Cytotoxic, Apoptotic and Genotoxic Effects of Lipid-Based and Polymeric Nano Micelles, an In Vitro Evaluation
Abstract
:1. Introduction
- “Identify the limitations of current test methods to assess the quality and safety of nano-particle-based therapeutics; and
- Evaluate the application of nanotechnology on product characteristics, including stability and content uniformity” [12].
2. Materials and Methods
2.1. Materials
Synthesis and Analysis of Purity of PCL2-PEG
2.2. Instruments
2.3. Methods
2.3.1. Preparation of Nano-Formulations
2.3.2. Particle Size Distribution and Zeta Potential Assays
2.3.3. Cell Culture and Maintenance
2.3.4. Cytotoxicity Assay
SRB
WST-1
2.3.5. Genotoxic Activity Assays (Comet Assay)
Sample Preparation
Cell Culture
Comet Assay
2.3.6. Detection of Apoptosis and Necrosis Using Acridine Orange/Ethidium Bromide (AO/EB) Staining
2.3.7. Statistical Analysis
3. Results
3.1. Particle Size Distribution and Zeta Potential Assays
3.2. Cytotoxicity Assay
3.2.1. SRB
3.2.2. WST-1
3.3. Genotoxic Activity Assays
3.4. Detection of Apoptosis and Necrosis Using Acridine Orange/Ethidium Bromide (AO/EB) Staining
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
- Maeda, H. Macromolecular therapeutics in cancer treatment: The EPR effect and beyond. J. Controll. Release 2012, 164, 138–144. [Google Scholar] [CrossRef] [PubMed]
- Comoglu, T.; Bahadori, F. NanoVectors for Neurotherapeutic Delivery, Part I: Liposomes and Micelles; OMICS International: Los Angeles, CA, USA, 2015. [Google Scholar]
- Rösler, A.; Vandermeulen, G.W.; Klok, H.-A. Advanced drug delivery devices via self-assembly of amphiphilic block copolymers. Adv. Drug Deliv. Rev. 2012, 64, 270–279. [Google Scholar] [CrossRef]
- Hafner, A.; Lovrić, J.; Lakoš, G.P.; Pepić, I. Nanotherapeutics in the EU: An overview on current state and future directions. Int. J. Nanomed. 2014, 9, 1005–1023. [Google Scholar]
- Malam, Y.; Loizidou, M.; Seifalian, A.M. Liposomes and nanoparticles: Nanosized vehicles for drug delivery in cancer. Trends Pharmacol. Sci. 2009, 30, 592–599. [Google Scholar] [CrossRef] [PubMed]
- Koudelka, Š.; Turánek, J. Liposomal paclitaxel formulations. J. Controll. Release 2012, 163, 322–334. [Google Scholar] [CrossRef] [PubMed]
- Kohli, A.G.; Kierstead, P.H.; Venditto, V.J.; Walsh, C.L.; Szoka, F.C. Designer lipids for drug delivery: From heads to tails. J. Controll. Release 2014, 190, 274–287. [Google Scholar] [CrossRef] [PubMed]
- Damoiseaux, R.; George, S.; Li, M.; Pokhrel, S.; Ji, Z.; France, B.; Xia, T.; Suarez, E.; Rallo, R.; Mädler, L. No time to lose—High throughput screening to assess nanomaterial safety. Nanoscale 2011, 3, 1345–1360. [Google Scholar] [CrossRef] [PubMed]
- Nel, A.; Grainger, D.; Alvarez, P.J.; Badesha, S.; Castranova, V.; Ferrari, M.; Godwin, H.; Grodzinski, P.; Morris, J.; Savage, N. Nanotechnology environmental, health, and safety issues. In Nanotechnology Research Directions for Societal Needs in 2020; Springer: Berlin/Heidelberg, Germany, 2011; pp. 159–220. [Google Scholar]
- FDA. FDA’s Approach to Regulation of Nanotechnology Products. Available online: http://www.fda.gov/ScienceResearch/SpecialTopics/Nanotechnology/ucm301114.htm#guidance (accessed on 15 December 2017).
- Pillay, V.; Choonara, Y.E. Advances in Neurotherapeutic Delivery Technologies; OMICS International: Los Angeles, CA, USA, 2015. [Google Scholar]
- U.S. Food and Drug. Center for Drug Evaluation and Research Nanotechnology Programs; FDA: Silver Spring, MD, USA, 2015.
- Bahadori, F.; Dag, A.; Durmaz, H.; Cakir, N.; Onyuksel, H.; Tunca, U.; Topcu, G.; Hizal, G. Synthesis and Characterization of Biodegradable Amphiphilic Star and Y-Shaped Block Copolymers as Potential Carriers for Vinorelbine. Polymers 2014, 6, 214–242. [Google Scholar] [CrossRef]
- Bahadori, F.; Topcu, G.; Eroglu, M.S.; Onyuksel, H. A New Lipid-Based Nano Formulation of Vinorelbine. AAPS Pharm. Sci. Tech. 2014, 15, 1138–1148. [Google Scholar] [CrossRef] [PubMed]
- Lim, S.B.; Banerjee, A.; Önyüksel, H. Improvement of drug safety by the use of lipid-based nanocarriers. J. Controll. Release 2012, 163, 34–45. [Google Scholar] [CrossRef] [PubMed]
- Altintas, O.; Hizal, G.; Tunca, U. Synthesis of an ABCD 4-miktoarm star quaterpolymer through a Diels–Alder click reaction. Des. Monomers Polym. 2009, 12, 83–98. [Google Scholar] [CrossRef]
- Lim, S.B.; Rubinstein, I.; Onyuksel, H. Freeze drying of peptide drugs self-associated with long-circulating, biocompatible and biodegradable sterically stabilized phospholipid nanomicelles. Int. J. Pharm. 2008, 356, 345–350. [Google Scholar] [CrossRef] [PubMed]
- Ashok, B.; Arleth, L.; Hjelm, R.P.; Rubinstein, I.; Onyuksel, H. In vitro characterization of PEGylated phospholipid micelles for improved drug solubilization: Effects of PEG chain length and PC incorporation. J. Pharm. Sci. 2004, 93, 2476–2487. [Google Scholar] [CrossRef] [PubMed]
- Wang, F.; Bronich, T.K.; Kabanov, A.V.; Rauh, R.D.; Roovers, J. Synthesis and Evaluation of a Star Amphiphilic Block Copolymer from Poly(ε-caprolactone) and Poly(ethylene glycol) as a Potential Drug Delivery Carrier. Bioconjug. Chem. 2005, 16, 397–405. [Google Scholar] [CrossRef] [PubMed]
- Ward, G.A. Measurements of binding thermodynamics in drug discovery. Drug Discov. Today 2005, 10, 1543–1551. [Google Scholar]
- Lv, Q.; Yu, A.; Xi, Y.; Li, H.; Song, Z.; Cui, J.; Cao, F.; Zhai, G. Development and evaluation of penciclovir-loaded solid lipid nanoparticles for topical delivery. Int. J. Pharm. 2009, 372, 191–198. [Google Scholar] [CrossRef] [PubMed]
- Zheng, C.; Qiu, L.; Yao, X.; Zhu, K. Novel micelles from graft polyphosphazenes as potential anti-cancer drug delivery systems: Drug encapsulation and in vitro evaluation. Int. J. Pharm. 2009, 373, 133–140. [Google Scholar] [CrossRef] [PubMed]
- Koo, O.M.Y.; Rubinstein, I.; Önyüksel, H. Actively targeted low-dose camptothecin as a safe, long-acting, disease-modifying nanomedicine for rheumatoid arthritis. Pharm. Res. 2011, 28, 776–787. [Google Scholar] [CrossRef] [PubMed]
- Banerjee, A.; Onyuksel, H. Human Pancreatic Polypeptide in a Phospholipid-Based Micellar Formulation. Pharm. Res. 2012, 29, 1698–1711. [Google Scholar] [CrossRef] [PubMed]
- Wu, L.; Zhao, W.; Yang, R.; Chen, X. Effects of pulsed electric fields processing on stability of egg white proteins. J. Food Eng. 2014, 139, 13–18. [Google Scholar] [CrossRef]
- Mehlen, P.; Preville, X.; Chareyron, P.; Briolay, J.; Klemenz, R.; Arrigo, A.-P. Constitutive expression of human HSP27, Drosophila HSP27, or human alpha B-crystallin confers resistance to TNF-and oxidative stress-induced cytotoxicity in stably transfected murine L929 fibroblasts. J. Immunol. 1995, 154, 363–374. [Google Scholar] [PubMed]
- Eldeniz, A.; Mustafa, K.; Ørstavik, D.; Dahl, J. Cytotoxicity of new resin-, calcium hydroxide-and silicone-based root canal sealers on fibroblasts derived from human gingiva and L929 cell lines. Int. Endod. J. 2007, 40, 329–337. [Google Scholar] [CrossRef] [PubMed]
- Kaga, M.; Noda, M.; Ferracane, J.; Nakamura, W.; Oguchi, H.; Sano, H. The in vitro cytotoxicity of eluates from dentin bonding resins and their effect on tyrosine phosphorylation of L929 cells. Dent. Mater. 2001, 17, 333–339. [Google Scholar] [CrossRef]
- Kirstein, M.; Fiers, W.; Baglioni, C. Growth inhibition and cytotoxicity of tumor necrosis factor in L929 cells is enhanced by high cell density and inhibition of mRNA synthesis. J. Immunol. 1986, 137, 2277–2280. [Google Scholar] [PubMed]
- Goossens, V.; Grooten, J.; Fiers, W. The oxidative metabolism of glutamine A modulator of reactive oxygen intermediate-mediated cytotoxicity of tumor necrosis factor in L929 fibrosarcoma cells. J. Biol. Chem. 1996, 271, 192–196. [Google Scholar] [CrossRef] [PubMed]
- Rao, S.; Ushida, T.; Tateishi, T.; Okazaki, Y.; Asao, S. Effect of Ti, Al, and V ions on the relative growth rate of fibroblasts (L929) and osteoblasts (MC3T3-E1) cells. Bio-Med. Mater. Eng. 1996, 6, 79–86. [Google Scholar]
- Vichai, V.; Kirtikara, K. Sulforhodamine B colorimetric assay for cytotoxicity screening. Nat. Protoc. 2006, 1, 1112–1116. [Google Scholar] [CrossRef] [PubMed]
- Sugiyama, T.; Yoneda, M.; Kuraishi, T.; Hattori, S.; Inoue, Y.; Sato, H.; Kai, C. Measles virus selectively blind to signaling lymphocyte activation molecule as a novel oncolytic virus for breast cancer treatment. Gene Ther. 2013, 20, 338–347. [Google Scholar] [CrossRef] [PubMed]
- Scaria, P.V.; Liu, Y.; Leng, Q.; Chou, S.-T.; Mixson, A.J.; Woodle, M.C. Enhancement of antifungal activity by integrin-targeting of branched histidine rich peptides. J. Drug Target. 2014, 22, 536–542. [Google Scholar] [CrossRef] [PubMed]
- Singh, N.P.; McCoy, M.T.; Tice, R.R.; Schneider, E.L. A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 1988, 175, 184–191. [Google Scholar] [CrossRef]
- Kocyigit, A.; Selek, S.; Celik, H.; Dikilitas, M. Mononuclear leukocyte DNA damage and oxidative stress: The association with smoking of hand-rolled and filter-cigarettes. Mutat. Res. Toxicol. Environ. Mutagen. 2011, 721, 136–141. [Google Scholar] [CrossRef] [PubMed]
- Collins, A.R.; Ai-Guo, M.; Duthie, S.J. The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mutat. Res. Repair 1995, 336, 69–77. [Google Scholar] [CrossRef]
- Dikilitas, M.; Kocyigit, A. Assessment of Computerized and Manual Analysis of Slides Processed in Single Cell Gel Electrophoresis ASSAY. Fresenius Environ. Bull. 2012, 21, 2981–2987. [Google Scholar]
- McGahon, A.J.; Martin, S.J.; Bissonnette, R.P.; Mahboubi, A.; Shi, Y.; Mogil, R.J.; Nishioka, W.K.; Green, D.R. The end of the (cell) line: Methods for the study of apoptosis in vitro. Methods Cell Biol. 1995, 46, 153–185. [Google Scholar] [PubMed]
- Kasibhatla, S.; Amarante-Mendes, G.P.; Finucane, D.; Brunner, T.; Bossy-Wetzel, E.; Green, D.R. Acridine orange/ethidium bromide (AO/EB) staining to detect apoptosis. CSH Protoc. 2006, 3. [Google Scholar] [CrossRef] [PubMed]
- Govender, T.; Riley, T.; Ehtezazi, T.; Garnett, M.C.; Stolnik, S.; Illum, L.; Davis, S.S. Defining the drug incorporation properties of PLA–PEG nanoparticles. Int. J. Pharm. 2000, 199, 95–110. [Google Scholar] [CrossRef]
- Brandenburg, K.S. Initial Studies for the Development of a Novel Antibacterial Nanomedicine; University of Illinois at Chicago: Chicago, IL, USA, 2009. [Google Scholar]
