Misinterpretation in Nanotoxicology: A Personal Perspective

Jun 1, 2016 - D. degree from Shiraz University of Medical Sciences in 2012. She is now a Ph.D. candidate in Pharmaceutical Nanotechnology at Tehran ...
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Misinterpretation in Nanotoxicology: A Personal Perspective Alaaldin M. Alkilany,*,† Nouf N. Mahmoud,† Fatemeh Hashemi,‡ Mohammad J. Hajipour,⊥ Fakhrosadat Farvadi,‡ and Morteza Mahmoudi*,‡,§ †

Department of Pharmaceutics & Pharmaceutical Technology, Faculty of Pharmacy, The University of Jordan, Amman 11942, Jordan Department of Nanotechnology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran § Department of Anesthesiology, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States ⊥ The Persian Gulf Marine Biotechnology Research Center, The Persian Gulf Biomedical Sciences Research Institute, Bushehr University of Medical Sciences, Bushehr, Iran ‡

ABSTRACT: As an emerging field, nanotoxicology is gaining significant interest from scientists as well as from international regulatory firms in an attempt to build accumulated knowledge on this topic, which will be the basis for regulatory codes and safer nanotechnology. However, conflicting results and findings are abundant in the literature calling for more careful experimental design, result interpretation, and detailed reporting. In this perspective, we focus on misinterpretation in nanotoxicology and highlight the importance of proper experimental practice to avoid artifacts by discussing various examples from the literature.

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CONTENTS

Introduction Nanotoxicology: The Supernatant Control Do Cationic NPs in Solution Stay Cationic in Cellular Media? Colloidal Stability of NPs in Altered Media Colloidal Stability of NPs upon Contact with Skin: Single NP versus NP Aggregate Cellular Uptake of NPs On Cell versus in Cell Protein Source Variation in Cell Responses to NPs: Cellular Heterogeneity and Cell Cycle Disease State and Pathophysiology: Important Factor Toxicity Assays of NPs: Interferences and Reliability Conclusions Author Information Corresponding Authors Notes Biographies Abbreviations References

experimental artifacts (here we mean misinterpretation) can be found in nanoscience, too. However, we may employ nanoscience in preparing beautiful artifacts, but as nanoscientists, we need to avoid undesired artifacts in nanoscience as much as possible, which in fact can be a major challenge. Such misinterpretations could cause severe issues in the progress of nanotoxicology, which put off the clinical use of nanoparticles (NPs). In this perspective, we discuss examples from various nanotechnology-based experimentations, in which we show how misinterpretation of experimental results and/or lack of proper controls may be crucial and result in improper conclusions regarding cellular uptake of NPs and their apparent toxicity. This perspective should help in building accumulated knowledge with more confidence and less debate. It is important to note that the provided examples and discussion herein describes “honest error” and not scientific “misconduct”.2 Nanotoxicology: The Supernatant Control. A major challenge in nanotechnology and nanomaterials is their associated toxicity to living systems and potential adverse effect on the environment.3,4 With this in mind, nanotoxicology is a rapidly growing discipline gaining significant research interest at both the academic and regulatory levels.5 Although the number of publications in the field is significantly increasing, serious concerns are emerging regarding the reproducibility and validity of published reports. The lack of

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INTRODUCTION While nanoscience can be found in artifacts (such as the famous Lycurgus cup and in stained-glass in European Churches),1 © XXXX American Chemical Society

