Reduced oxidative stress in primary human cells by antioxidant released from nanoporous alumina Shiuli Pujari-Palmer, Michael Pujari-Palmer, Marjam Karlsson Ott Department of Medical Sciences, Uppsala University, SE-75185, Uppsala, Sweden Received 14 October 2014; revised 9 March 2015; accepted 20 March 2015 Published online 7 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33427 Abstract: Nanoporous alumina elicits different inflammatory responses dependent on pore size, such as increased complement activation and reactive oxygen species (ROS) production, on 200 versus 20 nm pores. In this study, we attempt to further modulate inflammatory cell response by loading nanoporous alumina membranes (20, 100, and 200 nm pores), with an antioxidant, Trolox, for controlled drug release. For mononuclear cells (MNC) no difference in cell response, due to pore size, was seen when cultured on nonloaded membranes. However, when exposed to membranes loaded with Trolox, 100 uM was enough to quench ROS by more than 95% for all pore sizes. Polymorphonuclear cells (PMNC) produced significantly more ROS when exposed
to 20 versus 100 nm pores. For Trolox loaded membranes, this trend reversed, due to slower release of antioxidant from the 20 nm pores. Furthermore, Trolox exhibited a unique effect on PMNCs that has not previously been reported: It delayed the production of ROS in a manner distinct from antioxidant activity. The present study confirms that nanoporous alumina is a suitable vehicle for drug delivery, and that Trolox can successfully modulate the inflammatory response C 2015 Wiley Periodicals, Inc. J Biomed of both MNC and PMNCs. V Mater Res Part B: Appl Biomater, 104B: 568–575, 2016.
Key Words: nanoporous alumina, Trolox, mononuclear cells, polymorphonuclear cells, reactive oxygen species
How to cite this article: Pujari-Palmer S, Pujari-Palmer M, Karlsson Ott M. 2016. Reduced oxidative stress in primary human cells by antioxidant released from nanoporous alumina. J Biomed Mater Res Part B 2016:104B:568–575.
INTRODUCTION
Biomedical implants have become increasingly important as the age and lifetime of the current population expands. Implanted biomaterials can successfully improve the quality and length of life for patients suffering from disease or injury.1 Within the native cellular environment, the extracellular matrix contains biological and topographical features, on the nanometer scale, that facilitate cell-cell interactions and tissue integration during healing.2,3 There is, therefore, an increasing drive to design implants that mimic the native biological environment in order to promote and increase implant integration and healing. Anodized aluminum is a well characterized material.4 An ordered nanoporous pattern is fabricated through anodic oxidation of aluminum (anodization) in polyprotic acids.5,6 Nanoporous alumina is a promising candidate for bone implant coatings,7 immunoisolation devices,8 drug delivery devices for stent coatings,9 and for cocultivation of cells.10 Previous studies on nanoporous alumina have shown that 200 nm pores elicit a greater inflammatory response in vitro and in vivo, compared to 20 nm pores.11,12 Few studies, however, have focused on how to specifically decrease inflammation in response to this material. Vitamin E supplementation in vitro, resulted in a decrease in the production of proinflammory cytokines and adhesion molecules in
immune and endothelial cells.13–17 Trolox is a water soluble vitamin E derivative and a potent quencher of hydroxyl and peroxide free radicals. The hydrophilic nature of Trolox makes it an ideal antioxidant since it can readily disperse throughout the cytoplasm. It also exhibits minimal cytotoxicity,18,19 and is the standard by which antioxidant activity is measured (Trolox equivalent antioxidant capacity (TEAC))9.20 Furthermore, Trolox has shown to reduce lipid peroxidation after whole-body gamma irradiation21 and prevent liver necrosis.22 The aim of this study is (a) to compare the activation of primary human mononuclear (MNC) and polymorphonuclear (PMNC) cells when exposed to alumina with 20, 100, and 200 nm pores; (b) to determine whether an antioxidant, Trolox, can reduce or prevent inflammatory cell activation in response to nanoporosity, and finally, (c) to determine whether nanoporous alumina can be loaded with and deliver antioxidants to reduce PMNC and MNC activation. MATERIALS AND METHODS
Cell isolation Human MNC Isolation: Blood buffy coats were obtained from anonymous blood donors from the Uppsala University Hospital. MNCs were isolated using the Ficoll-Paque Plus density gradient centrifugation according to the
Correspondence to: M. Karlsson Ott; e-mail: [email protected] € r internationalisering av ho € gre utbildning och forskning) Contract grant sponsor: STINT (Stiftelsen fo
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manufacturer’s instructions. Briefly, blood was overlaid on top of the Ficoll-Paque Plus and centrifuged at 1200g for 20 min. The MNC layer was then collected and washed three times with phosphate buffered saline (PBS). The total cell number was determined using a hemocytometer (adding 0.4% trypan blue to determine viability). Cells were then seeded on the alumina membranes (400,000 cells/well). Human PMNC isolation.. PMNC isolation underwent 2 separation procedures. Briefly, buffy coat blood was subjected to Ficoll-Paque Plus density gradient centrifugation, as described previously. The blood pellet formed was then resuspended in 3% Dextran/0.9% saline solution for 25 min. The upper plasma layer was collected and centrifuged at 250g for 10 min. Contaminating erythrocytes were removed by adding 0.2% saline solution to the pellet for 20 s. An equal volume of 1.6% saline solution was then added and the cell suspension was centrifuged. The total cell number for each fraction was determined by counting on a hemocytometer with 0.4% trypan blue to determine viability, and 3% acetic acid to differentiate mononuclear from PMNC. Cells were then seeded on the alumina membranes (300,000 cells/well). Drug loading and release Drug loading. A 20 lL volume of a 60 mM ethanol solution of Trolox was added to each nanoporous alumina membrane (Anodisc Whatman International, Madison, England) and allowed to evaporate at room temperature for 30 min. Drug release. An elution volume of 500 lL of phosphate buffered saline was replaced every 10 min, for the first hour, and every hour thereafter for 5 h. The elution volume was also replaced after 16 and 24 h. Trolox release was measured with a UV Spectrophotometer at 290 