Received: 5 April 2016 Revised: 11 July 2016 Accepted: 2 August 2016

DOI 10.1002/bio.3207

RESEARCH ARTICLE

Enhancement of cell internalization and photostability of red

and green emitter quantum dots upon entrapment in novel
cationic nanoliposomes
Hamid Reza Samadikhah1 | Maryam Nikkhah1 | Saman Hosseinkhani2*

1
Department of Nanobiotechnology, Faculty
of Biological Sciences, Tarbiat Modares Abstract
University, Tehran, Iran Two quantum dots (QDs), a green emitter, CdSe and a red emitter, CdSe with ZnS shell are
2
Department of Biochemistry, Faculty of encapsulated into novel liposomes in two different formulations including cationic liposomes.
Biological Sciences, Tarbiat Modares Quantum dots have proven themselves as powerful inorganic fluorescent probes, especially for
University, Tehran, Iran
long‐term, multiplexed imaging and detection. Upon delivery into a cell, in endocytic vesicles
Correspondence
such as endosomes, their fluorescence is quenched. We have investigated the potential toxic
Saman Hosseinkhani, Department of
Biochemistry, Faculty of Biological Sciences, effects, photophysical properties and cell internalization of QDs in new formulation of liposomes
Tarbiat Modares University, Tehran, 14115‐ as an in vitro vesicle model. Entrapment of QDs into liposomes is brought about with a decrease
175, Iran.
in their intrinsic fluorescence and toxicities and an increase in their photostability and lifetime.
Email: saman_h@modares.ac.ir
The biomimetic lipid bilayer of liposomes provides high biocompatibility, thereby enhancing
the effectiveness of fluorescent nanoparticles for biological recognition in vitro and in vivo. The
prepared lipodots could effectively prevent QDs from photo‐oxidation during storage and when
exposed to ultraviolet (UV) light. Moreover, the flow cytometry of HEK 293 T cells showed that
the cell internalization of encapsulated QDs in (DSPC/CHO/DOPE/DOAB) liposome is enhanced
10 times compared with non‐encapsulated QD (bare QDs).

K E Y W O RD S

biocompatibility, liposome, photo‐oxidation, photoustability, quantum dots

1 | I N T RO D U CT I O N and optical) imaging[14–16] particularly in tumours[17,18] and

nanoparticle‐mediated drug delivery.
Recent progress in chemistry and material physics has provoked Most water‐soluble QDs have the negative charges on their sur-
[1]
molecular tracking of living organisms. Quantum dots (QDs) are face, which allows QDs to disperse in aqueous solution by electro-
one of the best tools for this purpose due to their specific proper- static repulsion.[19,20] QDs bind poorly to the surfaces of living
ties such as simplicity of management and structure.[2–4] QDs have cells, which results in these particles having low internalization effi-
some particular photophysical properties including high quantum ciency.[21,22] The delivery of QDs into a cell is a crucial requirement
yield. In comparison with other organic molecules, QDs have a for imaging purposes. Different methods have been used to improve
higher molar absorption coefficient, a broad excitation spectra and intracellular delivery of QD such as surface modifications with vari-
a narrow emission spectrum.[5,6] Moreover, they have shown molec- ous functional molecules such as liposome encapsulation or linking
ular orbitals and an electronic structure similar to other organic with cationic peptides.[23–26] Liposomes can act as a biological vehi-
molecules, plus ease of access of surface shell modifications with cle due to their excellent features such as biocompatibility, biode-
a variety of biologically active molecules and high resistance to gradability and lower toxicity.[27] Liposomes are recognized as a
chemical and photophysical deficiency.[7,8] Due to these mentioned clinical nanometer‐scale carrier for various drug and gene deliver-
special properties, QDs have been used for a wide range of ies.[28,29] They are also able to form self‐assembled vesicles with a
applications in biological research such as labelling for cellular lipid bilayer surrounding an internal aqueous cavity, thereby making
trafficking[9] membrane dynamics and cellular movements,[10,11] them an appropriate cargo for both hydrophobic and hydrophilic
single particle tracking[12,13] multicolor and multimodal (magnetic compounds. Several studies have been performed to encapsulate

Luminescence 2016; 1–12 wileyonlinelibrary.com/journal/bio Copyright © 2016 John Wiley & Sons, Ltd. 1
2 SAMADIKHAH ET AL.

the hydrophobic core/shell QDs in contrast with few studies on lipo- obtained. For preparation of the dried film, the lipid film was incubated
some encapsulation of hydrophilic QDs.[30] The loading capacity of at room temperature for 24 h. At the next step, distilled water at 50°C
the hydrophilic compounds in liposome vesicles is higher than that was added to the dried lipid film and this was mixed vigorously for
of hydrophobic ones. This difference is because the water phase 30 min by discontinuous vortexing to obtain dispersion. A bath
compounds are contained in the central cavity whose volume is sonicator was used for the primary homogenization (Soltec, Milan,
much higher than that of the lipid bilayer where the organic phase Italy) for 20 min and was followed by a 5 sec sonication with 10 sec
compounds are fixed. One of the most promising characteristics of intervals within 3 min using a microtip probe sonicator (Dr Hielscher,
QDs is their high loading efficiency, which is an important parameter Teltow, Germany). In the last stage, the homogeneous suspension
towards effective intracellular delivery. Each liposome can have up was lyophilized by lyophilizer (VDH‐2040, Snijders Scientific BV, Til-
to several million fluorescent dye molecules, thereby providing burg, The Netherlands). The suspension was frozen in liquid nitrogen,
greatly enhanced signals. Liposomes have been successfully used as and then dried in a lyophilizer at −40°C and under vacuum (0.4 milli-
reporter particles in bioassays. Moreover, the biomimetic lipid bilay- bars) conditions. The powder product of vesicles was kept at −20°C
ers of liposomes provide high biocompatibility, thereby enhancing until use.
the effectiveness of fluorescent nanoparticles for biological detec- In the second liposome formulation DSPC/CHO/DOPE/DOAB
tion in vitro and in vivo. lipids were mixed at a molar ratio of 3:3:3:1 and dissolved in chloro-
In this study, we have encapsulated green emitter and red emit- form solution[28] in a similar procedure as reported for the first
ter QDs into a novel liposome preparation (DPPC/CHO/DOAB lipo- formulation.
somes and DSPC/CHO/DOPE/DOAB liposomes). Prepared
liposomes enable the delivery of QD with modified surface negative
2.3 | Preparation of QDs loaded liposomes
charges through cell membranes. Endosomal entrapment is one of
problems that QDs encounter upon entry into a cell. This study For the lipodot preparation, we used a thin film hydration method.[9]
was also further focused on investigating photostability of QDs Briefly, DPPC/CHO/DOAB powders were mixed at a molar ratio of
encapsulated in lipid vesicles in comparison to intact QDs with the 7:2:1 and DSPC/CHO/DOPE/DOAB at a molar ratio of 3:3:3:1, which
goal of how to understand how to overcome fluorescence quenching were then dissolved in a chloroform 2:1 mixture. A rotary evaporator
of QDs. used for the evaporation of the organic solvent at 50°C. Solvent
traces were removed under vacuum for 6 h. Double‐distilled water
and high‐shear homogenization was used for lipid hydration for
2 | EXPERIMENTAL 10 min. Different concentrations of QDs (500 nM, 700 nM, 1 μM
and 3 μM) were added to the mixture. The dispersion was subjected
2.1 | Chemicals to probe sonication (VCX 600; Sonics and Materials, Newtown, CT,

DDAB (Didecyldimethylammonium bromide), DOPE (dioleoylphos USA) in the continuous mode for 30 min. The sonication energy was

phatidylethanolamine), DPPC (Dipalmitoylphosphatidylcholine), DOPE set at 35 W.

(dioleoylphosphatidylethanolamine) and DSPC (1,2‐Distearoyl‐sn‐

glycero‐3‐phosphocholine) was purchased from Avanti Polar Lipids 2.4 | Determination of zeta potential, particle size,
(Alabaster, AL). Cholestrol (CHO) and MTT reagent (3‐(4,5‐
and polydispersity index of liposomes
Dimethylthiazol‐2‐yl)‐2,5‐Diphenyltetrazolium Bromide) and QDs
(CdSe) and CdSe with a ZnS shell were purchased from Sigma Aldrich The average particle size and the polydispersity of the particle‐size dis-

(St Louis, MO, USA) and modified by addition of a carboxylate group tribution of the liposomes and lipodots were determined by dynamic

according to a reported procedure, [31]

Fetal bovine serum (FBS), light scattering using photon correlation spectroscopy. The measure-

Dulbecco's modified Eagle's medium (DMEM) were purchased from ments were performed at 25°C using a Zetasizer Nano ZS instrument

Gibco. All other chemicals were of commercial analytical grade and (Malvern Instruments Ltd, Malvern, Worcestershire, UK) equipped

were used without further purification. with a helium–neon laser and a scattering angle of 173°. For evaluation
of the extent of interaction of the liposomal surface cationic charges
with the anionic charges of QDs, the zeta potential of liposomes was
2.2 | Preparation of cationic liposomes
measured at 25°C with the same instrument by electrophoretic mobil-
For preparation of nanoliposome, two formulations were used: (1) ity. All samples were not further diluted for each particle size and zeta
DPPC/CHO/DOAB liposomes; and (2) DSPC/CHO/DOPE/DOAB potential measurement. A typical liposome refractive index of 1.45
liposomes. was used.
In this study we have used the freeze‐dried empty liposome
(FDEL) method for preparation of the DPPC/CHO/DOAB lipo-
somes.[32] One formulation was prepared from three lipids at a molar
2.5 | Cell culture
[27]
ratio of 7:2:1, lipids were dissolved in an organic phase (chloroform) Human embryonic kidney 293 T cells (HEK293T) were grown in
followed by evaporation (Heidolph, Schwabach, Germany) in order to medium supplemented with 10% FBS, penicillin (100 units/mL) and
exhaust the chloroform under reduced pressure and temperature streptomycin (100 mg/mL). Cells were grown in 25 cm2 polystyrene
(37°C), under vacuum, and at 40 rpm until a thin lipid film was tissue culture flasks in a humidified atmosphere of 5% CO2 in air and
SAMADIKHAH ET AL. 3