Received: April 1, 2016

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studies still ignore this important control, making the interpretation of toxicological results a challenging task. Do Cationic NPs in Solution Stay Cationic in Cellular Media? A general thought predicts a higher toxicity for cationic NPs compared to that of the anionic counterparts due to the ability of the former to disrupt the negatively charged cellular membrane by simple electrostatic interaction. Interestingly, we and others found that this thought is not necessarily true, as strong evidences suggested a spontaneous flip in the effective surface charge of cationic NPs upon contact with growth media due to the formation of a negatively charged protein corona.9,11−14 Indeed, the physiochemical properties of NPs in solution may differ dramatically from what cells see14 in biological fluids including cellular growth media (in our case, cells see anionic NPs due to adsorption of proteins and not the original cationic counterpart). With this in mind, changes in the effective surface charge of NPs upon exposure to biological media should be always evaluated as part of any toxicological study. Colloidal Stability of NPs in Altered Media. The colloidal stability of NPs has direct biological consequences, which is significantly changed in biological media.15 Depending on the type and amount of proteins bound to their surfaces, the NPs showed different colloidal stabilities in different media.16,17 The serum proteins adsorbed on the NP surface can intensify or reduce the NP aggregation via screening NP charge or enhancement of entropic/steric stability, respectively.15,17 For example, the amount of hemoglobin bound to the gold NPs determines their agglomeration level. The highest level of aggregation occurred when a monolayer of hemoglobin formed on the gold NP surface.17 In vitro studies showed that the composition of extracellular media is dynamically changed through biomolecule excretion/exhaust caused by cells.18 These changes affect the protein adsorption on the NP surface and consequent NP biological identity. Recently, it has been shown that cellular culture media are in dynamic change in composition and properties, which ultimately affect nanoparticle aggregation and influence their cytotoxic impact. Therefore, overlooking the colloidal stability of NPs in serum/extracellular environment/cellular growth media may result in contradictory data in nanotoxicology. NPs can aggregate in cellular media by various mechanisms such as electrolyte-induced charge screening. Aggregates of NPs have different sedimentation rates, and therefore, larger aggregates reach adherent cell layers quickly forming higher local concentration of NPs, which significantly affect the cellular uptake.19 Thus, the cellular uptake profiles of nanoformulations could be considerably different due to basically colloidal stability rather than other parameters such as particle size, surface charge, and the presence of targeting molecules and their density.20 Moreover, the uptake mechanisms of aggregates are totally different from single NPs which may result in artifacts in concluding regarding the mechanistic details on NP cellular entry as has been shown recently.21 Thus, the colloidal stability of NPs in cellular media is a critical parameter related to nanotoxicity and cellular uptake, which unfortunately, is usually ignored. Colloidal Stability of NPs upon Contact with Skin: Single NP versus NP Aggregate. Understanding the nanoskin interface is very essential to designing effective nanotherapeutics in order to treat various skin disorders as well as to develop dermal drug delivery systems.22 In many cases, nanobio contact may potentially disturb the colloidal stability of NPs

reproducibility of nanotoxicological results may occur for several reasons including (but not limited to) insufficient controls, inappropriate analysis techniques/software/toxicology-assays, small sample size, and inadequate pitfall reporting.6,7 Besides the predetermined reasons, which were fully discussed in recent publications,8 in this section, we are introducing the crucial effect of the supernatant control. Despite the extremely large number of scientific publications assessing NP toxicity, few studies compare the toxicity of NP solution with its corresponding supernatant solution as negative and essential controls. To resolve the contribution of cationic NP toxicity from the toxicity of leftover chemicals/ions freely present in the same solution, we compared the toxicity of purified cationic gold nanorod (GNR) solution with its supernatant (no GNR).9 Surprisingly, our results indicated that GNR solution and its supernatant have similar toxicity profiles to cultured cell lines, indicating that the toxicity of GNR solution originates from chemicals in the solution rather than the NPs themselves. Quantitatively, we determined a free cationic capping agent in the solution (cetyltrimethylammonium bromide, CTAB) to be the toxic part and not bound CTAB to GNRs. Unfortunately, we were unable to ultimately clean up our GNRs from free CTAB due to induced irreversible aggregation of GNRs during the purification process. Zubarev and co-workers used the thiolated version of the toxic CTAB to fix them on the surface of GNRs, which allowed for a more thorough cleanup process (very low levels of free CTAB) without aggregation.10 Interestingly, they showed a dramatic decrease in the toxicity of their GNRs. Their results also confirmed our finding that the toxicity of cationic NPs is not simply due to the positive surface charge (their GNRs showed a zeta potential of +55 mV) but mostly due to free toxic chemicals (free cationic CTAB). From these studies, we can extract a take home lesson considering the supernatant solution as an essential negative control that should be implemented in nanotoxicological studies to unveil the origin of nanoparticle toxicity (Figure 1). The significant effect of the free chemicals in solution is now widely accepted. However, a majority of

