Specifically designed and functionalized nanoparticles hold great promise for biomedical applications. Yet, the applicability of nanoparticles is critically predetermined by their surface functionalization and biodegradability. Here we demonstrate that amino-functionalized polystyrene nanoparticles (PS-NH2), but not amino- or hydroxyl-functionalized silica particles, trigger cell death in hepatocellular carcinoma Huh7 cells. Importantly, biodegradability of nanoparticles plays a crucial role in regulation of essential cellular processes. Thus, biodegradable silica nanoparticles having the same shape, size and surface functionalization showed opposite cellular effects in comparison with similar polystyrene nanoparticles. At the molecular level, PS-NH2 obstruct and amino-functionalized silica nanoparticles (Si-NH2) activate the mTOR signalling in Huh7 and HepG2 cells. PS-NH2 induced time-dependent lysosomal destabilization associated with damage of the mitochondrial membrane. Solely in PS-NH2-treated cells, permeabilization of lysosomes preceded cell death. Contrary, Si-NH2 nanoparticles enhanced proliferation of HuH7 and HepG2 cells. Our findings demonstrate complex cellular responses to functionalized nanoparticles and suggest that nanoparticles can be used to control activation of mTOR signaling with subsequent influence on proliferation and viability of HuH7 cells. The data provide fundamental knowledge which could help in developing safe and efficient nano-therapeutics.

Institute for Clinical and Experimental Medicine Prague Czech Republic

Institute of Experimental Medicine the Czech Academy of Sciences Prague Czech Republic

Institute of Physics of the Czech Academy of Sciences Prague Czech Republic

J Heyrovský Institute of Physical Chemistry AS CR v v i Dolejškova 2155 3 182 23 Prague 8 Czech Republic

See more in PubMed

Ferrari M. Cancer nanotechnology: Opportunities and challenges. Nat. Rev. Cancer. 2005;5:161–171. doi: 10.1038/nrc1566. PubMed DOI

Blanco E, Shen H, Ferrari M. Principles of nanoparticle design for overcoming biological barriers to drug delivery. Nat. Biotechnol. 2015;33:941–951. doi: 10.1038/nbt.3330. PubMed DOI PMC

Shi JJ, Votruba AR, Farokhzad OC, Langer R. Nanotechnology in Drug Delivery and Tissue Engineering: From Discovery to Applications. Nano Lett. 2010;10:3223–3230. doi: 10.1021/nl102184c. PubMed DOI PMC

Tukmachev D, et al. An effective strategy of magnetic stem cell delivery for spinal cord injury therapy. Nanoscale. 2015;7:3954–3958. doi: 10.1039/C4NR05791K. PubMed DOI

Buzea C, Pacheco II, Robbie K. Nanomaterials and nanoparticles: Sources and toxicity. Biointerphases. 2007;2:Mr17–Mr71. doi: 10.1116/1.2815690. PubMed DOI

Rothenberg ML, Carbone DR, Johnson DH. Improving the evaluation of new cancer treatments: challenges and opportunities. Nat. Rev. Cancer. 2003;3:303–309. doi: 10.1038/nrc1047. PubMed DOI

Schaue D, McBride WH. Opportunities and challenges of radiotherapy for treating cancer. Nat. Rev. Clin. Oncol. 2015;12:527–540. doi: 10.1038/nrclinonc.2015.120. PubMed DOI PMC

Shi J, Kantoff PW, Wooster R, Farokhzad OC. Cancer nanomedicine: progress, challenges and opportunities. Nat. Rev. Cancer. 2017;17:20–37. doi: 10.1038/nrc.2016.108. PubMed DOI PMC

Park K. Facing the truth about nanotechnology in drug delivery. ACS Nano. 2013;7:7442–7447. doi: 10.1021/nn404501g. PubMed DOI PMC

Matsumura Y, Maeda H. A new concept for macromolecular therapeutics in cancer-chemotherapy - mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res. 1986;46:6387–6392. PubMed

Gerlowski LE, Jain RK. Microvascular permeability of normal and neoplastic tissues. Microvasc. Res. 1986;31:288–305. doi: 10.1016/0026-2862(86)90018-X. PubMed DOI

Nel AE, et al. Understanding biophysicochemical interactions at the nano-bio interface. Nat. Mater. 2009;8:543–557. doi: 10.1038/nmat2442. PubMed DOI

Lunov O, et al. Differential uptake of functionalized polystyrene nanoparticles by human macrophages and a monocytic cell line. ACS Nano. 2011;5:1657–1669. doi: 10.1021/nn2000756. PubMed DOI

Gulbins E, Kolesnick RN. It takes a CAD to kill a tumor cell with a LMP. Cancer Cell. 2013;24:279–281. doi: 10.1016/j.ccr.2013.08.025. PubMed DOI

Saftig P, Sandhoff K. CANCER Killing from the inside. Nature. 2013;502:312–313. doi: 10.1038/nature12692. PubMed DOI

Kallunki T, Olsen OD, Jaattela M. Cancer-associated lysosomal changes: friends or foes? Oncogene. 2013;32:1995–2004. doi: 10.1038/onc.2012.292. PubMed DOI

Petersen NHT, et al. Transformation-associated changes in sphingolipid metabolism sensitize cells to lysosomal cell death induced by inhibitors of acid sphingomyelinase. Cancer Cell. 2013;24:379–393. doi: 10.1016/j.ccr.2013.08.003. PubMed DOI

Xia T, Kovochich M, Liong M, Zink JI, Nel AE. Cationic polystyrene nanosphere toxicity depends on cell-specific endocytic and mitochondrial injury pathways. ACS Nano. 2008;2:85–96. doi: 10.1021/nn700256c. PubMed DOI

Loos C, et al. Amino-functionalized nanoparticles as inhibitors of mTOR and inducers of cell cycle arrest in leukemia cells. Biomaterials. 2014;35:1944–1953. doi: 10.1016/j.biomaterials.2013.11.056. PubMed DOI

Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nat. Rev. Mol. Cell Bio. 2011;12:21–35. doi: 10.1038/nrm3025. PubMed DOI PMC

Easton JB, Houghton PJ. mTOR and cancer therapy. Oncogene. 2006;25:6436–6446. doi: 10.1038/sj.onc.1209886. PubMed DOI

Teachey DT, Grupp SA, Brown VI. Mammalian target of rapamycin inhibitors and their potential role in therapy in leukaemia and other haematological malignancies. Brit. J. Haematol. 2009;145:569–580. doi: 10.1111/j.1365-2141.2009.07657.x. PubMed DOI PMC

Chiu HW, et al. Cationic polystyrene nanospheres induce autophagic cell death through the induction of endoplasmic reticulum stress. Nanoscale. 2015;7:736–746. doi: 10.1039/C4NR05509H. PubMed DOI

Khan MI, et al. Induction of ROS, mitochondrial damage and autophagy in lung epithelial cancer cells by iron oxide nanoparticles. Biomaterials. 2012;33:1477–1488. doi: 10.1016/j.biomaterials.2011.10.080. PubMed DOI

Liu HL, et al. A functionalized single-walled carbon nanotube-induced autophagic cell death in human lung cells through Akt-TSC2-mTOR signaling. Cell Death Dis. 2011;2:e159. doi: 10.1038/cddis.2011.27. PubMed DOI PMC

Matter MS, Decaens T, Andersen JB, Thorgeirsson SS. Targeting the mTOR pathway in hepatocellular carcinoma: Current state and future trends. J. Hepatol. 2014;60:855–865. doi: 10.1016/j.jhep.2013.11.031. PubMed DOI PMC

Florence AT. Issues in oral nanoparticle drug carrier uptake and targeting. J Drug Target. 2004;12:65–70. doi: 10.1080/10611860410001693706. PubMed DOI

Tokiwa Y, Calabia BP, Ugwu CU, Aiba S. Biodegradability of plastics. Int J Mol Sci. 2009;10:3722–3742. doi: 10.3390/ijms10093722. PubMed DOI PMC