at 37°C. Cells were plated at a cell density of 70% in a 24‐well cell cul- different combinations of liposomes + QDs (DPPC/CHO/DOAB),
ture plate 1 day before transduction. and DSPC/CHO/DOPE/DOAB, with a different concentrations of
QDs, (500 nM, 700 nM, 1 μM, 3 μM) were placed in serum‐free
DMEM, added to the cells and the cells were then incubated for
2.6 | Cell viability assay
2 h. The cellular uptake experiment was initiated by adding liposomes
For the MTT assay; the HEK293T cells were seeded in 96‐well plates (liposomes + QDs) or QDs in PBS (200 mL) for 2 h at 37°C. The
and incubated for 24 h. After the cells were attached to the plate sur- experiment was terminated by washing the cells three times with
face then treated with cationic liposomes (CLs) and QDs + liposome PBS. After delivery, the incubation medium was replaced with DMEM
lipodots at the same concentrations used for cell internalization exper- supplemented with 10% FBS. Cell internalization efficiency was eval-
iments. Cell viability was assayed using MTT according to a reported uated using Cytation™ 3 Cell Imaging Multi‐Mode Reader (BioTek
method[24] with minor modifications. Briefly, MTT 10 μl (5 mg/mL) Instruments, Winooski, VT, USA).
was added to each well and the cells were incubated at 37°C for 4 h.
The formazan product was dissolved in 10% sodium dodecyl sulfate
(100 μL) containing hydrochloric acid 15 mM.[33] Colour intensity 3 | RESULTS
was measured using an absorbance microplate reader Cytation™ 3 Cell
Imaging Multi‐Mode Reader (BioTek Instruments, Winooski, VT, USA) 3.1 | Zeta potential, particle size, and polydispersity
at test and reference wavelengths of 570 nm. index of free liposomes and lipodots
In previous studies,[27,28] we showed that three component lipoplexes
2.7 | Transmission electron microscopy (DPPC/CHO/DDAB) and four component lipoplexes (DSPC/CHO/
The liposome and lipodots were observed under transmission electron DOPE/DOAB) at different molar ratios of cationic lipid to total lipid
microscopy (TEM; ZEISS902A, Oberkochen, Germany) to evaluate content (CL/TL) exhibited higher transfection efficiency than binary
their morphology. For this purpose, one drop of suspensions was component liposomes (DPPC/DDAB) that are usually employed for
placed on a copper grid and left for 2 min for attachment of liposome gene delivery purposes. Three component lipoplex (DPPC/CHO/
and lipodots to the grid. Any excess sample was removed using filter DDAB) at a molar ratio of 7:2:1 and four component lipoplex
paper and samples were then stained with 2% aqueous uranyl acetate (DSPC/CHO/DOPE/DOAB) at a molar ratio of 3:3:3:1 showed the
for 5 min. After complete air drying, the grids were screened in a trans- highest transfection efficiency and the lowest cytotoxic effects. Many
mission electron microscope at an acceleration voltage of 80.0 kV. investigators have shown that multi‐lipid carriers are more fusogenic
than single lipid carriers.[34] In this study; the reported multi‐compo-
nent liposomes[28] were used as the carrier for QDs delivery. Mean
2.8 | Flow cytometry analysis
particle size and zeta potential (surface charge potential) of lipodots
HEK293T (1.5 × 105 cells) were seeded in 12‐well plates (BD Biosci- with two different formulations of liposomes and a QD concentration
ences) with 500 mL of culture medium for 2 h and the cells were of 3 μM were measured. The two formulations with the highest
treated with QDs and lipodot cell internalization medium at 37°C. amount of fluorescence intensity were chosen for further
After internalization, the cells were washed three times by suspension experiments.
and centrifugation at 1200 rpm for 3 min. In the next step, the cells As shown (Table 1, Figure 1), the average size of lipodot particles
were suspended in 500 mL phosphate‐buffered saline (PBS) and used was larger than that of the free CLs. The zeta potential measurements
for flow cytometry analysis on a FACS caliber instrument (BD showed that the addition of QDs slightly decreased the zeta potential
Biosciences). to 51 mV compared with free liposomes (DPPC/CHO/DOAB). The
zeta potential for the free liposomes with the DSPC/CHO/DOPE/

2.9 | In vitro cell internalization experiment DOAB formulation was 30 mV, while the addition of QDs slightly
reduced this figure to 21 mV.
For the cell internalization, HEK293T cells were seeded in 24‐well
sterile culture plates at a density of 1 × 105 cells/well and were grown
overnight to approximately 80% confluency. For internalization of the
3.2 | Optimization of liposomes composition
lipodots, the growth medium was removed and then cells were Previous investigations into the preparation of liposome formulations for
washed twice with pre‐warmed PBS. The complexes with two gene delivery indicated that DPPC/CHO/DOAB liposomes at a molar

TABLE 1 The mean particle size and zeta potential of cationic liposome's (CLs) and liposomes + QDs (lipodots) with two different formulations of
liposomes and the same concentration of QDs (3 μM)
Group Sample Mean particle size (nm) Polydispersity index Zeta potential (mV)

1 DSPC/CHO/DOPE/DOAB 255 0.198 30

2 DSPC/CHO/DOPE/DOAB + QDS 354 0.339 23
3 DPPC/CHO /DOAB 76.95 0.280 55
4 DPPC/CHO /DOAB + QDS 155 0.260 51
4 SAMADIKHAH ET AL.

ratio of 7:2:1 showed a particle size of 76.0 nm, whereas the second for- encapsulation and different liposome concentrations. In order to retain
mulation (DSPC/CHO/DOPE/DOAB) at 3:3:3:1 molar ratio formed a the lowest cytotoxicity levels, we prepared formulations of lipodots with
255 nm liposome.[27,28] In this study, we have optimized the best lipo- a minimal concentration of cationic lipid. To accomplish this preparation,
some formulation, obtained from the previous study on QDs all CLs were formed at a constant molar ratio of CL/TL (1:10).

