- Email: [email protected]
Effect of serum on PEGylated quantum dots: Cellular uptake and intracellular distribution
Progress in Natural Science: Materials International 2013;23(6):566–572 Chinese Materials Research Society
Progress in Natural Science: Materials International www.elsevier.com/locate/pnsmi www.sciencedirect.com
ORIGINAL RESEARCH
Effect of serum on PEGylated quantum dots: Cellular uptake and intracellular distribution Yeting Jian, Xianghui Xu, Yunkun Li, Zhongwei Gun National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China Received 26 September 2013; accepted 16 October 2013 Available online 5 December 2013
KEYWORDS PEGylated QDs; Protein adsorption; Cellular uptake; Intracellular distribution
Abstract Protein adsorption is closely related with the interactions between nanoparticles and physiological systems, and further influences the cellular uptake and distribution of nanoparticles in cells. Although polyethylene glycol (PEG)ylation can largely reduce specific protein adsorption, some protein components in whole serum still interact with nanoparticles. In this work, PEGylated quantum dots (QDs) were used for investigating the quantitative and qualitative relationships of fetal bovine serum (FBS) and the cellular uptake/intracellular distribution in human hepatoma (HepG2) cell line. Nondenaturing polyacrylamide gel electrophoresis and two dimensional electrophoresis were used to analyze the adsorption of protein by PEGylated QDs. Quantitative cellular uptake of PEGylated QDs was determined by fluorescence activated cell sorting (FACS) with different FBS concentrations and incubating durations. The intracellular location of PEGylated QDs in HepG2 cells was observed using a confocal laser scanning microscope (CLSM) and a transmission electron microscope (TEM). This work will be helpful to understand the interaction between nanoparticles and cells with serum. & 2013 Chinese Materials Research Society. Production and hosting by Elsevier B.V. All rights reserved.
1.
Introduction
In recent years, advanced nanomaterials have largely promoted the development of disease diagnosis and treatment [1–3] and various nanoparticles are widely used in bioactive molecules delivery. Our n
Corresponding author. Fax: +028 85410653. E-mail address: [email protected] (Z. Gu). Peer review under responsibility of Chinese Materials Research Society.
group also reported a series of available nanoparticles for efficient therapeutic agents delivery [4–7]. However, there are a number of scientific problems on interactions between nanoparticles and physiological systems still to be resolved [8,9], because these interactions are able to determine the fate of nanoparticles and significantly influence the delivery efficiency of nanoparticles [10,11]. Therefore, the interactions between nanoparticles and physiological systems attract broad attentions in the field of nanomedicine. In order to improve the biomedical potentials of nanoparticles, extensive research activities are devoted to investigation of interaction between the nanoparticles (e.g., polymeric nanoparticles, inorganic nanoparticles) and physiological systems [12–14]. Most studies indicated that adsorption of proteins is one of the
1002-0071 & 2013 Chinese Materials Research Society. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.pnsc.2013.11.012
Effect of serum on PEGylated quantum dots: Cellular uptake and intracellular distribution most important factors affecting the properties of nanoparticles and further influencing the transport and metabolism of nanoparticles in physiological systems [15]. Some polyethylene glycol (PEG) ylated inorganic nanoparticles (e.g., magnetic nanoparticles, gold nanoparticles) are usually used for disclosing their behaviors in physiological systems due to the reduction of nonspecific interactions between nanoparticles and proteins (e.g., bovine serum albumin (BSA)) after PEGylation [16,17]. According to previous reports, researches focused on the interactions between nanoparticles and certain protein, such as BSA. Nevertheless, there are still some scientific problems on complex mixture of proteins impact on the interactions between PEGylated nanoparticles and physiological systems. Therefore, tracking PEGylated nanoparticles in physiological systems remains a serious task. And meanwhile, quantum dots (QDs), which become a promising nanoplatform for disease diagnosis and treatment, would become an inherent candidate for biological tracing due to their fluorescence. In the present work, we investigate that PEGylated QDs interact with fetal bovine serum (FBS), and further influence cellular uptake and distribution of PEGylated QDs in human hepatoma (HepG2) cell line. The interactions between PEGylated QDs and FBS were confirmed by nondenaturing polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The internalization of PEGylated QDs into cells was studied by fluorescence activated cell sorting (FACS) with different FBS concentrations and different monitoring durations. The cellular distribution of PEGylated QDs was analyzed by images obtained from a CLSM and a transmission electron microscope (TEM).
2.
electron microscope copper grids. Until the samples were dried out, they were observed by a Tecnai FEI GF20S-TWIN TEM. 2.3.
Nondenaturing polyacrylamide gel electrophoresis
The protein adsorbed PEGylated QDs samples were prepared by incubating 10 μL PEGylated QDs with the FBS for 30 min at 37 1C. 10% FBS diluted with water was used as control. Gels were hand-cast in Bio-Rad mini-gel cassettes. All the samples were loaded onto nondenaturing polyacrylamide with 25 mA constant current, until the bromophenol blue ran out of the gel. The PEGylated QDs were visualized by a Bio-Rad ChemiDoc XRSþ UV illuminator. Once the gel was stained by coomassie brilliant blue R250 staining buffer, it was destained for imaging. 2.4.
Two-dimensional electrophoresis
The two-dimensional electrophoresis processes were carried out as Manabe et al. described [18]. The samples were prepared as described above. A nondenaturing isoelectric focusing (IEF) was run with a 0.1 mA/tube constant current until a voltage of 300 V was reached. And the process was continued at a constant electric field of 300 V/cm for 50 min. The gel was equilibrated with 3% SDS, 0.01 M Tris and 0.076 M glycine buffer for 1 h. Then the IEF gel was transferred onto a second-dimensional slab gel. And electrophoresis was run for about 50 min in the micro-slab gels containing 0.1% SDS with 15 mA/slab constant current. The gel was stained and destained as per the steps described in the nondenaturing polyacrylamide gel electrophoresis process.
Material and method 2.5.
2.1.
567
Fluorescence activated cell sorting (FACS)
Materials and characterizations
Water-soluble PEGylated CdSe/ZnS QDs-525 (PEGylated QDs) samples were purchased from Wuhan Jiayuan Quantum Dots Co., Ltd. (Wuhan, China). LysoTracker Blue DND-22 and FM1-43 were obtained from Invitrogen (Carlsbad, CA). Phosphatebuffered saline (PBS, pH 7.4) and Coomassie brilliant blue R250 were purchased from Sigma-Aldrich. Dulbecco's minimal essential medium (DMEM), penicillin, streptomycin and FBS were obtained from Hyclone. All the electrophoresis processes were run by a Bio-Rad electrophoresis system. Transmission electron microscopy (TEM, Tecnai GF20S-TWIN, FEI, USA) was used to determine particle size and morphology. The size distributions of the nanoparticle were carried out using dynamic light scattering (DLS, Malvern Zetasizer Nano ZS). The cellular uptake was observed by CLSM (Leica TCP SP5, Germany) and FACS (Beckman Coulter Cytomics FC-500). The ultrathin section of HepG2 cells was observed with an H-600IV TEM (Hitachi, Japan). 2.2. Transmission electron microscope (TEM) and dynamic light scattering (DLS) Samples for DLS were prepared through incubating PEGylated QDs with 10% FBS for 30 min at room temperature. After incubating, the samples were centrifuged at 1 104g for 10 min. The supernate was discarded and the precipitate was re-dissolved with water. This solution was measured by a Malvern NANO ZSPO DLS. The samples for TEM images were dropped on
HepG2 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin) at 37 1C in 5% CO2 humidified atmosphere. In order to measure the cellular uptake of PEGylated QDs quantitatively, HepG2 cells were seeded into 12-well plates at an initial density of 1 105 cells/well. After 24 h, the cells were treated with PEGylated QDs with different FBS concentrations and different incubating durations 3 h, 6 h and 12 h. We removed the media and washed cells with PBS (pH 7.4) three times. Then the cells were trypsinized and resuspended in PBS (pH 7.4). The fluorescence of PEGylated QDs in cells was evaluated by FACS with 488 nm laser excitation [19]. 2.6.
Confocal laser scanning microscope (CLSM)
HepG2 cells were seeded in glass-bottomed dishes at a density of 1 104 cells/well 24 h before the experiment. We treated the cells with PEGylated QDs in FBS free (0% FBS) DMEM media and 10% FBS DMEM media for different durations. After incubation for 6 h, the media were removed and cells were washed twice with PBS (pH 7.4). The lysosomes were stained with LysoTracker Blue DND-22 (75 nM) in DMEM media for 60 min [20]. Then the cells were rinsed twice with PBS (pH 7.4). The cell membrane was stained by FM1-43 (1 mM) in PBS (pH 7.4) for 5 min [21]. The subcellular localization of PEGylated QDs was observed by CLSM with an exciting wavelength of 633 nm for FM1-43, 488 nm for QDs and 405 nm for LysoTracker Blue DND-22, and emission wavelength at 670 nm, 605 nm and 420 nm respectively.
568 2.7.
Y. Jian et al. Cell ultrathin sections for a TEM
The subcellular location of nanoparticles could be easily captured by a TEM [22]. HepG2 cells were seeded into 6-well plates at an
initial density of 2 105 cells/well. 24 h later, the cells were treated with PEGylated QDs in 0% FBS DMEM media and 10% FBS DMEM media, and incubated for another 6 h. Then cells were trypsinized and collected by centrifuging and fixed with 0.5%
Fig. 1 TEM images of PEGylated QDs (A) and PEGylated QDs adsorbed with FBS protein (B).
Fig. 2 Patterns of nondenaturing polyacrylamide gel electrophoresis: (A) proteins stained by coomassie brilliant blue and (B) fluorescence image of PEGylated QDs. Lane 1: 10% FBS; lane 2: PEGylated QDs incubated with 10% FBS.
Effect of serum on PEGylated quantum dots: Cellular uptake and intracellular distribution glutaraldehyde for 15 min at 4 1C. After centrifuging for 15 min at 1 104 rpm, cells were treated with 3% glutaraldehyde, and subsequently fixed with 1% osmium tetroxide followed by dehydration with ascending series of acetone. Then cells were embedded in Epon812, cut into ultrathin sections and doubly stained by lead citrate and uranyl acetate. The TEM images of cells were obtained from a Hitachi H-600IV TEM.
3. 3.1.
Results and discussion Size and morphology
First of all, the interaction between PEGylated QDs and FBS which was the most widely used in cell culture was studied by TEM and DLS. As shown in Fig. 1, the PEGylated QDs are with a well-defined nanostructure about 3 nm. The size of PEGylated QDs increased to approximately 6 nm after FBS adsorption. These results were confirmed by DLS. The size of PEGylated QDs is about 7 nm, while the size of PEGylated QDs mixed with FBS is about 10 nm. A bit bigger sizes of nanoparticles in water should be attributed to the stretch of PEG chains. These results demonstrated that the FBS protein indeed changed the size and morphology of PEGylated QDs after incubation.
Fig. 3 Two-dimensional electrophoresis patterns of (A) 10% FBS as control and (B) PEGylated QDs incubated with 10% FBS. Each sample contained the same FBS.
3.2.
569
Nondenaturing polyacrylamide gel electrophoresis results
In order to investigate the interaction between PEGylated QDs and FBS more directly, we utilized the inherent green fluorescence of PEGylated QDs to observe the position of QDs in a nondenaturing polyacrylamide gel electrophoresis [23,24]. As shown in Fig. 2A, there are five obvious bands on the gel loaded with FBS. They stood for five kinds of proteins with different molecular weights [25]. Compared to the FBS sample, the bands of PEGylated QDs mixed with FBS revealed some differences. The band of β globulin almost disappeared, while other bands presented almost no change. However, a new band, which exactly merged the fluorescence of PEGylated QDs in Fig. 2B, appeared on the gel. In general, bands in this position had larger molecular weight in comparison to the positions distant from the gel well. And in nondenaturing polyacrylamide gel electrophoresis conditions, the weak interactions between FBS proteins and PEGylated QDs
Fig. 4 FACS results of fluorescence positive cells incubated with PEGylated QDs mixed with (B) 0% FBS, (C) 2.5% FBS, (D) 5% FBS, (E) 7.5%, (F) 10%, (G) 15%, and (H) 20% FBS for 24 h. (A) Cells incubated with DMEM media were used as control.
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were not destroyed. Thus, we supposed that β globulin adsorbed on PEGylated QDs to form complex so that the shift of β globulin in gel became much smaller. 3.3.
Two-dimensional electrophoresis patterns
We knew that one band in the nondenaturing polyacrylamide gel electrophoresis contained several proteins with different isoelectric points although the proteins had a similar molecular weight. So we used the two dimensional electrophoresis to detect details of the adsorbed proteins. Fig. 3A shows a pattern of pure FBS. Compared to this result, some blots in Fig. 3B are different and they are marked with red circles. From the pattern we knew that these different blots had a similar molecular weight, some of them disappeared and the
others just became faded. This result confirmed the adsorption of protein on PEGylated QDs and found that β globulin was the major protein adsorbed on PEGylated QDs rather than BSA. In other words, through TEM, DLS, nondenaturing polyacrylamide gel electrophoresis and two-dimensional electrophoresis, we demonstrated that FBS proteins indeed adsorbed on PEGylated QDs. 3.4.
FACS results
After confirming the interactions between some components of FBS and PEGylated QDs, we turned to investigate the cellular uptake of PEGylated QDs with different incubation conditions. Based on the native fluorescence of PEGylated QDs, FACS assay could provide quantitative information on cellular uptake with
Fig. 5 FACS results of fluorescence positive cells with different FBS concentrations at several time points.
Fig. 6 Intracellular distribution of PEGylated QDs and protein adsorbed PEGylated QDs. (A) HepG2 cells incubated with PEGylated QDs in 0% FBS media. (B) HepG2 cells incubated with PEGylated QDs in media with 10% FBS. The cells membrane was stained by FM1-43 (red), the lysosome was stained by LysoTracker Blue (blue) and the PEGylated QDs were marked as green. Bar: 10 μm.
Effect of serum on PEGylated quantum dots: Cellular uptake and intracellular distribution different concentrations of FBS. It was found that PEGylated QDs were internalized into 32.6% HepG2 cells for 24 h without FBS. With increased concentration of FBS, the cells located with PEGylated QDs reduced significantly (Fig. 4). When the cellular uptake experiment was carried out with 10%, 15% and 20% FBS conditions, few PEGylated QDs were internalized into HepG2 cells as compared to blank control. Subsequently, we inspected how incubating time affected the cellular uptake of PEGylated QDs with or without FBS. As shown in Fig. 5, with extending of incubating time, PEGylated QDs were enveloped into more and more cells without FBS, and PEGylated QDs started enriching in HepG2 cells accompanying the enhancement of fluorescence. There was small increase in the number of HepG2 cells with PEGylated QDs with 2.5% FBS concentrations. With 10% FBS concentrations, no significant increase of fluorescence intensity was detected in cells. These results confirmed that β globulin in FBS would hinder the cellular uptake of PEGylated QDs.
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Previous researches were focused on BSA protein, which was one of major proteins in FBS, and some results showed that the adsorption of BSA protein influenced cellular uptake and distribution of nanoparticles [9]. In this work, we found that FBS as a protein mixture also changed the fate of PEGylated QDs. However, combined with electrophoresis results, it was clear that the adsorption of β globulin was obviously more than other protein components, such as BSA. β globulin would be a key protein determining the behaviors of nanoparticles in physiological systems. The above-mentioned FACS results indicated the cellular uptake of PEGylated QDs would be inhibited by the adsorption of the proteins. 3.5.
CLSM images for intracellular distribution
In order to study the intracellular distribution influenced by protein adsorption, HepG2 cells were treated with PEGylated QDs with or without FBS for CLSM. The PEGylated QDs were able to be observed as green fluorescence by a CLSM to obtain clear cell location, the lysosome was labeled with LysoTracker (blue), and the cell membrane was labeled with FM1-43 (red). There was a wide intracellular distribution of PEGylated QDs without FBS (Fig. 6A) as compared to the control group with 10% FBS (Fig. 6B). Moreover, after internalization of PEGylated QDs without FBS, these green fluorescent nanoparticles were enveloped into the lysosome [26]. In contrast, a small quantity of the PEGylated QDs with FBS conditions just adsorbed onto cell membranes. The green fluorescence of PEGylated QDs provided direct evidence that intracellular distribution would be influenced by FBS. 3.6.
