In vitro uptake of apoptotic body mimicking phosphatidylserine-quantum dot micelles by monocytic cell line
© Maiseyeu and Bagalkot; licensee Springer. 2014
Received: 24 January 2014
Accepted: 31 March 2014
Published: 11 April 2014
A new quantum dot (QD) PEGylated micelle laced with phosphatidylserine (PS) for specific scavenger receptor-mediated uptake by macrophages is reported. The size and surface chemistry of PS-QD micelles were characterized by standard methods and the effects of their physicochemical properties on specific targeting and uptake were comprehensively studied in a monocytic cell line (J774A.1).
KeywordsPhosphatidylserine Micelles Quantum dots Macrophages
Macrophage plays an important role in the destabilization of atherosclerotic lesions. Molecular imaging approaches that target and image macrophages may be potentially useful towards predicting plaque vulnerability during the natural history of the disease [1–5]. Macrophages are effective efferocytes with the ability to recognize the externalized phosphatidylserine (PS) on the plasma membrane surface of apoptotic cells via the scavenger receptors and remove them from circulation and the arterial wall [6–9]. Phosphatidylserine is a naturally occurring phospholipid (PL) and its use for targeting macrophages may improve the biocompatibility of the contrast agent and avoid the use of exogenous targeting agents such as antibodies and peptides. This approach of using phosphatidylserine for targeting macrophages has been reported previously for magnetic resonance imaging of macrophage contents in atheroma with gadolinium-containing liposomes  but PS-containing micelles have not been reported. Lipid-polyethylene glycol (PEG) micelles have traditionally been used to solubilize hydrophobic drugs and solubilize hydrophobic nanoparticles into discrete clusters that can include either single or multiple nanoparticles in their cores and thus can achieve size tunability for particular application . Micelles formed from lipid-PEG are biocompatible, non-toxic, and stable in vivo [11–16]. For the purpose of tracking the uptake of micelles by macrophages, QDs were incorporated into the micelle preparations because of its extreme brightness and photostability in real time imaging. Furthermore, QDs can be substituted by other inorganic nanoparticles such as gadolinium, iron oxide, gold, and tantalum for clinical translation. The PS micelles were further assembled with an amphiphilic polymeric surfactant, phospholipid conjugated to polyethylene glycol (PL-PEG) for the solubilization of hydrophobic nanoparticles (QD), improved dispersibility of micelles in physiological buffers and prolonged circulation in vivo . However, PEGylation can potentially interfere with the interactions between ligand and cell surface receptor and reduce cellular uptake [17, 18], a fine balance between stability and targeting for PEGylated nanoparticles were extensively studied. We hypothesize that the ratio of PL-PEG and PS shell coverage for 6- to 8-nm hydrophobic trioctylphosphine oxide (TOPO) quantum dot (QD) could be optimized for colloidal stability and targeting efficacy.
L-α-phosphatidylserine (PS), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (ammonium salt) (DSPE-mPEG, 2kDa) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). All other chemicals were obtained from Sigma-Aldrich Corporation (St. Louis, MO, USA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin, and hydrophobic trioctylphosphine oxide (TOPO) QDs (QD 620nm) were purchased from Ocean Nanotech, Corp (Carlsbad, CA, USA). MTT assay kit was purchased from Roche Applied Science (Indianapolis, IN, USA). Lab-TekTM chamber slide system was purchased from Thermo Scientific/Nalgene Nunc International (Rochester, NY, USA). Vectashield mounting medium with DAPI was purchased from Vector Laboratories, Inc. (Burlingame, CA, USA). J774A.1 monocytic cell line was obtained from American Type Cell line Collection (ATCC) (ATCC® TIB67™). A 100-kD dialysis membrane was purchased from Spectrum Laboratories (Irvine, CA, USA).
Preparation of PS-QD micelles
Preparation and physico-chemical characteristics of PS-QD micelles
Polar lipids (mg)
QD (620 nm; 2-μM concentration)
Clarity of emulsion
Stability of flourescence
Average size (by intensity; in nm)
Polydispersity index (PDI)
Zeta potential charge (in mV)
QD-PEG-PS mole ratio
DSPE-PEG (2000) methoxy
100:0, PS (0)
Quenched after 45 days
60:40, PS (40)
50:50, PS (50)
40:60, PS (60)
0:100, PS (100)
DSPE-PEG (2000) carboxylic acid
Physico-chemical characterization of PS-QD micelles
The mean hydrodynamic diameter, polydispersity index and zeta potential charge of PS-QD micelles was measured using a Zeta Nanosizer ZS (Malvern Instruments Ltd, Worcestershire, UK; Table 1). For size measurements, the PS-QD micelles were diluted (1:100) in 100-mM PBS buffer and for zeta potential measurements the PS-QD micelles were diluted (1:1,000) in 10-mM PBS buffer. All samples were measured in triplicate. The morphology of PS-QD micelles was analyzed by transmission electron microscopy (TEM; JEM1010; JEOL, Tokyo, Japan) operating at 60kV. For the preparation of PS-QD micelles for TEM, PS-QD micelles were diluted in distilled water and dropped on Formvar-coated copper grids. Samples were examined with and without negatively staining with osmium tetroxide.
In vitro stability of PS-QD micelles
The colloidal stability of PS-QD micelles was analyzed by incubating PS-QD micelles in cell culture medium containing 10% fetal bovine serum (FBS). Four-hundred microliters of PS-QD micelles (QD concentration 1 μM) were diluted in 800 μL of cell culture media and placed in a 37°C water bath for 24 h. After 24 h, 0.5 mL of the micelle solution in media was diluted twice with PBS buffer (0.1M) for particle size analysis using a Zeta Nanosizer ZS.
In vitro cell uptake (fluorescence microscopy and flow cytometry studies)
The cellular uptake and distribution of PS-QD micelles were semiquantitated by fluorescence microscopy and flow cytometry. After the J774A.1 cells reached 80% confluency, the cells were detached by a scraper and seeded onto a 6-well plate at a density of 2 × 104 cells per well and incubated overnight. The culture medium was removed and PS-QD micelles PS (0), (40), (50), (60), and (100) at 10-nM concentration were added and incubated for 4h at 37°C. After incubation, the solution was removed and the cells were washed with PBS for at least three times. After washing with PBS, cells were scraped and centrifuged, the supernatant was carefully removed. PBS buffer containing 2% (v/v) FBS was added to the cell pellet and resuspended. The cells were analyzed using a FACS Calibur fluorescence-activated cell sorter (FACS™) equipped with Cell Quest software (Becton Dickinson Biosciences, San Jose, CA, USA). For flourescence microscopy, J774A.1 cells were seeded onto 4-well chamber slides at a density of 4.0 × 103 per well (surface area of 1.7cm2 per well, 4-chamber slides) and incubated for 24h at 37°C. The PS-QD micelles PS (0), (40), (50), (60), and (100) at 10-nM concentration were added to the cells and incubated for 4h at 37°C. After incubation, the solution was removed and the cells were washed with PBS for at least three times. The cells were fixed with 4% formalin for 10min and washed with PBS and mounted with the DAPI mounting medium for nuclear staining. The cells were examined by an epifluorescence microscope (NIKON Eclipse) using separate filters for nuclei, DAPI filter (blue), and for QD (620); TRITC filter (red).
J774A.1 macrophage cells were cultured with DMEM supplemented with 10% FBS, 100 U/mL penicillin, and 100μg/mL streptomycin in a 5% CO2 atmosphere at 37°C. The cytotoxicity of PS-QD micelles on J774A.1 cells was evaluated using a colorimetric MTT assay kit. After the cells achieved 80% confluency, the cells were scraped and seeded onto a 96-well plate at a density of 1.5 × 104 cells per well. After 24h of incubation, the cell culture medium was removed. All PS-QD micelles were filtered using a 0.45-μM syringe filter before addition to the cell culture medium. PS-QD micelles PS (0), (40), (50), (60), and (100) at concentrations of 1-, 5-, 10-, and 50-nM concentrations were incubated with the cells for 24 h at 37°C under a 5% CO2 atmosphere. After incubation, the medium was removed and the cells were washed with PBS three times. Fresh medium was added to the wells with 10 μL of MTT reagent at 37°C for 4 h according to the manufacturer's protocol. The absorbance was read at a wavelength of 550 nm with a spectramax microplate reader (Molecular Devices, Sunnyvale, CA, USA). The assay was run in triplicates.
Results and discussion
Next, the PEG packing density of PS (50) micelles was controlled by tuning the homogenization speed of the micro-emulsion that resulted in the preparation of micelles of two different sizes of approximately 40-nm PS (50-1) and approximately 100-nm PS (50-2) micelles. When tested for macrophage-specific targeting, it was found that PS (50-1) micelles with a size of approximately 40 nm were not uptaken by macrophages (incubated at 25 pM) and at different micelle concentrations (Additional file 1: Figure S6), while PS (50-2) micelles with a size of approximately 100 nm in size are avidly uptaken by macrophages (MFI 15.1 versus 5.6) (Figure 2B). Further, the possibility that the uptake of larger-sized PS (50-2) micelles by macrophages were indeed correlated to the surface coverage of PS in the micelles and independent of surface negative charge was also investigated. For this purpose, the amount of PS in the PS (50-2) micelles was varied by substituting PS with a negatively charged lipid: 1,2-dipalmitoyl-sn-glycero-3-phospho-(glycerol) (DPPG) at two PS-DPPG molar ratios (40:10 and 30:20) but keeping the overall molar ratio constant at 50 mol%). As shown in Figure 2C, PS-PG (40:10) micelles containing more PS than PS-PG (30:20) micelles were taken up to a higher degree by macrophages, suggesting macrophage uptake of micelles was dependent on the PS content in micelles and independent of the surface charge. The above results show that PEG coverage and size can be fine-tuned to influence the surface exposure of PS and thus permit or block the ligand receptor recognition and cell uptake.
In conclusion, a size-dependent uptake of approximately 100-nm PS-QD micelles that resemble dead/apoptotic cells and recognized as ‘self’ are detected and uptaken by macrophage-like cells, whereas PS-QD micelles that are intermediate in size (approximately 40 nm) and recognized as ‘non-self’ are not uptaken by macrophage-like cells. The importance of this study based on the size and phospholipid coating of equal molar ratio of PS and PL-PEG for nanoparticles can be further extended to targeted delivery of inorganic particles for imaging or drug delivery applications.
We deeply thank Dr. Patrick Kee for helpful discussions through the work and in preparation of this manuscript. This work is supported by National Institutes of Health (NIH), National Heart Lung Blood Institute (NHLBI) R21Grant (Grant # 8226385). Dr. Maiseyeu was supported by American Heart Association NCRP Scientist Development Grant 13SDG14500015.
- Moore KJ, Tabas I: Macrophages in the pathogenesis of atherosclerosis. Cell 2011, 145: 341–355. 10.1016/j.cell.2011.04.005View ArticleGoogle Scholar
- Saha P, Modarai B, Humphries J, Mattock K, Waltham M, Burnand KG, Smith A: The monocyte/macrophage as a therapeutic target in atherosclerosis. Curr Opin Pharmacol 2009, 9: 109–118. 10.1016/j.coph.2008.12.017View ArticleGoogle Scholar
- Jaffer FA, Libby P, Weissleder R: Optical and multimodality molecular imaging: insights into atherosclerosis. Circulation 2007, 116: 1052–1061. 10.1161/CIRCULATIONAHA.106.647164View ArticleGoogle Scholar
- Shaw SY: Molecular imaging in cardiovascular disease: targets and opportunities. Nat Rev Cardiol 2009, 6: 569–579. 10.1038/nrcardio.2009.119View ArticleGoogle Scholar
- Desai MY, Schoenhagen P: Emergence of targeted molecular imaging in atherosclerotic cardiovascular disease. Expert Rev Cardiovasc Ther 2009, 7: 197–203. 10.1586/14779072.7.2.197View ArticleGoogle Scholar
- Krahling S, Callahan MK, Williamson P, Schlegel RA: Exposure of phosphatidylserine is a general feature in the phagocytosis of apoptotic lymphocytes by macrophages. Cell Death Differ 1999, 6: 183–189. 10.1038/sj.cdd.4400473View ArticleGoogle Scholar
- Fadok VA, Bratton DL, Rose DM, Pearson A, Ezekewitz RA, Henson PM: A receptor for phosphatidylserine-specific clearance of apoptotic cells. Nature 2000, 405: 85–90. 10.1038/35011084View ArticleGoogle Scholar
- Moghimi SM, Hunter AC: Recognition by macrophages and liver cells of opsonized phospholipid vesicles and phospholipid headgroups. Pharm Res 2001, 18: 1–8. 10.1023/A:1011054123304View ArticleGoogle Scholar
- Fadok VA, Voelker DR, Campbell PA, Cohen JJ, Bratton DL, Henson PM: Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J Immunol 1992, 148: 2207–2216.Google Scholar
- Maiseyeu A, Mihai G, Roy S, Kherada N, Simonetti OP, Sen CK: Detection of macrophages via paramagnetic vesicles incorporating oxidatively tailored cholesterol ester: an approach for atherosclerosis imaging. Nanomedicine (Lond) 2010, 5: 1341–1356. 10.2217/nnm.10.87View ArticleGoogle Scholar
- Torchilin VP: Recent advances with liposomes as pharmaceutical carriers. Nat Rev Drug Discov 2005, 4: 145–160. 10.1038/nrd1632View ArticleGoogle Scholar
- Owens DE, Peppas NA: Opsonization, biodistribution, and pharmacokinetics of polymeric nanoparticles. Int J Pharm 2006, 307: 93–102. 10.1016/j.ijpharm.2005.10.010View ArticleGoogle Scholar
- Torchilin VP: Micellar nanocarriers: pharmaceutical perspectives. Pharm Res 2007, 24: 1–16.View ArticleGoogle Scholar
- Torchilin VP: PEG-based micelles as carriers of contrast agents for different imaging modalities. Adv Drug Deliv Rev 2002, 54: 235–252. 10.1016/S0169-409X(02)00019-4View ArticleGoogle Scholar
- Cormode DP, Skajaa T, Schooneveld MMV, Koole R, Jarzyna P, Lobatto ME, Calcagno C, Barazza A, Gordon RE, Zanzonico P, Fisher EA, Fayad ZA, Mulder WJM: Nanocrystal core high-density lipoproteins: A multimodality contrast agent platform. Nanoletters 2008, 8: 3715–3723. 10.1021/nl801958bView ArticleGoogle Scholar
- Andrew MS, Hongwei D, Aaron MM, Nie S: Bioconjugated quantum dots for in vivo molecular and cellular imaging. Adv Drug Deliv Rev 2008, 60: 1226–1240. 10.1016/j.addr.2008.03.015View ArticleGoogle Scholar
- Bocca C, Caputo O, Cavalli R, Gabriel L, Miglietta A, Gasco MR: Phagocytic uptake of fluorescent stealth and non-stealth solid lipid nanoparticles. Int J Pharm 1998, 175: 185–193. 10.1016/S0378-5173(98)00282-8View ArticleGoogle Scholar
- Gabizon A, Shmeeda H, Horowitz AT, Zalipsky S: Tumor cell targeting of liposome-entrapped drugs with phospholipid-anchored folic acid-PEG conjugates. Adv Drug Deliv Rev 2004, 56: 1177–1192. 10.1016/j.addr.2004.01.011View ArticleGoogle Scholar
- Walkey CD, Olsen JB, Guo NH, Emili A, Chan WC: Nanoparticle size and surface chemistry determine serum protein adsorption and macrophage uptake. J Am Chem Soc 2012, 134: 2139–2147. 10.1021/ja2084338View ArticleGoogle Scholar
- Hagan SA, Coombes AGA, Garnet MC, Dunn SE, Davies MC, Illum L, Davis SS: Polylactide - Poly (ethylene glycol) Copolymers as Drug Delivery Systems. 1. Characterization of Water Dispersible Micelle-Forming Systems. Langmuir 1996, 12: 2153–2161. 10.1021/la950649vView ArticleGoogle Scholar
- Bazile D, Prudhomme C, Bassoullet MT, Marlard M, Spenlehauer G, Veillard M: Stealth Me. PEG-PLA nanoparticles avoid uptake by the mononuclear phagocytes system. J Pharm Sci 1995, 84: 493–498. 10.1002/jps.2600840420View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited.