- Email: [email protected]
Optimized bio-nano interface using peptide modified colloidal gold nanoparticles
Colloids and Interface Science Communications 1 (2014) 54–56
Contents lists available at ScienceDirect
Colloids and Interface Science Communications journal homepage: www.elsevier.com/locate/colcom
Rapid Communication
Optimized bio-nano interface using peptide modified colloidal gold nanoparticles Celina Yang a, Darren Yohan a, Devika B. Chithrani a,b,⁎ a b
Department of Physics, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, 30 Bond Street, Toronto, ON M5B 1W8, Canada
a r t i c l e
i n f o
Article history: Received 29 May 2014 Accepted 8 July 2014 Available online xxxx Keywords: Colloidal gold nanoparticles Nucleus Peptides Endosomes Lysosomes
a b s t r a c t The interactions of synthetically produced colloidal gold nanoparticles (GNPs) with living organisms are gaining much attention in the biomedical sciences. While non-viral carriers, such as GNPs, are safer and more costefficient as compared to viral vectors, the probability of cell internalization is lower. In this paper, we discussed a method to increase cell internalization of GNPs using a specific peptide known as “RME-Peptide”. Our results showed an enhancement in cell uptake of peptide-capped GNPs. Furthermore, we investigated the exocytosis process of peptide-capped GNPs. Our results showed that the peptide-modified GNPs had a lower number of NPs exocytosed as compared to citrate-capped or as-made colloidal GNPs. Our study shows that bio-nano interface can be improved using specially designed peptides on colloidal GNPs. The outcome of this research will be beneficial for their applications in therapeutics and imaging. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Nanotechnology-based approaches facilitate further development of safer yet more effective diagnostic and therapeutic modalities for cancer therapy [1,2]. The primary goal of nanoparticle (NP)-based platforms will be the optimized delivery of therapeutics to tumors while causing minimal damage to normal tissue and side effects to the patients [3–7]. Among other NPs, Gold NPs (GNPs) are being used in cancer research due to their biocompatibility and ability to act as both a radiosensitizer and drug carrier in cancer therapy [8–12]. The design of smart multifunctional NPs in order to improve current therapeutic applications requires a thorough understanding of the mechanisms behind nanoparticles (NPs) entering and leaving the cells. For drug delivery, radiation therapy, and imaging applications, it is necessary to control and manipulate the accumulation of NPs for an extended period of time within the cell. Previous studies have shown that both uptake and removal mechanisms are dependent on the size, shape, and surface properties of NPs. Among NP sizes between 13 and 100 nm, NPs of diameter 50 nm showed the highest cell uptake [13]. Elucidating the mechanism of uptake and removal of NPs in cells could lead to a better understanding of NP toxicity. For example, if the NPs are trapped in vesicles and leave the cells intact, they are unlikely to induce cellular toxicity. Theoretical calculations support the size dependent NP uptake [14]. Chithrani et al. have put forward a theoretical calculation to
⁎ Corresponding author at: Department of Physics, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada. Tel.: +1 416 979 5000x4115; fax: +1 416 979 5000. E-mail address: [email protected] (D.B. Chithrani).
support the NP removal process [15]. In most of these studies, NPs took the endo-lyso path where NPs enter the cell through receptormediated endocytosis (RME), are trapped in endosomes, fuse with lysosomes for processing, and leave the cell via the exocytosis process. During the endo-lyso path, NPs were localized in either endosomes or lysosomes. The cell cytoplasm and nucleus were free of NPs. Nanoparticles localized in the lysosomes exit the cell through exocytosis process. Previous studies have shown that the path of NPs conjugated with certain peptides is different from a regular endo-lyso path [16,17]. Nanoparticles conjugated with peptides containing integrin binding domain can enter the cell via the endocytosis process followed by escaping into the cytoplasm from endosomes [17,18]. These peptides are also referred to as RME peptides since it enhances the RME process [17,18]. Brust and co-workers suggested the approach of evading the well-established endo-lyso pathway of uptake to a significant extent, either via the delivery of NPs by liposomes or by surface modification of the NPs with the supposed cell penetrating peptides (CPPs) [19]. The exocytosis process of NPs modified with peptides is not yet known entirely. In this study, we used two peptides: 1) Pentapeptide (sequence: H-Cys-Ala-Leu-Asn-Asn-OH) was used for stabilizing NPs for peptide conjugation [20], and 2) RME peptide (sequence: CKKKKKKGGRGDMFG) was used for endosomal escape and also to improve NP uptake [17]. Our transmission electron microscopy (TEM) images show that the RME peptide allows NPs to be released into the cytoplasm (see Fig. 1). The endosomal escape of NPs reduced the rate of the exocytosis process (as discussed later in this article). This increase in residence time of GNPs within the cell may lead to improved therapeutic applications.