Figure 1. Supernatant control in nanotoxicological evaluation. The conclusion on the toxicity of NP solution should be coupled with a comparative toxicological evaluation between the original solution (left) and its corresponding supernatant (right). This negative control eliminates possible false positive toxicity of NPs that may arise from leftover free chemicals/impurities in the original solution. B

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since it is the basis of nanomedicine and nanotoxicity.1,24 A large body of research is devoted to understand the mechanism of NP entry to cells as a function of NP physiochemical properties and exposure variables.25,26 Analytical tools to quantify the cellular uptake do not distinguish between internalized fractions of NPs in cell from associated NPs with cell surfaces on cell (Figure 3).27,28 For example, quantifying the cellular uptake of metallic NPs is usually carried out by collecting cell mass or isolated organs followed by acid digestion and analysis using inductively coupled plasma spectrometry/spectroscopy techniques such as ICP-MS or ICP-OES. Despite their excellent selectivity and sensitivity, these analytical techniques are destructive in nature and cannot distinguish between NPs in or on cells. Few experimental procedures were developed to address this problem. For example, comparison between cellular uptakes at low (4 °C) or nominal (37 °C) temperature is used to estimate the contribution of adsorbed NPs (at low temperature) on cells versus those internalized at nominal temperature. 29−31 However, we need to note here that the temperature approach should be used with caution since even very slight changes in temperature may change the protein corona decoration at the surface of NPs and affect their uptake profiles.32,33 Another approach was used for gold NPs by chemically selective itching of adsorbed NPs using KI/I2 solution prior to total analysis using ICP-MS.27 Unfortunately, an effort to differentiate between in and on cells is often overlooked in various research reports. Protein Source. The protein source is an important factor that can significantly affect the protein corona structure on the surface of NPs and thus can substantially change their uptakes by cells.34−36 Recent findings demonstrated that the type of human diseases can significantly change the protein source, and therefore, their corona structures (so-called patient-specific or personalized protein coronas) can change the biological fate of NPs in a patient-specific manner.37,38 The findings clearly reveal that a considerable part of the available in vitro results in the protein corona field is valid for healthy human cases (with the hypothesis that the employed human serum/plasma was extracted from healthy individuals) and could not be assumed for patients (as their protein source variation may cause significant changes in biological responses). In order to have more meaningful and precise corona data, the authors should provide the exact information with regard to the human serum/ plasma source. Variation in Cell Responses to NPs: Cellular Heterogeneity and Cell Cycle. Our previous studies demonstrated that various cells differently respond to identical NPs.39 The exact same NPs showed different uptake levels, intracellular localization, and toxic effects when they were exposed to different cells.6,40Therefore, the results of nanotoxicological studies achieved for specific cells could not be assumed for others. This fact should be considered to reduce the possible contradictory results. In addition to cell type, cellular heterogeneity affects the accuracy of nanotoxicology assays. Indeed, the heterogeneity at the single-cell level is the main cause of variability in cell population average responses to the NPs.41 Cell cycle is another parameter that influences cell response to the NPs. The cellular uptake efficacy of NPs is, in part, dependent on different phases of cell cycles. For example, the highest and the least NP uptake levels were at G2/M and G0/G1 phases, respectively.42 Overlooking the hidden factors

and increase the tendency of NPs to aggregate. This is very important because aggregated NPs behave in a different way from an individual nanoparticle upon contact with tissues or cells. The uptake or diffusion of such aggregated NPs into cells or tissues will ultimately be under or overestimated and definitely will result in serious data misinterpretation and artifacts. For example, the stability of NPs upon skin contact is usually overlooked and ignored in the majority of published studies investigating the penetration of NPs through skin. This ignored parameter contributes to the current conflicting results and debatable findings regarding the penetration of NPs into skin. We recently investigated the colloidal stability of GNR solutions upon exposure to human skin.23 Interestingly, we found that GNRs coated with a cationic polymer (poly(allylamine hydrochloride), PAH) aggregated severely upon contact to skin compared to those coated with an anionic polymer (poly acrylic acid, PAA) or a neutrally thiolated polymer (poly ethylene glycol, PEG). We have found also that negatively charged skin-secreted biomolecules (e.g., proteins) were released from skin, adsorbed on the surface of cationic GNRs, and induced GNR aggregation. Collectively, we did conclude that in order to precisely compare the tendency of different NPs to penetrate the skin, a systematic evaluation of the colloidal stability of each type upon contact with skin should be carefully and thoroughly addressed to prevent result misinterpretation and artifacts (Figure 2). For example, when

Figure 2. Cartoon demonstrates skin penetration by nonaggregated nanorods (left) versus retarder penetration of aggregated clusters of nanorods (right).

we consider reported results concluding that anionic-NPs were superior to cationic counterparts in targeting skin and hair follicles, it is hard to tell if this observation originates from superior colloidal stability of the anionic NPs or due to the intrinsic effect of the charge itself. In this section, we discussed an example related to skin, but we do believe that evaluation of colloidal stability of nanomaterials upon exposure to other tissues/organs is critical and should not be overlooked. Cellular Uptake of NPs. On Cell versus in Cell. Cellular uptake of NPs is a critical parameter to be evaluated precisely C

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Figure 3. (A) Cartoon demonstrates nonaggregated NPs versus aggregated clusters of NPs in biological medium such as cell growth media. (B) NPs adsorbed on cells versus NPs internalized into cellular compartments. (C) TEM images of vascular endothelial cell exposed to cationic gold nanoparticles followed by thorough washing and imaging (note the presence of both adsorbed and internalized NPs). Scale bar = 0.5 μm.

assays needs to be evaluated case by case to select a suitable assay for each NP type. Moreover, essential modifications on the conventional toxicity approaches should be applied.49

which affect the cell response to NPs may result in conflicting reports in future studies. Disease State and Pathophysiology: Important Factor. For in vivo studies, the progress and pathophysiology of diseases should be considered in nanotoxicology assays.43 A deep knowledge on the disease state/progress provides a unique chance to assess the real toxic/therapeutic impacts of NPs. The susceptibility and amenability of patients to NPs is dependent on the disease stage. In the early stages of Alzheimer’s disease, the NPs can be more effective rather than at late stages. This is mainly because the amounts of the amyloid beta proteins, which experience conformational changes in favor of fibrillation processes, are much lower in early stages compared to that at the late stage of the disease. Indeed, the oligomers and preformed fibrils should be targeted in the first and end stages of Alzheimer’s disease, respectively.44 It is well recognized that interstitial fluid pressure increase occurred after cancer growth, hindering NP distribution to the tumor area.45,46 Moreover, the NPs circulating in blood have more accessibility to cancer cells at early stages of cancer. Therefore, it can be concluded that the NPs may show more cytotoxic effects against early stage tumors rather than late stage tumors. Toxicity Assays of NPs: Interferences and Reliability. The NPs can interfere with current nanotoxicity assays due to their intrinsic properties such as optical and magnetic properties, high adsorption capability, and catalytic activity. For instance, NPs can change the cell medium composition leading to the entrance of large error in cytotoxicity monitoring approaches such as 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide (MTT) assay.6,47 For example, Goss and coworkers reported that CdSe 100 and helical rosette nanotubes interfered with the MTS assay (assay that is used widely to assess the toxicity of nanoparticles similar to that of MTT with higher water solubility). Interestingly, the authors reported that the presence of these nanoparticles resulted in false positive calculation of 547 ± 224 (CdSe) and 1118 ± 89 cells (rosette nanotubes) in a cell-free assay.48 In order to minimize misinterpretation of the results, the accuracy of NP toxicity

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CONCLUSIONS In summary, numerous research studies in nanotechnology and nanotoxicology have identified many potential experimental artifacts. Such artifacts usually lead to the increased risk of incorrect conclusions and data misinterpretations regarding understanding the nano−bio interface and toxicology. Proper experimental design and inclusion of well-designed control experiments will help to prevent such artifacts and misinterpretation of results and definitely increase the reliability of conclusions and test findings.