nm. Delivery methods for in vitro studies. Trolox was delivered either by direct external addition or preloaded in the alumina membranes, hereafter referred to as preloaded. For external addition, 100 lM of Trolox was added. To simulate a dynamic release of 100 lM of preloaded Trolox, 60 mM was loaded in the membranes. The membranes were soaked in PBS for 15 min (prior to the cell studies) releasing excess Trolox, thus leaving 100 lM in the membranes. Chemiluminescence To quantify the generation of reactive oxygen species (ROS) released by the cells, luminol amplified chemiluminescence (CL) was used.23 All measurements were performed in white 24 well optiplates (Perkin Elmer) at 37 C in medium containing a 4:1 ratio of PBS to RPMI-1640 medium containing100 mM glucose. The luminol solution (500 lM) was prepared by incorporating 1% of luminol taken from the stock solution, 2% horse radish peroxidase (2 lg/mL), and a 1:100 ratio of 0.1M NaOH into the 4:1 PBS to RPMI-1640 containing100 mM glucose medium. The luminol stock solution was prepared by dissolving 50 mM of luminol into 0.2M NaOH. 400,000 MNCs/well
and 300,000 PMNCs/well were seeded on the membranes and measurements were taken every 2 min for up to 2 h using a plate reader in the luminescence mode (Tecan). MNCs were activated using 0.5 lM phorbol myristate acetate (PMA) while PMNCs were activated by 0.25 lM PMA. Total ROS was quantified by integrating the total area under the CL kinetic curve (AUC) using Origin Software. Tissue culture polystyrene was used in all experiments as the control. MNC and PMNC morphology After 2 hours of incubation, MNCs and PMNCs were fixed in 1.5% glutaraldehyde. The samples underwent a series of alcohol dehydration steps (10, 30, 50, 70, 90, and 99%). Hexamethyl dixilazane was used in the last dehydration step, followed by air drying. The samples were then coated with Au/Pd for scanning electron microscopy (SEM, AS02 SEM/EDS 1550, Zeiss) analysis. Statistics and data analysis All statistics was evaluated using Microsoft Excel 2000/XL Stat. The cumulative percentage of release was compared between pore sizes with a student’s t test. CL experiments were analyzed with ANOVA and Tukey’s HSD post hoc analysis. Three samples per experiment were used and each study was performed at least 3 times. RESULTS
ROS release in response to pore size The ROS released by MNCs seeded on different pore sizes was not significantly different [Figure 1(A)]. However, in contrast, PMNC activation (ROS production) was affected by pore size [Figure 1(B)]. There was a significant difference in ROS production between alumina pore sizes in PMNCs [Figure 1(B)]. PMNCs produced more ROS when exposed to 20 nm pores, as compared with 100 nm pores. PMNCs seeded on 200 nm membranes also consistently more ROS compared with 100 nm membranes, though this difference was only statistically significant in one of three experiments (p 5 0.006, 0.094, 0.365). The CL peak time was comparable between pore sizes for both MNCs and PMNCs. Peak CL typically occurred within 2–4 min after PMA exposure for both MNCs and PMNCs. Scavenging effect of Trolox on polystyrene (dose response) Tissue culture treated polystyrene was included as a control in all experiments. MNCs and PMNCs, seeded on polystyrene, exhibited a dose dependent reduction in ROS with increasing concentrations of Trolox (Figure 2). MNCs [Figure 2(A)] required 10-fold less Trolox compared to PMNCs [Figure 2(B)]. The 100 lM dose was the most effective and was, therefore, selected for subsequent experiments. The tangent lines drawn in Figure 2 describe the initial rate of ROS production in phagocytes. Trolox reduces ROS without changing the rate of production in MNCs, while in PMNCs the rate and amount of ROS is reduced.
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FIGURE 1. Differences in CL based on pore size. ROS released from MNC (A) and PMNC (B) on 20, 100, and 200 nm alumina. MNCs generated comparable ROS on all pore sizes. PMNCs released significantly greater ROS upon exposure to 20 nm as compared to 100 nm pores (p 5 0.0061).
Drug release The release profile for Trolox is shown in Figure 3. Within 15 min 80% of the loaded Trolox was released from the membranes with 20 nm pores, while a significantly greater amount (90%) was released from the 100 and 200 nm pore membranes.
External addition of Trolox The ROS released from MNCs was reduced by 97% with the addition of 100 lM of Trolox, [Figure 5(A)] for all pore sizes. No change in peak time was seen. When adding a100 lM of Trolox, the ROS was reduced by 20% for PMNCs
seeded on 20 nm pores. For 100 and 200 nm pores, no change was seen as compared with the control [Figure 5(B)]. Trolox caused a delay in CL peak time for all pore sizes, comparable to preloaded Trolox [Figure 6(D)]. Interestingly, the delay in peak time was found to be dose dependent on all pore sizes, as represented by 200 nm [Figure 5(C)] and on polystyrene [Figure 2(B)].
Preloaded Trolox When Trolox was preloaded, ROS production in MNCs [Figure 6(A,C)] was significantly reduced for all pore sizes. No
FIGURE 2. ROS release from MNCs (A) and PMNCs (B) seeded on polystyrene, with increasing concentrations of Trolox. ROS decreased with an increase in Trolox concentration. The 100 lM concentration was selected for the subsequent experiments.
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DISCUSSION
FIGURE 3. Trolox release from 20, 100, and 200 nm alumina pores. At 15 and 30 min, 20 nm membranes released significantly less drug than 100 (p 5 0.012), and 200 nm pores (p 5 0.008).
shift in peak time was seen. PMNCs [Figure 6(B,D)] exhibited significantly lower ROS production for all pore sizes compared to unloaded controls, with an average 30% reduction in ROS for 20 nm, 8% for 100 nm, and 15% for 200 nm pores. A delay in peak CL time for PMNCs was seen for all pore sizes in the presence of Trolox [Figure 6(D)]. Cell morphology PMNC [Figure 7(A)] and MNC [Figure 7(B)] morphology were both recorded after 2 hours. PMNCs cultured on the 20 nm pores, adopted a more spread morphology, with noticeable lamellopodial extensions, (typical for activation) as compared to a more round PMNC morphology seen on 100 and 200 nm pores. For Trolox preloaded membranes, PMNCs showed a similar rounded morphology, independent of pore size. For MNCs cultured on membranes without Trolox, a similar morphology was seen, independent of pore size. For preloaded membranes, cells on all pore sizes also showed a similar morphology, however, more rounded (less activated) as compared to unloaded membranes.