http://dx.doi.org/10.1016/j.colcom.2014.07.003 2215-0382/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
C. Yang et al. / Colloids and Interface Science Communications 1 (2014) 54–56
The size of the GNPs used in this study was approximately 15 nm in diameter. The citrate reduction method was used to synthesize GNPs. Stock solutions of 1% HAuCl4 were prepared and stored at room temperature. Stock solutions of 1% sodium citrate were prepared fresh each time. In the first step, 300 μL of 1% chloroauric acid solution was added to 30 mL of deionized water in a round bottom flask. The mixture was brought to boiling on a hot plate while being stirred with a magnetic stir bar. In the second step, 600 μL of 1% citrate solution was added to the solution mixture and left on the hot plate for 5 min to complete the reaction. The color of the solution changed from clear to dark blue to red. The mixture was brought to room temperature while stirring. Peptide-gold particle complexes were assembled by first conjugating the GNPs with the pentapeptide with approximately 300 peptides/ GNP ratio for stabilization purposes. Then the RME peptide was added with 8 to 10 peptide/GNP ratio. Each peptide was prepared with a terminal cystine group since cystine-terminated peptides can directly bind to the GNP surface via a covalent sulfur\gold bond [16,21]. NP complexes were characterized using UV visible spectroscopy, Dynamic Light Scattering (DLS), and zeta potential measurements (see Supplementary Section S1). There was no significant increase in terms of the size of NPs following conjugation with the peptide according to the DLS measurements. This is because the peptides were only few nanometers in size. However, the zeta potential changes from a negative value to a positive value. The negative charge of the nonconjugated (citrate-capped) GNPs is due to citrate molecules on the surface. Peptides are positively charged, and GNPs were positively charged following conjugation with the peptides. The peptide-GNP complexes were tested for their stability in cell culture media before using them for cell uptake and transport studies. The cellular uptake of peptide-GNP complexes was characterized by using HeLa cells, a well-known human fibroblast epithelial cell line. For tissue culture studies, cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO2. The cells were incubated with GNPs when the tissue culture dishes were 70–80% confluent. We have investigated the variation of NP distribution in cells internalized with citrate-capped and peptide-capped NPs using TEM imaging. The
Fig. 1. Nanoparticle transport pathway for peptide-capped NPs. A. Schematic showing the endosomal escape of peptide-capped NPs into the cytoplasm. B. TEM images showing localization of citrate-capped NPs in endosomes. C. Peptide-capped NPs escaped from endosomes were localized in the cytoplasm. The scale bar is 100 nm.
55
citrate-capped GNPs travel through the regular endo-lyso path causing NPs to become trapped in either endosomes or lysosomes [13] while most of the peptide-capped GNPs escaped from the endosomes and got localized in the cytoplasm (see Fig. 1). For quantification of the GNP uptake, the cells were washed three times with PBS and trypsinized. Cells were counted and then treated with HNO3 at 200 °C in an oil bath for an Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) analysis. Details of the quantification process are given in Supplementary Section S2. The cellular uptake data proved that the cells targeted with a RME peptide had the highest NP uptake, as compared to cells targeted with citrate-capped NPs (Fig. 2). It is believed that the synthetic RME peptide with an integrin binding domain (RGD) supplemented an additional driving force for the cell entry of NPs. In addition, positively charged NPs have a higher uptake compared to negatively charged (citrated-capped) NPs [22]. Zeta potential measurements showed that peptide-capped GNPs were positively charged (Supplementary Section S1). Hence, RME peptide and the positive charge of the NPs may have caused the increased uptake of peptide-modified NPs as compared to citrate-capped NPs. Previous work has shown that the exocytosis process depends on the cell type, NP size, and surface properties of NPs [15,23]. The exocytosis of GNPs functionalized with nuclear targeting peptides is not properly known yet. Our studies have shown for the first time that the fraction of NPs exocytosed was lower by two-fold for cells targeted with peptide-conjugated NPs (Fig. 3). Citrate-capped NPs were localized in endosomes followed by processing via fusion with lysosomes. NPs localized in lysosomes were excreted into the extracellular matrix through fusing with the cell membrane. However, most of the GNPs capped with peptides were able to enter the cytoplasm. This increase in the residence time in the cytoplasm could lead to a slower exocytosis process. Our results are in agreement with previously published work for CuO NPs and silica NPs. For example, Wang et al. evaluated the excretion of CuO NPs in A549 cells and discovered that a portion of NPs, which were located in mitochondria and nucleus, could not be excreted by the cells [24]. Similarly, based on findings by Chu et al., clusters of silica NPs in lysosomes are more easily exocytosed by H1299 cells as compared to single NPs in the cytoplasm [25]. We evaluated the exocytosis process as a function of time. To quantify NP removal, cells pre-incubated with NPs for eight hours were washed with PBS three times, introduced to fresh media supplemented with FBS, and left in the incubator for monitoring the exocytosis process. After one and six hour time points, the cell were rinsed three times with PBS and trypsinized for quantification of GNPs present per cell. As illustrated in Fig. 3A, a significant portion of the NPs were able to re-enter the cells. The percentage of NPs re-entering the cells was 10% and 15% respectively, for citrate-capped (non-targeted) and peptidecapped NPs. Nuclear targeted NPs appeared to aggregate less within the cell. Hence, it is possible for these NPs to re-enter cells once excreted. This could be one of the reasons for an increase in NPs present in cells targeted with peptides following a prolonged exocytosis process. The fraction of the GNPs exocytosed was calculated as illustrated in
Fig. 2. Nanoparticle uptake variation for citrate-capped and peptide-capped nanoparticles.
56
C. Yang et al. / Colloids and Interface Science Communications 1 (2014) 54–56
References
Fig. 3. Exocytosis of peptide-capped GNPs. Dynamics of exocytosis process following one and six hours. A. Number of GNPs exocytosed per cell after one hour and 6 h. B. Fraction of GNPs exocytosed per cell after 6 h.
Fig. 3B for six hour time point. Fraction of NPs exocytosed is lower for cells targeted with peptide-capped GNPs. Hence, the enhanced retention of NPs within cells can be used for improved therapeutics in radiation therapy and chemotherapy. In conclusion, our results showed that the functionalization of GNPs with RME peptides enhanced the NP uptake by five-fold. It was also found that the fraction of NPs exocytosed was lower by a factor of two for cells targeted with peptide-capped NPs in comparison to citrate-capped (non-targeted) NPs. This prolonged intracellular retention due to peptide functionalization would allow NPs to exert their desired applications more efficiently in cells, especially during drug delivery. Hence, a proper understanding of the endocytosis and exocytosis dynamics will shed more light on the design of GNPs in demand for cancer therapeutic applications. The authors would like acknowledge Natural Sciences and Engineering Research Council of Canada (NSERC) and Ryerson University for their financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.colcom.2014.07.003.
[1] A.G. Cuenca, H. Jiang, S.N. Hochwald, M. Delano, W.G. Cance, S.R. Grobmyer, Emerging implications of nanotechnology on cancer diagnostics and therapeutics, Cancer 107 (2006) 459–466. [2] J. Rao, Shedding light on tumors using nanoparticles, ACS Nano 2 (2008) 1984–1986. [3] P. Alivisatos, The use of nanocrystals in biological detection, Nat. Biotechnol. 22 (2003) 47–51. [4] M. Liong, J. Lu, M. Kovochich, T. Xia, S.G. Ruehm, A.E. Nel, F. Tamanoi, J.I. Zink, Multifunctional inorganic nanoparticles for imaging targeting, and drug delivery, ACS Nano 2 (2008) 889–896. [5] S. Langereis, J. Keupp, J.L.J. van Velthoven, I.H.C. de Roos, D. Burdinski, J.A. Pikkemaat, H. Grüll, A temperature-sensitive liposomal 1H CEST and 19 F contrast agent for MR image-guided drug delivery, J. Am. Chem. Soc. 9 (2009) 1380–1381. [6] S.D. Perrault, C. Walkey, T. Jennings, H.C. Fischer, W.C.W. Chan, Mediating tumor targeting efficiency of nanoparticles through design, Nano Lett. 9 (2009) 1909–1915. [7] J.E. Lee, N. Lee, H. Kim, J. Kim, S.H. Choi, J.H. Kim, T. Kim, I.C. Song, S.P. Park, W.K. Moon, T. Hyeon, Uniform mesoporous dye-doped silica nanocrystals for simultaneous enhanced magnetic resonance imaging, fluorescence imaging, and drug delivery, J. Am. Chem. Soc. 132 (2010) 552–557. [8] B.D. Chithrani, Intracellular uptake, transport, and processing of gold nanostructures, Mol. Membr. Biol. 27 (2010) 299–311. [9] B.D. Chithrani, Optimization of bio-nano interface using gold nanostructures as a model nanoparticle system, Insciences J. 1 (2011) 136–156. [10] J.M. Bergen, H.A. van Recum, T.T. Goodman, A.P. Massey, S.H. Pun, Gold nanoparticles as a versatile platform for optimizing physicochemical parameters for targeted drug delivery, Macromol. Biosci. 6 (2006) 506–516. [11] R. Shukla, V. Bansal, M. Chaudhary, A. Basu, R.R. Bhonde, M. Sastry, Biocompatibility of gold nanoparticles and their endocytotic fate inside the cellular compartment: a microscopic overview, Langmuir 21 (2005) 10644–10654. [12] S. Jelveh, B.D. Chithrani, Gold nanostructures as a platform for combinational therapy in future cancer therapeutics, Cancers 3 (2011) 1081–1110. [13] B.D. Chithrani, A.A. Ghazani, W.C.W. Chan, Determining the size and shape dependence of gold nanoparticle uptake into mammalian cells, Nano Lett. 6 (2006) 662–668. [14] H. Gao, W. Shi, L.B. Freund, Mechanics of receptor-mediated endocytosis, Proc. Natl. Acad. Sci. U. S. A. 102 (2005) 9469–9474. [15] B.D. Chithrani, W.C.W. Chan, Elucidating the mechanism of cellular uptake and removal of protein-coated gold nanoparticles of different sizes and shapes, Nano Lett. 7 (2007) 1542–1550. [16] A.G. Tkachenko, H. Xie., Y. Liu, D. Coleman, J. Ryan, W.R. Glomm, M.K. Shipton, S. Franzen, D.L. Feldheim, Cellular trajectories of peptide-modified gold particle complexes: comparison of nuclear localization signals and peptide transduction domains, Bioconjug. Chem. 15 (2004) 482–490. [17] C. Yang, M. Neshatian, M. Van Prooijen, B.D. Chithrani, Cancer nanotechnology: enhanced therapeutic response using peptide-modified gold nanoparticles, J. Nanosci. Nanotechnol. 14 (2014) 4813–4819. [18] C. Yang, B.D. Chithrani, Peptide-modified smaller nanoparticles for improved cancer therapy, J. Pharm. Pharmacol. (2014) (In press). [19] P. Nativo, I.A. Prior, M. Brust, Uptake and intracellular fate of surface-modified gold nanoparticles, ACS Nano 2 (2008) 1639–1644. [20] R. Lévy, N.T.K. Thanh, R.C. Doty, I. Hussain, R.J. Nichols, D.J. Schiffrin, M. Brust, D.G. Fernig, Rational and combinatorial design of peptide capping ligands for gold nanoparticles, J. Am. Chem. Soc. 126 (2004) 10076–10084. [21] Y. Zhao, F. Zhou, H. Zhou, H. Su, The structural and bonding evolution in cysteine– gold cluster complexes, Phys. Chem. Chem. Phys. 15 (2013) 1690–1698. [22] E. Frohlich, The role of surface charge in cellular uptake and cytotoxicity of medical nanoparticles, Int. J. Nanomedicine 7 (2012) 5577–5591. [23] D. Bartczak, T. Sanchez-Elsner, F. Louafi, T.M. Millar, A.G. Kanaras, Receptormediated interactions between colloidal gold nanoparticles and human umbilical vein endothelial cells, Small 7 (2011) 388–394. [24] Z. Wang, N. Li, J. Zhao, J.C. White, P. Qu, B. Xing, CuO nanoparticle interaction with human epithelial cells: cellular uptake, location, export, and genotoxicity, Chem. Res. Toxicol. 25 (2012) 1512–1521. [25] Z. Chu, Y. Huang, Q. Tao, Q. Li, Cellular uptake evolution, and excretion of silica nanoparticles in human cells, Nanoscale 3 (2011) 3291–3299.