TEM images for cellular distribution
Next, TEM was used to obtain the subcellular location of the PEGylated QDs with or without FBS. As shown in Fig. 7, large amount of PEGylated QDs dispersed in the cytoplasm of HepG2 cells and mainly accumulated on endocytic organelles after incubation for 6 h without FBS. In contrast, after exposure to PEGylated QDs with FBS, few PEGylated QDs were observed in the cytoplasm, and a bit of PEGylated QDs just attached onto cell membrane. The significant differences of the cellular uptake and distribution of PEGylated QDs were induced by some protein adsorption with PEGylated QDs in a FBS-containing medium. The TEM images coupled with FACS analysis and CLSM images indicated that the FBS could largely influence cellular uptake and distribution of PEGylated QDs [21,27]. It was known that an ideal intracellular distribution played a very important role for theranostic agent delivery, such as drug, gene and imaging probes. β globulin affected cellular uptake and intracellular location of PEGylated QDs in cells. However, PEG was widely used to resist protein adsorption by coating PEG to nanoparticles. Therefore, the improvement of the resistant properties of PEGylated materials to β globulin could indeed develop delivery efficiency of nanoparticles. 4.
Fig. 7 Intracellular distribution of (A) PEGylated QDs and (B) protein adsorbed PEGylated QDs in a HepG2 cell. Arrow: PEGylated QDs. Bar: 500 nm.
Conclusions
In this study, we investigated the interactions between PEGylated QDs and FBS which would influence the cellular uptake and intracellular distribution of PEGylated QDs. The electrophoresis result indicated that β globulin was major protein adsorbed onto the PEGylated QDs. Furthermore, it was found that the adsorption
572 of β globulin in FBS would largely reduce the cellular uptake of PEGylated QDs in varying degrees. The CLSM images combined with TEM images demonstrated that the adsorption of β globulin in FBS would influence intracellular distribution. Very few PEGylated QDs were located on the cell membrane under FBS conditions, while the PEGylated QDs were widely distributed in cytoplasm without FBS. These results indicated that the adsorption of some component proteins in FBS would influence the cellular uptake and intracellular distribution of nanoparticles. Lots of studies, such as molecular biology, need to be carried out to disclose the mechanism on fetal bovine serum influencing the interactions between nanoparticles and physiological systems. We hope this work stimulates the research on resistance adsorption of some component proteins in FBS to improve delivery efficiency. Acknowledgments This work was supported by National Science Foundation of China (NSFC, No. 51133004), National Basic Research Program of China (National 973 program, No. 2011CB606206), and Joint Sino-German Research Project (No. GZ756). References [1] S.H. Cheng, C.H. Lee, M.C. Chen, J.S. Souris, F.G. Tseng, C.S. Yang, C.Y. Mou, C.T. Chen, L.W. Lo, J. Mater. Chem. 20 (2010) 6149–6157. [2] Y.P. Ho, K.W. Leong, Nanoscale 2 (2010) 60–68. [3] E. Lukianova-Hleb, E. Hanna, J. Hafner, D. Lapotko, Nanotechnology 21 (2010) 085102. [4] X. Xu, H. Yuan, J. Chang, B. He, Z. Gu, Angew. Chem. Int. Ed. 51 (2012) 3130–3133. [5] J. Chang, X. Xu, H. Li, Y. Jian, G. Wang, B. He, Z. Gu, Adv. Funct. Mater. 23 (2013) 2691–2699. [6] H. Li, X. Xu, Y. Li, Y. Geng, B. He, Z. Gu, Polym. Chem. 4 (2013) 2235–2238.
Y. Jian et al. [7] X. Xu, C. Li, H. Li, R. Liu, C. Jiang, Y. Wu, B. He, Z. Gu, Sci. China Chem. 54 (2011) 326–333. [8] M. Yoshida, K.-H. Roh, J. Lahann, Biomaterials 28 (2007) 2446–2456. [9] C.D. Walkey, J.B. Olsen, H. Guo, A. Emili, W.C. Chan, J. Am. Chem. Soc. 134 (2012) 2139–2147. [10] P.P. Karmali, D. Simberg, Expert Opin. Drug Deliv. 8 (2011) 343–357. [11] S. Kim, Y. Shi, J.Y. Kim, K. Park, J.X. Cheng, Expert Opin. Drug Deliv. 7 (2010) 49–62. [12] K. Avgoustakis, A. Beletsi, Z. Panagi, P. Klepetsanis, A. Karydas, D. Ithakissios, J. Control. Release 79 (2002) 123–135. [13] J. Tan, D. Butterfield, C. Voycheck, K. Caldwell, J. Li, Biomaterials 14 (1993) 823–833. [14] R. Liu, L. Yin, Y. Pu, G. Liang, J. Zhang, Y. Su, Z. Xiao, B. Ye, Proc. Nat. Acad. Sci. USA 19 (2009) 573–579. [15] S. Patil, A. Sandberg, E. Heckert, W. Self, S. Seal, Biomaterials 28 (2007) 4600–4607. [16] H. Lee, E. Lee, D.K. Kim, N.K. Jang, Y.Y. Jeong, S. Jon, J. Am. Chem. Soc. 128 (2006) 7383–7389. [17] H. Otsuka, Y. Nagasaki, K. Kataoka, Adv. Drug Deliv. Rev. 64 (2012) 246–255. [18] T. Manabe, H. Mizuma, K. Watanabe, Electrophoresis 20 (1999) 830–835. [19] B. Ferrari, P. Bergquist, Cytometry Part A 71 (2007) 265–271. [20] X. Gao, T. Wang, B. Wu, J. Chen, J. Chen, Y. Yue, N. Dai, H. Chen, X. Jiang, Biochem. Biophys. Res. Commun. 377 (2008) 35–40. [21] Q. Zhang, Y.Q. Cao, R.W. Tsien, Proc. Nat. Acad. Sci. USA 104 (2007) 17843–17848. [22] R.t. Liu, J. Liu, J.q. Tong, T. Tang, W.C. Kong, X.w. Wang, Y. Li, J.t. Tang, Prog. Nat. Sci. Mater. Int. 22 (2012) 31–39. [23] A. Raab, B. Pioselli, C. Munro, J. Thomas-Oates, J. Feldmann, Electrophoresis 30 (2009) 303–314. [24] G. Vicente, L.A. Colón, Anal. Chem. 80 (2008) 1988–1994. [25] S.R. Haider, B.L. Sharp, H.J. Reid, J. Sep. Sci. 34 (2011) 2463–2467. [26] Y. Zhang, L. Mi, P.N. Wang, S.J. Lu, J.Y. Chen, J. Guo, W.L. Yang, C.C. Wang, Small 4 (2008) 777–780. [27] S.K. Lai, K. Hida, S.T. Man, C. Chen, C. Machamer, T.A. Schroer, J. Hanes, Biomaterials 28 (2007) 2876–2884.
Progress in Natural Science: Materials International www.elsevier.com/locate/pnsmi www.sciencedirect.com
ORIGINAL RESEARCH
Effect of serum on PEGylated quantum dots: Cellular uptake and intracellular distribution Yeting Jian, Xianghui Xu, Yunkun Li, Zhongwei Gun National Engineering Research Center for Biomaterials, Sichuan University, 29 Wangjiang Road, Chengdu 610064, China Received 26 September 2013; accepted 16 October 2013 Available online 5 December 2013
KEYWORDS PEGylated QDs; Protein adsorption; Cellular uptake; Intracellular distribution
Abstract Protein adsorption is closely related with the interactions between nanoparticles and physiological systems, and further influences the cellular uptake and distribution of nanoparticles in cells. Although polyethylene glycol (PEG)ylation can largely reduce specific protein adsorption, some protein components in whole serum still interact with nanoparticles. In this work, PEGylated quantum dots (QDs) were used for investigating the quantitative and qualitative relationships of fetal bovine serum (FBS) and the cellular uptake/intracellular distribution in human hepatoma (HepG2) cell line. Nondenaturing polyacrylamide gel electrophoresis and two dimensional electrophoresis were used to analyze the adsorption of protein by PEGylated QDs. Quantitative cellular uptake of PEGylated QDs was determined by fluorescence activated cell sorting (FACS) with different FBS concentrations and incubating durations. The intracellular location of PEGylated QDs in HepG2 cells was observed using a confocal laser scanning microscope (CLSM) and a transmission electron microscope (TEM). This work will be helpful to understand the interaction between nanoparticles and cells with serum. & 2013 Chinese Materials Research Society. Production and hosting by Elsevier B.V. All rights reserved.
1.
Introduction
In recent years, advanced nanomaterials have largely promoted the development of disease diagnosis and treatment [1–3] and various nanoparticles are widely used in bioactive molecules delivery. Our n
Corresponding author. Fax: +028 85410653. E-mail address: [email protected] (Z. Gu). Peer review under responsibility of Chinese Materials Research Society.
group also reported a series of available nanoparticles for efficient therapeutic agents delivery [4–7]. However, there are a number of scientific problems on interactions between nanoparticles and physiological systems still to be resolved [8,9], because these interactions are able to determine the fate of nanoparticles and significantly influence the delivery efficiency of nanoparticles [10,11]. Therefore, the interactions between nanoparticles and physiological systems attract broad attentions in the field of nanomedicine. In order to improve the biomedical potentials of nanoparticles, extensive research activities are devoted to investigation of interaction between the nanoparticles (e.g., polymeric nanoparticles, inorganic nanoparticles) and physiological systems [12–14]. Most studies indicated that adsorption of proteins is one of the
1002-0071 & 2013 Chinese Materials Research Society. Production and hosting by Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.pnsc.2013.11.012
Effect of serum on PEGylated quantum dots: Cellular uptake and intracellular distribution most important factors affecting the properties of nanoparticles and further influencing the transport and metabolism of nanoparticles in physiological systems [15]. Some polyethylene glycol (PEG) ylated inorganic nanoparticles (e.g., magnetic nanoparticles, gold nanoparticles) are usually used for disclosing their behaviors in physiological systems due to the reduction of nonspecific interactions between nanoparticles and proteins (e.g., bovine serum albumin (BSA)) after PEGylation [16,17]. According to previous reports, researches focused on the interactions between nanoparticles and certain protein, such as BSA. Nevertheless, there are still some scientific problems on complex mixture of proteins impact on the interactions between PEGylated nanoparticles and physiological systems. Therefore, tracking PEGylated nanoparticles in physiological systems remains a serious task. And meanwhile, quantum dots (QDs), which become a promising nanoplatform for disease diagnosis and treatment, would become an inherent candidate for biological tracing due to their fluorescence. In the present work, we investigate that PEGylated QDs interact with fetal bovine serum (FBS), and further influence cellular uptake and distribution of PEGylated QDs in human hepatoma (HepG2) cell line. The interactions between PEGylated QDs and FBS were confirmed by nondenaturing polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The internalization of PEGylated QDs into cells was studied by fluorescence activated cell sorting (FACS) with different FBS concentrations and different monitoring durations. The cellular distribution of PEGylated QDs was analyzed by images obtained from a CLSM and a transmission electron microscope (TEM).
2.
electron microscope copper grids. Until the samples were dried out, they were observed by a Tecnai FEI GF20S-TWIN TEM. 2.3.
Nondenaturing polyacrylamide gel electrophoresis
The protein adsorbed PEGylated QDs samples were prepared by incubating 10 μL PEGylated QDs with the FBS for 30 min at 37 1C. 10% FBS diluted with water was used as control. Gels were hand-cast in Bio-Rad mini-gel cassettes. All the samples were loaded onto nondenaturing polyacrylamide with 25 mA constant current, until the bromophenol blue ran out of the gel. The PEGylated QDs were visualized by a Bio-Rad ChemiDoc XRSþ UV illuminator. Once the gel was stained by coomassie brilliant blue R250 staining buffer, it was destained for imaging. 2.4.
Two-dimensional electrophoresis
The two-dimensional electrophoresis processes were carried out as Manabe et al. described [18]. The samples were prepared as described above. A nondenaturing isoelectric focusing (IEF) was run with a 0.1 mA/tube constant current until a voltage of 300 V was reached. And the process was continued at a constant electric field of 300 V/cm for 50 min. The gel was equilibrated with 3% SDS, 0.01 M Tris and 0.076 M glycine buffer for 1 h. Then the IEF gel was transferred onto a second-dimensional slab gel. And electrophoresis was run for about 50 min in the micro-slab gels containing 0.1% SDS with 15 mA/slab constant current. The gel was stained and destained as per the steps described in the nondenaturing polyacrylamide gel electrophoresis process.
Material and method 2.5.
2.1.
567
Fluorescence activated cell sorting (FACS)
Materials and characterizations
Water-soluble PEGylated CdSe/ZnS QDs-525 (PEGylated QDs) samples were purchased from Wuhan Jiayuan Quantum Dots Co., Ltd. (Wuhan, China). LysoTracker Blue DND-22 and FM1-43 were obtained from Invitrogen (Carlsbad, CA). Phosphatebuffered saline (PBS, pH 7.4) and Coomassie brilliant blue R250 were purchased from Sigma-Aldrich. Dulbecco's minimal essential medium (DMEM), penicillin, streptomycin and FBS were obtained from Hyclone. All the electrophoresis processes were run by a Bio-Rad electrophoresis system. Transmission electron microscopy (TEM, Tecnai GF20S-TWIN, FEI, USA) was used to determine particle size and morphology. The size distributions of the nanoparticle were carried out using dynamic light scattering (DLS, Malvern Zetasizer Nano ZS). The cellular uptake was observed by CLSM (Leica TCP SP5, Germany) and FACS (Beckman Coulter Cytomics FC-500). The ultrathin section of HepG2 cells was observed with an H-600IV TEM (Hitachi, Japan). 2.2. Transmission electron microscope (TEM) and dynamic light scattering (DLS) Samples for DLS were prepared through incubating PEGylated QDs with 10% FBS for 30 min at room temperature. After incubating, the samples were centrifuged at 1 104g for 10 min. The supernate was discarded and the precipitate was re-dissolved with water. This solution was measured by a Malvern NANO ZSPO DLS. The samples for TEM images were dropped on
HepG2 cells were cultured in DMEM supplemented with 10% heat-inactivated FBS and antibiotics (100 U/mL penicillin G and 100 mg/mL streptomycin) at 37 1C in 5% CO2 humidified atmosphere. In order to measure the cellular uptake of PEGylated QDs quantitatively, HepG2 cells were seeded into 12-well plates at an initial density of 1 105 cells/well. After 24 h, the cells were treated with PEGylated QDs with different FBS concentrations and different incubating durations 3 h, 6 h and 12 h. We removed the media and washed cells with PBS (pH 7.4) three times. Then the cells were trypsinized and resuspended in PBS (pH 7.4). The fluorescence of PEGylated QDs in cells was evaluated by FACS with 488 nm laser excitation [19]. 2.6.
Confocal laser scanning microscope (CLSM)
HepG2 cells were seeded in glass-bottomed dishes at a density of 1 104 cells/well 24 h before the experiment. We treated the cells with PEGylated QDs in FBS free (0% FBS) DMEM media and 10% FBS DMEM media for different durations. After incubation for 6 h, the media were removed and cells were washed twice with PBS (pH 7.4). The lysosomes were stained with LysoTracker Blue DND-22 (75 nM) in DMEM media for 60 min [20]. Then the cells were rinsed twice with PBS (pH 7.4). The cell membrane was stained by FM1-43 (1 mM) in PBS (pH 7.4) for 5 min [21]. The subcellular localization of PEGylated QDs was observed by CLSM with an exciting wavelength of 633 nm for FM1-43, 488 nm for QDs and 405 nm for LysoTracker Blue DND-22, and emission wavelength at 670 nm, 605 nm and 420 nm respectively.
568 2.7.
Y. Jian et al. Cell ultrathin sections for a TEM
The subcellular location of nanoparticles could be easily captured by a TEM [22]. HepG2 cells were seeded into 6-well plates at an
initial density of 2 105 cells/well. 24 h later, the cells were treated with PEGylated QDs in 0% FBS DMEM media and 10% FBS DMEM media, and incubated for another 6 h. Then cells were trypsinized and collected by centrifuging and fixed with 0.5%
Fig. 1 TEM images of PEGylated QDs (A) and PEGylated QDs adsorbed with FBS protein (B).
Fig. 2 Patterns of nondenaturing polyacrylamide gel electrophoresis: (A) proteins stained by coomassie brilliant blue and (B) fluorescence image of PEGylated QDs. Lane 1: 10% FBS; lane 2: PEGylated QDs incubated with 10% FBS.
Effect of serum on PEGylated quantum dots: Cellular uptake and intracellular distribution glutaraldehyde for 15 min at 4 1C. After centrifuging for 15 min at 1 104 rpm, cells were treated with 3% glutaraldehyde, and subsequently fixed with 1% osmium tetroxide followed by dehydration with ascending series of acetone. Then cells were embedded in Epon812, cut into ultrathin sections and doubly stained by lead citrate and uranyl acetate. The TEM images of cells were obtained from a Hitachi H-600IV TEM.
3. 3.1.
Results and discussion Size and morphology
First of all, the interaction between PEGylated QDs and FBS which was the most widely used in cell culture was studied by TEM and DLS. As shown in Fig. 1, the PEGylated QDs are with a well-defined nanostructure about 3 nm. The size of PEGylated QDs increased to approximately 6 nm after FBS adsorption. These results were confirmed by DLS. The size of PEGylated QDs is about 7 nm, while the size of PEGylated QDs mixed with FBS is about 10 nm. A bit bigger sizes of nanoparticles in water should be attributed to the stretch of PEG chains. These results demonstrated that the FBS protein indeed changed the size and morphology of PEGylated QDs after incubation.
Fig. 3 Two-dimensional electrophoresis patterns of (A) 10% FBS as control and (B) PEGylated QDs incubated with 10% FBS. Each sample contained the same FBS.
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Nondenaturing polyacrylamide gel electrophoresis results
In order to investigate the interaction between PEGylated QDs and FBS more directly, we utilized the inherent green fluorescence of PEGylated QDs to observe the position of QDs in a nondenaturing polyacrylamide gel electrophoresis [23,24]. As shown in Fig. 2A, there are five obvious bands on the gel loaded with FBS. They stood for five kinds of proteins with different molecular weights [25]. Compared to the FBS sample, the bands of PEGylated QDs mixed with FBS revealed some differences. The band of β globulin almost disappeared, while other bands presented almost no change. However, a new band, which exactly merged the fluorescence of PEGylated QDs in Fig. 2B, appeared on the gel. In general, bands in this position had larger molecular weight in comparison to the positions distant from the gel well. And in nondenaturing polyacrylamide gel electrophoresis conditions, the weak interactions between FBS proteins and PEGylated QDs
Fig. 4 FACS results of fluorescence positive cells incubated with PEGylated QDs mixed with (B) 0% FBS, (C) 2.5% FBS, (D) 5% FBS, (E) 7.5%, (F) 10%, (G) 15%, and (H) 20% FBS for 24 h. (A) Cells incubated with DMEM media were used as control.
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were not destroyed. Thus, we supposed that β globulin adsorbed on PEGylated QDs to form complex so that the shift of β globulin in gel became much smaller. 3.3.
Two-dimensional electrophoresis patterns
We knew that one band in the nondenaturing polyacrylamide gel electrophoresis contained several proteins with different isoelectric points although the proteins had a similar molecular weight. So we used the two dimensional electrophoresis to detect details of the adsorbed proteins. Fig. 3A shows a pattern of pure FBS. Compared to this result, some blots in Fig. 3B are different and they are marked with red circles. From the pattern we knew that these different blots had a similar molecular weight, some of them disappeared and the
others just became faded. This result confirmed the adsorption of protein on PEGylated QDs and found that β globulin was the major protein adsorbed on PEGylated QDs rather than BSA. In other words, through TEM, DLS, nondenaturing polyacrylamide gel electrophoresis and two-dimensional electrophoresis, we demonstrated that FBS proteins indeed adsorbed on PEGylated QDs. 3.4.
FACS results
After confirming the interactions between some components of FBS and PEGylated QDs, we turned to investigate the cellular uptake of PEGylated QDs with different incubation conditions. Based on the native fluorescence of PEGylated QDs, FACS assay could provide quantitative information on cellular uptake with
Fig. 5 FACS results of fluorescence positive cells with different FBS concentrations at several time points.
Fig. 6 Intracellular distribution of PEGylated QDs and protein adsorbed PEGylated QDs. (A) HepG2 cells incubated with PEGylated QDs in 0% FBS media. (B) HepG2 cells incubated with PEGylated QDs in media with 10% FBS. The cells membrane was stained by FM1-43 (red), the lysosome was stained by LysoTracker Blue (blue) and the PEGylated QDs were marked as green. Bar: 10 μm.
Effect of serum on PEGylated quantum dots: Cellular uptake and intracellular distribution different concentrations of FBS. It was found that PEGylated QDs were internalized into 32.6% HepG2 cells for 24 h without FBS. With increased concentration of FBS, the cells located with PEGylated QDs reduced significantly (Fig. 4). When the cellular uptake experiment was carried out with 10%, 15% and 20% FBS conditions, few PEGylated QDs were internalized into HepG2 cells as compared to blank control. Subsequently, we inspected how incubating time affected the cellular uptake of PEGylated QDs with or without FBS. As shown in Fig. 5, with extending of incubating time, PEGylated QDs were enveloped into more and more cells without FBS, and PEGylated QDs started enriching in HepG2 cells accompanying the enhancement of fluorescence. There was small increase in the number of HepG2 cells with PEGylated QDs with 2.5% FBS concentrations. With 10% FBS concentrations, no significant increase of fluorescence intensity was detected in cells. These results confirmed that β globulin in FBS would hinder the cellular uptake of PEGylated QDs.
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Previous researches were focused on BSA protein, which was one of major proteins in FBS, and some results showed that the adsorption of BSA protein influenced cellular uptake and distribution of nanoparticles [9]. In this work, we found that FBS as a protein mixture also changed the fate of PEGylated QDs. However, combined with electrophoresis results, it was clear that the adsorption of β globulin was obviously more than other protein components, such as BSA. β globulin would be a key protein determining the behaviors of nanoparticles in physiological systems. The above-mentioned FACS results indicated the cellular uptake of PEGylated QDs would be inhibited by the adsorption of the proteins. 3.5.
CLSM images for intracellular distribution
In order to study the intracellular distribution influenced by protein adsorption, HepG2 cells were treated with PEGylated QDs with or without FBS for CLSM. The PEGylated QDs were able to be observed as green fluorescence by a CLSM to obtain clear cell location, the lysosome was labeled with LysoTracker (blue), and the cell membrane was labeled with FM1-43 (red). There was a wide intracellular distribution of PEGylated QDs without FBS (Fig. 6A) as compared to the control group with 10% FBS (Fig. 6B). Moreover, after internalization of PEGylated QDs without FBS, these green fluorescent nanoparticles were enveloped into the lysosome [26]. In contrast, a small quantity of the PEGylated QDs with FBS conditions just adsorbed onto cell membranes. The green fluorescence of PEGylated QDs provided direct evidence that intracellular distribution would be influenced by FBS. 3.6.
TEM images for cellular distribution
Next, TEM was used to obtain the subcellular location of the PEGylated QDs with or without FBS. As shown in Fig. 7, large amount of PEGylated QDs dispersed in the cytoplasm of HepG2 cells and mainly accumulated on endocytic organelles after incubation for 6 h without FBS. In contrast, after exposure to PEGylated QDs with FBS, few PEGylated QDs were observed in the cytoplasm, and a bit of PEGylated QDs just attached onto cell membrane. The significant differences of the cellular uptake and distribution of PEGylated QDs were induced by some protein adsorption with PEGylated QDs in a FBS-containing medium. The TEM images coupled with FACS analysis and CLSM images indicated that the FBS could largely influence cellular uptake and distribution of PEGylated QDs [21,27]. It was known that an ideal intracellular distribution played a very important role for theranostic agent delivery, such as drug, gene and imaging probes. β globulin affected cellular uptake and intracellular location of PEGylated QDs in cells. However, PEG was widely used to resist protein adsorption by coating PEG to nanoparticles. Therefore, the improvement of the resistant properties of PEGylated materials to β globulin could indeed develop delivery efficiency of nanoparticles. 4.
Fig. 7 Intracellular distribution of (A) PEGylated QDs and (B) protein adsorbed PEGylated QDs in a HepG2 cell. Arrow: PEGylated QDs. Bar: 500 nm.
Conclusions
In this study, we investigated the interactions between PEGylated QDs and FBS which would influence the cellular uptake and intracellular distribution of PEGylated QDs. The electrophoresis result indicated that β globulin was major protein adsorbed onto the PEGylated QDs. Furthermore, it was found that the adsorption
572 of β globulin in FBS would largely reduce the cellular uptake of PEGylated QDs in varying degrees. The CLSM images combined with TEM images demonstrated that the adsorption of β globulin in FBS would influence intracellular distribution. Very few PEGylated QDs were located on the cell membrane under FBS conditions, while the PEGylated QDs were widely distributed in cytoplasm without FBS. These results indicated that the adsorption of some component proteins in FBS would influence the cellular uptake and intracellular distribution of nanoparticles. Lots of studies, such as molecular biology, need to be carried out to disclose the mechanism on fetal bovine serum influencing the interactions between nanoparticles and physiological systems. We hope this work stimulates the research on resistance adsorption of some component proteins in FBS to improve delivery efficiency. Acknowledgments This work was supported by National Science Foundation of China (NSFC, No. 51133004), National Basic Research Program of China (National 973 program, No. 2011CB606206), and Joint Sino-German Research Project (No. GZ756). References [1] S.H. Cheng, C.H. Lee, M.C. Chen, J.S. Souris, F.G. Tseng, C.S. Yang, C.Y. Mou, C.T. Chen, L.W. Lo, J. Mater. Chem. 20 (2010) 6149–6157. [2] Y.P. Ho, K.W. Leong, Nanoscale 2 (2010) 60–68. [3] E. Lukianova-Hleb, E. Hanna, J. Hafner, D. Lapotko, Nanotechnology 21 (2010) 085102. [4] X. Xu, H. Yuan, J. Chang, B. He, Z. Gu, Angew. Chem. Int. Ed. 51 (2012) 3130–3133. [5] J. Chang, X. Xu, H. Li, Y. Jian, G. Wang, B. He, Z. Gu, Adv. Funct. Mater. 23 (2013) 2691–2699. [6] H. Li, X. Xu, Y. Li, Y. Geng, B. He, Z. Gu, Polym. Chem. 4 (2013) 2235–2238.
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