The purpose of this study was to determine the effect of nanotopography on mononcuclear and PMNCs, and to evaluate the feasibility of antioxidant delivery from a nanoporous vehicle. An antioxidant, Trolox, was selected as a model drug aiming to reduce the excessive inflammation that occur after a device is implanted in the body,24 as well as treat diseases stemming from extensive release of ROS.25 A device that locally delivers an antioxidant can potentially extend the lifetime of an implanted device by reducing inflammation at the site of implantation. The release profile observed in this study, and in other studies using nanoporous alumina,26 nanoporous titanium,27 oxidized aluminum,28 and mesoporous silica,29 is predominantly a burst release. [mt]60% of the loaded drug is released within 20 min, thus diffusion based models, such Peppas-Korsmeyer,30 or Higuchi,31 cannot be applied. While there appears to be significantly slower release from the smaller pore size (20 nm, p < 0.05), which is in agreement with previous studies28,32,33 the poorly defined nature of burst release prevents us from further investigating the mechanism underlying this difference. Kumeria et al. has recently reported that under conditions that allow rapid, precise, repeat measurements using a microfluidic device, over small measurement times, burst release from nanoporous material is actually Fickian with a linear release profile that could accurately be described with a modified Higuchi equation.32 Gultepe et al. reported similar findings, with the burst release from nanoporous alumina reduced to simple Fickian diffusion during the early stage of release.34 However, the creation of a novel micromeasurement device, and flow chamber were outside the scope of this study. Thus, we have limited our analysis to the type of release profile (burst), under static conditions when Trolox is loaded by simple surface evaporation/adsorption. Under these conditions, Trolox is released for 1 h after loading into nanoporous alumina. While the release rate in this study was rapid, with complete release within 1 h, we report that nanoporous alumina does not appear to irreversibly sequester or detrimentally alter drug activity. Subsequent studies should focus on extending the release time through common
FIGURE 4. Principle sketches detailing the experimental set up for adding Trolox externally (A) and preloading Trolox into the alumina membranes (B).
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FIGURE 5. When 100 lM of Trolox was added, the total ROS released from MNCs (A) was reduced by 97% on all pore sizes. For PMNCs (B), 100 lM concentration of Trolox reduced ROS by 20% on 20 nm membranes (p 5 0.024), but was unchanged on 100 and 200 nm pores. The kinetic dose response curve (C) for PMNCs on 200 nm pores indicated that increasing concentrations of Trolox led to a delay in peak CL time (representative for all 3 membranes).
approaches such as capping the pores with a slowly degrading polymer after drug loading, using the same size pore diameter but longer pore depth (greater storage capacity), drug carriers that increase drug affinity for the alumina surface. The production of ROS from activated MNCs and PMNCs plays an important role in host defense.35 Although useful in the host defense, over production of free radicals can damage the surrounding tissues.36,37 In this study the nanopore size affected the ROS production for PMNCs but not MNCs. The trend observed in the present study is in agreement with previous studies, reporting a greater inflammatory response to alumina with 20 versus 200 nm pores.38 The amount of ROS produced by MNCs and PMNCs depend upon the priming state of NADPH oxidase,39which, in turn, depends upon the cytoskeletal organization within the cell. NADPH oxidase produces more ROS when the cytoskeleton is stretched and less when it is rounded.40,41 Inflammatory cells seeded onto biomaterials with different surface roughness showed different levels of activation and thus, ROS production.42–44 Furthermore, NADPH oxidase cannot produce ROS until the cell adheres to a surface and the cytoskeleton is reorganized. Prior studies have shown that smaller pore sizes tend to increase adhesion45,46 and engage
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the lamellopodia,47 which lead to increased signaling to NADPH oxidase. In this study, the morphology of PMNCs on the 20 nm pores was more spread, with greater density of lamellopodia as compared with cells on 100 and 200 nm pores, a sign of both activation and specific reorganization of the cytoskeleton.48In contrast, the morphology of MNCs was comparable between the different pore sizes. Additionally, MNCs produced the same amount of ROS independent of pore size. Prior studies have reported that 200 nm pores promote greater MNC and macrophage cytokine production and more spread morphology as compared to 20 nm pores. The difference between these studies and this investigation, is that MNCs were stimulated with PMA immediately, rather than 24 h after seeding49 or not at all.11 Trolox is a strong antioxidant, capable of scavenging a wide variety of free radicals.50,51 On polystyrene, Trolox concentrations as low as 5 lM, significantly quenched ROS for MNCs, while PMNCs required doses in excess of 50 lM to achieve significant reduction. Trolox was a much less effective ROS quencher for PMNCs than MNCs, perhaps because PMNCs typically produce much greater respiratory bursts than MNCs. On alumina membranes, Trolox was equally effective at quenching ROS on all pore sizes, for
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FIGURE 6. When Trolox was preloaded, total ROS released from MNCs (A) was significantly reduced in response to 20 nm (0.0002), 100 nm (0.0023), and 200 nm (0.0013) pores as compared with unloaded controls. PMNCs (B) also, exhibited significantly less ROS on 20 nm (p 5 0.0001), 100 nm (p 5 0.0117), and 200 nm (p 5 0.0443) pores. The MNC kinetic curve (C) shows immediate quenching of ROS while PMNCs (D) shows a delay in ROS release as well as a shift in peak time in response to preloaded Trolox.
FIGURE 7. PMNC and MNC Cell Morphology. PMNCs (A top) seeded without Trolox exhibited a more flattened and spread morphology for the 20 nm pores, compared to the 100 and 200 nm pores. Magnification is at 8.62KX. The morophology of MNCs (B top) seeded without Trolox was comparable for all pore sizes. For preloaded Trolox PMNCs (A bottom) and MNCs (B bottom), both exhibited a more rounded morphology. Magnification is at 5 KX.
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MNCs. However, for PMNCs, a lower amount of ROS was detected when exposed to 20 nm pores in combination with Trolox. We report for the first time that the efficacy of Trolox depends on the material and nanotopography that PMNCs encounter. Although PMNCs consistently produce less ROS on alumina than on polystyrene, the scavenging effect of Trolox was less effective on all membranes as compared with polystyrene. The kinetic peak [Figure 5(C)] indicates that Trolox is not altering the rate or amount of ROS production on 200 nm pores, thus Trolox must either be sequestered or an alternate ROS source is depleting Trolox. The kinetic curve in response to the 200 nm pores show a unique effect: the ROS burst associated with NADPH oxidase activation is delayed by Trolox in a dose dependent manner, though the amount of ROS remains unchanged. This effect was observed for all pore sizes. Two key observations from this study provide a potential explanation: first MNCs do not respond to Trolox with a peak delay or to a smaller pore size with greater ROS, which suggest a difference in adhesion signaling between MNCs and PMNCs. Second, the ROS production trend in PMNCs is reversed by Trolox: PMNCs produce the most ROS in response to 20 nm pores, but the least on these pores when Trolox is present. This trend reversal suggests that Trolox may be a more effective quencher by inhibiting the source or signal causing ROS. One important difference in NADPH oxidase/adhesion signaling between MNCs and PMNCs is the form of Rac, a rho GTPase that transduces cytoskeletal changes, NADPH oxidase activity, and lamelopodia activation.48,52–54 In human MNC 95% of the Rac present is in the Rac1 isoform, while 95% of the Rac present in human PMNCs is in the Rac2 isoform. Rac2 knockouts display delayed NADPH oxidase kinetics and impaired ROS production, similar to those seen in Trolox PMNC samples.55 Thus, we hypothesize that the two key differences mentioned above are a result of Trolox interacting with Rac2 in PMNCs, but not in MNCs. This explanation is consistent with the observed reduction in lamellopodia and spreading for Trolox treated PMNCs. Alternatively, ROS production may be needed to stimulate a feedback loop that leads to NADPH Oxidase assembly. If Trolox quenches this preliminary ROS production, it would explain why we see a delay in peak CL. Rac activation has recently been shown to rely on ROS even prior to NADPH Oxidase assembly. If ROS is suppressed Rac fails to activate NADPH Oxidase and cytoskeletal reorganization is prevented.56 For PMNCs, Trolox released from the membranes (preloaded) was more efficient in scavenging ROS as compared to manually added Trolox. It is possible that a thin coating of Trolox on the preloaded surface of alumina places it into close prolonged proximity with the cellular adhesion and the NADPH oxidase apparatus of the cell, thereby increasing the effect of Trolox. However, despite the increase in antioxidant efficacy of presoaked Trolox on alumina membranes, compared to externally added, there was no significant difference in peak time between the two drug delivery models.
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It should be noted while luminol CL is a sensitive detector of hydrogen peroxide ROS, the measurements reported in this study are an indirect result of superoxide production. CONCLUSION
Biomaterial implantation triggers inflammation. An overproduction of ROS can lead to tissue damage and subsequently implant failure. We have demonstrated that nanoporous alumina can be loaded with and deliver Trolox, scavenging ROS from human primary inflammatory cells. PMNCs react to Trolox differently than MNCs. We speculate that for PMNCs Trolox delays the activity of NADPH oxidase, distinct from its antioxidant activity. We also report that Trolox is a less effective ROS quencher when added externally as compared to preloaded ACKNOWLEDGMENTS
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15. Faruqi R, Delamotte C, Dicorleto PE. Alpha-tocopherol inhibits agonist-induced monocytic cell-adhesion to cultured human endothelial-cells. J Clin Invest 1994;94:592–600. 16. Martin A, Foxall T, Blumberg JB, Meydani M. Vitamin E inhibits low-density lipoprotein-induced adhesion of monocytes to human aortic endothelial cells in vitro. Arterioscler Thromb Vasc Biol 1997;17:429–436. 17. Wu DY, Koga T, Martin KR, Meydani M. Effect of vitamin E on human aortic endothelial cell production of chemokines and adhesion to monocytes. Atherosclerosis 1999;147:297–307. 18. Satoh K, Kadofuku T, Sakagami H. Effect of Trolox, a synthetic analog of alpha-tocopherol, on cytotoxicity induced by UV irradiation and antioxidants. Anticancer Res 1997;17:2459–2463. 19. Majd ES, Goldberg M, Stanislawski L. In vitro effects of ascorbate and Trolox on the biocompatibility of dental restorative materials. Biomaterials 2003;24:3–9. 20. Miller NJ, Sampson J, Candeias LP, Bramley PM, RiceEvans CA. Antioxidant activities of carotenes and xanthophylls. Febs Lett 1996;384:240–242. 21. Thiriot C, Durand P, Jasseron MP, Kergonou JF, Ducousso R. Radiosensitive antioxidant membrane-bound factors in rat-liver microsomes .1. the roles of glutathione and vitamin-E. Biochem Int 1987;14:1–8. 22. Casini AF, Pompella A, Comporti M. Liver glutathione depletion induced by bromobenzene, iodobenzene, and diethylmaleate poisoning and its relation to lipid-peroxidation and necrosis. Am J Pathol 1985;118:225–237. 23. Dahlgren C, Karlsson A. Respiratory burst in human neutrophils. J Immunol Methods 1999;232:3–14. 24. Anderson JM, Rodriguez A, Chang DT. Foreign body reaction to biomaterials. Semin Immunol 2008;20:86–100. 25. Mirshafiey A, Mohsenzadegan M. The role of reactive oxygen species in immunopathogenesis of rheumatoid arthritis. Iran J Allergy Asthma Immunol 2008;7:195–202. 26. Kennard R, DeSisto WJ, Mason MD. Molecular diffusivity measurement through an alumina membrane using time-resolved fluorescence imaging. Appl Phys Lett 2010;97. 27. Ayon AA, Cantu M, Chava K, Agrawal CM, Feldman MD, Johnson D, Patel D, Marton D, Shi E. Drug loading of nanoporous TiO2 films. Biomed Mater 2006;1:L11–L15. 28. Kwak DH, Yoo JB, Kim DJ. Drug release behavior from nanoporous anodic aluminum oxide. J Nanosci Nanotechnol 2010;10:345–348. 29. Izquierdo-Barba I, Sousa E, Doadrio JC, Doadrio AL, Pariente JP, Martinez A, Babonneau F, Vallet-Regi M. Influence of mesoporous structure type on the controlled delivery of drugs: Release of ibuprofen from MCM-48, SBA-15 and functionalized SBA-15. J SolGel Sci Technol 2009;50:421–429. 30. Ritger PL, Peppas NA. A simple equation for description of solute release. I. Fickian and non-Fickian release from non-swellable devices in the form of slabs, spheres, cylinders or discs. J. Control Rel 1987;5:23–36. 31. Higuchi T. Mechanism of sustained-action medication. theoretical analysis of rate of release of solid drugs dispersed in solid matrices. J Pharm Sci 1963;52:1145–1149. 32. Kumeria T, Gulati K, Santos A, Losic D. Real-time and in situ drug release monitoring from nanoporous implants under dynamic flow conditions by reflectometric interference spectroscopy. Acs Appl Mater Interfaces 2013;5:5436–5442. 33. Martin F, Walczak R, Boiarski A, Cohen M, West T, Cosentino C, Shapiro J, Ferrari M. Tailoring width of microfabricated nanochannels to solute size can be used to control diffusion kinetics (vol 102, pg 123, 2005). J Controlled Release 2005;107:183–183. 34. Gultepe E, Nagesha D, Menon L, Busnaina A, Sridhar S. Highthroughput assembly of nanoelements in nanoporous alumina templates. Appl Phys Lett 2007;90. 35. Quinn MT, Gauss KA. Structure and regulation of the neutrophil respiratory burst oxidase: Comparison with nonphagocyte oxidases. J Leukoc Biol 2004;76:760–781.
36. Pelletier M, Roberge CJ, Gauthier M, Vandal K, Tessier PA, Girard D. Activation of human neutrophils in vitro and dieldrin-induced neutrophilic inflammation in vivo. J Leukoc Biol 2001;70:367–373. 37. Yang KD, Hill HR. Neutrophil function disorders: pathophysiology, prevention, and therapy. J Pediatr 1991;119:343–354. 38. Karlsson M, Johansson A, Tang L, Boman M. Nanoporous aluminum oxide affects neutrophil behaviour. Microscopy Res Tech 2004;63:259–265. 39. Wang Q, Doerschuk CM. Neutrophil-induced changes in the biomechanical properties of endothelial cells: Roles of ICAM-1 and reactive oxygen species. J Immunol 2000;164:6487–6494. 40. Berton G, Laudanna C, Sorio C, Rossi F. Generation of signals activating neutrophil functions by leukocyte integrins - Lfa-1 and Gp150/95, but not Cr3, are able to stimulate the respiratory burst of human neutrophils. J Cell Biol 1992;116:1007–1017. 41. Nathan CF. Neutrophil activation on biological surfaces - massive secretion of hydrogen-peroxide in response to products of macrophages and lymphocytes. J Clin Invest 1987;80:1550–1560. 42. Galli C, Macaluso GM, Piemontese M, Passeri G. Titanium topography controls FoxO/beta-catenin signaling. J Dent Res 2011;90: 360–364. 43. Chang S, Popowich Y, Greco RS, Haimovich B. Neutrophil survival on biomaterials is determined by surface topography. J Vasc Surg 2003;37:1082–1090. 44. Bryan N, Ashwin H, Smart N, Bayon Y, Scarborough N, Hunt JA. The innate oxygen dependant immune pathway as a sensitive parameter to predict the performance of biological graft materials. Biomaterials 2012;33:6380–6392. 45. Mohiuddin M, Pan HA, Hung YC, Huang GS. Control of growth and inflammatory response of macrophages and foam cells with nanotopography. Nanoscale Res Lett 2012;7:1-9. 46. Cavalcanti-Adam EA, Volberg T, Micoulet A, Kessler H, Geiger B, Spatz JP. Cell spreading and focal adhesion dynamics are regulated by spacing of integrin ligands. Biophys J 2007;92:2964–2974. 47. Passeri G, Cacchioli A, Ravanetti F, Galli C, Elezi E, Macaluso GM. Adhesion pattern and growth of primary human osteoblastic cells on five commercially available titanium surfaces. Clin Oral Implants Res 2010;21:756–765. 48. Cicchetti G, Allen PG, Glogauer M. Chemotactic signaling pathways in neutrophils: From receptor to actin assembly. Crit Rev Oral Biol Med 2002;13:220–228. 49. Ferraz N, Hong J, Santin M, Karlsson Ott M. Nanoporosity of alumina surfaces induces different patterns of activation in adhering monocytes/macrophages. Int J Biomater 2010;2010:402715. 50. Aruoma OI, Evans PJ, Kaur H, Sutcliffe L, Halliwell B. An evaluation of the antioxidant and potential pro-oxidant properties of food additives and of trolox C, vitamin E and probucol. Free Radic Res Commun 1990;10:143–157. 51. Cadenas E, Merenyi G, Lind J. Pulse radiolysis study of the reactivity of Trolox C phenoxyl radical with superoxide anion. Febs Lett 1989;253:235–238. 52. Dharmawardhane S, Bokoch GM. Rho GTPases and leukocyte cytoskeletal regulation. Curr Opin Hematol 1997;4:12–18. 53. Knaus UG, Heyworth PG, Evans T, Curnutte JT, Bokoch GM. Regulation of phagocyte oxygen radical production by the GTPbinding protein Rac 2. Science 1991;254:1512–1515. 54. Roberts AW, Kim C, Zhen L, Lowe JB, Kapur R, Petryniak B, Spaetti A, Pollock JD, Borneo JB, Bradford GB, Atkinson SJ, Dinauer MC, Williams DA. Deficiency of the hematopoietic cell-specific Rho family GTPase Rac2 is characterized by abnormalities in neutrophil function and host defense. Immunity 1999;10:183–196. 55. Glogauer M, Marchal CC, Zhu F, Worku A, Clausen BE, Foerster I, Marks P, Downey GP, Dinauer M, Kwiatkowski DJ. Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions. J Immunol 2003;170:5652–5657. 56. Kuiper JWP, Sun CX, Magalhaes MAO, Glogauer M. Rac regulates PtdInsP(3) signaling and the chemotactic compass through a redox-mediated feedback loop. Blood 2011;118:6164–6171.
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to 20 versus 100 nm pores. For Trolox loaded membranes, this trend reversed, due to slower release of antioxidant from the 20 nm pores. Furthermore, Trolox exhibited a unique effect on PMNCs that has not previously been reported: It delayed the production of ROS in a manner distinct from antioxidant activity. The present study confirms that nanoporous alumina is a suitable vehicle for drug delivery, and that Trolox can successfully modulate the inflammatory response C 2015 Wiley Periodicals, Inc. J Biomed of both MNC and PMNCs. V Mater Res Part B: Appl Biomater, 104B: 568–575, 2016.
Key Words: nanoporous alumina, Trolox, mononuclear cells, polymorphonuclear cells, reactive oxygen species
How to cite this article: Pujari-Palmer S, Pujari-Palmer M, Karlsson Ott M. 2016. Reduced oxidative stress in primary human cells by antioxidant released from nanoporous alumina. J Biomed Mater Res Part B 2016:104B:568–575.