Hardy CL, et al. Inert 50-nm polystyrene nanoparticles that modify pulmonary dendritic cell function and inhibit allergic airway inflammation. Journal of Immunology. 2012;188:1431–1441. doi: 10.4049/jimmunol.1100156. PubMed DOI

Fifis T, et al. Size-dependent immunogenicity: therapeutic and protective properties of nano-vaccines against tumors. J Immunol. 2004;173:3148–3154. doi: 10.4049/jimmunol.173.5.3148. PubMed DOI

Johrden L, et al. Comparison of polystyrene nanoparticles and UV-inactivated antigen-displaying adenovirus for vaccine delivery in mice. Virol J. 2013;10:108. doi: 10.1186/1743-422X-10-108. PubMed DOI PMC

Park JH, et al. Biodegradable luminescent porous silicon nanoparticles for in vivo applications. Nat. Mater. 2009;8:331–336. doi: 10.1038/nmat2398. PubMed DOI PMC

de Oliveira LF, et al. Functionalized silica nanoparticles as an alternative platform for targeted drug-delivery of water insoluble drugs. Langmuir. 2016;32:3217–3225. doi: 10.1021/acs.langmuir.6b00214. PubMed DOI

Vivero-Escoto JL, Slowing II, Trewyn BG, Lin VSY. Mesoporous silica nanoparticles for intracellular controlled drug delivery. Small. 2010;6:1952–1967. doi: 10.1002/smll.200901789. PubMed DOI

Salvati A, et al. Transferrin-functionalized nanoparticles lose their targeting capabilities when a biomolecule corona adsorbs on the surface. Nat. Nanotechnol. 2013;8:137–143. doi: 10.1038/nnano.2012.237. PubMed DOI

Docter D, et al. The nanoparticle biomolecule corona: lessons learned - challenge accepted? Chem. Soc. Rev. 2015;44:6094–6121. doi: 10.1039/C5CS00217F. PubMed DOI

Bertoli F, Garry D, Monopoli MP, Salvati A, Dawson KA. The intracellular destiny of the protein corona: a study on its cellular internalization and evolution. ACS Nano. 2016;10:10471–10479. doi: 10.1021/acsnano.6b06411. PubMed DOI

Lunov O, et al. Amino-functionalized polystyrene nanoparticles activate the NLRP3 inflammasome in human macrophages. ACS Nano. 2011;5:9648–9657. doi: 10.1021/nn203596e. PubMed DOI

Loos, C. et al. Amino-functionalized nanoparticles inhibit mTOR and induce cell cycle arrest and apoptosis in leukemia cells. FASEB J. 27, Supplement 575.577 (2013).

Alkilany AM, Murphy CJ. Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? J Nanopart Res. 2010;12:2313–2333. doi: 10.1007/s11051-010-9911-8. PubMed DOI PMC

Sabella S, et al. A general mechanism for intracellular toxicity of metal-containing nanoparticles. Nanoscale. 2014;6:7052–7061. doi: 10.1039/c4nr01234h. PubMed DOI PMC

Barnard PJ, Berners-Price SJ. Targeting the mitochondrial cell death pathway with gold compounds. Coordination Chemistry Reviews. 2007;251:1889–1902. doi: 10.1016/j.ccr.2007.04.006. DOI

Rocker C, Potzl M, Zhang F, Parak WJ, Nienhaus GU. A quantitative fluorescence study of protein monolayer formation on colloidal nanoparticles. Nat. Nanotechnol. 2009;4:577–580. doi: 10.1038/nnano.2009.195. PubMed DOI

Li Y, et al. Probing of the assembly structure and dynamics within nanoparticles during interaction with blood proteins. ACS Nano. 2012;6:9485–9495. doi: 10.1021/nn302317j. PubMed DOI PMC

Reddi KK, Holland JF. Elevated serum ribonuclease in patients with pancreatic cancer. P. Natl. Acad. Sci. USA. 1976;73:2308–2310. doi: 10.1073/pnas.73.7.2308. PubMed DOI PMC

Ries, J., Weidemann, T. & Schwille, P. In Comprehensive Biophysics 210–245 (Elsevier, 2012).

Cheng CJ, Tietjen GT, Saucier-Sawyer JK, Saltzman WM. A holistic approach to targeting disease with polymeric nanoparticles. Nat. Rev. Drug Discov. 2015;14:239–247. doi: 10.1038/nrd4503. PubMed DOI PMC

Almeida PV, et al. Amine-modified hyaluronic acid-functionalized porous silicon nanoparticles for targeting breast cancer tumors. Nanoscale. 2014;6:10377–10387. doi: 10.1039/C4NR02187H. PubMed DOI PMC

Yu MK, Park J, Jon S. Targeting strategies for multifunctional nanoparticles in cancer imaging and therapy. Theranostics. 2012;2:3–44. doi: 10.7150/thno.3463. PubMed DOI PMC

Chakravarty R, et al. Hollow mesoporous silica nanoparticles for tumor vasculature targeting and PET image-guided drug delivery. Nanomedicine (Lond) 2015;10:1233–1246. doi: 10.2217/nnm.14.226. PubMed DOI PMC

McCarthy JR, Bhaumik J, Karver MR, Sibel Erdem S, Weissleder R. Targeted nanoagents for the detection of cancers. Mol Oncol. 2010;4:511–528. doi: 10.1016/j.molonc.2010.08.003. PubMed DOI PMC

Gonzalez-Moragas L, Yu SM, Carenza E, Laromaine A, Roig A. Protective effects of bovine serum albumin on superparamagnetic iron oxide nanoparticles evaluated in the nematode caenorhabditis elegans. ACS Biomater. Sci. Eng. 2015;1:1129–1138. doi: 10.1021/acsbiomaterials.5b00253. PubMed DOI

Docter D, et al. The protein corona protects against size- and dose-dependent toxicity of amorphous silica nanoparticles. Beilstein J. Nanotech. 2014;5:1380–1392. doi: 10.3762/bjnano.5.151. PubMed DOI PMC

Leland PA, Schultz LW, Kim BM, Raines RT. Ribonuclease A variants with potent cytotoxic activity. P. Natl. Acad. Sci. USA. 1998;95:10407–10412. doi: 10.1073/pnas.95.18.10407. PubMed DOI PMC

Turci F, et al. An integrated approach to the study of the interaction between proteins and nanoparticles. Langmuir. 2010;26:8336–8346. doi: 10.1021/la904758j. PubMed DOI

Lee CS, Belfort G. Changing activity of ribonuclease-a during adsorption - a molecular explanation. P. Natl. Acad. Sci. USA. 1989;86:8392–8396. doi: 10.1073/pnas.86.21.8392. PubMed DOI PMC

Gotte G, et al. Cross-linked trimers of bovine ribonuclease A: activity on double-stranded RNA and antitumor action. FEBS Lett. 1997;415:308–312. doi: 10.1016/S0014-5793(97)01147-2. PubMed DOI

Libonati M, Bertoldi M, Sorrentino S. The activity on double-stranded RNA of aggregates of ribonuclease A higher than dimers increases as a function of the size of the aggregates. Biochem. J. 1996;318:287–290. doi: 10.1042/bj3180287. PubMed DOI PMC

Fujisawa A, et al. Disease-associated mutations in CIAS1 induce cathepsin B-dependent rapid cell death of human THP-1 monocytic cells. Blood. 2007;109:2903–2911. PubMed

Guicciardi ME, Leist M, Gores GJ. Lysosomes in cell death. Oncogene. 2004;23:2881–2890. doi: 10.1038/sj.onc.1207512. PubMed DOI

Gallud A, et al. Macrophage activation status determines the internalization of mesoporous silica particles of different sizes: Exploring the role of different pattern recognition receptors. Biomaterials. 2017;121:28–40. doi: 10.1016/j.biomaterials.2016.12.029. PubMed DOI

Ba XL, et al. The role of moderate static magnetic fields on biomineralization of osteoblasts on sulfonated polystyrene films. Biomaterials. 2011;32:7831–7838. doi: 10.1016/j.biomaterials.2011.06.053. PubMed DOI

Loos C, et al. Functionalized polystyrene nanoparticles as a platform for studying bio-nano interactions. Beilstein J. Nanotech. 2014;5:2403–2412. doi: 10.3762/bjnano.5.250. PubMed DOI PMC

Tang FQ, Li LL, Chen D. Mesoporous silica nanoparticles: synthesis, biocompatibility and drug delivery. Adv. Mater. 2012;24:1504–1534. doi: 10.1002/adma.201104763. PubMed DOI

Petros RA, DeSimone JM. Strategies in the design of nanoparticles for therapeutic applications. Nat. Rev. Drug Discov. 2010;9:615–627. doi: 10.1038/nrd2591. PubMed DOI

Singh R, Lillard JW. Nanoparticle-based targeted drug delivery. Exp. Mol. Pathol. 2009;86:215–223. doi: 10.1016/j.yexmp.2008.12.004. PubMed DOI PMC

Jiang BH, Liu LZ. Role of mTOR in anticancer drug resistance: Perspectives for improved drug treatment. Drug Resist. Update. 2008;11:63–76. doi: 10.1016/j.drup.2008.03.001. PubMed DOI PMC

Gulati P, et al. Amino acids activate mTOR Complex 1 via Ca2+/CaM signaling to hVps34. Cell Metab. 2008;7:456–465. doi: 10.1016/j.cmet.2008.03.002. PubMed DOI PMC

Wullschleger S, Loewith R, Hall MN. TOR signaling in growth and metabolism. Cell. 2006;124:471–484. doi: 10.1016/j.cell.2006.01.016. PubMed DOI

Copp J, Manning G, Hunter T. TORC-Specific Phosphorylation of Mammalian Target of Rapamycin (mTOR): Phospho-Ser(2481) Is a Marker for Intact mTOR Signaling Complex 2. Cancer Res. 2009;69:1821–1827. doi: 10.1158/0008-5472.CAN-08-3014. PubMed DOI PMC

Villanueva A, et al. Pivotal role of mTOR signaling in hepatocellular carcinoma. Gastroenterology. 2008;135:1972–1983. doi: 10.1053/j.gastro.2008.08.008. PubMed DOI PMC

Olson OC, Joyce JA. Cysteine cathepsin proteases: regulators of cancer progression and therapeutic response. Nat. Rev. Cancer. 2015;15:712–729. doi: 10.1038/nrc4027. PubMed DOI

Chantranupong L, et al. The CASTOR proteins are arginine sensors for the mTORC1 pathway. Cell. 2016;165:153–164. doi: 10.1016/j.cell.2016.02.035. PubMed DOI PMC

Sancak Y, et al. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell. 2010;141:290–303. doi: 10.1016/j.cell.2010.02.024. PubMed DOI PMC

Efeyan A, et al. Regulation of mTORC1 by the Rag GTPases is necessary for neonatal autophagy and survival. Nature. 2013;493:679–683. doi: 10.1038/nature11745. PubMed DOI PMC

Wang SY, et al. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1. Science. 2015;347:188–194. doi: 10.1126/science.1257132. PubMed DOI PMC

Kim E, Goraksha-Hicks P, Li L, Neufeld TP, Guan KL. Regulation of TORC1 by Rag GTPases in nutrient response. Nat. Cell Biol. 2008;10:935–945. doi: 10.1038/ncb1753. PubMed DOI PMC

Sancak Y, et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science. 2008;320:1496–1501. doi: 10.1126/science.1157535. PubMed DOI PMC

Farazi PA, DePinho RA. Hepatocellular carcinoma pathogenesis: from genes to environment. Nat. Rev. Cancer. 2006;6:674–687. doi: 10.1038/nrc1934. PubMed DOI

Llovet JM, Villanueva A, Lachenmayer A, Finn RS. Advances in targeted therapies for hepatocellular carcinoma in the genomic era. Nat. Rev. Clin. Oncol. 2015;12:408–424. doi: 10.1038/nrclinonc.2015.103. PubMed DOI

Prieto J, Melero I, Sangro B. Immunological landscape and immunotherapy of hepatocellular carcinoma. Nat. Rev. Gastro. Hepat. 2015;12:681–700. doi: 10.1038/nrgastro.2015.173. PubMed DOI

Benda A, et al. How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy. Langmuir. 2003;19:4120–4126. doi: 10.1021/la0270136. DOI

Wien RW, Mcconnell HM, Morrisett JD. Spin-label-induced nuclear relaxation - distances between bound saccharides, histidine-15, and tryptophan-123 on lysozyme in solution. Biochemistry. 1972;11:3707–3716. doi: 10.1021/bi00770a008. PubMed DOI

Gavet O, Pines J. Progressive activation of cyclinB1-Cdk1 coordinates entry to mitosis. Dev. Cell. 2010;18:533–543. doi: 10.1016/j.devcel.2010.02.013. PubMed DOI PMC

Lunov O, et al. The effect of carboxydextran-coated superparamagnetic iron oxide nanoparticles on c-Jun N-terminal kinase-mediated apoptosis in human macrophages. Biomaterials. 2010;31:5063–5071. doi: 10.1016/j.biomaterials.2010.03.023. PubMed DOI

Lunov O, et al. Lysosomal degradation of the carboxydextran shell of coated superparamagnetic iron oxide nanoparticles and the fate of professional phagocytes. Biomaterials. 2010;31:9015–9022. doi: 10.1016/j.biomaterials.2010.08.003. PubMed DOI

Zimmermann H, Gerhard D, Dingermann T, Hothorn LA. Statistical aspects of design and validation of microtitre-plate-based linear and non-linear parallel in vitro bioassays. Biotechnol. J. 2010;5:62–74. doi: 10.1002/biot.200900146. PubMed DOI

Waters JC. Accuracy and precision in quantitative fluorescence microscopy. J. Cell Biol. 2009;185:1135–1148. doi: 10.1083/jcb.200903097. PubMed DOI PMC

Hamilton N. Quantification and its applications in fluorescent microscopy imaging. Traffic. 2009;10:951–961. doi: 10.1111/j.1600-0854.2009.00938.x. PubMed DOI

Dell RB, Holleran S, Ramakrishnan R. Sample size determination. ILAR J. 2002;43:207–213. doi: 10.1093/ilar.43.4.207. PubMed DOI PMC

Weissgerber TL, Milic NM, Winham SJ, Garovic VD. Beyond bar and line graphs: time for a new data presentation paradigm. PLoS Biol. 2015;13:e1002128. doi: 10.1371/journal.pbio.1002128. PubMed DOI PMC

Mechanical Regulation of Mitochondrial Dynamics and Function in a 3D-Engineered Liver Tumor Microenvironment

The interactions between DNA nanostructures and cells: A critical overview from a cell biology perspective

Biological Evaluation of Photodynamic Effect Mediated by Nanoparticles with Embedded Porphyrin Photosensitizer

Protein Corona Inhibits Endosomal Escape of Functionalized DNA Nanostructures in Living Cells

Expression of Interferons Lambda 3 and 4 Induces Identical Response in Human Liver Cell Lines Depending Exclusively on Canonical Signaling

Light-induced modulation of the mitochondrial respiratory chain activity: possibilities and limitations

Iron Oxide Nanoparticle-Induced Autophagic Flux Is Regulated by Interplay between p53-mTOR Axis and Bcl-2 Signaling in Hepatic Cells

Remote Actuation of Apoptosis in Liver Cancer Cells via Magneto-Mechanical Modulation of Iron Oxide Nanoparticles

Preliminary Study of Ge-DLC Nanocomposite Biomaterials Prepared by Laser Codeposition

Targeting the mTOR Signaling Pathway Utilizing Nanoparticles: A Critical Overview

A Critical Review on Selected External Physical Cues and Modulation of Cell Behavior: Magnetic Nanoparticles, Non-thermal Plasma and Lasers

Manipulating the mitochondria activity in human hepatic cell line Huh7 by low-power laser irradiation