilized vials, 3 mL of complete medium was added to each container and the particles were incubated overnight in the dark in a humidified incubator at 378C. As a control, a piece of silicon wafer (60 mg) was also incubated with complete medium. After overnight incubation, the contents of each vial were centrifuged at 7000 g to pellet any remaining particles; 1 mL of the supernatant was placed onto the cultured human lens epithelial cells and incubated for further 24 h at 378C. A well with no cells, and a well containing cells, were used as a negative and positive control, respectively. Fresh complete medium was added to the control wells. After this incubation step, the medium was removed and the cells were washed with sterile PBS three times to remove any residual medium; 500 lL of a 10% (v/v) of Alamar Blue in fresh complete medium was subsequently placed into each well. The cells were incubated with the Journal of Biomedical Materials Research Part A

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Alamar Blue for 2 h at 378C; 100 lL of this solution was removed and placed into a 96-well plate for spectrophotometric analysis at 570 nm and 600 nm. Cell viability was determined by the concentration of Alamar Blue reduced by live cells and was calculated using the formula provided by the supplier. For the indirect viability assay using phenol red-free medium, DMEM without phenol red but with antibiotics, L-glutamine, and FBS was used. The assay was carried out in the dark as described earlier and phenol red-free medium was also used for the Alamar Blue assay. For the indirect viability assay using PBS, the porous silicon microparticles were incubated in PBS rather than cell culture medium. The PBS was then incubated with cells as described earlier with a change to the length of incubation time. As cells were not expected to survive in PBS for 24 h,20 the cells were incubated with the PBS solution for 1.5 h in the dark. The remainder of the assay followed the method described for the indirect assay using complete medium containing phenol red. As a control, particles in complete medium were subjected to the same treatment and were run alongside the assay conducted in PBS.

Preparation of DCFH solution A variation of the method by Cathcart et al.21 and Wilson et al.22 was used to prepare 20 ,70 -dichlorofluorescin (DCFH), a non-fluorescent compound. A 1 mM stock solution of 20 ,70 -dichlorofluorescin diacetate (DCFH-DA) was prepared in ethanol; 0.5 mL of the stock solution was added to 2 mL of 10 mM NaOH to induce ester hydrolysis. The solution was allowed to sit at room temperature in the dark for 30 min. The NaOH was neutralized by the addition of 10 mL phosphate buffered saline with a final pH of 7.1. The final DCFH concentration was 10 lM. This solution was kept in the dark and discarded at the end of each day. DCFH can be oxidized by ROS species to yield 20 ,70 -dichlorofluorescein (DCF), a fluorescent product.23

Detection of ROS The porous silicon particles were used at a ratio of 20 mg to 1 mL of medium in the indirect assay. Eighteen milligram of either non-treated or thermally-oxidized porous silicon particles were added to 900 lL of cell culture medium. The mixture was sonicated for 5 min to ensure mixing; 300 lL of prepared DCFH solution was then added and the mixture was vortexed and incubated for 1 min at room temperature. As the presence of microparticles impedes fluorescence output, the particles were removed from the solution by filtration through a 0.22 lm mesh filter. Fluorescence was read on a LS-55 fluorimeter (Perkin Elmer) using an excitation at 485 nm and collecting emission at 520 nm over a 100 s integration time. Samples were tested in triplicate.

Statistical analysis Statistical analysis of the Alamar Blue viability assay results was conducted using KaleidaGraph software verJournal of Biomedical Materials Research Part A

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sion 4.02 (Synergy Software). An independent t-test was performed on all samples against the positive control, considering p 0.05 as statistically significant.

RESULTS The surface chemistry of porous silicon was determined using transmission-IR of porous silicon membranes. Non-treated porous silicon displayed spectra consistent with a hydride-terminated surface (Fig. 1). The distinctive stretching modes at 2088 cm21, 2116 cm21, and 2137 cm21 all relate to SiH surface bonds.24,25 Upon thermal oxidation, the bands corresponding to Si H stretching and bending modes disappeared and were replaced with a broad band at 3400 cm21 attributed to SiOH bonds. A smaller SiOH bending mode at 1640 cm21 and the large peak at 1070 cm21 were attributed to Si O Si stretching modes.26 The appearance of an OSiH stretching mode at 2256 cm21 is characteristic of a thermally-oxidized porous silicon surface.25 Few human lens epithelial cells adhered to nontreated porous silicon membranes, whereas the thermally-oxidized membranes displayed a large number of attached cells (Fig. 2). The cells on the non-treated membranes did not display spreading morphology and remained small and rounded, indicative of poor attachment. In contrast, on the thermally-oxidized membrane, the cells appeared well-spread and displayed normal morphology.27 It is unclear whether the lack of attachment to the nontreated membrane was a result of direct toxicity of the membrane or due to the instability of the rapidly degrading surface.5 Thermally-oxidized and non-treated microparticles were incubated in cell culture medium. The particles were then removed and the medium placed onto an established cell layer. We used a human lens epithelial cell line here because we are pursuing applications of porous silicon membranes for ocular cell expansion in regenerative medicine. Interestingly, a significant loss in cell viability was observed in cells in indirect contact with non-treated porous silicon particles (Fig. 3, black bars). The result was statistically significantly different to the positive control (p 5 0.04). In contrast, neither the indirect assay with thermally-oxidized particles nor the indirect assay with the silicon wafer sample showed a significant loss in cell viability (p 5 0.32 and p 5 0.47, respectively). During the course of this experiment, it was noted that the color of the cell culture medium changed when incubated with the non-treated porous silicon particles. The phenol red present in DMEM is used as a pH indicator, giving the medium a rose pink color. When the medium was incubated with nontreated porous silicon particles, the medium turned


Figure 1. Transmission FTIR spectra of porous silicon and thermally-oxidized porous silicon.

colorless. We initially considered that a change in pH was causing this color change and that such a pH change might cause cell death. However, when tested, we found that the pH was maintained at pH 7.2 across all samples. We next investigated whether the interaction of phenol red with the porous silicon microparticles caused the loss in cell viability. DMEM without phenol red was used according to the same protocol as in the previous experiment. The Alamar Blue assay, used to determine viability, is a colorimetric assay and the absence of phenol red in the solution gave absorbance values different to results observed with phenol red-containing medium. Therefore, we could not compare directly the results obtained using phenol red-containing and phenol red-free medium. Our results demonstrated that in the absence of phenol red, a loss in cell viability was still observed with the non-treated porous silicon samples in

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relation to the control sample (Fig. 3, white bars). If anything, the absence of phenol red appeared to acerbate indirect toxicity. Independent of the presence of phenol red, no loss of viability was observed for cells in indirect contact with thermally-oxidized porous silicon particles. Short exposure (2 h) indirect viability assays were conducted comparing incubations in cell culture medium and PBS to determine if toxic components that were leaching from the porous silicon microparticles would interact with components in the cell culture medium, producing secondary species that resulted in cell death. Incubations in cell culture medium and PBS were carried out side-byside and the results showed that there was a significant loss in cell viability when using cell culture medium that had been incubated with porous silicon particles (Fig. 4). On the contrary, PBS incubated with porous silicon particles did not exert significant toxic effects. Investigating the origin of cytotoxicity, a fluorogenic molecular probe was used that is known to be oxidized in the presence of ROS to form the fluorescent DCF.28 High fluorescence intensity is an indication of the presence of ROS in the solution. When DCFH was applied to cell culture medium incubated with either thermally-oxidized or nontreated particles, significantly higher fluorescence intensity was observed for the non-oxidized particles (Fig. 5), strongly suggesting that non-treated particles indeed generate ROS in the presence of cell culture medium. DISCUSSION The aim of this study was to determine the biocompatibility of porous silicon microparticles. Porous silicon is currently being investigated as an implantable biomaterial for tissue engineering and as a drug-delivery platform. These applications

Figure 2. Human lens epithelial cells on a non-treated porous silicon membrane (A) and on a thermally-oxidized porous silicon membrane (B) after a 24 h incubation. Cells were stained with DiOC5(3). Scale bar: 100 lm. [Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.] Journal of Biomedical Materials Research Part A

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Figure 3. Indirect Alamar Blue cell viability assay results for human lens epithelial cells cultured in complete medium containing phenol red (black bars) or no phenol red (white bars), that had previously been incubated with porous silicon particles. Silicon: silicon wafer; pSi: nontreated porous silicon particles; Thermally-oxidized pSi: 6008C thermally-oxidized porous silicon particles. þControl: Cells with medium and 2Control: no cells, fresh medium only. Pre-incubated medium was cultured with the cells for 24 h before application of Alamar Blue. Statistics were performed via a paired independent t-test. All p values are from comparisons made between the þControl sample and the other samples. * Denotes significant difference (p 0.05). Error bars: standard deviation of the mean, n 5 3.

exploit the tunable porosity and pore size of this material, the high surface area, but also the degradability of the material in aqueous solutions into non-toxic, small molecular mass silicic acid.29 The rate of degradation can be controlled by changing the surface chemistry, such as by oxidizing the surface. After etching, the porous silicon surface is hydride terminated.25 Heating the porous silicon to high temperatures incorporates oxygen into the silicon backbonds replacing silicon hydride bonds with silicon oxygen species30 generating a thermally-oxidized surface. Thermal oxidation makes the surface resistant to hydrolytic attack29 and significantly more stable when immersed in aqueous solution in comparison to a non-treated surface.5 We used an indirect assay to determine the biocompatibility of porous silicon particles, and as a control, thermally-oxidized particles were used. In this instance, an indirect assay was utilized, as previous studies have shown that porous silicon is capable of acting as a reducing agent and is therefore able to reduce compounds used in various colorimetric viability assays, leading to false results.5,19 When cells were seeded on a non-treated porous silicon membrane, we observed lack of cell attachment to the surface (Fig. 2). Poor attachment sugJournal of Biomedical Materials Research Part A

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Figure 4. Indirect Alamar Blue viability assay on human lens epithelial cells in cell culture medium (black bars) or in PBS (white bars). Pre-incubated medium and PBS were cultured with the cells for 2 h before the application of Alamar Blue. * Denotes significant difference (p 0.05). Error bars: standard deviation of the mean.

gested direct porous silicon toxicity or a rapidly eroding surface unable to sustain cell attachment.5 Interestingly, on a thermally-oxidized membrane, cells behaved very differently. Cells adhered readily to this membrane, appeared well-spread, and displayed normal cell morphology. The absence of cell attachment on the non-treated membrane prompted us to carry out an indirect assay on porous silicon

Figure 5. DCFH-mediated detection of ROS generation with porous silicon particles in cell culture medium. Results are expressed as mean fluorescence units at 100 s integration time point. Fluorescence intensity is significantly higher for porous silicon particles (Porous silicon) in comparison to thermally-oxidized porous silicon particles (thermally-oxidized pSi) and to the no particles sample (p < 0.05). n 5 3 for thermally-oxidized and no-particle samples and n 5 4 for non-treated samples.


particles to investigate toxic effects on human lens epithelial cells. This indirect assay showed that whilst thermally-oxidized porous silicon microparticles did not affect cell viability, non-oxidized particles reduced viability significantly. An interesting observation was that non-treated particles decolourized standard tissue culture medium containing phenol red. We determined that this phenomenon was not due to a change in pH. Phenol red has been shown to be decolourized in the presence of certain oxidants31 and is commonly employed in assays to detect the presence of hydrogen peroxide.32 Since porous silicon itself cannot act as an oxidant, another species produced upon reaction of the cell culture medium with porous silicon must be responsible for phenol red oxidation. We further investigated if the interaction of the dye in the medium with porous silicon was the culprit of the observed cell toxicity in the indirect assay. Using phenol red-free medium, we found that toxicity for mammalian cells was still evident, suggesting that the non-treated particles were exerting toxicity that did not involve phenol red but that oxidative decolourization may be caused by the toxic species or an intermediate species. Since phenol red is known to act as a scavenger of ROS,32,33 we decided to investigate if ROS are possible sources of oxidants in the medium. ROS 2 include superoxide, O 2 , the hydroxyl radical, HO 1 hydrogen peroxide, H2O2, and singlet oxygen, O2. Superoxide is associated with the activation of apoptosis.17,33 The cytotoxicity of hydrogen peroxide is well established.16 Hydrogen peroxide in turn can mediate the formation of the reactive hydroxyl radical, which can attack and damage DNA and proteins.15 Singlet oxygen has been shown to induce cytotoxicity via degradation of the guanine bases of DNA and RNA.34 Studies investigating the photoluminescence properties of porous silicon12,35–37 have demonstrated singlet oxygen generation from hydride-terminated (Si H, non-treated) porous silicon under illumination.13,38,39 Under illuminated conditions, photo-generated electron and electron hole pairs (excitons) are produced in porous silicon. Excitons remain trapped within the silicon crystal structure, but are able to react and exchange electrons with oxygen molecules present on the surface, converting the oxygen molecules into singlet oxygen40 and possibly other ROS. On oxidized porous silicon surfaces, ROS generation is no longer evident.40,41 In contrast to these studies, we had carried out our experiments in the dark, without deliberate illumination; although some light exposure occurred during sample handling. Singlet oxygen generation is generally assumed to be negligible in the absence of illumination.42 As a second key difference,

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although most studies on singlet oxygen generation from porous silicon use films or membranes,37,39,41,43 we used high-porosity microparticles with diameters ranging from 20 to 60 lm. Recent evidence suggests that particles are more efficient than planar porous silicon in electron transfer to surface oxygen molecules, permitting efficient energy transfer at room temperature.44 Kovalev et al. also reported on a light-independent mechanism of singlet oxygen formation, albeit in different conditions from the ones used here.45 The group proposed that surface silicon atoms interact directly with molecular oxygen, and that this interaction results in the creation of new free radicals including the ROS singlet oxygen and hydroxyl radicals. We used the fluorogen DCFH, which is oxidized by various ROS to the fluorescent DCF. DCFH has been used as a marker for oxidative stress and to detect the presence of H2O2 in the presence of peroxidize,23 NO,46 singlet oxygen,47 and the hydroxyl radical.48 Our results showed that the non-oxidized porous silicon particles were able to convert DCFH to DCF. This is significant since it provides further evidence that some form of ROS is being generated by porous silicon particles without direct illumination. DCF fluorescence intensity for the thermally-oxidized particles was similar to the control, which did not contain any particles. This suggested that ROS was not generated from thermally-oxidized particles. This is in agreement with literature results, where the thick oxide layer on the surface of the crystal structure reduces the efficiency of electron transfer from the excitons to the surface molecules, eliminating ROS generation from oxidized samples.40,41 When this experiment was repeated using PBS instead of medium, we did not observe any fluorescence increase from either thermally-oxidized or untreated particles (data not shown), indicating that components in the cell culture medium may be required for DCFH oxidation. This is consistent with our observation that the viability of human lens epithelial cells was not significantly compromised in indirect contact when PBS was used instead of cell culture medium. So far we have shown evidence for ROS generation by porous silicon particles in combination with cell culture medium. In the present study, porous silicon particles were in contact with cell culture medium for 24 h before the particles were removed and the remaining cell culture medium was then incubated with cells. During this time, most generated ROS and in particular singlet oxygen and hydroxyl radicals would have undergone further reactions as they have an extremely short lifespan.49,50 The short-lived nature of ROS and the fact that DCFH oxidation did not occur in the abJournal of Biomedical Materials Research Part A

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Figure 6. Generation of singlet oxygen by Si H terminated porous silicon. Excitons, which remain trapped in the crystal and are able to transfer electrons to oxygen molecules adsorbed on the surface, converting them into singlet oxygen or other ROS which are short-lived and react with the components of the cell culture medium to generate long-lived toxic species.

sence of cell culture medium, makes us speculate that components of the cell culture medium are activated upon contact with porous silicon forming long-lived toxic products (Fig. 6). For example, singlet oxygen has been shown to be able to directly oxidize amino acids on proteins and peptides to form reactive side chain peroxides, in particular with tryptophan, histidine, and tyrosine.51 Protein peroxides are still detectable after 24 h in solution, indicating that they were relatively stable in solution.52 Protein peroxides are not easily detoxified by cells, generating oxidative stress and cellular toxicity.53 Protein peroxides can also release hydrogen peroxide.51 At the same time, compounds such as riboflavin and tryptophan, present in DMEM, are able to store and release ROS.34,54 Our results show that cell culture medium incubated with non-treated porous silicon microparticles is cytotoxic to human lens epithelial cells. The species responsible for indirect cytotoxicity are still unknown, but ROS production by porous silicon microparticles in the presence of cell culture medium appears to be involved. The observed toxic effect can be completely mitigated by protective surface treatments of porous silicon microparticles including thermal oxidation. More work is required to identify the mechanism for ROS generation and to reveal the identity of the toxic species. However, given the extensive and still growing use of porous silicon particles as drugdelivery vehicles and as biomaterials, our findings are highly relevant. In particular, our work has established that attention to the toxicity of non-oxidized porous silicon microparticles is warranted. Whether the observed in vitro toxicity translates into in vivo toxicity remains to be seen. Finally, we should point out that the behavior of cells is dependent on the level of ROS. Although high ROS levels invariably cause cell death, low levels of ROS may be beneficial since they are known to induce mitogenic responses and increase growth of mammalian cells.55 This raises the possibility of Journal of Biomedical Materials Research Part A

exploiting controlled ROS production from porous silicon biomaterials.

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