FIGURE 1 Size distribution profile of (a) DSPC/CHO/DOPE/DOAB liposomes, amd (b) DSPC/CHO/DOPE/DOAB liposomes + QDs (3 μM). (c)
Free DPPC/CHO/DOAB liposomes. (d) DPPC/CHO/DOAB lipodots + QDs (3 μM)
SAMADIKHAH ET AL. 5

FIGURE 2
Fluorescence spectra of the QDs before and after encapsulation in (a) liposomes (DSPC/CHO/DOPE/DOAB) and (b) liposomes (DPPC/
CHO/DOAB) of different concentrations

3.3 | Optimization of CLs/QDs ratio fluorescence intensity was kept compared with free QDs. In order to
prove that QD fluorescence is conserved after incorporation into the
The effects of different concentrations of QDs (500 nM, 700 nM,
lipid bilayer environment, QD fluorescence intensity was monitored
1 μM and 3 μM) on cell delivery efficiency by lipodots were studied.
upon exposure to UV light over a period of time. Samples were located
The highest emission spectrum was obtained with a QD concentration
under a UV lamp (312 nm wavelength) immediately upon lipodots for-
of 3 μM while encapsulated in both DPPC/CHO/DOAB and DSPC/
mation (Figures 4 and 5).
CHO/DOPE/DOAB (Figure 2). It should be noted that at a constant
concentration four component lipodots (DSPC/CHO/DOPE/DOAB)
showed higher fluorescence intensity when compared (Figure 3) with
three component lipodots (DPPC/CHO/DOAB).

3.4 | Photostability of lipodots

In order to confirm that QDs fluorescence was preserved after incor-
poration into the liposome, incorporation of QDs within the liposomes
to form lipid–QDs vesicles led to improved photostability compared
with free QDs at the same concentration, after 5 h of exposure to air
at room temperature. There was no significant photobleaching when
the QDs were embedded within the lipid bilayer, compared with loss
of fluorescence intensity for free QDs. Furthermore, the effect of UV
radiation (Hg vapour lamp with λ = 312 nm; Biotek Company) was
studied by exposing QDs and lipid–QDs to a UV light source for 5 h
after preparation. The QDs were photochemically unstable when
exposed to UV light as evidenced by a sharp reduction in fluorescence
intensity. Conversely, the lipid–QDs exhibited improved photostability FIGURE 3 Comparison of fluorescence intensity of two formulations
after 5 h of UV exposure and a substantial amount of the initial of lipodots in different concentration of QDs
6 SAMADIKHAH ET AL.

3.5 | pH Stability of QD fluorescence CHO/DOPE/DOAB liposome was ~255 nm which corresponds prac-
[35]
tically to the size determined by the transmission electron micros-
It is well known that different cell organelles have different pH to
copy. Transmission electron microscope (TEM) images shows that
sustain certain pH‐sensitive enzyme activity.[36] The mechanisms
the size of empty liposomes (Figure 7a, c); and lipodots containing
responsible for intracellular vesicle trafficking by an acidic pH
QD dots (CdSe) are comparable with those reported by DLS experi-
remained unclear. However, the estimated intravesicular pH values
ments (Figure 7b, d). TEM images of CLs showed mainly small vesicles
are recognized to be around 5.5–6.5 for the endosomes, lysosomes,
that separated from each other well due to their positive surface
and trans‐Golgi network and 7.2 for the endoplasmic reticulum and
charge, so repulsive forces between the liposomes prevent their
cytosol. In the present study, we have thus investigated whether fluo-
aggregation. However, encapsulation of QDs within liposome struc-
rescence quenching in living cells could be explained by the acidic envi-
ture increased the tendency of them towards aggregation, as indi-
ronment in endosomes and lysosomes.
cated in Figure 7.
As indicated in Figure 6, the QD fluorescence profile is pH depen-
dent with maximal photoluminescence intensity at pH 7.0 correspond-
ing to that of cytosol. Intensity of bare QDs at pH 5.0 and pH 6.0 was
3.7 | Cell internalization efficiency of QDs using
decreased.
liposomes
To establish the cell internalization efficiency of QDs into HEK293T,
3.6 | Transmission electron microscopy images of
the cells was internalized with QDs (CdSe) using liposomes for 2 h.
lipodots The internalization of QDs was estimated and compared using flow
Dynamic light scattering (DLS) measurements show that the average cytometry analysis. The internalization efficiency of 3 μM of bare
size of the DPPC/CHO/DOAB) liposome was ~76 and for DSPC/ QDs after 2 h was 2.02% (Figure 8a, b), for three component liposomes

FIGURE 4 Comparison of optical stability of QDs and lipodot at the same total concentration 3 μM during storage (a) and exposure to UV irradi-
ation with 365 nm (b)

FIGURE 5 Comparisons of QDs optical stability and lipodot exposure to UV irradiation with 312 nm (a) immediately upon formation (b) after a
period of time
SAMADIKHAH ET AL. 7