INTRODUCTION
Biomedical implants have become increasingly important as the age and lifetime of the current population expands. Implanted biomaterials can successfully improve the quality and length of life for patients suffering from disease or injury.1 Within the native cellular environment, the extracellular matrix contains biological and topographical features, on the nanometer scale, that facilitate cell-cell interactions and tissue integration during healing.2,3 There is, therefore, an increasing drive to design implants that mimic the native biological environment in order to promote and increase implant integration and healing. Anodized aluminum is a well characterized material.4 An ordered nanoporous pattern is fabricated through anodic oxidation of aluminum (anodization) in polyprotic acids.5,6 Nanoporous alumina is a promising candidate for bone implant coatings,7 immunoisolation devices,8 drug delivery devices for stent coatings,9 and for cocultivation of cells.10 Previous studies on nanoporous alumina have shown that 200 nm pores elicit a greater inflammatory response in vitro and in vivo, compared to 20 nm pores.11,12 Few studies, however, have focused on how to specifically decrease inflammation in response to this material. Vitamin E supplementation in vitro, resulted in a decrease in the production of proinflammory cytokines and adhesion molecules in
immune and endothelial cells.13–17 Trolox is a water soluble vitamin E derivative and a potent quencher of hydroxyl and peroxide free radicals. The hydrophilic nature of Trolox makes it an ideal antioxidant since it can readily disperse throughout the cytoplasm. It also exhibits minimal cytotoxicity,18,19 and is the standard by which antioxidant activity is measured (Trolox equivalent antioxidant capacity (TEAC))9.20 Furthermore, Trolox has shown to reduce lipid peroxidation after whole-body gamma irradiation21 and prevent liver necrosis.22 The aim of this study is (a) to compare the activation of primary human mononuclear (MNC) and polymorphonuclear (PMNC) cells when exposed to alumina with 20, 100, and 200 nm pores; (b) to determine whether an antioxidant, Trolox, can reduce or prevent inflammatory cell activation in response to nanoporosity, and finally, (c) to determine whether nanoporous alumina can be loaded with and deliver antioxidants to reduce PMNC and MNC activation. MATERIALS AND METHODS
Cell isolation Human MNC Isolation: Blood buffy coats were obtained from anonymous blood donors from the Uppsala University Hospital. MNCs were isolated using the Ficoll-Paque Plus density gradient centrifugation according to the
Correspondence to: M. Karlsson Ott; e-mail: [email protected] € r internationalisering av ho € gre utbildning och forskning) Contract grant sponsor: STINT (Stiftelsen fo
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manufacturer’s instructions. Briefly, blood was overlaid on top of the Ficoll-Paque Plus and centrifuged at 1200g for 20 min. The MNC layer was then collected and washed three times with phosphate buffered saline (PBS). The total cell number was determined using a hemocytometer (adding 0.4% trypan blue to determine viability). Cells were then seeded on the alumina membranes (400,000 cells/well). Human PMNC isolation.. PMNC isolation underwent 2 separation procedures. Briefly, buffy coat blood was subjected to Ficoll-Paque Plus density gradient centrifugation, as described previously. The blood pellet formed was then resuspended in 3% Dextran/0.9% saline solution for 25 min. The upper plasma layer was collected and centrifuged at 250g for 10 min. Contaminating erythrocytes were removed by adding 0.2% saline solution to the pellet for 20 s. An equal volume of 1.6% saline solution was then added and the cell suspension was centrifuged. The total cell number for each fraction was determined by counting on a hemocytometer with 0.4% trypan blue to determine viability, and 3% acetic acid to differentiate mononuclear from PMNC. Cells were then seeded on the alumina membranes (300,000 cells/well). Drug loading and release Drug loading. A 20 lL volume of a 60 mM ethanol solution of Trolox was added to each nanoporous alumina membrane (Anodisc Whatman International, Madison, England) and allowed to evaporate at room temperature for 30 min. Drug release. An elution volume of 500 lL of phosphate buffered saline was replaced every 10 min, for the first hour, and every hour thereafter for 5 h. The elution volume was also replaced after 16 and 24 h. Trolox release was measured with a UV Spectrophotometer at 290 nm. Delivery methods for in vitro studies. Trolox was delivered either by direct external addition or preloaded in the alumina membranes, hereafter referred to as preloaded. For external addition, 100 lM of Trolox was added. To simulate a dynamic release of 100 lM of preloaded Trolox, 60 mM was loaded in the membranes. The membranes were soaked in PBS for 15 min (prior to the cell studies) releasing excess Trolox, thus leaving 100 lM in the membranes. Chemiluminescence To quantify the generation of reactive oxygen species (ROS) released by the cells, luminol amplified chemiluminescence (CL) was used.23 All measurements were performed in white 24 well optiplates (Perkin Elmer) at 37 C in medium containing a 4:1 ratio of PBS to RPMI-1640 medium containing100 mM glucose. The luminol solution (500 lM) was prepared by incorporating 1% of luminol taken from the stock solution, 2% horse radish peroxidase (2 lg/mL), and a 1:100 ratio of 0.1M NaOH into the 4:1 PBS to RPMI-1640 containing100 mM glucose medium. The luminol stock solution was prepared by dissolving 50 mM of luminol into 0.2M NaOH. 400,000 MNCs/well
and 300,000 PMNCs/well were seeded on the membranes and measurements were taken every 2 min for up to 2 h using a plate reader in the luminescence mode (Tecan). MNCs were activated using 0.5 lM phorbol myristate acetate (PMA) while PMNCs were activated by 0.25 lM PMA. Total ROS was quantified by integrating the total area under the CL kinetic curve (AUC) using Origin Software. Tissue culture polystyrene was used in all experiments as the control. MNC and PMNC morphology After 2 hours of incubation, MNCs and PMNCs were fixed in 1.5% glutaraldehyde. The samples underwent a series of alcohol dehydration steps (10, 30, 50, 70, 90, and 99%). Hexamethyl dixilazane was used in the last dehydration step, followed by air drying. The samples were then coated with Au/Pd for scanning electron microscopy (SEM, AS02 SEM/EDS 1550, Zeiss) analysis. Statistics and data analysis All statistics was evaluated using Microsoft Excel 2000/XL Stat. The cumulative percentage of release was compared between pore sizes with a student’s t test. CL experiments were analyzed with ANOVA and Tukey’s HSD post hoc analysis. Three samples per experiment were used and each study was performed at least 3 times. RESULTS
ROS release in response to pore size The ROS released by MNCs seeded on different pore sizes was not significantly different [Figure 1(A)]. However, in contrast, PMNC activation (ROS production) was affected by pore size [Figure 1(B)]. There was a significant difference in ROS production between alumina pore sizes in PMNCs [Figure 1(B)]. PMNCs produced more ROS when exposed to 20 nm pores, as compared with 100 nm pores. PMNCs seeded on 200 nm membranes also consistently more ROS compared with 100 nm membranes, though this difference was only statistically significant in one of three experiments (p 5 0.006, 0.094, 0.365). The CL peak time was comparable between pore sizes for both MNCs and PMNCs. Peak CL typically occurred within 2–4 min after PMA exposure for both MNCs and PMNCs. Scavenging effect of Trolox on polystyrene (dose response) Tissue culture treated polystyrene was included as a control in all experiments. MNCs and PMNCs, seeded on polystyrene, exhibited a dose dependent reduction in ROS with increasing concentrations of Trolox (Figure 2). MNCs [Figure 2(A)] required 10-fold less Trolox compared to PMNCs [Figure 2(B)]. The 100 lM dose was the most effective and was, therefore, selected for subsequent experiments. The tangent lines drawn in Figure 2 describe the initial rate of ROS production in phagocytes. Trolox reduces ROS without changing the rate of production in MNCs, while in PMNCs the rate and amount of ROS is reduced.
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FIGURE 1. Differences in CL based on pore size. ROS released from MNC (A) and PMNC (B) on 20, 100, and 200 nm alumina. MNCs generated comparable ROS on all pore sizes. PMNCs released significantly greater ROS upon exposure to 20 nm as compared to 100 nm pores (p 5 0.0061).
Drug release The release profile for Trolox is shown in Figure 3. Within 15 min 80% of the loaded Trolox was released from the membranes with 20 nm pores, while a significantly greater amount (90%) was released from the 100 and 200 nm pore membranes.
External addition of Trolox The ROS released from MNCs was reduced by 97% with the addition of 100 lM of Trolox, [Figure 5(A)] for all pore sizes. No change in peak time was seen. When adding a100 lM of Trolox, the ROS was reduced by 20% for PMNCs
seeded on 20 nm pores. For 100 and 200 nm pores, no change was seen as compared with the control [Figure 5(B)]. Trolox caused a delay in CL peak time for all pore sizes, comparable to preloaded Trolox [Figure 6(D)]. Interestingly, the delay in peak time was found to be dose dependent on all pore sizes, as represented by 200 nm [Figure 5(C)] and on polystyrene [Figure 2(B)].
Preloaded Trolox When Trolox was preloaded, ROS production in MNCs [Figure 6(A,C)] was significantly reduced for all pore sizes. No
FIGURE 2. ROS release from MNCs (A) and PMNCs (B) seeded on polystyrene, with increasing concentrations of Trolox. ROS decreased with an increase in Trolox concentration. The 100 lM concentration was selected for the subsequent experiments.
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DISCUSSION
FIGURE 3. Trolox release from 20, 100, and 200 nm alumina pores. At 15 and 30 min, 20 nm membranes released significantly less drug than 100 (p 5 0.012), and 200 nm pores (p 5 0.008).
shift in peak time was seen. PMNCs [Figure 6(B,D)] exhibited significantly lower ROS production for all pore sizes compared to unloaded controls, with an average 30% reduction in ROS for 20 nm, 8% for 100 nm, and 15% for 200 nm pores. A delay in peak CL time for PMNCs was seen for all pore sizes in the presence of Trolox [Figure 6(D)]. Cell morphology PMNC [Figure 7(A)] and MNC [Figure 7(B)] morphology were both recorded after 2 hours. PMNCs cultured on the 20 nm pores, adopted a more spread morphology, with noticeable lamellopodial extensions, (typical for activation) as compared to a more round PMNC morphology seen on 100 and 200 nm pores. For Trolox preloaded membranes, PMNCs showed a similar rounded morphology, independent of pore size. For MNCs cultured on membranes without Trolox, a similar morphology was seen, independent of pore size. For preloaded membranes, cells on all pore sizes also showed a similar morphology, however, more rounded (less activated) as compared to unloaded membranes.
The purpose of this study was to determine the effect of nanotopography on mononcuclear and PMNCs, and to evaluate the feasibility of antioxidant delivery from a nanoporous vehicle. An antioxidant, Trolox, was selected as a model drug aiming to reduce the excessive inflammation that occur after a device is implanted in the body,24 as well as treat diseases stemming from extensive release of ROS.25 A device that locally delivers an antioxidant can potentially extend the lifetime of an implanted device by reducing inflammation at the site of implantation. The release profile observed in this study, and in other studies using nanoporous alumina,26 nanoporous titanium,27 oxidized aluminum,28 and mesoporous silica,29 is predominantly a burst release. [mt]60% of the loaded drug is released within 20 min, thus diffusion based models, such Peppas-Korsmeyer,30 or Higuchi,31 cannot be applied. While there appears to be significantly slower release from the smaller pore size (20 nm, p < 0.05), which is in agreement with previous studies28,32,33 the poorly defined nature of burst release prevents us from further investigating the mechanism underlying this difference. Kumeria et al. has recently reported that under conditions that allow rapid, precise, repeat measurements using a microfluidic device, over small measurement times, burst release from nanoporous material is actually Fickian with a linear release profile that could accurately be described with a modified Higuchi equation.32 Gultepe et al. reported similar findings, with the burst release from nanoporous alumina reduced to simple Fickian diffusion during the early stage of release.34 However, the creation of a novel micromeasurement device, and flow chamber were outside the scope of this study. Thus, we have limited our analysis to the type of release profile (burst), under static conditions when Trolox is loaded by simple surface evaporation/adsorption. Under these conditions, Trolox is released for 1 h after loading into nanoporous alumina. While the release rate in this study was rapid, with complete release within 1 h, we report that nanoporous alumina does not appear to irreversibly sequester or detrimentally alter drug activity. Subsequent studies should focus on extending the release time through common
FIGURE 4. Principle sketches detailing the experimental set up for adding Trolox externally (A) and preloading Trolox into the alumina membranes (B).
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FIGURE 5. When 100 lM of Trolox was added, the total ROS released from MNCs (A) was reduced by 97% on all pore sizes. For PMNCs (B), 100 lM concentration of Trolox reduced ROS by 20% on 20 nm membranes (p 5 0.024), but was unchanged on 100 and 200 nm pores. The kinetic dose response curve (C) for PMNCs on 200 nm pores indicated that increasing concentrations of Trolox led to a delay in peak CL time (representative for all 3 membranes).
approaches such as capping the pores with a slowly degrading polymer after drug loading, using the same size pore diameter but longer pore depth (greater storage capacity), drug carriers that increase drug affinity for the alumina surface. The production of ROS from activated MNCs and PMNCs plays an important role in host defense.35 Although useful in the host defense, over production of free radicals can damage the surrounding tissues.36,37 In this study the nanopore size affected the ROS production for PMNCs but not MNCs. The trend observed in the present study is in agreement with previous studies, reporting a greater inflammatory response to alumina with 20 versus 200 nm pores.38 The amount of ROS produced by MNCs and PMNCs depend upon the priming state of NADPH oxidase,39which, in turn, depends upon the cytoskeletal organization within the cell. NADPH oxidase produces more ROS when the cytoskeleton is stretched and less when it is rounded.40,41 Inflammatory cells seeded onto biomaterials with different surface roughness showed different levels of activation and thus, ROS production.42–44 Furthermore, NADPH oxidase cannot produce ROS until the cell adheres to a surface and the cytoskeleton is reorganized. Prior studies have shown that smaller pore sizes tend to increase adhesion45,46 and engage
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the lamellopodia,47 which lead to increased signaling to NADPH oxidase. In this study, the morphology of PMNCs on the 20 nm pores was more spread, with greater density of lamellopodia as compared with cells on 100 and 200 nm pores, a sign of both activation and specific reorganization of the cytoskeleton.48In contrast, the morphology of MNCs was comparable between the different pore sizes. Additionally, MNCs produced the same amount of ROS independent of pore size. Prior studies have reported that 200 nm pores promote greater MNC and macrophage cytokine production and more spread morphology as compared to 20 nm pores. The difference between these studies and this investigation, is that MNCs were stimulated with PMA immediately, rather than 24 h after seeding49 or not at all.11 Trolox is a strong antioxidant, capable of scavenging a wide variety of free radicals.50,51 On polystyrene, Trolox concentrations as low as 5 lM, significantly quenched ROS for MNCs, while PMNCs required doses in excess of 50 lM to achieve significant reduction. Trolox was a much less effective ROS quencher for PMNCs than MNCs, perhaps because PMNCs typically produce much greater respiratory bursts than MNCs. On alumina membranes, Trolox was equally effective at quenching ROS on all pore sizes, for
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FIGURE 6. When Trolox was preloaded, total ROS released from MNCs (A) was significantly reduced in response to 20 nm (0.0002), 100 nm (0.0023), and 200 nm (0.0013) pores as compared with unloaded controls. PMNCs (B) also, exhibited significantly less ROS on 20 nm (p 5 0.0001), 100 nm (p 5 0.0117), and 200 nm (p 5 0.0443) pores. The MNC kinetic curve (C) shows immediate quenching of ROS while PMNCs (D) shows a delay in ROS release as well as a shift in peak time in response to preloaded Trolox.
FIGURE 7. PMNC and MNC Cell Morphology. PMNCs (A top) seeded without Trolox exhibited a more flattened and spread morphology for the 20 nm pores, compared to the 100 and 200 nm pores. Magnification is at 8.62KX. The morophology of MNCs (B top) seeded without Trolox was comparable for all pore sizes. For preloaded Trolox PMNCs (A bottom) and MNCs (B bottom), both exhibited a more rounded morphology. Magnification is at 5 KX.
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MNCs. However, for PMNCs, a lower amount of ROS was detected when exposed to 20 nm pores in combination with Trolox. We report for the first time that the efficacy of Trolox depends on the material and nanotopography that PMNCs encounter. Although PMNCs consistently produce less ROS on alumina than on polystyrene, the scavenging effect of Trolox was less effective on all membranes as compared with polystyrene. The kinetic peak [Figure 5(C)] indicates that Trolox is not altering the rate or amount of ROS production on 200 nm pores, thus Trolox must either be sequestered or an alternate ROS source is depleting Trolox. The kinetic curve in response to the 200 nm pores show a unique effect: the ROS burst associated with NADPH oxidase activation is delayed by Trolox in a dose dependent manner, though the amount of ROS remains unchanged. This effect was observed for all pore sizes. Two key observations from this study provide a potential explanation: first MNCs do not respond to Trolox with a peak delay or to a smaller pore size with greater ROS, which suggest a difference in adhesion signaling between MNCs and PMNCs. Second, the ROS production trend in PMNCs is reversed by Trolox: PMNCs produce the most ROS in response to 20 nm pores, but the least on these pores when Trolox is present. This trend reversal suggests that Trolox may be a more effective quencher by inhibiting the source or signal causing ROS. One important difference in NADPH oxidase/adhesion signaling between MNCs and PMNCs is the form of Rac, a rho GTPase that transduces cytoskeletal changes, NADPH oxidase activity, and lamelopodia activation.48,52–54 In human MNC 95% of the Rac present is in the Rac1 isoform, while 95% of the Rac present in human PMNCs is in the Rac2 isoform. Rac2 knockouts display delayed NADPH oxidase kinetics and impaired ROS production, similar to those seen in Trolox PMNC samples.55 Thus, we hypothesize that the two key differences mentioned above are a result of Trolox interacting with Rac2 in PMNCs, but not in MNCs. This explanation is consistent with the observed reduction in lamellopodia and spreading for Trolox treated PMNCs. Alternatively, ROS production may be needed to stimulate a feedback loop that leads to NADPH Oxidase assembly. If Trolox quenches this preliminary ROS production, it would explain why we see a delay in peak CL. Rac activation has recently been shown to rely on ROS even prior to NADPH Oxidase assembly. If ROS is suppressed Rac fails to activate NADPH Oxidase and cytoskeletal reorganization is prevented.56 For PMNCs, Trolox released from the membranes (preloaded) was more efficient in scavenging ROS as compared to manually added Trolox. It is possible that a thin coating of Trolox on the preloaded surface of alumina places it into close prolonged proximity with the cellular adhesion and the NADPH oxidase apparatus of the cell, thereby increasing the effect of Trolox. However, despite the increase in antioxidant efficacy of presoaked Trolox on alumina membranes, compared to externally added, there was no significant difference in peak time between the two drug delivery models.
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It should be noted while luminol CL is a sensitive detector of hydrogen peroxide ROS, the measurements reported in this study are an indirect result of superoxide production. CONCLUSION
Biomaterial implantation triggers inflammation. An overproduction of ROS can lead to tissue damage and subsequently implant failure. We have demonstrated that nanoporous alumina can be loaded with and deliver Trolox, scavenging ROS from human primary inflammatory cells. PMNCs react to Trolox differently than MNCs. We speculate that for PMNCs Trolox delays the activity of NADPH oxidase, distinct from its antioxidant activity. We also report that Trolox is a less effective ROS quencher when added externally as compared to preloaded ACKNOWLEDGMENTS
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JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | APR 2016 VOL 104B, ISSUE 3
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