Contents lists available at ScienceDirect
Colloids and Interface Science Communications journal homepage: www.elsevier.com/locate/colcom
Rapid Communication
Optimized bio-nano interface using peptide modified colloidal gold nanoparticles Celina Yang a, Darren Yohan a, Devika B. Chithrani a,b,⁎ a b
Department of Physics, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, 30 Bond Street, Toronto, ON M5B 1W8, Canada
a r t i c l e
i n f o
Article history: Received 29 May 2014 Accepted 8 July 2014 Available online xxxx Keywords: Colloidal gold nanoparticles Nucleus Peptides Endosomes Lysosomes
a b s t r a c t The interactions of synthetically produced colloidal gold nanoparticles (GNPs) with living organisms are gaining much attention in the biomedical sciences. While non-viral carriers, such as GNPs, are safer and more costefficient as compared to viral vectors, the probability of cell internalization is lower. In this paper, we discussed a method to increase cell internalization of GNPs using a specific peptide known as “RME-Peptide”. Our results showed an enhancement in cell uptake of peptide-capped GNPs. Furthermore, we investigated the exocytosis process of peptide-capped GNPs. Our results showed that the peptide-modified GNPs had a lower number of NPs exocytosed as compared to citrate-capped or as-made colloidal GNPs. Our study shows that bio-nano interface can be improved using specially designed peptides on colloidal GNPs. The outcome of this research will be beneficial for their applications in therapeutics and imaging. © 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Nanotechnology-based approaches facilitate further development of safer yet more effective diagnostic and therapeutic modalities for cancer therapy [1,2]. The primary goal of nanoparticle (NP)-based platforms will be the optimized delivery of therapeutics to tumors while causing minimal damage to normal tissue and side effects to the patients [3–7]. Among other NPs, Gold NPs (GNPs) are being used in cancer research due to their biocompatibility and ability to act as both a radiosensitizer and drug carrier in cancer therapy [8–12]. The design of smart multifunctional NPs in order to improve current therapeutic applications requires a thorough understanding of the mechanisms behind nanoparticles (NPs) entering and leaving the cells. For drug delivery, radiation therapy, and imaging applications, it is necessary to control and manipulate the accumulation of NPs for an extended period of time within the cell. Previous studies have shown that both uptake and removal mechanisms are dependent on the size, shape, and surface properties of NPs. Among NP sizes between 13 and 100 nm, NPs of diameter 50 nm showed the highest cell uptake [13]. Elucidating the mechanism of uptake and removal of NPs in cells could lead to a better understanding of NP toxicity. For example, if the NPs are trapped in vesicles and leave the cells intact, they are unlikely to induce cellular toxicity. Theoretical calculations support the size dependent NP uptake [14]. Chithrani et al. have put forward a theoretical calculation to
⁎ Corresponding author at: Department of Physics, Ryerson University, 350 Victoria Street, Toronto, ON M5B 2K3, Canada. Tel.: +1 416 979 5000x4115; fax: +1 416 979 5000. E-mail address: [email protected] (D.B. Chithrani).
support the NP removal process [15]. In most of these studies, NPs took the endo-lyso path where NPs enter the cell through receptormediated endocytosis (RME), are trapped in endosomes, fuse with lysosomes for processing, and leave the cell via the exocytosis process. During the endo-lyso path, NPs were localized in either endosomes or lysosomes. The cell cytoplasm and nucleus were free of NPs. Nanoparticles localized in the lysosomes exit the cell through exocytosis process. Previous studies have shown that the path of NPs conjugated with certain peptides is different from a regular endo-lyso path [16,17]. Nanoparticles conjugated with peptides containing integrin binding domain can enter the cell via the endocytosis process followed by escaping into the cytoplasm from endosomes [17,18]. These peptides are also referred to as RME peptides since it enhances the RME process [17,18]. Brust and co-workers suggested the approach of evading the well-established endo-lyso pathway of uptake to a significant extent, either via the delivery of NPs by liposomes or by surface modification of the NPs with the supposed cell penetrating peptides (CPPs) [19]. The exocytosis process of NPs modified with peptides is not yet known entirely. In this study, we used two peptides: 1) Pentapeptide (sequence: H-Cys-Ala-Leu-Asn-Asn-OH) was used for stabilizing NPs for peptide conjugation [20], and 2) RME peptide (sequence: CKKKKKKGGRGDMFG) was used for endosomal escape and also to improve NP uptake [17]. Our transmission electron microscopy (TEM) images show that the RME peptide allows NPs to be released into the cytoplasm (see Fig. 1). The endosomal escape of NPs reduced the rate of the exocytosis process (as discussed later in this article). This increase in residence time of GNPs within the cell may lead to improved therapeutic applications.
http://dx.doi.org/10.1016/j.colcom.2014.07.003 2215-0382/© 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
C. Yang et al. / Colloids and Interface Science Communications 1 (2014) 54–56
The size of the GNPs used in this study was approximately 15 nm in diameter. The citrate reduction method was used to synthesize GNPs. Stock solutions of 1% HAuCl4 were prepared and stored at room temperature. Stock solutions of 1% sodium citrate were prepared fresh each time. In the first step, 300 μL of 1% chloroauric acid solution was added to 30 mL of deionized water in a round bottom flask. The mixture was brought to boiling on a hot plate while being stirred with a magnetic stir bar. In the second step, 600 μL of 1% citrate solution was added to the solution mixture and left on the hot plate for 5 min to complete the reaction. The color of the solution changed from clear to dark blue to red. The mixture was brought to room temperature while stirring. Peptide-gold particle complexes were assembled by first conjugating the GNPs with the pentapeptide with approximately 300 peptides/ GNP ratio for stabilization purposes. Then the RME peptide was added with 8 to 10 peptide/GNP ratio. Each peptide was prepared with a terminal cystine group since cystine-terminated peptides can directly bind to the GNP surface via a covalent sulfur\gold bond [16,21]. NP complexes were characterized using UV visible spectroscopy, Dynamic Light Scattering (DLS), and zeta potential measurements (see Supplementary Section S1). There was no significant increase in terms of the size of NPs following conjugation with the peptide according to the DLS measurements. This is because the peptides were only few nanometers in size. However, the zeta potential changes from a negative value to a positive value. The negative charge of the nonconjugated (citrate-capped) GNPs is due to citrate molecules on the surface. Peptides are positively charged, and GNPs were positively charged following conjugation with the peptides. The peptide-GNP complexes were tested for their stability in cell culture media before using them for cell uptake and transport studies. The cellular uptake of peptide-GNP complexes was characterized by using HeLa cells, a well-known human fibroblast epithelial cell line. For tissue culture studies, cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% CO2. The cells were incubated with GNPs when the tissue culture dishes were 70–80% confluent. We have investigated the variation of NP distribution in cells internalized with citrate-capped and peptide-capped NPs using TEM imaging. The
Fig. 1. Nanoparticle transport pathway for peptide-capped NPs. A. Schematic showing the endosomal escape of peptide-capped NPs into the cytoplasm. B. TEM images showing localization of citrate-capped NPs in endosomes. C. Peptide-capped NPs escaped from endosomes were localized in the cytoplasm. The scale bar is 100 nm.
55
citrate-capped GNPs travel through the regular endo-lyso path causing NPs to become trapped in either endosomes or lysosomes [13] while most of the peptide-capped GNPs escaped from the endosomes and got localized in the cytoplasm (see Fig. 1). For quantification of the GNP uptake, the cells were washed three times with PBS and trypsinized. Cells were counted and then treated with HNO3 at 200 °C in an oil bath for an Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) analysis. Details of the quantification process are given in Supplementary Section S2. The cellular uptake data proved that the cells targeted with a RME peptide had the highest NP uptake, as compared to cells targeted with citrate-capped NPs (Fig. 2). It is believed that the synthetic RME peptide with an integrin binding domain (RGD) supplemented an additional driving force for the cell entry of NPs. In addition, positively charged NPs have a higher uptake compared to negatively charged (citrated-capped) NPs [22]. Zeta potential measurements showed that peptide-capped GNPs were positively charged (Supplementary Section S1). Hence, RME peptide and the positive charge of the NPs may have caused the increased uptake of peptide-modified NPs as compared to citrate-capped NPs. Previous work has shown that the exocytosis process depends on the cell type, NP size, and surface properties of NPs [15,23]. The exocytosis of GNPs functionalized with nuclear targeting peptides is not properly known yet. Our studies have shown for the first time that the fraction of NPs exocytosed was lower by two-fold for cells targeted with peptide-conjugated NPs (Fig. 3). Citrate-capped NPs were localized in endosomes followed by processing via fusion with lysosomes. NPs localized in lysosomes were excreted into the extracellular matrix through fusing with the cell membrane. However, most of the GNPs capped with peptides were able to enter the cytoplasm. This increase in the residence time in the cytoplasm could lead to a slower exocytosis process. Our results are in agreement with previously published work for CuO NPs and silica NPs. For example, Wang et al. evaluated the excretion of CuO NPs in A549 cells and discovered that a portion of NPs, which were located in mitochondria and nucleus, could not be excreted by the cells [24]. Similarly, based on findings by Chu et al., clusters of silica NPs in lysosomes are more easily exocytosed by H1299 cells as compared to single NPs in the cytoplasm [25]. We evaluated the exocytosis process as a function of time. To quantify NP removal, cells pre-incubated with NPs for eight hours were washed with PBS three times, introduced to fresh media supplemented with FBS, and left in the incubator for monitoring the exocytosis process. After one and six hour time points, the cell were rinsed three times with PBS and trypsinized for quantification of GNPs present per cell. As illustrated in Fig. 3A, a significant portion of the NPs were able to re-enter the cells. The percentage of NPs re-entering the cells was 10% and 15% respectively, for citrate-capped (non-targeted) and peptidecapped NPs. Nuclear targeted NPs appeared to aggregate less within the cell. Hence, it is possible for these NPs to re-enter cells once excreted. This could be one of the reasons for an increase in NPs present in cells targeted with peptides following a prolonged exocytosis process. The fraction of the GNPs exocytosed was calculated as illustrated in
Fig. 2. Nanoparticle uptake variation for citrate-capped and peptide-capped nanoparticles.
56
C. Yang et al. / Colloids and Interface Science Communications 1 (2014) 54–56
References
Fig. 3. Exocytosis of peptide-capped GNPs. Dynamics of exocytosis process following one and six hours. A. Number of GNPs exocytosed per cell after one hour and 6 h. B. Fraction of GNPs exocytosed per cell after 6 h.
Fig. 3B for six hour time point. Fraction of NPs exocytosed is lower for cells targeted with peptide-capped GNPs. Hence, the enhanced retention of NPs within cells can be used for improved therapeutics in radiation therapy and chemotherapy. In conclusion, our results showed that the functionalization of GNPs with RME peptides enhanced the NP uptake by five-fold. It was also found that the fraction of NPs exocytosed was lower by a factor of two for cells targeted with peptide-capped NPs in comparison to citrate-capped (non-targeted) NPs. This prolonged intracellular retention due to peptide functionalization would allow NPs to exert their desired applications more efficiently in cells, especially during drug delivery. Hence, a proper understanding of the endocytosis and exocytosis dynamics will shed more light on the design of GNPs in demand for cancer therapeutic applications. The authors would like acknowledge Natural Sciences and Engineering Research Council of Canada (NSERC) and Ryerson University for their financial support. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.colcom.2014.07.003.
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