+ QDs (DPPC/CHO/DOAB) was 2.62% (Figure 8c) and for four com- exposure to lipodot with four lipid (DSPC/CHO/DOPE/DOAB)
ponent liposomes + QDs (DSPC/CHO/DOPE/DOAB) was more than and for the other lipodot with three lipid component (DPPC/
35% (Figure 8d). This is also shown in Figure 9(a, b). CHO/DOAB) were 85% and 70%, respectively. In comparison with
bare QDs (25%), a less cytotoxic effect with our lipodots prepara-
tions was observed.
3.8 | Fluorescence imaging of living cells
To study the compatibility and interaction of novel lipid–QDs vesicles
with live cells, the lipid–QDs were incubated at 37°C with HEK293T 4 | DISCUSSION
cells for 120 min. The cellular uptake was investigated using Cytation™
3 Cell Imaging Multi‐Mode Reader (BioTek Instruments, Winooski, VT, Transfection efficiency of cationic liposomes is under the control of
USA). Figure 10 depicts images of HEK293T cells with liposomes some physicochemical factors including the size of the complexes,
encapsulating (CdSe) QDs. the charge of CLs, and the total lipid/particles ratio. These parameters
can affect stability and reproducibility of used liposomes in internaliza-
tion.[37] As the use of QD particles changes the charge and size of CLs,
3.9 | Cell viability assay the amount of QDs incorporated into liposomes should be low enough
One of the most important aspects of delivery reagents is their to inhibit a drastic change in charge and size and high enough for bright
toxicity. The cytotoxic effect of different concentrations of QDs fluorescence intensity. The increasing average particle size of lipodots
and lipodots on HEK293T cells was investigated. Lower toxic compared with CLs could be due to adhesion and fusion of QDs to
effects on HEK293T cells were observed for DSPC/CHO/DOPE/ liposomes. Conversely, the addition of QDs slightly reduced the zeta
DOAB and DPPC/CHO/DOAB lipodots with the same concentra- potential.
tion of QDs (3 μM) while a major reduction in viable cells was When the complexes show an extra of positive or negative charge,
observed for bare QDs treatment. The survival percentages after electrostatic repulsive forces avoid extensive aggregation and fusion,

FIGURE 6 Stability of QD fluorescence at different pH (a) pH 5, (b) pH 6, (c) pH 7

8 SAMADIKHAH ET AL.

FIGURE 7 TEM images of the nanoparticles. (a) Empty liposomes with the formulation (DSPC/CHO/DOPE/DOAB) as unilamellar and
multilamellar vesicles of variable sizes with different magnification. (b) Lipodots (DSPC/CHO/DOPE/DOAB) with QD concentration of 3 μM. (c)
Empty liposome (DPPC/CHO/DOAB). (d) Common view of the lipodots (DPPC/CHO/DOAB) + QDs (3 μM)

thus leading to the formation of smaller complexes. In contrast, broad internalization efficiency than binary complexes. In the present study,
aggregations are favored in the case of approximately neutral com- we have investigated some parameters that might account for the
plexes in which repulsive forces are minimal. In a recent report[27] we delivery efficiency and photostability of QDs such as lipid composition,
have shown that two component liposomes demonstrated higher cell charge ratio of cationic lipid to QDs, size and influence of QDs

FIGURE 8 Flow cytometry analysis of QD and lipodot transduction in HEK293T cells. (a) Untreated cells as a control. (b) Bare QDs (3 μM). (c) Lipo-
somes (DPPC/CHO/DOAB) + QDs (3 μM). (d) Liposome's (DSPC/CHO/DOPE/DOAB) + QDs (3 μM)
SAMADIKHAH ET AL. 9