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AUTHOR INFORMATION

Corresponding Authors

*(A.M.A.) E-mail: [email protected]. *(M.M.) E-mail: [email protected]. Notes

The authors declare no competing financial interest. Biographies Dr. Alaaldin M. Alkilany obtained his Ph.D. degree in Chemistry under the supervision of Professor Catherine Murphy at the University of Illinois Urbana-Champaign with research focus on gold nanotechnology and its interactions with cells. Afterward, Dr. Alkilany accepted a postdoctoral research position at the Augusta University, U.S.A. to perform a multidisciplinary research on understanding the nanoparticle−vascular tissue interactions. At present, Dr. Alkilany is an Assistant Professor of Pharmaceutics and Nanotechnology at the University of Jordan (Faculty of Pharmacy) with a research focus on the synthesis of novel nanomaterials to address challenges in pharmaceutical and biomedical fields. Nouf N. Mahmoud received her B.Sc. in Pharmacy from the University of Jordan, Jordan, in 2003, and M.Sc. in Clinical Pharmacy from the same university in 2007. She worked at Dar-Al-Dawa Pharmaceutical Company, Jordan, in the R&D department before beginning the M.Sc. program. In 2008, she joined the faculty of D

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(5) Krug, H. F. (2014) Nanosafety research–are we on the right track? Angew. Chem., Int. Ed. 53, 12304−12319. (6) Laurent, S., Burtea, C., Thirifays, C., Hafeli, U. O., and Mahmoudi, M. (2012) Crucial ignored parameters on nanotoxicology: the importance of toxicity assay modifications and ″cell vision″. PLoS One 7, e29997. (7) Vaux, D. L. (2012) Research methods: Know when your numbers are significant. Nature 492, 180−181. (8) Poland, C. A., Miller, M. R., Duffin, R., and Cassee, F. (2014) The elephant in the room: reproducibility in toxicology. Part. Fibre Toxicol. 11, 42. (9) Alkilany, A. M., Nagaria, P. K., Hexel, C. R., Shaw, T. J., Murphy, C. J., and Wyatt, M. D. (2009) Cellular uptake and cytotoxicity of gold nanorods: molecular origin of cytotoxicity and surface effects. Small 5, 701−708. (10) Vigderman, L., Manna, P., and Zubarev, E. R. (2012) Quantitative replacement of cetyl trimethylammonium bromide by cationic thiol ligands on the surface of gold nanorods and their extremely large uptake by cancer cells. Angew. Chem., Int. Ed. 51, 636− 641. (11) Monopoli, M. P., Aberg, C., Salvati, A., and Dawson, K. A. (2012) Biomolecular coronas provide the biological identity of nanosized materials. Nat. Nanotechnol. 7, 779−786. (12) Cho, E. C., Liu, Y., and Xia, Y. (2010) A simple spectroscopic method for differentiating cellular uptakes of gold nanospheres and nanorods from their mixtures. Angew. Chem., Int. Ed. 49, 1976−1980. (13) Casals, E., Pfaller, T., Duschl, A., Oostingh, G. J., and Puntes, V. (2010) Time evolution of the nanoparticle protein corona. ACS Nano 4, 3623−3632. (14) Walczyk, D., Bombelli, F. B., Monopoli, M. P., Lynch, I., and Dawson, K. A. (2010) What the cell ″sees″ in bionanoscience. J. Am. Chem. Soc. 132, 5761−5768. (15) Ehrenberg, M. S., Friedman, A. E., Finkelstein, J. N., Oberdörster, G., and McGrath, J. L. (2009) The influence of protein adsorption on nanoparticle association with cultured endothelial cells. Biomaterials 30, 603−610. (16) Bharti, B., Meissner, J., and Findenegg, G. H. (2011) Aggregation of silica nanoparticles directed by adsorption of lysozyme. Langmuir 27, 9823−9833. (17) Moerz, S. T., Kraegeloh, A., Chanana, M., and Kraus, T. (2015) Formation Mechanism for Stable Hybrid Clusters of Proteins and Nanoparticles. ACS Nano 9, 6696−6705. (18) Albanese, A., Walkey, C. D., Olsen, J. B., Guo, H., Emili, A., and Chan, W. C. (2014) Secreted biomolecules alter the biological identity and cellular interactions of nanoparticles. ACS Nano 8, 5515−5526. (19) Cho, E. C., Zhang, Q., and Xia, Y. (2011) The effect of sedimentation and diffusion on cellular uptake of gold nanoparticles. Nat. Nanotechnol. 6, 385−391. (20) Mahmoudi, M., Meng, J., Xue, X., Liang, X. J., Rahman, M., Pfeiffer, C., Hartmann, R., Gil, P. R., Pelaz, B., Parak, W. J., Del Pino, P., Carregal-Romero, S., Kanaras, A. G., and Tamil Selvan, S. (2014) Interaction of stable colloidal nanoparticles with cellular membranes. Biotechnol. Adv. 32, 679−692. (21) Albanese, A., and Chan, W. C. (2011) Effect of gold nanoparticle aggregation on cell uptake and toxicity. ACS Nano 5, 5478−5489. (22) Labouta, H. I., and Schneider, M. (2013) Interaction of inorganic nanoparticles with the skin barrier: current status and critical review. Nanomedicine 9, 39−54. (23) Mahmoud, N. N., Al-Qaoud, K. M., Al-Bakri, A. G., Alkilany, A. M., and Khalil, E. A. (2016) Colloidal stability of gold nanorod solution upon exposure to excised human skin: Effect of surface chemistry and protein adsorption. Int. J. Biochem. Cell Biol. 16, 30044− 30049. (24) Alkilany, A. M., Lohse, S. E., and Murphy, C. J. (2013) The gold standard: gold nanoparticle libraries to understand the nano-bio interface. Acc. Chem. Res. 46, 650−661. (25) Kettler, K., Veltman, K., van de Meent, D., van Wezel, A., and Hendriks, A. J. (2014) Cellular uptake of nanoparticles as determined

