in vivo nirf imaging of tumor targetability of nanosized liposomes in tumor-bearing mice

Full Paper

In vivo NIRF Imaging of Tumor Targetability ofNanosized Liposomes in Tumor-Bearing Micea

Sangmin Lee, Seung-Young Lee, Sangjin Park, Ju Hee Ryu, Jin Hee Na,Heebeom Koo, Kyung Eun Lee, Hyesung Jeon, Ick Chan Kwon,Kwangmeyung Kim,* Seo Young Jeong*

To optimize tumor targetability of nanosized liposomes for application as drug carriers,various liposomes are prepared by incorporating different amounts (10, 30, and 50 wt%) ofcationic, anionic, and PEGylated lipids into neutral lipid. In vivo near-infrared fluorescenceimages reveal that PEG-PE/PC liposomes display hightumor accumulation in tumor-bearing mice, while largeamounts of DOTAP/PC liposomes are rapidly captured inthe liver, resulting in poor tumor accumulation. Theseresults demonstrate that optimization of the surface prop-erties of liposomes is very important for their tumor tar-getability, and that in vivo imaging techniques are usefulin developing and optimizing nanosized liposome-baseddrug carriers.

1. Introduction

Liposomes have been long considered promising drug

delivery carriers for treatment of various diseases.[1]

Liposomes are phospholipid bilayer vesicles composed of

an aqueous interior and a biocompatible lipid exterior; they

have great potential for the targeted delivery of therapeutic

agents, like chemical drugs or genes, to disease sites while

S. Lee, J. H. Na, Prof. S. Y. JeongDepartment of Life and Nanopharmaceutical Science, Kyung HeeUniversity, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701,Republic of KoreaE-mail: [email protected]. Lee, Dr. S. Park, J. H. Ryu, Dr. H. Koo, Dr. K. E. Lee, Dr. H. Jeon,Dr. I. C. Kwon, Dr. K. KimCenter for Theragnosis, Biomedical Research Institute, KoreaInstitute of Science and Technology (KIST), Hwarangno 14-gil 6,Seongbuk-gu, Seoul 136-791, Republic of KoreaE-mail: [email protected]

a Supporting Information for this article is available from the WileyOnline Library or from the author.

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reducing systemic toxicity and immunogenicity.[2,3] More-

over, the prolonged blood circulation of nanosized lipo-

somes is expected to prevent the fast excretion of drugs

from the body and enhance their accumulation in tumor

tissue through the defective vascular structure, which is

referred to the enhanced permeability and retention (EPR)

effect.[4] However, the recognition and unintended entrap-

ment of liposomes by the reticuloendothelial system (RES),

including the liver and spleen, is still a critical hurdle to their

use as drug carriers.[5]

To analyze the in vivo behavior of liposomes, many

studies have been performed focusing on their interaction

with cells, and their clearance and distribution in the body,

using animal models.[6–8] However, the obtained informa-

tion is insufficient for their optimization as tumor-targeting

drug carriers, and there is still a need to study the in vivo fate

of liposomes more precisely. Recently, various nanoparti-

cles have been non-invasively assayed by a live animal

imaging system that offers valuable information on both

the in vivo biodistribution and the tumor targetabilty in

tumor-bearing mice.[9,10] We thought that the in vivo fate of

liposomes labeled with near-infrared fluorescence (NIRF)

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dye, Cy5.5, might be monitored with an NIRF imaging

system, because the absorbance of light from hemoglobin,

water, lipids, and other tissues is minimal in the NIRF

region.[11]

Herein, in order to evaluate and optimize the tumor

targeting characteristics of liposomes, we prepared various

liposomes with different phospholipid compositions

based on a neutral lipid (1,2-dipalmitoyl-sn-glycero-3-

phosphocholine; PC), a cationic lipid (1,2-dioleoyl-3-

trimethylammonium-propane; DOTAP), an anionic lipid

(1,2-dioleoyl-sn-glycero-3-phospho-L-serine; PS), and a

PEGylated lipid (1,2-distearoyl-sn-glycero-3-phosphoeth-

anolamine-N-[methoxy[poly(ethylene glycol)]2000]; PEG-PE).

In order to determine their physicochemical properties, the

particle size, polydispersity, surface charge, and stability of

various liposomes in aqueous condition were characterized.