of QDs upon their entry into cells. To prepare model membranes, a

fast, low cost, and convenient method was employed as described ear-
lier by MacDonald et al.[42] It has been shown that small unilamellar
vesicles (SUVs) or multilamellar vesicles (MLVs) of variable sizes can
be created simply by extruding hydrated phospholipids through a poly-
carbonate filter.[43]
Cell internalization efficiency was also investigated which that
showed the lipodots with the formulation of DSPC/CHO/DOPE/
DOAB + QDs (3 μM) increased the cell uptake efficiency of QDs
approximately 10–15 times compared with bare QDs at the same con-
centration (Figure 9a, b). This agrees with previous reports that QDs
alone are rarely taken up by cells.[42]
Delivery of QD by encapsulated liposomes was performed using a
fluorescence imager. We found that QD‐loaded liposome vesicles can
effectively enhance the intracellular delivery of QDs in a human
embryonic kidney cell line (HEK293T). The photobleaching of encapsu-
lated QDs in cells was also reduced compared with non‐capsulated
QDs; measured by the photoluminescence (PL) decay of cellular QDs
with a continuous laser irradiation in the microscope. The flow
cytometric measurements further showed that the enhancing ratios
of encapsulated QDs on cell uptake are about 10–15 times in 293 T
cells. These results suggest that the CLs encapsulation is an effective
modality to enhance the intracellular delivery of green emitter QDs
(CdSe) and red emitter QDs: CdSe with ZnS shell QDs.
Fluorescence light was observed in the cells, which suggests that
uptake of fluorescent vesicles by the cells. Both types of QDs
exhibited an intense fluorescent signal, that represented that the living
cells were successfully in taking up the probe and fluorescence of the
probes was not quenched in the interior of cells (Figure 10c–f). Control
FIGURE 9 Transduction efficiency with two formulations of lipo-
experiments using bare QDs were also performed. The negative
somes and bare QDs with the same concentration (3 μM). (a) Overlay
charges on the bare QDs limit their cellular uptake, thereby hindering
histogram: control (black), bare QDs (green), liposomes (DPPC/CHO/
DOAB) + QDs (3 μM) (blue), liposomes (DSPC/CHO/DOPE/ their potential application in cell imaging. This liposome system
DOAB) + QDs (3 μM) (red). (b) The mean fluorescence units of increased biocompatibility and stability of QDs, thus improving the
transduction imaging effects for cancer cell labelling. The lipid–QDs were efficiently
internalized by HEK293T cells in a time‐dependent manner. After
entrapment. CLs with a mixture of DDAB as a cationic lipid and DOPE, 120 min incubation, lipid–QDs vesicles were capable of intracellular
DPPC, DSPC and CHO as helper lipids were prepared to test the inter- trafficking and could be imaged throughout the entire cell. A major fac-
nalization efficiency of QDs into HEK293T cells. tor limiting in vivo use of QDs is the putative toxicity. Heavy metal ions
Measurement of photostability of QDs showed that the protec- released from unchanged QDs are potentially toxic to living cells. Sur-
tion of novel lipid composition could effectively prevent photo‐oxida- face coating of QDs, however, seems to prevent direct contact
tion of QDs (Figure 4a) and also protect against UV light exposure ( between QDs and the cells. In our study, the lipid–QD vesicles did
Figure 4b), which can be attributed to the tight packing of QDs within not show morphological change in the cells during the incubation
the liposomes. The increased photostability of liposomes–encapsu- period (Figure 10).
lated QDs might be expected to improve their performance as novel Lack of cytotoxic effects after incubation with living cells sug-
fluorescence markers. This was also confirmed by an increase in fluo- gests that the lipid–QD vesicles are biocompatible, and capable for
rescence intensity after irradiation (Figure 5a, b). Conversely, fluores- cellular uptake as well as intracellular trafficking (Figure 11). Differ-
cence intensity was not completely quenched at lower pH (Figure 6a, ent methods have been used for ex vivo cell labelling by QDs includ-
b) compared with at pH 7.0 (Figure 6c) which can be attributed to ing non‐specific endocytosis, microinjection, liposome‐mediated
the protective role of liposomes at lower pH as reported earlier for uptake, and electroporation. Previous studies showed that
[38,39]
pH stability of QDs within cellular organelles. lipofectamine 2000 has the highest delivery efficiency, but the
TEM images (Figure 7) confirmed comparable sizes for liposomes encapsulated QDs were aggregates.[42] Electroporation also delivered
and lipodots as indicated by DLS experiments (Figure 1). Along the QDs in aggregates,[44] and may cause cell death. However, in this
endosomal pathway, large multilamellar/multivesicular granules (late study; the novel nanoliposomes deliver QDs into the live cells with-
endosomes and lysosomes) may be formed.[40,41] Therefore, incorpora- out visible aggregates in cell culture medium and they have shown
tion of QDs in a liposome model probably resembles the real situation to be suitable tools for studying live cell imaging (Figure 10). It
10 SAMADIKHAH ET AL.

FIGURE 10 Microscopic images of endocytosis of HEK293T cells after 2 h of incubation of (a) and (b) Bare QDs, (c, d) liposomes (DPPC/CHO/
DOAB) + QDs, (e, f) liposomes (DSPC/CHO/DOPE/DOAB) + QDs, (g, h) liposomes (DSPC/CHO/DOPE/DOAB) + red emitter QDs (CdSe with
ZnS shell). Note: The left panels are the observations under visible (white) light. The right panels are the observations under fluorescence
microscopy

should be noted that TEM images show aggregation of liposomes were used in labelling cells for in vivo applications (Figure 11). In fact,
upon encapsulation of QDs (Figure 7) which disappeared in the pres- we examined HEK293T cell proliferation and viability at only one QD
ence of living cells. concentration (3 μM) and observed no significant changes between
Earlier studies have shown that QD toxicity is dose dependent QD labeled cells and control unlabeled cells.
with increasing concentrations affecting cell growth and viability.[45] Cell internalization of QD into living cells has been hindered by
However, no significant toxicity was observed at concentrations that some barriers such as agglomeration at the extracellular surface and
SAMADIKHAH ET AL. 11

QDs delivery with a new lipid composition to the HEK293T for diag-
nostic and imaging aims. Clearly, the potential use of QD‐based lipo-
somes as nanosensors for drug release has just begun to be realized
and its application to pharmaceutical analysis is still in its infancy. Fur-
ther work, such as the quantitative analysis of fluorescence intensity in
liposomes imaging in vivo can support their functional applications.

ACKNOWLEDGMENTS
Research Council of Tarbiat Modares University is acknowledged for
financial support of this work.

ABBR E VI AT IONS USE D

FIGURE 11 Viability of HEK293T cells treated with cationic liposomes
(CLs), magnetic cationic, bare QDs and lipodot with two different for-
mulations. Cells were seeded at 105 cell/mL in a 96‐well plate and CL cationic liposome
incubated at 37°C. The percentage cell viability was determined fol- DLS dynamic light scattering
lowing 2‐h exposure to different formulations. 1: Control, 2: Bare QDs, DMEM Dulbecco's modified Eagle's medium
3: Liposome's (DPPC/CHO/DOAB) + QDs, 4: Liposomes (DSPC/CHO/ FBS fetal bovine serum
DOPE/DOAB) + QDs. Note: Data represent the percentage cell via-
FDEL freeze‐dried empty liposome
bility compared with untreated cells
PBS phosphate‐buffered saline
[45–48] PL photoluminescence
within cells such as endosomal entrapment. Despite mentioned
QD quantum dots
previous reports, we have found that internalization of our hydrophilic
SUV small unilamellar vesicles
QDs into HEK 293 T cells was obtained without entrapment within
TEM transmission electron microscope
endosomes as confirmed by flow cytometry assay (Figure 8) and cell
UV ultraviolet
imaging (Figure 10) and lack of significant cell toxicity as confirmed
by MTT assay (Figure 11). The present study using an in vitro liposomal
RE FE RE NC ES
model has demonstrated that QDs lose their fluorescence and
[1] S. J. Leung, M. Romanowski, Theranostics 2012, 2, 1020.
photosensitizing action when entrapped in phospholipid vesicles. In
[2] B. O. Dabbousi, J. Rodriguez‐Viejo, F. V. Mikulec, J. R. Heine, H.
addition, acidic environment (pH 6.0) also leads to quenching of QD Mattoussi, R. Ober, K. F. Jensen, M. G. Bawendi. J. Phys. Chem. B
fluorescence. Our model mimics the effect of QD fluorescence 1997, 101, 9463.
quenching and reappearance as it happens in endocytic vesicles of liv- [3] J. A. Hollingsworth, V. I. Klimov, Soft Chemical Synthesis and Manipula-
ing cells. Design of QDs that can either escape endosomes or lyso- tion of Semiconductor Nanocrystals, Marcel Dekker, New York/Basel,
Switzerland 2004.
somes, or are routed through other internalization pathways to avoid
[4] A. P. Alivisatos, Science 1996, 271(5251), 933.
entrapment in endocytic vesicles, is desirable to make them suitable
[5] T. Kippeny, L. A. Swafford, S. J. Rosenthal, J. Chem. Educ. 2002,
for photodynamic applications.[49] 79, 1094.
[6] A. M. Smith, H. Duan, A. M. Mohs, S. Nie, Adv. Drug Delivery Rev. 2008,
60, 1226.
5 | C O N CL U S I O N [7] A. P. Alivisatos, J. Phys. Chem. 1996, 100, 13226.
[8] A. P. Alivisatos, W. Gu, C. Larabell, Annu. Rev. Biomed. Eng. 2005, 7, 55.
According to the results presented in this manuscript, novel and highly [9] C.‐J. Wen, L.‐W. Zhang, S. A. Al‐Suwayeh, T.‐C. Yen, J.‐Y. Fang, Int. J.
luminescent QD‐loaded liposomes were prepared via a fast and conve- Nanomed. 2012, 7, 1599.

nient method. These studies demonstrated that both green and red [10] H. Chen, I. Titushkin, M. Stroscio, M. Cho, Biophys. J. 2007, 92, 1399.

emitter quantum dot nanoparticles could be successfully encapsulated [11] K. Gonda, T. M. Watanabe, N. Ohuch, H. Higuchi, J. Biol. Chem. 2010,
285, 2750.
into liposomes. As the synthesis of hydrophilic QDs was simple, low
[12] V. Levi, E. Gratton, 2007, Exploring dynamics in living cells by tracking
cost and less polluting, the critical factor for QD‐loaded liposome syn-
single particles. Cell biochem. and biophys. 48, 1.
thesis involves the transfer of hydrophilic QDs. The protection pro-
[13] D. M. Warshaw, G. G. Kennedy, S. S. Work, E. B. Krementsova, S.
vided by the lipid bilayer could effectively prevent QDs from photo‐ Beck, K. M. Trybus, Biophys. J. 2005, 88, L30.
oxidation during storage or exposure to UV light. Prepared QDs are [14] A. M. Smith, S. Dave, S. Nie, L. True, X. Gao, Expert Rev. Mol. Diagn.
encapsulated in liposome complexes and were stable, biocompatible 2006, 6, 231.
and maintained fluorescence capacity in vitro and in vivo. We studied [15] X. Michalet, F. F. Pinaud, L. A. Bentolila, J. M. Tsay, S. Doose, J. J. Li, G.
Sundaresan, A. M. Wu, S. S. Gambhir, S. Weiss. Science 2005, 307
the ability of the fluorescent lipid vesicles to label living cells in vitro.
(5709), 538.
The self‐organized lipid–QDs with small sizes showed improved bio-
[16] A. M. Smith, G. Ruan, M. N. Rhyner, S. Nie, Annals Biomed. Eng. 2006,
compatibility and were qualified for imaging living cells. The systems 34, 3.
developed in this study can be used to deliver QDs without the need [17] Y. Ghasemi, P. Peymani, S. Afifi, Acta Biomed. 2009, 80, 156.
for conjugation chemistry. Most liposomes–QDs hybrids were [18] S. Jiang, M. K. Gnanasammandhan, Y. Zhang, J. Roy. Soc. Interf. 2010,
designed for diagnosis. The present work shows novel liposomes– 7, 3.
12 SAMADIKHAH ET AL.

[19] S. Lin, X. Xie, M. R. Patel, Y.‐H. Yang, Z. Li, F. Cao, et al., BMC [36] Y. Maeda, T. Kinoshita, Methods Enzymol. 2010, 480, 495.
Biotechnol. 2007, 7, 1. [37] L. Mayer, M. Hope, P. Cullis, BBA Biomembranes. 1986, 858, 161.
[20] D. R. Larson, W. R. Zipfel, R. M. Williams, S. W. Clark, M. P. Bruchez, F. [38] P. Paroutis, N. Touret, S. Grinstein, Physiology 2004, 19, 207.
W. Wise, W. W. Webb. Science 2003, 300(5624), 1434.
[39] Y. Sun, Y. Liu, P. Vernier, C. Liang, S. Chong, L. Marcu, M. Gundersen.
[21] Y. Wang, C. Ye, L. Wu, Y. Hu, J. Pharmaceut. Biomed. Anal. 2010, 53, 235. Nanotechnology 2006, 17, 4469.
[22] C. Ye, Y. Wang, C. Li, J. Yu, Y. Hu, Microchim. Acta 2013, 180(1–2), [40] N. Oku, D. A. Kendall, R. C. MacDonald, BBA Biomembranes 1982,
117. 691, 332.
[23] Z. Zhelev, D. Kokuryo, I. Aoki, Int. J. Nanomed. 2011, 6, 1719. [41] J. Chevallier, J. Gruenberg, Lipid Membrane Domains in Endosomes.
[24] W. Liu, M. Howarth, A. B. Greytak, Y. Zheng, D. G. Nocera, A. Y. Ting, Endosomes, Springer, New York 2006 14.
M. G. Bawendi. J. Am. Chem. Soc. 2008, 130, 1274. [42] R. C. MacDonald, R. I. MacDonald, B. P. M. Menco, K. Takeshita, N. K.
[25] G. Gopalakrishnan, C. Danelon, P. Izewska, M. Prummer, P. Y. Bolinger, Subbarao, L.‐R. Hu, BBA Biomembranes 1991, 1061, 297.
I. Geissbühler, D. Demurtas, J. Dubochet, H. Vogel. Angew. Chem. Int. [43] W. T. Al‐Jamal, K. Kostarelos, Account Chem. Res. 2011, 44, 1094.
Edit. 2006, 45, 5478.
[44] B. Chen, Q. Liu, Y. Zhang, L. Xu, X. Fang, Langmuir 2008, 24, 11866.
[26] W. T. Al‐Jamal, K. T. Al‐Jamal, B. Tian, A. Cakebread, J. M. Halket, K.
[45] A. M. Derfus, W. C. Chan, S. N. Bhatia, Nano Lett. 2004, 4, 11.
Kostarelos, Mol. Pharmaceut. 2009, 6, 520.
[46] G. D. Bothun, A. E. Rabideau, M. A. Stoner, J. Phys. Chem. B 2009, 113,
[27] H. R. Samadikhah, A. Majidi, M. Nikkhah, S. Hosseinkhani, Int. J.
7725.
Nanomed. 2011, 6, 2275.
[47] V. Dudu, M. Ramcharan, M. L. Gilchrist, E. C. Holland, M. Vazquez,
[28] M. Ghanbari Safari, S. Hosseinkhani, J. Lipos. Res. 2013, 23, 174.
J. Nanosci. Nanotechnol. 2008, 8, 2293.
[29] A. Bangham, M. M. Standish, J. Watkins, J. Mol. Biol. 1965, 13, 238.
[48] V. Sigot, D. J. Arndt‐Jovin, T. M. Jovin, Bioconjugate Chem. 2010, 21,
[30] K. Tahara, S. Fujimoto, F. Fujii, Y. Tozuka, T. Jin, H. Takeuchi, 1465.
J. Pharmaceut. 2013, 6, 2044.
[49] T. Wang, J. Bai, X. Jiang, G. U. Nienhaus, ACS Nano 2012, 6, 1251.
[31] G. Mandal, S. Bhattacharya, T. Ganguly, Chem. Phys. Lett. 2009, 478, 271.
[32] H. Kikuchi, N. Suzuki, K. Ebihara, H. Morita, Y. Ishii, A. Kikuchi,
S. Sugaya, T. Serikawa, K. Tanaka. J. Controlled Release 1999, 62, 269. How to cite this article: Samadikhah, H. R., Nikkhah, M., and
[33] H. Garn, H. Krause, V. Enzmann, K. Dröβler, J. Immunol. Methods 1994, Hosseinkhani, S. (2016), Enhancement of cell internalization
168, 253.
and photostability of red and green emitter quantum dots upon
[34] S. Pautot, B. J. Frisken, D. Weitz, Proc. Natl. Acad. Sci. 2003, 100,
entrapment in novel cationic nanoliposomes. Luminescence, doi:
10718.
10.1002/bio.3207
[35] J. A. Heyes, D. Niculescu‐Duvaz, R. G. Cooper, C. J. Springer, J. Med.
Chem. 2002, 45, 99.