pharmacy at Al-Zaytoonah University of Jordan, Jordan, as a lecturer for five years. She is currently a Ph.D. candidate in the faculty of pharmacy at the University of Jordan studying the penetration of gold nanorods into human skin and hair follicles and their antibacterial activity. Fatemeh Hashemi was born in 1985. Fatemeh received her Pharm.D. degree from Shiraz University of Medical Sciences in 2012. She is now a Ph.D. candidate in Pharmaceutical Nanotechnology at Tehran University of Medical Sciences. She joined Nano-Bio Interactions Lab (www.biospion.com) in the winter of 2013. Her work is focused on exploring “hidden factors” in protein corona composition to achieve more precise information on nanotoxicology. Dr. Mohammad Javad Hajipour obtained his Ph.D. from National Institute of Genetic Engineering and Biotechnology (NIGEB) with specialization on the personalized protein corona formation and the biological impacts of personalized disease-specific protein corona in different patients. He joined Proessor. Mahmoudi’s research team as a research scientist; his research at the Laboratory of BioNano Interactions (www.biospion.com) is focused on the hidden factors at the nano-bio interfaces and personalized nanomedicine. Currently, he is an assistant professor at the Persian Gulf Biomedical Sciences Research Institute of Bushehr University of Medical Sciences. Fakhrosadat Farvadi is a Ph.D. candidate in Pharmaceutical Nanotechnology at Tehran University of Medical Sciences. Farzaneh was born in 1985. She received her Pharm.D. degree in 2012 at Shiraz University of Medical Sciences. Farzaneh joined the NanoBio Interactions laboratory in 2013. Her research is focused on developing smart pseudo-3D-substrates for high-throughput cytotoxicity evaluations. Dr. Morteza Mahmoudi is the director of NanoBio Interaction Laboratory at Tehran University of Medical Sciences. He received his B.Sc. in Materials Science and Engineering from the University of Tehran, his M.Sc. in Biomedical Engineering from Amirkabir University of Technology, and his Ph.D. in Nanoscience and Nanotechnology from Sharif University of Technology. His current research involves the control of protein corona decoration at the surface of nanoparticles and hidden parameters that affect the nanobio interfaces.

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ABBREVIATIONS CdSe, cadmium selenide; CTAB, cetyltrimethylammonium bromide; GNRs, gold nanorods; ICP-MS, inductively coupled plasma-mass spectrometry; ICP-OES, inductively coupled plasma optical emission spectroscopy; MTS, 3-(4, 5-dimethylthiazol-2-yl)-5 (3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium; MTT, 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide; NPs, nanoparticles; PAA, poly acrylic acid; PAH, poly allylamine hydrochloride; PEG, poly ethylene glycol

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DOI: 10.1021/acs.chemrestox.6b00108 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX