Abstract
A set of biomedically relevant iron oxide nanoparticles with systematically modified polymer surfaces was investigated regarding their interaction with the first contact partners after systemic administration such as blood cells, blood proteins, and the endothelial blood vessels, to establish structure–activity relationships. All nanoparticles were intensively characterized regarding their physicochemical parameters. Cyto- and hemocompatibility tests showed that (1) the properties of the core material itself were not relevant in short-term incubation studies, and (2) toxicities increased with higher polymer mass, neutral = anionic < cationic surface charge and charge density, as well as agglomeration. Based on this, it was possible to classify the nanoparticles in three groups, to establish structure–activity relationships and to predict nanosafety. While the results between cyto- and hemotoxicity tests correlated well for the polymers, data were not fully transferable for the nanoparticles, especially in case of cationic low molar mass polymer coatings. To evaluate the prediction efficacy of the static in vitro models, the results were compared to those obtained in an ex ovo shell-less hen’s egg test after microinjection under dynamic flow conditions. While the polymers demonstrated hemotoxicity profiles comparable to the in vitro tests, the size-dependent risks of nanoparticles could be more efficiently simulated in the more complex ex ovo environment, making the shell-less egg model an efficient alternative to animal studies according to the 3R concept.
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Ahmad Khanbeigi R, Kumar A, Sadouki F, Lorenz C, Forbes B, Dailey LA, Collins H (2012) The delivered dose: Applying particokinetics to in vitro investigations of nanoparticle internalization by macrophages. J Controll Releas 162:259–266. doi:10.1016/j.jconrel.2012.07.019
Alexiou C, Arnold W, Klein RJ et al (2000) Locoregional cancer treatment with magnetic drug targeting. Cancer Res 60:6641–6648
ASTM (2013) ASTM F756-13, standard practice for assessment of hemolytic properties of materials. ASTM Int doi:10.1520/F0756
Bähring F, Schlenk F, Wotschadlo J et al (2013) Suitability of viability assays for testing biological effects of coated superparamagnetic nanoparticles. IEEE Trans Magn 49:383–388. doi:10.1109/TMAG.2012.2222635
Barrow M, Taylor A, Murray P, Rosseinsky MJ, Adams DJ (2015) Design considerations for the synthesis of polymer coated iron oxide nanoparticles for stem cell labelling and tracking using MRI. Chem Soc Rev 44:6733–6748. doi:10.1039/C5CS00331H
Bauer M, Lautenschlaeger C, Kempe K, Tauhardt L, Schubert US, Fischer D (2012) Poly(2-ethyl-2-oxazoline) as alternative for the stealth polymer poly(ethylene glycol): comparison of in vitro cytotoxicity and hemocompatibility. Macromol Biosci 12:986–998. doi:10.1002/mabi.201200017
Bhirde AA, Hassan SA, Harr E, Chen XY (2014) Role of albumin in the formation and stabilization of nanoparticle aggregates in serum studied by continuous photon correlation spectroscopy and multiscale computer simulations. J Phys Chem C 118:16199–16208. doi:10.1021/jp5034068
Briley-Saebo KC, Johansson LO, Hustvedt SO, Haldorsen AG, Bjornerud A, Fayad ZA, Ahlstrom HK (2006) Clearance of iron oxide particles in rat liver: effect of hydrated particle size and coating material on liver metabolism. Invest Radiol 41:560–571. doi:10.1097/01.rli.0000221321.90261.09
Calatayud MP, Sanz B, Raffa V, Riggio C, Ibarra MR, Goya GF (2014) The effect of surface charge of functionalized Fe3O4 nanoparticles on protein adsorption and cell uptake. Biomaterials 35:6389–6399. doi:10.1016/j.biomaterials.2014.04.009
Cardoso AV, Pereira MH, de Araújo Marcondes G, Ferreira AR, de Araújo PR (2007) Microplate reader analysis of triatomine saliva effect on erythrocyte aggregation. Mater Res 10:31–36. doi:10.1590/S1516-14392007000100008
