Internalization of paramagnetic phosphatidylserine-containing liposomes by macrophages
© Geelen et al.; licensee BioMed Central Ltd. 2012
Received: 21 June 2012
Accepted: 23 August 2012
Published: 28 August 2012
Inflammation plays an important role in many pathologies, including cardiovascular diseases, neurological conditions and oncology, and is considered an important predictor for disease progression and outcome. In vivo imaging of inflammatory cells will improve diagnosis and provide a read-out for therapy efficacy. Paramagnetic phosphatidylserine (PS)-containing liposomes were developed for magnetic resonance imaging (MRI) and confocal microscopy imaging of macrophages. These nanoparticles also provide a platform to combine imaging with targeted drug delivery.
Incorporation of PS into liposomes did not affect liposomal size and morphology up to 12 mol% of PS. Liposomes containing 6 mol% of PS showed the highest uptake by murine macrophages, while only minor uptake was observed in endothelial cells. Uptake of liposomes containing 6 mol% of PS was dependent on the presence of Ca2+ and Mg2+. Furthermore, these 6 mol% PS-containing liposomes were mainly internalized into macrophages, whereas liposomes without PS only bound to the macrophage cell membrane.
Paramagnetic liposomes containing 6 mol% of PS for MR imaging of macrophages have been developed. In vitro these liposomes showed specific internalization by macrophages. Therefore, these liposomes might be suitable for in vivo visualization of macrophage content and for (visualization of) targeted drug delivery to inflammatory cells.
KeywordsInflammation Liposomes MRI Phosphatidylserine Cell internalization
Inflammation plays a crucial role in many pathologies, including cardiovascular diseases, neurological disorders and oncology, and is generally considered as an important predictor for disease progression and outcome [1, 2]. Therefore, modulation of the inflammatory response by dedicated therapy is of particular interest.
The efficacy of traditional therapeutic compounds of low molecular weight is often limited by short blood circulation half-lives and adverse side effects due to non-specific systemic distribution and accumulation. Additionally, it is difficult to obtain quantitative information on the amount of drug accumulating in the diseased tissue. Drug delivery via a nanocarrier system provides an attractive alternative to alleviate these drawbacks. For example, Doxil is a clinically approved nanocarrier system for cancer treatment, which consists of doxorubicin encapsulated in liposomes [3, 4]. This formulation limits cardiotoxicity and prolongs the blood circulation half-life compared to free doxorubicin, which results in an enhanced time window for drug delivery and extravasation of the liposomes through the leaky tumor vasculature.
The surface composition of nanocarriers containing drugs can be tailored to tune clearance kinetics, for instance polyethylene glycol (PEG) is often incorporated to prolong the blood half-life . Furthermore, the larger size of nanocarriers promotes a higher level of uptake in diseased tissues by the enhanced permeability and retention (EPR) effect [6, 7]. Importantly, to address the inflammatory response in cardiovascular disease, the drug-containing nanocarriers should be delivered with high specificity to inflammatory cells in the diseased tissue. This can be achieved by introducing ligands that mediate nanocarrier recognition and internalization by the inflammatory cells.
An attractive route to target macrophages is by incorporation of the lipid phosphatidylserine (PS) in lipid-based nanoparticles, such as liposomes. In mammalian cells, PS is predominantly present in the inner leaflet of cell membranes. When a cell becomes apoptotic, PS is exposed on the outer leaflet of the cell membrane, which serves as a trigger for phagocytosis by macrophages [8, 9]. The incorporation of PS in the liposomal membrane can therefore promote uptake by macrophages. Previously it was shown that incorporation of PS in liposomes indeed resulted in enhanced uptake by macrophages [10, 11].
Also, magnetic resonance imaging (MRI) contrast agents can be incorporated to image drug delivery and obtain quantitative information on the local concentration of drugs at the target site [12, 13]. Previously, Harel-Ader et al. developed liposomes with PS containing iron-oxides for MRI visualization of inflammatory cells in myocardial infarction  and Maiseyeu et al. described liposomes with PS containing Gd-DTPA-distearylamide for MR imaging of macrophages in atherosclerotic plaques . However, a detailed characterization and optimization of MRI-detectable PS-containing liposomes, including the conditions under which they most effectively target macrophages and induce strongest contrast in MRI, is still lacking.
In this study, we therefore describe the design and characterization of MRI-detectable liposomes that are targeted to macrophages using PS. Liposomes containing different molar percentages of PS were prepared and liposome size and morphology were studied by dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryoTEM). Fluorescent labels, incorporated in the liposomes, enabled detailed analysis of liposome binding and internalization by macrophages using confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS). The ability of the liposomes to induce contrast changes in MR images was studied in macrophages and quantified on the basis of the measured changes in T1 and T2 relaxation times.
Characterization of liposomes
Mol% of lipids present in different liposome formulations
Characterization of liposome formulations
Hydrodynamic diameter (nm) a
136 ± 3
120 ± 7
111 ± 10
97 ± 4*
PDI (−) a
0.13 ± 0.03
0.28 ± 0.05*
0.30 ± 0.02*
0.30 ± 0.19*
r 1 (mM -1 ·s -1 ) b
3.0 ± 0.1
3.0 ± 0.3
3.4 ± 0.4
4.0 ± 0.1
r 2 (mM -1 ·s -1 ) b
42.3 ± 5.5
51.5 ± 4.5
60.7 ± 5.1
46.7 ± 4.2
r 2 /r 1 b
14.4 ± 2.0
18.1 ± 2.6
18.2 ± 0.8
11.8 ± 0.9
The ability of the liposomes to generate contrast in MRI is determined by their potency to change the longitudinal (T1) and transversal (T2) relaxation times, which is expressed by the longitudinal (r1) and transversal (r2) relaxivity. The r1 and r2 of the liposomes at 9.4 T and room temperature, normalized to Gd concentration, were 3.0-4.0 mM-1·s-1 and 42–60 mM-1·s-1, respectively (Table 2). Incorporation of PS did not significantly affect the longitudinal and transversal relaxivity. All liposome formulations displayed a similar relatively high r2/r1 ratio.
Association of PS-containing liposomes with macrophages
In vitro experiments were performed to determine which formulation of PS-containing liposomes resulted in highest association with mouse macrophages (RAW cells). RAW cells were incubated with PC-L, PS-6-L, PS-12-L and PS-37-L and association of liposomes with the macrophages was characterized by several readouts exploiting the various components of the liposomes, including quantitative T1 and T2 mapping with MRI, quantitative Gd determinations by inductively coupled plasma mass spectrometry (ICP-MS), and CLSM.
MRI measurements were performed at 9.4 T on the same cell pellets as for ICP-MS. MRI consisted of quantitative T1 and T2 mapping. The cells incubated with PS-6-L could clearly be distinguished from untreated cells and cells incubated with other types of liposomes (Figure 2b). Average R1 (=1/T1) and R2 (=1/T2) values for the different groups are summarized in Figure 2c and 2d, respectively. Incubation with liposomes always resulted in enhanced R1 and R2 values (p <0.05 vs. no L). In agreement with quantitative Gd determinations, however less pronounced, both R1 and R2 were highest for the incubations with PS-6-L (0.942 ± 0.004 s-1 and 37.3 ± 1.2 s-1, respectively, p <0.05 vs. all).
Relaxivities of the liposomes in the cellular environment
1.9 ± 0.3
32.5 ± 3.6
0.8 ± 0.4
16.3 ± 5.8
1.4 ± 0.5
19.1 ± 6.9
1.3 ± 0.1
22.0 ± 8.1
CLSM imaging of the near-infrared (NIR)-labeled lipids incorporated in the liposomal membrane revealed association of all types of liposomes with RAW cells (Figure 2e). No NIR autofluorescence signal was detected in RAW cells incubated without liposomes. In agreement with MRI, NIR fluorescence and therefore liposome association was highest for cells incubated with PS-6-L and intermediate for PC-L, while PS-12-L and PS-37-L showed similarly low levels of NIR fluorescence.
Divalent cation dependency of liposome association with macrophages
The above-described experiments suggested that liposomes with 6 mol% DSPS (PS-6-L) were the most optimal formulation for targeting of macrophages. Therefore, PS-6-L was used in the experiments described from here. Association of PS-containing vesicles with the macrophage cell membrane depends on the presence of divalent cations such as Ca2+ and Mg2+. To test whether PS-6-L binding depended on the presence of divalent cations, which could be indicative for an interaction between these ions and PS resulting in membrane binding, RAW cells were incubated with PC-L and PS-6-L in Hank’s buffered salt solution (HBSS) with 1.26 mM Ca2+ and 0.90 mM Mg2+ (HBSS+) or without Ca2+ and Mg2+ (HBSS-). Furthermore, samples from RAW cells incubated in incubation medium with intermediate Ca2+ and Mg2+ concentrations (0.424 mM and 0.407 mM, respectively), identical to the ones used in previous experiments, were included. Cellular association was quantified with FACS.
DLS showed that the diameter of both PC-L and PS-6-L increased after 2 h of incubation in HBSS+(Figure 4d). For PC-L the average hydrodynamic diameter changed from 164.3 ± 0.9 nm in HBSS- to 209.5 ± 25.2 nm, while for PS-6-L the diameter increased from 104.6 ± 19.4 nm to 170.8 ± 33.5 nm. This size increase could additionally enhance the uptake of both types of liposomes by the cells.
Binding versus internalization
CLSM confirmed the FACS measurements (Figure 5c). Incubation of RAW cells with PC-L and PS-6-L at 4°C resulted in minor association of liposomes. CLSM using higher laser intensities showed that the liposomes appeared as a rim around every cell, bound to the cell membrane. No significant internalization was observed. For incubations with PC-L at 37°C CLSM images were comparable to incubations at 4°C, with minor association of liposomes, and higher laser intensities revealed that PC-L were mainly bound to the cell membrane. CLSM confirmed that incubation with PS-6-L at 37°C resulted in massive internalization of the liposomes, as shown by the high NIR signal inside RAW cells.
Macrophages play a decisive role in several cardiovascular diseases. For example, in atherosclerosis high macrophage content is one of the hallmarks of plaque vulnerability . The inflammatory response after myocardial infarction is important for cardiac remodeling and outcome . Therefore, macrophages form a significant therapeutic target in cardiovascular diseases and tools for noninvasive MR imaging of macrophages are highly desired. Iron oxides have been successfully applied for the MR visualization of macrophages in cardiovascular diseases [15–17]. Nevertheless, targeting of iron oxides to CD11b/CD18, which is expressed on macrophages, did not improve specificity for MR imaging of macrophages in a mouse model of atherosclerosis . Recently, Gd-labeled liposomes were used to visualize monocytes and/or macrophages infiltration in the mouse myocardium up to 7 days after myocardial infarction .
In this study, we describe the design and characterization of paramagnetic liposomes targeted to macrophages by incorporation of PS in the liposomal membrane. The liposomes contained Gd-DOTA-DSPE for MRI detection. Gd-DOTA-DSPE is a phospholipid that presents a high r1 and the Gd-DOTA complex displays a high thermodynamic and kinetic stability . As expected, at 9.4 T, the longitudinal relaxivity is not as high as at lower, clinical field strengths [20, 21]. Importantly, incorporation of PS did not significantly affect liposomal r1 and r2 values. The r2/r1 ratio of the liposome formulations at 9.4 T was relatively high, which means that the liposomes will display a significant T2 effect as well. Nevertheless, by appropriately choosing the MRI sequence parameters, the T1 effect of the liposomes can be effectively exploited (Figure 2).
A distinct difference between the PS-containing liposomes used in this study and previously reported formulations for use in in vivo MRI studies is the incorporation of 5 mol% polyethylene glycol (PEG) lipids in the liposomal membrane. PEG reduces the interactions between the liposomes, reducing aggregation and ensuring a monodisperse formulation (Table 2 and Figure 1). Additionally, PEG increases the in vivo blood circulation half-life by reducing the interactions with plasma proteins, assuring a longer interaction time with macrophages . According to previous studies, incorporation of 5 mol% PEG in PS-containing liposomes is not impeding the interaction of PS with macrophages, since at least 10–15 mol% PEG would be needed to completely shield the liposomes from any interactions with proteins [22–24]. We therefore did not expect a decrease in the uptake by shielding of the PS.
Liposomes containing 6 mol% PS resulted in the highest uptake by RAW murine macrophages (Figure 2). Maiseyeu et al. and Rimle et al. have observed optimal uptake by macrophages of liposomes without PEG when these contained 5–12 mol% PS [10, 11]. Interestingly, these experimentally determined optimal concentrations are in the range of 2–10 mol% PS found in the membranes of mammalian cells , which suggests that macrophages are optimally equipped to recognize and phagocytose nanoparticles that express approximate physiological concentrations of PS. Association was specific for macrophages as uptake by endothelial H5V cells was significantly lower (Figure 3).
Uptake of PS-containing liposomes by macrophages was stimulated by the presence of divalent cations (Figure 4). Higher uptake was not primarily caused by divalent cation-mediated clustering of the liposomes, since incubation of liposomes in HBSS+ resulted in moderate changes in liposome size for both PC-L and PS-6-L. The HBSS+ buffer contained a physiologically relevant concentration of 1.26 mM Ca2+, compared to for example approximately 1.24 mM Ca2+ in mouse blood . For the PS-mediated recognition of apoptotic cells by macrophages, different engulfment receptors have been identified, such as scavenger receptors, oxidized low-density lipoproteins recognizing receptors and CD68 , which for the LOX-1 scavenger receptor has been proven to be Ca2+-dependent . Which of these receptors are important for PS-mediated uptake of liposomes remains unknown.
With respect to MR imaging of liposome uptake, a relatively high association of PS-6-L with macrophages, as determined with ICP-MS, resulted only in a modest increase in R1 (Figure 2). This is probably related to compartmentalization of PS-6-L in intracellular vesicles after phagocytosis, which limits effective access of bulk water protons to the Gd contrast agent [29, 30]. T1 shortening requires direct physical contact between Gd and water protons to be most effective. This interpretation is corroborated by the observation that the estimated cellular relaxivity of PS-6-L (r1 = 0.8 ± 0.4 mM-1·s-1) was lower than the one of PS-6-L in aqueous solution (r1 = 3.0 ± 0.3 mM-1·s-1). Furthermore, internalization of PS-6-L was observed by CLSM for incubations at 37°C (Figure 5).
The next step will be to apply and study the uptake of PS-6-L in a relevant animal model of cardiovascular inflammation, for example in atherosclerosis or myocardial infarction. Christiansen et al. have shown that echocardiography of PS-containing microbubbles trapped in infarcted myocardium correlated moderately well with MPO activity, which are excreted by inflammatory cells .
Apart from use in imaging applications, PS-containing liposomes are a promising vehicle for targeted drug delivery. Liposomes loaded with Q10, ATP or adenosine delivered to infarct myocardium were demonstrated to reduce infarct size and salvage ischemic myocardium [32–34]. Also, liposomes have been used as a vehicle for delivery of glucocorticoids drugs to perform anti-inflammatory cancer therapy . Targeting could enhance the specificity of drug delivery to macrophages. Alternatively, PS-liposomes themselves can be used for therapy of inflammation as well [14, 35–37]. As PS-liposomes mimic apoptotic cells, they inhibit pro-inflammatory cytokines release and promote secretion of anti-inflammatory cytokines. However, for therapy purposes higher PS-concentrations (up to 30 mol%) were used [14, 36], which in this study did not enhance uptake by macrophages.
In summary, paramagnetic liposomes, containing 6 mol% of PS, showed enhanced uptake by macrophages compared to liposomes without PS, while significantly less uptake was observed for non-phagocytic cells. Association of PS-containing liposomes to macrophages was increased by the presence of divalent cations in the incubation medium and resulted mainly in internalization of liposomes, whereas only minor binding was observed. Therefore, these liposomes can be used for molecular MR imaging of macrophages and might as well be suitable for targeted drug delivery to macrophages in cardiovascular diseases.
Preparation of PS-containing liposomes
Liposomes containing different mole percentages of PS were prepared by modification of the protocol described by Hak et al.. In short, lipid film hydration of a lipid mixture was performed (typically 50 μmol of total lipid). The lipid mixture, consisting of 1,2-distearoyl-sn-glycero-3-phospho-L-serine (DSPS, Avanti Polar Lipids, Alabaster, USA), 2-distearoyl-sn-glycero-3-phosphocholine (DSPC, Lipoid, Steinhausen, Switzerland), Gd-DOTA-1,2-distearoyl-sn-glycero-3-phospoethanolamine (Gd-DOTA-DSPE, SyMO-Chem BV, Eindhoven, the Netherlands), cholesterol (Avanti Polar Lipids) and 1,2-distearoyl-sn-glycero-3-phospoethanolamine-N-[methoxy(poly(ethylene glycol))-2000] (PEG2000-DSPE, Lipoid), was dissolved in chloroform and methanol (8:1 v/v) at molar percentages as shown in Table 1. For liposomes containing DSPS, the mixture was heated to dissolve the DSPS (to maximally 65°C). Additionally, 0.1 mol% near-infrared664-1,2-distearoyl-sn-glycero-3-phospoethanolamine (NIR664-DSPE, SyMO-Chem BV) was incorporated. After rotary evaporation at 30°C and overnight drying under a nitrogen flow, the lipid film was hydrated in HEPES-buffered saline (HBS, 10 mM HEPES, 135 mM NaCl, pH 7.4) at 65°C. The resulting multilamellar vesicles were sized by extrusion through 400 nm filters (2 times) and 200 nm filters (8 times). Finally, the liposomes were concentrated using ultracentrifugation (45 min, 55,000 rpm, 4°C) and resuspended in HBS at a concentration of approximately 70 mM total lipid.
Characterization of liposomes
Total lipid concentrations of the final liposome formulations were determined by a phosphate determination according to Rouser . Hydrodynamic number-weighted size and size distribution were assessed with dynamic light scattering (DLS, ZetaSizer NanoS, Malvern Instruments, Worcestershire, UK) at 23°C.
To confirm the presence of DSPS lipids in the PS-containing liposomes, thin layer chromatography (TLC) was performed on an aluminum sheet coated with silica gel 60 F254 (Merck BV, Schiphol-Rijk, the Netherlands) . As eluent a mixture of chloroform, methanol, glacial acetic acid and water (65:25:8:4 v/v) was used. Liposomes were applied (expected concentrations of DSPS: PC-L 0 mg/mL, PS-6-L 2 mg/mL, PS-12-L 4 mg/mL and PS-37-L 6 mg/mL) and allowed to migrate for 30 min. As controls standard solutions of DSPS (0.5, 1, 2, 4 and 8 mg/mL) were used. Finally, primary and secondary amines in DSPS and Gd-DOTA-DSPE were detected with ninhydrin.
Liposomal morphology was evaluated with cryogenic transmission electron microscopy (cryoTEM). Samples were vitrified on carbon-coated cryoTEM grids with a vitrification robot (Vitrobot Mark III, FEI, Hillsboro, USA). Imaging was performed on a Tecnai 20 Sphera TEM instrument (FEI) equipped with a LaB6 filament (200 kV) and Gatan cryoholder (approximately -170°C) at 25,000x magnification.
Liposomal longitudinal and transversal relaxation times (T1 and T2) were determined with a 9.4 T small animal MR scanner (Bruker Biospin GmbH, Ettlingen, Germany) equipped with a 35-mm-diameter quadrature birdcage RF coil (Rapid Biomedical, Rimpar, Germany). For T1 measurements an inversion recovery fast low angle shot (FLASH) sequence was used, with the following parameters: overall repetition time (TR) 15 sec, TR 4 ms, echo time (TE) 2 ms, flip angle (α) 15o, number of excitations (NEX) 4, field of view (FOV) 3x3 cm2, matrix 128x128, 1 mm slice thickness, 32 segments and 60 inversion times ranging from 72 to 4792 ms. T2 relaxation times were determined using a multi-slice multi-echo sequence with the following parameters: TR 2000 ms, 32 TEs ranging from 9 to 288 ms, α 180o, NEX 4, FOV 3x3 cm2, matrix 128x128 and 1 mm slice thickness. T1 and T2 relaxation times were calculated by mono-exponential fitting with a custom-built fitting program (Mathematica 6, Wolfram Research Europe, Oxfordshire, UK). Relaxivities r1 and r2 (in mM-1·s-1) were determined from Ri = Ri,0 + ri·[Gd], with i = 1,2, Ri = 1/Ti, Ri,0 the relaxation rate of a sample without liposomes and [Gd] between 0.001 and 1 mM Gd.
Mouse macrophages, RAW264.7 (European Collection of Animal Cell Cultures (ECACC)), were cultured in RPMI medium (phenol-red free), supplemented with 10% FBS, 2 mM L-glutamine and 100 U/mL penicillin/streptomycin. As non-phagocytic control cells, murine heart endothelioma cells, H5V (kindly provided by prof. dr. G. Molema, University of Groningen, the Netherlands), were maintained in DMEM medium, containing 10% FBS, 2 mM L-glutamine and 100 U/mL penicillin/streptomycin .
Association of PS-containing liposomes with RAW cells
To determine the mol% of PS present in liposomes resulting in maximal uptake by macrophages, RAW cells were incubated with PC-L, PS-6-L, PS-12-L and PS-37-L for 2 h at 37°C (1 mM total lipid). For MRI and inductively coupled plasma mass spectrometry (ICP-MS), cells were harvested by scraping and non-bound liposomes were removed by centrifugation (3x5 min, 500 g, RPMI medium at 37°C). Cells were fixed in 4% PFA (250 μL) and a loosely packed cell pellet was allowed to form by storage at 4°C (>2 days). For confocal laser scanning microscopy (CLSM), cells were cultured on coverslips. After incubation with the liposome formulations, cells were fixed with 4% PFA (20 min). Finally cells were washed with and stored in phosphate bufferd saline (PBS).
Association of PS-containing liposomes with H5V cells
To confirm that PS-containing liposomes were not taken up by endothelial cells, H5V cells were incubated with PC-L, PS-6-L, PS-12-L and PS-37-L for 2 h at 37°C (1 mM total lipid). For MRI, cells were washed with medium (37°C) and PBS (37°C). Afterwards, cells were harvested with trypsin/EDTA, fixed with 4% PFA and a loosely packed pellet was allowed to form. For CLSM, cells were cultured on gelatin-coated coverslips and handled as described above.
Divalent cation dependency of liposome association with RAW cells
The association of liposomes to RAW cells under different calcium and magnesium concentrations was studied. RAW cells were incubated with PC-L and PS-6-L (2 h, 37°C, 1 mM total lipid) in Hank’s buffered salt solution (HBBS) containing 1.26 mM Ca2+ and 0.90 mM Mg2+ (HBSS+), HBSS without Ca2+ and Mg2+ (HBSS-) and RPMI medium (0.424 mM Ca2+ and 0.407 mM Mg2+). Afterwards, cells were harvested by scraping, washed in the appropriate medium (HBSS+, HBSS- or RPMI, 37°C), fixed in 4% PFA (20 min) and stored in 0.01% sodium-azide in PBS for FACS.
To investigate possible clustering of PC-L and PS-6-L under high calcium and magnesium concentrations, liposomes were incubated in HBSS+ or HBSS- (2 h, 37°C). Changes in hydrodynamic number-weighted diameter and size distribution were measured with DLS as described above at 37°C.
Binding versus internalization
To evaluate phagocytosis of PS-containing liposomes by macrophages, RAW cells were incubated with PC-L or PS-6-L at 4°C or at 37°C (1 mM total lipid in HBSS+, 2 h). Incubation at 4°C inhibits phagocytosis. For FACS, cells were incubated with liposomes in HBSS+ and harvested and washed as described above. For CLSM, cells were cultured and incubated with liposomes in microscopy chambers (Ibidi GmbH, München, Germany). Afterwards, cells were washed with HBSS+ (4°C or 37°C), fixed with 4% PFA (20 min), washed and stored in PBS.
Cellular relaxation rates and relaxivities
The cellular relaxation rates of cell pellets (R1 and R2) were determined at 9.4 T using the MRI protocol as described above. Furthermore, the cell pellet volume was determined using a 3D FLASH sequence with the following parameters: TR 25 ms, TE 3.7 ms, α 30o, NEX 1, FOV 25.6x25.6x25.6 mm3 and matrix 256x256x256. Cell pellets were segmented with OsiriX Imaging Software (http://www.osirix-viewer.com) and pellet volumes were calculated. The Gd content of cell pellets was determined with ICP-MS (DRCII, Perkin Elmer, Waltham, USA) after destruction in nitric acid and perchloric acid (1:2 v/v) at 180°C. Next, gadolinium concentrations were derived using the cell pellet volume. Cellular relaxivities were calculated from Ri = Ri,0 + ri·[Gd], with i = 1,2, and Ri,0 the relaxation rate of untreated cells.
Cellular fluorescence quantification
The fluorescence intensity of NIR664-lipids, present in the liposomes associated with RAW cells, was quantified by fluorescence activated cell sorting (FACS) on a Guava Easycyte 8HT (Millipore, Billerica, USA). NIR664 was excited with a 640 nm laser and detected using a 661/19 nm band-pass filter. Mean cellular fluorescence intensity was calculated with GuavaSoft 1.0 software (Millipore) and was corrected for autofluorescence as detected by measurement of untreated cells.
The cellular distribution of liposomes was studied with CLSM. Cell membranes of RAW cells were labeled with rat anti-mouse CD68-fluorescein isothiocynate (CD68-FITC, 4 μg/mL, AbD Serotec, Dusseldorf, Germany). H5V cells were labeled with rat anti-mouse CD31 (10 μg/mL, BioLegend, Uithoorn, The Netherlands) conjugated to goat anti-rat FITC (Invitrogen, Bleiswijk, The Netherlands). Nuclei were stained with 4′6-diamidino-2-phenylindole dihydrochloride (DAPI, 0.1 μg/mL, Invitrogen).
A Zeiss LSM META system (Carl Zeiss BV, Sliedrecht, the Netherlands) was used for acquisition of CLSM images. NIR664 present in liposomes was excited with a 633 nm HeNe laser (5.0 mW) and the emission was filtered with a 680/60 nm band-pass filter. Cell membranes labeled with FITC were excited with a 488 nm Ar laser and the emission was filtered with a 525/50 nm band-pass filter. For two-photon excitation of DAPI, a Ti:Sapphire laser tuned to 780 nm was used and emission was captured with a 460/50 band-pass filter. All images were acquired with a 63x/1.4 oil immersion objective, a matrix of 2048x2048, resulting in a resolution of 0.07x0.07 μm2, and 4 averages.
All data are presented as mean ± standard error of the mean (SEM). To test for significant differences between groups, one-way analysis of variance (ANOVA) with Bonferroni correction for multiple group comparisons or a Student’s t-test for independent samples was applied. All statistical analyses were performed in PASW Statistics 18.02 (IBM Corporation, Armonk, NY, USA) and P <0.05 was considered significant.
Analysis of variance
Confocal laser scanning microscopy
Cryogenic transmission electron microscopy
Dynamic light scattering
Enhanced permeability and retention
Fluorescence activated cell sorting
Fetal bovine serum
HEPES buffered saline
Hank’s buffered salt solution
HBSS without Ca2+ and Mg2+
HBSS with Ca2+ and Mg2+
Inductively coupled plasma mass spectrometry
Magnetic resonance imaging
Phosphate buffered saline
Liposomes containing x mol% of PS
Longitudinal relaxation rate
Transversal relaxation rate
Standard error of the mean
Longitudinal relaxation time
Transversal relaxation time
Thin layer chromatography.
This work is supported by the Dutch Technology Foundation STW, applied science division of NWO and the Technology Program of the Ministry of Economic Affairs; Grant number: 07952. We thank Henk Keijzer (SyMO-Chem BV, Eindhoven, the Netherlands) for assistance with the TLC measurements.
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