Cellular uptake profiles of different liposomes were also

observed in HeLa cells using confocal microscopy. Finally,

the in vivo biodistribution and tumor targetability of

liposomes were visualized and analyzed in tumor-bearing

mice models by the non-invasive NIRF imaging system.[12]

2. Experimental Section

2.1. Materials

PC, 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), PS, and

PEG-PE were purchased from Avanti Polar Lipids (Alabaster, AL).

4-(Dimethylamino)pyridine (DMAP) and 4-methylmorpholine

(NMM) were obtained from Sigma (St. Louis, MO). The mono-

reactive hydroxysuccinimide ester of Cy5.5 (Cy5.5-NHS) was from

Amersham Biosciences (Piscataway, NJ). Methanol, chloroform,

and ether were purchased from Merck (Darmstadt, Germany). All

other chemicals were of analytical grade and used without further

purification.

2.2. Preparation and Characterization of Liposomes

Various liposomes were prepared by incorporating 10, 30, and

50 wt% of DOTAP, PS, and PEG-PE into the basic neutral PC liposome

for differently charged outer lipid layer or without/with PEGylated

surface. In brief, pure PC lipid and lipid mixtures were dissolved in

chloroform/methanol (2:1) co-solvent at predetermined weight

ratio, and dried under nitrogen. The obtained film was added to PBS

(pH¼7.4), and then sonicated three times using a probe-type

sonifier (Ultrasonic Processor, Cole-Parmer Inst. co. USA) at 90 W for

2 min. Next, the liposomes were passed through a syringe filter

(0.8 mm, Milipore). The morphology of liposomes was characterized

by cryogenic transmission electron microscopy (cryo-TEM). Each

sample was prepared as a thin aqueous film supported on a holey

carbon grid. Cryo-TEM images were obtained at a temperature of

approximately�170 8C with a 200 kV Tecnai F20 (FEI, Netherlands).

The average diameter and size distribution of liposomes was

measured via dynamic light scattering (DLS; 90 Plus Particle Size

Analyzer, Brookhaven Instruments. Co., NY, USA) at 633 nm

and 25 8C. We evaluated the polydispersity factor, presented as

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m2/G2, using the cumulant analysis method.[13] The surface charge

of liposomes (1 mg �mL�1) in distilled water was determined using

a zeta-potential analyzer (Zeta Plus, Brookhaven Instrument. Co.,

NY, USA). To assay the stability of liposomes in serum condition,

each liposome (1 mg �mL�1) was incubated in PBS (pH¼7.4)

containing human serum albumin (20 mg �mL�1) at room tem-

perature, and the mean particle sizes were measured by DLS for up

to 48 h.

2.3. Synthesis of Cy5.5-Labeled Liposomes

To label liposomes with Cy5.5 for in vitro and in vivo NIRF imaging,

PE (20 mg, 28 mmol) dissolved in chloroform/methanol (2:1) co-

solvent (7 mL) was chemically conjugated with Cy5.5-NHS (20 mg,

17 mmol) by the aid of DMAP (7 mg, 56 mmol) and NMM (15 mL,

136 mmol). After overnight reaction at room temperature in the

dark, the solvent was removed using a rotary evaporator, and the

obtained Cy5.5-PE was precipitated in ether. Then, it was further

purified by dialysis against distilled water for 2 d, and dried under

vacuum for 3 d. Cy5.5-labeled liposomes were prepared by addition

of the resulting Cy5.5-labeled PE (0.05 wt%) to each lipid mixture

before film casting during fabrication, wherein Cy5.5-labeled PE did

not change the physicochemical properties of the original

liposomes.

2.4. Cell Culture and Cellular Uptake Studies

Human cervical cancer (HeLa) cells were purchased from the

American Type Culture Collection (ATCC; Manassas, VA, USA). The

cells were cultivated in Dulbecco’s modified Eagle’s medium,

supplemented with 10% FBS, 100 U �mL�1 penicillin G, and

100 mg �mL�1 streptomycin at 37 8C using a humidified 5% CO2

incubator. Cells were seeded onto 35 mm glass-bottom dish and

allowed to grow until a confluence of 60–80%. Then, the cells were

washed twice with PBS (pH¼ 7.4) to remove the remnant growth

medium. For cellular uptake study, the cells were incubated with

PE-Cy5.5 incorporated liposomes (PC, DOTAP, PS, and PEG-PE with

different weight percentages) at concentration of 25 mg �mL�1 for

up to 60 min at 37 8C in 2 mL serum-free transfection medium.

Then, they were washed twice with PBS containing Ca2þ and Mg2þ,

fixed with formaldehyde–glutaraldehyde combined fixative for

15 min at room temperature, and then stained with 4’,6-diamidino-

2-phenylindole (DAPI, Invitrogen, Carlsbad, CA, USA) to label nuclei.

NIRF images of cells was obtained using a FluoView FV1000

confocal laser scanning microscope (Olympus, Tokyo, Japan)

equipped with 405 diode (405 nm) and He-Ne red (633 nm) lasers.

2.5. In vivo Non-Invasive NIRF Imaging of Liposomein Tumor-Bearing Mice

All experiments with live animals were performed in compliance

with the relevant laws and institutional guidelines of the Korea

Institute of Science and Technology (KIST); institutional commit-

tees approved the experiments. To make tumor-bearing mice

models, squamous cell carcinoma (SCC7) tumors were induced into

5-week-old male athymic nude mice (Institute of Medical Science,

Tokyo) by subcutaneous injection of 1.0�106 SCC7 cells. When the

tumor diameter grew to about 10 mm, mice were divided into ten

groups for PC (100 wt%), DOTAP/PC (10, 30, and 50 wt%), PS/PC (10,

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Figure 1. Illustration and chemical structures of liposomes withdifferent lipid compositions.

In vivo NIRF Imaging of Tumor Targetability of Nanosized Liposomes in . . .

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30, and 50 wt%), and PEG-PE/PC (10, 30, and 50 wt%) liposomes,

respectively, and 0.05 wt% of Cy5.5-labeled PE were incorporated to

all liposomes. Then, each sample (10 mg � kg�1) was intravenously

injected into tumor-bearing mice, and their biodistribution and

tumor targetability were imaged 1, 3, 5, and 7 h post-injection by

the eXplore Optix system (ART Advanced Research Technologies

Inc., Montreal, Canada; n¼3, for each group).[14] Laser power and

count time settings were optimized at 9 mW and 0.3 s per point.

Excitation and emission spots were raster-scanned in 1 mm steps

over the selected region of interest to generate emission

wavelength scans. A 670 nm pulsed laser diode was used to excite

Cy5.5 molecules. NIRF emission at 700 nm was collected and

detected with a fast photomultiplier tube (Hamamatsu, Japan) and

a time-correlated single photon counting system (Becker and Hickl

GmbH, Berlin, Germany). Furthermore, the tumor accumulations of

samples were confirmed by measuring the NIRF intensity at the

tumor site. All data were processed using the region of interest (ROI)

function of the Analysis Workstation software (ART Advanced

Research Technologies Inc., Montreal, Canada).[15]

2.6. Ex vivo Organ Analysis

After intravenous injection of Cy5.5 labeled liposomes

(10 mg � kg�1), the major organs and tumors were dissected from

mice 7 h post injection. NIRF images of dissected organs (liver, lung,

spleen, heart, and kidney) and tumors were obtained with a Kodak

Image Station 4000 MM (New Haven, CT). The image station was

equipped with a 12-bit CCD camera, halogen lamp (150 W), and

excitation/emission filter sets of Cy 5.5 (600–700 nm; Omega

Optical).[16] The amounts of accumulated liposomes in the liver and

tumor were quantified by measuring the ratio of NIRF intensity of

tissues from liposome-treated mice (per mm2) to that from non-

treated mice (per mm2).

Table 1. Size distribution and zeta potential value of liposomes.

Samplea) m2/G2 Size

[nm]

z Potential

[mV]

PC(100) 0.31 126� 6 0.25� 1.55

DOTAP/PC (10) 0.21 103� 3 22.48� 2.00

DOTAP/PC (30) 0.30 92� 6 32.43� 5.52

DOTAP/PC (50) 0.26 85� 3 42.69� 6.53

PS/PC (10) 0.23 114� 3 �25.67� 5.52

PS/PC (30) 0.19 116� 7 �38.18� 3.05

PS/PC (50) 0.27 132� 8 �50.08� 4.43

PEG-PE/PC (10) 0.17 97� 4 2.33� 2.42

PEG-PE/PC (30) 0.25 112� 3 7.93� 2.71

PEG-PE/PC (50) 0.22 111� 5 1.31� 0.39

a)Numbers in parentheses indicate content of the last component

(in wt%).

3. Results and Discussion

We fabricated various liposomes with different surface

properties by changing their lipid composition, and

evaluated their in vivo tumor targeting ability. The

liposomes were fabricated by traditional film-casting

methods in aqueous condition, and their composition

was differentiated by the incorporation of charged (DOTAP,

PS) or PEGylated lipid (PEG-PE) into neutral lipid (PC) at

different weight percentages, in resulting pure PC, DOTAP/

PC (10–50 wt%), PS/PC(10–50 wt%), and PEG-PE/PC

(10–50 wt%) (Figure 1). The size of resulting liposomes

was determined by DLS (see Table 1). The sizes of pure PC,

DOTAP/PC, PS/PC, and PEG-PE/PC were about 100 nm, and

they were not significantly different in aqueous condition

upon different composition of phospholipids. The low

polydispersity factors (m2/G2) of all liposomes indicated

their narrow size distribution. On the other hands, the zeta

potential values of liposomes were differentiated upon

their chemical end-groups, which were located on the

surface of liposomes. PC liposomes have both anionic

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Macromol. Biosci. 2012, DOI: 10.1



phosphate group and cationic tertiary amine group in their

chemical structure, and displayed almost neutral surfaces

(0.25 mV). DOTAP/PC liposomes with cationic tertiary

amine groups became more cationic with increasing

percentage of DOTAP in the liposomes (from 22.48 to

42.69 mV). In case of PS/PC liposomes with anionic

carboxylic acid groups, a negatively charged surface was

observed as increasing the percentage of PS (from�25.67 to

�50.08 mV). The incorporation of PEG-PE did not affect the

zeta-potential value of liposomes. Although PEG-PE has one

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anionic charge, the zeta potential values of PEG-PE/PC

liposomes were almost neutral may due to the shielding

effect of PEG chains on surfaces.[17] These results demon-

strated the surface properties of liposomes were dependent

on their phospholipid composition, as expected.

CryoTEM images also showed that all liposomes have

spherical shapes and nanosized structures in aqueous

condition, regardless of their phospholipid composition

(Figure 2a). However, their stability under serum conditions

depended on their lipid composition. After incubation in

human serum albumin PBS solution (20 mg �mL�1), the size

of DOTAP/PC liposomes greatly increased over time to

around micrometer scale after 48 h (Figure 2b). This was

deduced to result from the interaction between lipid

carriers and albumin; it should be considered important

for in vivo drug delivery.[18] Even though size is less than

100 nm, this could be greatly increases with strong

aggregates forming in the blood stream.[19]

On the other hand, the average size and size distribution

of PEG-PE/PC showed negligible changes under the same

Figure 2. In vitro characterization of liposomes: a) CryoTEMimages of liposomes; and, b) Stability of liposomes in PBS with20 mg �mL�1 human serum albumin.




conditions. This result shows that the introduction of PEG

groups into liposomes could prevent aggregation with

serum proteins and can be expected to improve their in vivo

stability, as previously reported.[20] In the case of PEG-PE/PC

liposomes, we used 30 wt% liposomes since this ratio was

found to be optimal for tumor targetability in vivo (see

below).

To assess the cellular uptake of liposomes, human Hela

cells were incubated with 0.05 wt% of Cy5.5 labeled

liposomes up to 1 h at 37 8C. Their localization was tracked

based on the NIRF intensity of Cy5.5 molecules in the

liposomes using confocal microscopy (Figure 3). Nanosized

neutrally charged pure PC liposomes showed red fluor-

escent spots throughout the cells showing their intracel-

lular localization in tumor cells. The cellular uptake of

liposomes was greatly enhanced by incorporation of

cationic DOTAP lipid. DOTAP/PC (50 wt%) liposomes

showed very strong fluorescence intensity and most cells

contained liposomes in their cytosol. However, in the case

of PS/PC liposomes, the incorporation of anionic PS lipid

resulted in reduced cellular uptake, which may be due to the

charge repulsion between anionic liposome and anionic

polysaccharides on the cell surface. It is evident that the

surface charge of liposomes is a critical factor to facilitate

efficient uptake of the liposomes across the cellular

membranes. As shown in the images of PEG-PE/PC

liposomes treated cells, increase of the PEG groups in

liposome also reduced their cellular uptake showing the

anti-fouling surface of PEGylated liposomes in a cell culture

system.[21].Therefore, when we use anionic or PEGylated

liposomes, targeting molecules such as peptide, antibody,

or aptamer on the surface could enhance the cellular uptake

of liposomes.[22,23]

Non-invasive and real-time imaging systems were

successfully employed to visualize and quantify the tissue

distribution of drug carriers in vivo.[24,25] We utilized this

imaging system to obtain direct and real-time information

about the biodistribution of our liposomes in tumor-

bearing mice after intravenous injection. The effect of

incorporated lipids on tumor-targeting was evaluated with

the comparison of the NIRF intensity in tumor region,

showing the time-dependent tumor accumulation profiles

of liposomes (Figure 4a). The NIRF signals in the whole body

were shown to have significant correlation with the blood

concentration of Cy5.5-labeled materials in our previous

papers.[15,26] Therefore, the circulation time of labeled

liposomes can be deduced based on the NIRF signals in body.

All liposomes maintained their NIRF intensities in the

whole body up to at least 7 h post injection. However, the

tumor accumulation pattern of liposomes was significantly

different upon the phospholipid compositions of liposomes.

First, there was remarkable NIRF signal in tumor region of

PC liposome-treated mice over 7 h. This result showed the

high accumulation of liposomes in tumor tissue compared

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Figure 3. Cellular uptake of liposomes. Fluorescence images of HeLa cells after 60 min incubation of Cy5.5-labeled liposomes.


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to other sites to be mainly due to EPR effects, as mentioned

above. However, DOTAP/PC displayed stronger intensity in

the liver area with increasing cationic DOTAP concentra-

tion. High accumulation of DOTAP/PC in the liver might be

induced by their aggregation with anionic serum proteins

in the blood flow and recognition by the RES system.[5] With

increasing PS or PEG-PE lipid percentages in the liposomes,

the liposomes exhibited stronger NIRF intensity in tumor

tissue, implying enhanced tumor-targetability. In particu-

lar, the NIRF intensity of PEG-PE/PC (30 wt%) in tumor tissue

was the highest, showing this composition is advantageous

for in vivo tumor-targeting. In addition, the reduced tumor

accumulation of PEG-PE/PC (50 wt%) could be explained by

the reduced stability of excessive PEGylation.[27] Based on

the whole body images in Figure 4a, the NIRF intensities of

liposomes in the tumor area 7 h post injection were

quantified using the analysis software (Figure 4b). The

intensity in the tumor region was about 2.5 fold higher


Macromol. Biosci. 2012, DOI: 10.1



in the case of PEG-PE/PC (30 wt%) than that of DOTAP/PC

(50 wt%) showing their high tumor accumulation.

Based on the imaging data, the long circulation and the

EPR effect are more important factors in tumor tissue

accumulation, while fast cellular uptake due to cationic

charge ironically results in a negative effect for in vivo

tumor targetability.

The organ distributions of liposomes were evaluated in

the NIRF images of tumors and organs (liver, lung, spleen,

heart, and kidney) dissected 7 h post injection (Figure 5a). In

ex vivo organ images, strong NIRF intensity for PC

liposomes was observed in the liver, implying their high

accumulation in liver tissue. DOTAP/PC liposomes showed

even higher fluorescence intensity in the liver and spleen

than in the tumor, showing significant entrapment of

cationic liposomes by the RES system. Interestingly, on

increasing the incorporation percentage of PS, the fluores-

cence intensities in tumor were simultaneously increased.

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Figure 4. In vivo distribution of liposomes in tumor-bearing mice models: a) Whole-body NIRF images of tumor-bearing mice models afterintravenous injection of Cy5.5-labeled liposomes; and, b) NIRF intensity in tumor tissues of (a).

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Despite the liver uptake, anionic and stable PS/PC liposomes

accumulated in tumor tissue by preventing aggregation

with anionic serum proteins, resulting in an enhanced EPR

effect. The circulation time of PS liposomes without PEG

was unexpectedly long. We think that it might be due to the

suppressed immune system and reduced macrophage

activity in nude mice. We expect that they may show

more accumulation in liver and spleen in case of normal

(not nude) mice models. For the mice treated with PEG-PE/

PC liposomes, the fluorescence intensity in the liver was

gradually minimized, which clearly demonstrated the

superior anti-fouling effect of PEG molecules on the

liposomal surface, which could prevent aggregation in

the blood flow and escape the RES in the liver. PEG on the

surface enabled the escape of liposomes from RES capture

by the liver and consequently high accumulation in the

tumor, in accordance with the results for PEGylated drugs,

proteins, and carriers shown by other researchers.[20,28,29]

However, in spite of reduced uptake in the liver, PEG-PE/PC




(50 wt%) liposomes showed lower tumor accumulation

than PEG-PE/PC (30 wt%) liposomes. This result shows that

excessive PEGylation might reduce the stability of lipo-

somes and their tumor targeting efficiency, in accordance

with the results of in vivo imaging images.[27] The

quantification data of the organ distribution of liposomes

showed that the incorporation of charged (DOTAP and PS)

and PEG-modified phospholipids into liposomes affected

the liver and tumor accumulation profiles (Figure 5b).

After 7 h post injection, the ratio of fluorescence intensity

of PEG-PE/PC (30 wt%) in the liver was particularly lower

than that of pure PC liposome, and the intensity in the

tumor was highest among all liposomes. PS/PC (50 wt%)

displayed a higher ratio of fluorescent intensities in the

liver and tumor (up to 1.5-fold), compared to pure PC

liposome. On the other hand, DOTAP/PC (50 wt%) showed

the highest intensity in the liver and poor tumor target-

ability. These results significantly demonstrate that surface

properties of liposomes can be determined by their lipid

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Figure 5. Ex vivo organ distribution of liposomes in tumor-bearing mice models: a) NIRFimages of excised organs from tumor-bearing mice models 7 h post injection of Cy5.5-labeled liposomes; and, b) Relative ratios of NIRF intensity in the liver and tumor to thatof PC liposome-treated groups.


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composition and are a critical factor for their in vivo tumor

targeting.

4. Conclusion

We prepared various liposomes incorporated with different

weight percentages of charged and PEGylated phospholi-

pids to evaluate their biodistribution and tumor accumula-

tion in vivo. The mean diameters of all liposomes were

about 100 nm and were not significantly different. The

surface charges of liposomes were largely depended on

the chemical end-groups of incorporated phospholipids. All

liposomes showed similar size distribution under physio-

logical conditions, but PEG-PE incorporation into liposomes

showed enhanced stability under serum conditions with-

out aggregation. Under cell culture conditions, the posi-

tively charged DOTAP/PC liposomes showed the highest

cellular uptake due to interaction with anionic cell

membranes. However, in vivo imaging data revealed that


Macromol. Biosci. 2012, DOI: 10.1002/mabi.201200001

� 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinh

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PEG-PE/PC liposomes circulated in the

blood for longer periods of time and

displayed higher tumor accumulation

because of the low liver uptake and

shielding effect of the PEGlated surface.

DOTAP/PC liposomes demonstrated the

lowest tumor-targeting efficiency and the

highest liver accumulation may due to

entrapment by the RES systems in spite of

their fast cellular uptake in vitro. Through

in vivo and ex vivo NIRF imaging of

animals, we could clearly understand the

effect of surface charges and PEGylation

of liposomes upon their in vivo tumor-

targeting. We expect that these overall

results can provide valuable information

for further application of liposomes as

tumor-targeting drug carriers.

Acknowledgements: This research was sup-ported by the Global Research Laboratory (GRL)Project, the Fusion Technology Project (2009-0081876) of MEST, and the IntramuralResearch Program (Theragnosis) of KIST.

Received: January 4, 2012; Revised: February29, 2012; Published online: DOI: 10.1002/mabi.201200001

Keywords: drug delivery systems; imaging;liposomes; PEGylation; tumor targeting

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