Cedervall T, Lynch I, Lindman S et al (2007) Understanding the nanoparticle-protein corona using methods to quantify exchange rates and affinities of proteins for nanoparticles. Proc Natl Acad Sci U S A 104:2050–2055. doi:10.1073/pnas.0608582104
Choksakulnimitr S, Masuda S, Tokuda H, Takakura Y, Hashida M (1995) In vitro cytotoxicity of macromolecules in different cell culture systems. J Controll Release 34:233–241. doi:10.1016/0168-3659(95)00007-U
Chouly C, Pouliquen D, Lucet I, Jeune JJ, Jallet P (1996) Development of superparamagnetic nanoparticles for MRI: effect of particle size, charge and surface nature on biodistribution. J Microencapsul 13:245–255. doi:10.3109/02652049609026013
Clancy AA, Gregoriou Y, Yaehne K, Cramb DT (2010) Measuring properties of nanoparticles in embryonic blood vessels: Towards a physicochemical basis for nanotoxicity. Chem Phys Lett 488:99–111. doi:10.1016/j.cplett.2010.02.016
Cromer Berman SM, Walczak P, Bulte JW (2011) Tracking stem cells using magnetic nanoparticles. Wiley Interdiscip Rev: Nanomed Nanobiotechnol 3:343–355. doi:10.1002/wnan.140
Deng ZJ, Liang M, Toth I, Monteiro M, Minchin RF (2012) Plasma protein binding of positively and negatively charged polymer-coated gold nanoparticles elicits different biological responses. Nanotoxicology 7:314–322. doi:10.3109/17435390.2012.655342
DIN (2009) Biological evaluation of medical devices—Part 5: tests for in vitro cytotoxicity ISO 10993-5
Domey J, Bergemann C, Bremer-Streck S, Krumbein I, Reichenbach JR, Teichgraber U, Hilger I (2015) Long-term prevalence of NIRF-labeled magnetic nanoparticles for the diagnostic and intraoperative imaging of inflammation. Nanotoxicology. doi:10.3109/17435390.2014.1000413
Dunn BE (1974) Technique of shell-less culture of the 72-hour avian embryo. Poult Sci 53:409–412
Eberbeck D, Kettering M, Bergemann C, Zirpel P, Hilger I, Trahms L (2010) Quantification of the aggregation of magnetic nanoparticles with different polymeric coatings in cell culture medium. J Phys D: Appl Phys 43:405002. doi:10.1088/0022-3727/43/40/405002
EDQM (2014) European pharmacopoeia 8.0 2.6.1 Sterility. European Council. European Pharmacopoeia Commission
Ehrenberg MS, Friedman AE, Finkelstein JN, Oberdörster G, McGrath JL (2009) The influence of protein adsorption on nanoparticle association with cultured endothelial cells. Biomaterials 30:603–610. doi:10.1016/j.biomaterials.2008.09.050
Fernandes JC, Eaton P, Nascimento H et al (2008) Effects of chitooligosaccharides on human red blood cell morphology and membrane protein structure. Biomacromolecules 9:3346–3352. doi:10.1021/bm800622f
Fischer D, von Harpe A, Kunath K, Petersen H, Li YX, Kissel T (2002) Copolymers of ethylene imine and N-(2-hydroxyethyl)-ethylene imine as tools to study effects of polymer structure on physicochemical and biological properties of DNA complexes. Bioconjugate Chem 13:1124–1133. doi:10.1021/bc025550w
Fischer D, Li Y, Ahlemeyer B, Krieglstein J, Kissel T (2003) In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis. Biomaterials 24:1121–1131. doi:10.1016/S0142-9612(02)00445-3
Fleischer CC, Payne CK (2014) Nanoparticle–cell interactions: molecular structure of the protein corona and cellular outcomes. Acc Chem Res 47:2651–2659. doi:10.1021/ar500190q
Gebauer JS, Malissek M, Simon S et al (2012) Impact of the nanoparticle-protein corona on colloidal stability and protein structure. Langmuir 28:9673–9679. doi:10.1021/la301104a
Gessner A, Lieske A, Paulke BR, Müller RH (2002) Influence of surface charge density on protein adsorption on polymeric nanoparticles: analysis by two-dimensional electrophoresis. Eur J Pharm Biopharm 54:165–170. doi:10.1016/S0939-6411(02)00081-4
Ghosh S, Jiang W, McClements JD, Xing B (2011) Colloidal stability of magnetic iron oxide nanoparticles: influence of natural organic matter and synthetic polyelectrolytes. Langmuir 27:8036–8043. doi:10.1021/la200772e
Grund S, Bauer M, Fischer D (2011) Polymers in drug delivery—state of the art and future trends. Adv Eng Mater 13:61–87. doi:10.1002/adem.201080088
Grüttner C, Teller J (1999) New types of silica-fortified magnetic nanoparticles as tools for molecular biology applications. J Magn Magn Mater 194:8–15. doi:10.1016/S0304-8853(98)00561-7
Hamburger V, Hamilton HL (1992) A series of normal stages in the development of the chick embryo. Dev Dyn 195:231–272. doi:10.1002/aja.1001950404
Harisinghani MG, Barentsz J, Hahn PF et al (2003) Noninvasive detection of clinically occult lymph-node metastases in prostate cancer. N Engl J Med 348:2491–2499. doi:10.1056/NEJMoa022749
Hildebrandt B, Wust P, Ahlers O et al (2002) The cellular and molecular basis of hyperthermia. Crit Rev Oncol Hematol 43:33–56. doi:10.1016/S1040-8428(01)00179-2
Hilger I (2013) In vivo applications of magnetic nanoparticle hyperthermia. Int J Hyperthermia 29:828–834. doi:10.3109/02656736.2013.832815
Hilger I, Kaiser WA (2012) Iron oxide-based nanostructures for MRI and magnetic hyperthermia. Nanomedicine 7:1443–1459. doi:10.2217/nnm.12.112
Hilger I, Andrä W, Hergt R, Hiergeist R, Schubert H, Kaiser WA (2001) Electromagnetic heating of breast tumors in interventional radiology: in vitro and in vivo studies in human cadavers and mice. Radiology 218:570–575. doi:10.1148/radiology.218.2.r01fe19570
Hirsch V, Kinnear C, Moniatte M, Rothen-Rutishauser B, Clift MJ, Fink A (2013) Surface charge of polymer coated SPIONs influences the serum protein adsorption, colloidal stability and subsequent cell interaction in vitro. Nanoscale 5:3723–3732. doi:10.1039/c2nr33134a
ICCVAM (2006) ICCVAM test method evaluation report: appendix G-ICCVAM recommended HET-CAM test method protocol. National Institute of Environmental Health Sciences. National Institutes of Health
ICCVAM (2010) ICCVAM test method evaluation report: current validation status of in vitro test methods proposed for identifying eye injury hazard potential of chemicals and products Appendix B3. National Institute of Environmental Health Sciences. National Institutes of Health
Jin R, Lin B, Li D, Ai H (2014) Superparamagnetic iron oxide nanoparticles for MR imaging and therapy: design considerations and clinical applications. Curr Opin Pharmacol 18:18–27. doi:10.1016/j.coph.2014.08.002
Joris F, Manshian BB, Peynshaert K, De Smedt SC, Braeckmans K, Soenen SJ (2013) Assessing nanoparticle toxicity in cell-based assays: influence of cell culture parameters and optimized models for bridging the in vitro–in vivo gap. Chem Soc Rev 42:8339–8359. doi:10.1039/c3cs60145e
Kafil V, Omidi Y (2011) Cytotoxic impacts of linear and branched polyethylenimine nanostructures in a431 cells. Bioimpacts 1:23–30. doi:10.5681/bi.2011.004
Klueh U, Dorsky DI, Moussy F, Kreutzer DL (2003) Ex ova chick chorioallantoic membrane as a novel model for evaluation of tissue responses to biomaterials and implants. J Biomed Mater Res, Part A 67:838–843. doi:10.1002/jbm.a.10059
Landgraf L, Müller I, Ernst P et al (2015) Comparative evaluation of the impact on endothelial cells induced by different nanoparticle structures and functionalization. Beilstein J Nanotechnol 6:300–312. doi:10.3762/bjnano.6.28
Ludwig R, Stapf M, Dutz S, Muller R, Teichgraber U, Hilger I (2014) Structural properties of magnetic nanoparticles determine their heating behavior—an estimation of the in vivo heating potential. Nanoscale Res Lett 9(1):602. doi:10.1186/1556-276X-9-602
Ma Z, Bai J, Wang Y, Jiang X (2014) Impact of shape and pore size of mesoporous silica nanoparticles on serum protein adsorption and RBCs hemolysis. ACS Appl Mater Interfaces 6:2431–2438. doi:10.1021/am404860q
Maffre P, Brandholt S, Nienhaus K, Shang L, Parak WJ, Nienhaus GU (2014) Effects of surface functionalization on the adsorption of human serum albumin onto nanoparticles—a fluorescence correlation spectroscopy study. Beilstein J Nanotechnol 5:2036–2047. doi:10.3762/bjnano.5.212
Mahmoudi M, Hofmann H, Rothen-Rutishauser B, Petri-Fink A (2012) Assessing the in vitro and in vivo toxicity of superparamagnetic iron oxide nanoparticles. Chem Rev 112:2323–2338. doi:10.1021/Cr2002596
Monopoli MP, Åberg C, Salvati A, Dawson KA (2012) Biomolecular coronas provide the biological identity of nanosized materials. Nat Nanotechnol 7:779–786. doi:10.1038/nnano.2012.207
Mosesson MW (2005) Fibrinogen and fibrin structure and functions. J Thromb Haemost 3:1894–1904. doi:10.1111/j.1538-7836.2005.01365.x
Müller R, Stranik O, Schlenk F, Werner S, Malsch D, Fischer D, Fritzsche W (2015) Optical detection of nanoparticle agglomeration in a living system under the influence of a magnetic field. J Magn Magn Mater 380:61–65. doi:10.1016/j.jmmm.2014.10.043
Neu B, Wenby R, Meiselman HJ (2008) Effects of dextran molecular weight on red blood cell aggregation. Biophys J 95:3059–3065. doi:10.1529/biophysj.108.130328
Ogris M, Brunner S, Schuller S, Kircheis R, Wagner E (1999) PEGylated DNA/transferrin-PEI complexes: reduced interaction with blood components, extended circulation in blood and potential for systemic gene delivery. Gene Ther 6:595–605. doi:10.1038/sj.gt.3300900
Olsvik Ø, Popovic T, Skjerve E, Cudjoe KS, Hornes E, Ugelstad J, Uhlén M (1994) Magnetic separation techniques in diagnostic microbiology. Clin Microbiol Rev 7:43–54
Öztürk N, Bereli N, Akgöl S, Denizli A (2008) High capacity binding of antibodies by poly(hydroxyethyl methacrylate) nanoparticles. Colloids Surf, B 67:14–19. doi:10.1016/j.colsurfb.2008.07.005
Peng Q, Zhang S, Yang Q et al (2013) Preformed albumin corona, a protective coating for nanoparticles based drug delivery system. Biomaterials 34:8521–8530. doi:10.1016/j.biomaterials.2013.07.102
Perez JM, Josephson L, O’Loughlin T, Högemann D, Weissleder R (2002) Magnetic relaxation switches capable of sensing molecular interactions. Nat Biotechnol 20:816–820. doi:10.1038/nbt720
Rajan B, Sathish S, Balakumar S, Devaki T (2015) Synthesis and dose interval dependent hepatotoxicity evaluation of intravenously administered polyethylene glycol-8000 coated ultra-small superparamagnetic iron oxide nanoparticle on Wistar rats. Environ Toxicol Pharmacol 39:727–735. doi:10.1016/j.etap.2015.01.018
Reddy LH, Arias JL, Nicolas J, Couvreur P (2012) Magnetic nanoparticles: design and characterization, toxicity and biocompatibility, pharmaceutical and biomedical applications. Chem Rev 112:5818–5878. doi:10.1021/cr300068p
Reimer P, Balzer T (2003) Ferucarbotran (Resovist): a new clinically approved RES-specific contrast agent for contrast-enhanced MRI of the liver: properties, clinical development, and applications. Eur Radiol 13:1266–1276. doi:10.1007/s00330-002-1721-7
Röcker C, Pötzl M, Zhang F, Parak WJ, Nienhaus GU (2009) A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles. Nat Nanotechnol 4:577–580. doi:10.1038/nnano.2009.195
Rosenbruch M (1994) Early stages of the incubated chicken egg as a model in experimental biology and medicine. ALTEX 11:199–206
Santhosh PB, Ulrih NP (2013) Multifunctional superparamagnetic iron oxide nanoparticles: promising tools in cancer theranostics. Cancer Lett 336:8–17. doi:10.1016/j.canlet.2013.04.032
Saraswathy A, Nazeer SS, Nimi N, Arumugam S, Shenoy SJ, Jayasree RS (2014) Synthesis and characterization of dextran stabilized superparamagnetic iron oxide nanoparticles for in vivo MR imaging of liver fibrosis. Carbohydr Polym 101:760–768. doi:10.1016/j.carbpol.2013.10.015
Schlenk F, Grund S, Fischer D (2013) Recent developments and perspectives on gene therapy using synthetic vectors. Ther Deliv 4:95–113. doi:10.4155/tde.12.128
Schroeder HW, Cavacini L (2010) Structure and function of immunoglobulins. J Allergy Clin Immunol 125:41–52. doi:10.1016/j.jaci.2009.09.046
Setyawati MI, Tay CY, Docter D, Stauber RH, Leong DT (2015) Understanding and exploiting nanoparticles’ intimacy with the blood vessel and blood. Chem Soc Rev 44:8174–8199. doi:10.1039/c5cs00499c
Stins MF, Badger J, Kim KS (2001) Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells. Microb Pathogenesis 30:19–28. doi:10.1006/mpat.2000.0406
Subramanian A, Rau AV, Kaligotla H (2006) Surface modification of chitosan for selective surface-protein interaction. Carbohydr Polym 66:321–332. doi:10.1016/j.carbpol.2006.03.022
Tenzer S, Docter D, Kuharev J et al (2013) Rapid formation of plasma protein corona critically affects nanoparticle pathophysiology. Nat Nanotechnol 8:772–781. doi:10.1038/nnano.2013.181
Vargas A, Zeisser-Labouèbe M, Lange N, Gurny R, Delie F (2007) The chick embryo and its chorioallantoic membrane (CAM) for the in vivo evaluation of drug delivery systems. Adv Drug Deliv Rev 59:1162–1176. doi:10.1016/j.addr.2007.04.019
Verma A, Stellacci F (2010) Effect of surface properties on nanoparticle–cell interactions. Small 6:12–21. doi:10.1002/smll.200901158
Wang YX (2011) Superparamagnetic iron oxide based MRI contrast agents: current status of clinical application. Quant Imaging Med Surg 1:35–40. doi:10.3978/j.issn.2223-4292.2011.08.03
Wen G, Zhang XL, Chang RM, Xia Q, Cang P, Zhang Y (2002) Superparamagnetic iron oxide (Feridex)-enhanced MRI in diagnosis of focal hepatic lesions. Di Yi Jun Yi Da Xue Xue Bao 22:451–452
Woodard H, White D (1986) The composition of body tissues. Br J Radiol 59:1209–1218. doi:10.1259/0007-1285-59-708-1209
Yaehne K, Tekrony A, Clancy A et al (2013) Nanoparticle accumulation in angiogenic tissues: towards predictable pharmacokinetics. Small 9:3118–3127. doi:10.1002/smll.201201848
Zhao M, Josephson L, Tang Y, Weissleder R (2003) Magnetic sensors for protease assays. Angew Chem Int Ed 42:1375–1378. doi:10.1002/anie.200390352
Zhu M, Nie G, Meng H, Xia T, Nel A, Zhao Y (2012) Physicochemical properties determine nanomaterial cellular uptake, transport, and fate. Acc Chem Res 46:622–631. doi:10.1021/ar300031y
Zwadlo-Klarwasser G, Görlitz K, Hafemann B, Klee D, Klosterhalfen B (2001) The chorioallantoic membrane of the chick embryo as a simple model for the study of the angiogenic and inflammatory response to biomaterials. J Mater Sci: Mater Med 12:195–199. doi:10.1023/A:1008950713001
Acknowledgements
We thank Götz Nowak and Daniela Rosner for introduction into the hen’s egg model as well as Angela Herre and Ramona Brabetz for their excellent technical assistance. Furthermore, we are grateful to Harald Schubert from the Institute of Laboratory Animal Science for providing the sheep blood.
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This study was funded by the Federal Ministry of Education and Research (Grant Numbers BMBF 03X0104D and BMBF 03XP0003).
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This article does not contain any studies with human participants performed by any of the authors. This article does not contain any studies with animals performed by any of the authors. The ex ovo experiments performed with chicken embryos for up to 96 h are no animal experiments in compliance with national and international law.
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Schlenk, F., Werner, S., Rabel, M. et al. Comprehensive analysis of the in vitro and ex ovo hemocompatibility of surface engineered iron oxide nanoparticles for biomedical applications. Arch Toxicol 91, 3271–3286 (2017). https://doi.org/10.1007/s00204-017-1968-z
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DOI: https://doi.org/10.1007/s00204-017-1968-z