- Keepers, Y.P.; Pizao, P.E.; Peters, G.J.; van Ark-Otte, J.; Winograd, B.; Pinedo, H.M. Comparison of the sulforhodamine B protein and tetrazolium (MTT) assays for in vitro chemosensitivity testing. Eur. J. Cancer Clin. Oncol. 1991, 27, 897–900. [Google Scholar] [CrossRef]
- Peskin, A.V.; Winterbourn, C.C. A microtiter plate assay for superoxide dismutase using a water-soluble tetrazolium salt (WST-1). Clin. Chim. Acta 2000, 293, 157–166. [Google Scholar] [CrossRef]
- Tice, R.; Agurell, E.; Anderson, D.; Burlinson, B.; Hartmann, A.; Kobayashi, H.; Miyamae, Y.; Rojas, E.; Ryu, J.; Sasaki, Y. Single cell gel/comet assay: Guidelines for in vitro and in vivo genetic toxicology testing. Environ. Mol. Mutagen. 2000, 35, 206–221. [Google Scholar] [CrossRef]
- Darakhshan, S.; Ghanbari, A. Tranilast enhances the anti-tumor effects of tamoxifen on human breast cancer cells in vitro. J. Biomed. Sci. 2013, 20, 76. [Google Scholar] [CrossRef] [PubMed]
- Williams, D.F. On the mechanisms of biocompatibility. Biomaterials 2008, 29, 2941–2953. [Google Scholar] [CrossRef] [PubMed]
- Bianco, A.; Kostarelos, K.; Prato, M. Making carbon nanotubes biocompatible and biodegradable. Chem. Commun. 2011, 47, 10182–10188. [Google Scholar] [CrossRef] [PubMed]
- Ludatscher, R.M.; Stehbens, W.E. Vesicles of fenestrated and non-fenestrated endothelium. Zeitschrift für Zellforschung und Mikroskopische Anatomie 1969, 97, 169–177. [Google Scholar] [CrossRef] [PubMed]
- Ryan, U.S.; Ryan, J.; Smith, D.S.; Winkler, H. Fenestrated endothelium of the adrenal gland: Freeze-fracture studies. Tissue Cell 1975, 7, 181–190. [Google Scholar] [CrossRef]
- Alexis, F.; Pridgen, E.; Molnar, L.K.; Farokhzad, O.C. Factors affecting the clearance and biodistribution of polymeric nanoparticles. Mol. Pharm. 2008, 5, 505–515. [Google Scholar] [CrossRef] [PubMed]
- Spagnou, S.; Miller, A.D.; Keller, M. Lipidic carriers of siRNA: Differences in the formulation, cellular uptake, and delivery with plasmid DNA. Biochemistry 2004, 43, 13348–13356. [Google Scholar] [CrossRef] [PubMed]
- Dokka, S.; Toledo, D.; Shi, X.; Castranova, V.; Rojanasakul, Y. Oxygen radical-mediated pulmonary toxicity induced by some cationic liposomes. Pharm. Res. 2000, 17, 521–525. [Google Scholar] [CrossRef] [PubMed]
- Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. Toxicity of cationic lipids and cationic polymers in gene delivery. J. Controll. Release 2006, 114, 100–109. [Google Scholar] [CrossRef] [PubMed]
- Sharma, A.; Madhunapantula, S.V.; Robertson, G.P. Toxicological considerations when creating nanoparticle-based drugs and drug delivery systems. Expert Opin. Drug Metab. Toxicol. 2012, 8, 47–69. [Google Scholar] [CrossRef] [PubMed]
- Vukovic, L.; Khatib, F.A.; Drake, S.P.; Madriaga, A.; Brandenburg, K.S.; Král, P.; Onyuksel, H. Structure and dynamics of highly PEG-ylated sterically stabilized micelles in aqueous media. J. Am. Chem. Soc. 2011, 133, 13481–13488. [Google Scholar] [CrossRef] [PubMed]
- Pai, A.S.; Rubinstein, I.; Önyüksel, H. PEGylated phospholipid nanomicelles interact with β-amyloid (1–42) and mitigate its β-sheet formation, aggregation and neurotoxicity in vitro. Peptides 2006, 27, 2858–2866. [Google Scholar] [CrossRef] [PubMed]
- Rubinstein, I.; Krishnadas, A.; Peddakota, L.; Önyüksel, H. Nanoparticulate paclitaxel loaded into sterically stabilized mixed phospholipid micelles to improve chemotherapy of breast cancer. Breast Cancer Res. 2005, 7 (Suppl. S1), 4. [Google Scholar] [CrossRef]
- Stern, S.T.; Adiseshaiah, P.P.; Crist, R.M. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Part. Fibre Toxicol. 2012, 9, 20. [Google Scholar] [CrossRef] [PubMed]
- De Stefano, D.; Carnuccio, R.; Maiuri, M.C. Nanomaterials toxicity and cell death modalities. J. Drug Deliv. 2012, 2012, 167896. [Google Scholar] [CrossRef] [PubMed]
- Elbert, D.L.; Hubbell, J.A. Surface treatments of polymers for biocompatibility. Ann. Rev. Mater. Sci. 1996, 26, 365–394. [Google Scholar] [CrossRef]
- Yin, H.; Gong, C.; Shi, S.; Liu, X.; Wei, Y.; Qian, Z. Toxicity evaluation of biodegradable and thermosensitive PEG-PCL-PEG hydrogel as a potential in situ sustained ophthalmic drug delivery system. J. Biomed. Mater. Res. Part B Appl. Biomater. 2010, 92, 129–137. [Google Scholar] [CrossRef] [PubMed]
- Porter, C.J.; Moghimi, S.M.; Illum, L.; Davis, S.S. The polyoxyethylene/polyoxypropylene block co-polymer Poloxamer-407 selectively redirects intravenously injected microspheres to sinusoidal endothelial cells of rabbit bone marrow. FEBS Lett. 1992, 305, 62–66. [Google Scholar] [CrossRef]
- Panyam, J.; Labhasetwar, V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Deliv. Rev. 2003, 55, 329–347. [Google Scholar] [CrossRef]
- Song, M.-M.; Song, W.-J.; Bi, H.; Wang, J.; Wu, W.-L.; Sun, J.; Yu, M. Cytotoxicity and cellular uptake of iron nanowires. Biomaterials 2010, 31, 1509–1517. [Google Scholar] [CrossRef] [PubMed]
- Singh, N.; Jenkins, G.J.; Asadi, R.; Doak, S.H. Potential toxicity of superparamagnetic iron oxide nanoparticles (SPION). Nano Rev. 2010, 1. [Google Scholar] [CrossRef] [PubMed]
- Buja, L.; Eigenbrodt, M.L.; Eigenbrodt, E.H. Apoptosis and necrosis. Basic types and mechanisms of cell death. Arch. Pathol. Lab. Med. 1993, 117, 1208–1214. [Google Scholar] [PubMed]
- Shin, S.W.; Song, I.H.; Um, S.H. Role of physicochemical properties in nanoparticle toxicity. Nanomaterials 2015, 5, 1351–1365. [Google Scholar] [CrossRef] [PubMed]
Functional Groups | Chemical Shifts | Multiplicities |
---|---|---|
ArH of anthracene | 8.30 | d * |
ArH of anthracene | 8.03 | d * |
ArH of anthracene | 7.60–7.47 | m * |
CH2–anthracene | 6.2 | s * |
CH2OC=O of PCL | 4.05–4.02 | br * |
CH2OH of PCL end-group | 3.60 | bs * |
C=OCH2 of PCL | 2.20 | br * |
CH2CH2CH2 of PCL | 1.80–1.20 | m * |
Media | PBS | Complete Culture Media | |||||||
---|---|---|---|---|---|---|---|---|---|
% Volume | SD | % Number | SD | % Intensity | SD | PDI | Zeta Potential (mV) | % Intensity | |
SSM | 13.45 | 3.754 | 11.62 | 2.754 | 16.37 | 4.389 | 0.194 | 0.01 | 16.25 |
PM | 112.7 | 39.41 | 87.92 | 25.39 | 128.9 | 38.36 | 0.242 | -26.3 | 132.56 |
© 2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Share and Cite
Bahadori, F.; Kocyigit, A.; Onyuksel, H.; Dag, A.; Topcu, G. Cytotoxic, Apoptotic and Genotoxic Effects of Lipid-Based and Polymeric Nano Micelles, an In Vitro Evaluation. Toxics 2018, 6, 7. https://doi.org/10.3390/toxics6010007
Bahadori F, Kocyigit A, Onyuksel H, Dag A, Topcu G. Cytotoxic, Apoptotic and Genotoxic Effects of Lipid-Based and Polymeric Nano Micelles, an In Vitro Evaluation. Toxics. 2018; 6(1):7. https://doi.org/10.3390/toxics6010007
Chicago/Turabian StyleBahadori, Fatemeh, Abdurrahim Kocyigit, Hayat Onyuksel, Aydan Dag, and Gulacti Topcu. 2018. "Cytotoxic, Apoptotic and Genotoxic Effects of Lipid-Based and Polymeric Nano Micelles, an In Vitro Evaluation" Toxics 6, no. 1: 7. https://doi.org/10.3390/toxics6010007