Original Paper
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Acta Biochim Biophys
Sin 2006, 38: 646-652
doi:10.1111/j.1745-7270.2006.00211.X
Atomic force microscopy-based
cell nanostructure for ligand-conjugated
quantum dot endocytosis
Yun-Long PAN1*,
Ji-Ye CAI2, Li QIN3, and Hao WANG4
1 The First Affiliated
Hospital of Jinan University, Guangzhou 510632, China;
2 College of Life Science
and Technology, Jinan University, Guangzhou 510632, China;
3
Department of Histology and Embryology, Medical College of Jinan University,
Guangzhou 510632, China;
4 School
for Information and Optoelectronic Science and Engineering, South China Normal
University, Guangzhou 510631, China
Received: May 8,
2006
Accepted: June 27,
2006
This work was supported
by the grants from the Major State Basic Research Development Program of China
(2001CB510101), the National Natural Science Foundation of China (No. 30230350,
60278014 and 60578025), and the doctor
innovation fund of Jinan University
*
Corresponding author: Tel, 86-20-38688609; Fax, 86-20-38688000; E-mail,
Abstract While it has been well demonstrated that quantum dots (QDs)
play an important role in biological labeling both in vitro and in
vivo, there is no report describing the cellular nanostructure basis of
receptor-mediated endocytosis. Here, nanostructure evolution responses to the
endocytosis of transferrin (Tf)-conjugated QDs were characterized by atomic
force microscopy (AFM). AFM-based nanostructure analysis demonstrated that the
Tf-conjugated QDs were highly specifically and tightly bound to the cell
receptors and the nanostructure evolution is highly correlated with the cell
membrane receptor-mediated transduction. Consistently, confocal microscopic and
flow cytometry results have demonstrated the specificity and dynamic property
of Tf-QD binding and internalization. We found that the internalization of
Tf-QD is linearly related to time. Moreover, while the nanoparticles on the
cell membrane increased, the endocytosis was still very active, suggesting that
QD nanoparticles did not interfere sterically with the binding and function of
receptors. Therefore, ligand-conjugated QDs are potentially useful in
biological labeling of cells at a nanometer scale.
Key words quantum dot; endocytosis; nanostructure;
atomic force microscopy
Recently, our understanding of cancer
diagnosis and biological imaging has been significantly extended by the application
of fluorescent semiconductor nanocrystals (quantum dots, QDs) because of their
unique optical properties and outstanding biocompatibility [1–13]. It is interesting to note that one of
the most important applications of ligand-conjugated QDs is to target them to
the cell surface receptors. For example, Chan and Nie [11] showed that QD
bio-conjugates were transported into the cells by receptor-mediated endocytosis
and detected the clusters and aggregates on the cell surface. Osaki et al.
[12] revealed the size effect of endocytosis in the subviral region and
demonstrated that the endocytosis is highly size-dependent because the size
complementarity governed the molecular recognition in small host-guest systems.
Data of Rosenthal et al. [13] suggested that serotonin-labeled
nanocrystals (SNACs) interacted with the serotonin transporter protein (SERT)
in transfected HeLa cells and oocytes in vitro. They also showed that
fluorescent SNACs could be used to visualize SERTs expressed in human
epithelial kidney cells in an antidepressant-sensitive manner [13].
Although the potential of QDs in biological
labeling and cancer diagnosis has been well described, little is known about
the cellular nanostructures response to the QD labeling and receptor-mediated endocytosis
of the QD-ligand. It is important to point out that cell surface structures at
the nanometer scale may have important roles in shaping the function of the
particular molecules on the cell surface. By monitoring the binding,
internalization and function of ligand-conjugated QDs at the nanometer scale,
ligand-conjugated QDs may help to advance the applications of QDs in targeting
drug delivery, protein transportation, ligand-receptor binding and signal
transduction in biology and immunology. Furthermore, atomic force microscopy
(AFM) has emerged as a powerful tool in cell biology studies at the nanometer
scale under near physiological condition [14]. Therefore, AFM visualization and
determination of responses in cells at the nanometer scale may elicit
particular molecular recognition and offer new insights into the binding and
internalization of ligand-conjugated QDs.
In this paper, AFM was used to characterize
the cellular nanostructure evolution when HepG2 cells were cultured in
ligand-conjugated QDs and free QDs for different time periods. At the same
time, confocal laser scanning microscopy images, mean fluorescent intensity
(MFI) and percentages of positive cells obtained by flow cytometry were used to
reveal the fluorescent characteristics at different stages of endocytosis. As a
result, the combined methods of AFM, confocal laser scanning microscopy and
flow cytometry demonstrated that transferrin (Tf)-conjugated QDs (Tf-QDs)
interact specifically with the transferrin receptor (TfR) in human hepatic
cancer cells. The internalization of ligand-conjugated QDs during endocytosis
appears to be a linear process with time. These experiments suggested that
AFM-based nanostructure analysis should provide novel insights into
receptor-mediated transductions and fluorescent nanocrystal-based molecule
detection.
Materials and methods
Reagents
Biotin-Tf was purchased from Molecular
Probes (Eugene, USA). Streptavidin-conjugated 605 nm Qdots was purchased from Quantum
Dot (Hayward, USA). All other reagents were of analytical grade.
Cell culture and incubation of
ligand-conjugated quantum dot
HepG2 cells were seeded in a 6-well Lab-Tek
chamber (Nalge Nunc, Naperville, USA) and grown in RPMI 1640 medium with glutamax
supplemented with 10% fetal calf serum in 5% CO2 at 37 ?C.
Two activation methods were applied to activate TfR: (1) first, the
biotinylated Tf were added directly in cell culture well to activate the TfR on
cell surface, and then streptavidin-QDs were loaded into cell culture well to
bind Tf; (2) the Tf-QD conjugates were prepared in test tube using the strong
binding between streptavidin and biotin and then Tf-QD conjugates were loaded
into cell culture well. Tf-QD complexes were formed by incubating biotin-Tf (47
ng/ml) and streptavidin-QDs at 4 ?C with mixture for 30 min and added to the
cell culture. Cells were centrifuged to remove unbounded Tf-QDs and fixed by 4%
formaldehyde. Consecutive binding of biotin-Tf and streptavidin-QDs was
conducted with cells labeled by 1.9 ng/ml biotin-Tf in culture medium and then incubated with 200 nM QDs for different
durations. Then the cells were washed, centrifuged and fixed for imaging.
Microscopy and flow cytometry
Atomic force microscopy was performed in
contact mode with a commercial atomic force microscope (AutoProbe CP, Veeco, USA) at room
temperature. The AFM images were planar-leveled using the software
(Thermomicroscopes Proscan Image Processing Software Version 2.1) provided by
the manufacturer. The contact angles were measured by this software directly.
Here, the contact angles mean the shrinking degree of cell. The larger contact
angels mean the higher degree of cellular shrinkage. Confocal laser scanning microscopy
was performed with a Bio-Rad MRC600 microscopy system (Bio-Rad, Hercules, USA)
using a 488-nm line excitation of an air-cooled 100 mW argon laser. The emitted
fluorescence was detected through the combination of the appropriate AG2 filter
set with a high pass at 605 nm. Cell suspension with a density of about 5105 cells per milliliter was processed for
flow cytometry under commercial flow cytometry facility (FACSCalibur; BD
Bioscience, Franklin Lakes, USA).
Results
Fine AFM images (Fig. 1) of individual
cells without Tf-QD endocytosis at the nanometer scale were readily observable.
These images showed the characteristic features of cells with an average
diameter of about 16 mm. The
cell membrane showed a smooth feature with several holes, which allowed
endocytosis of nutrients. The contact angle between the cell and the substrate
was about 27?.
Cells were exposed to biotinylated Tf and
TfRs were activated to bind streptavidin-QDs, allowing specific binding of
biotin-Tf and streptavidin-QDs. After exposing to streptavidin-QDs for about 1
min, the filopodia of cells became larger (Fig. 2) probably due to
binding of QDs to Tf. This result clearly demonstrated that Tf activated the
TfR within 1 min. It should be noted that Lidke et al. [4] also reported
similar results using confocal laser scanning microscopy, suggesting that the
QDs bind to the cell filopodia. Importantly, this consistency revealed the high
sensitivity and reliability of AFM-based nanostructure analysis. After about
15-min incubation, the cell edge tended to shrink and the cell body expanded
significantly, suggesting that rapid and extensive endocytosis of Tf-QD
occurred. Collectively, these results demonstrated that the nanoparticles
activate the cell receptor readily and do not damage the cellular receptor
activity and the endocytic pathways. Also, the changes in cell nanostructures
at different time points suggested that endocytosis might be a dynamic process.
To determine whether the receptor
activation pathway was involved in endocytosis at the nanometer scale, we
activated the cell receptors using an alternative approach. We preloaded the
streptavidin-QDs with biotin-Tf to obtain a Tf-QD complex. Importantly, after
culturing the cells in Tf-QDs for 15 min, the cell body expanded and the cell
edge shrank, supporting the feasibility of the idea that ligand-conjugate QDs
preloading and the desired receptor-mediated transportation of ligand-conjugate
QDs could be achieved by such strategy as well. Interestingly, these two
different receptor activation pathways give similar results on cellular
nanostructure (Figs. 3 and 4). It should be noted that bulges
could be observed on the cell edge and these bulges may be cell vesicles or
nanocrystals of Tf-QDs.
there are at least two differences between
control cells and cells that have undergone endocytosis of ligand-conjugated
QDs. First, cells with QDs endocytosis showed expansive body and contractive
edge. As a result, the shrinkage has led to an increase in the contact angles
between the edge and substrate. Second, the cell surface has provided more
space to allow more ligand-conjugated QDs to bind onto the cell membrane, which
have resulted in a dramatic increase of nanoparticles on the cell surface (Fig.
5). The differences in size and shape between these cells could be
attributed to the following factors. First, the binding and internalization of
ligand-conjugated QDs resulted in the expanse of cell body. Second, the
nonspecific endocytosis Tf-QD and aggregation property of QDs after leveling
the storage buffer condition may also result in an increase in the nanoclusters
on the cell surface.
In order to confirm that the differences
were the results of the receptor-mediated Tf-QD endocytosis rather than
unspecific QD binding, we cultured the cells in the presence of free QDs. As
expected, the cell membrane remained almost intact and relatively unaffected
although there were some particles on the cell surface (Fig. 6). It
should be pointed out that such particles [Fig. 6(B)] were likely the
results of nonspecific binding and aggregation. This result was further
confirmed by flow cytometry (Fig. 7). Compared with the blank cells,
there is no significant increase in MFI, but the significant positive cell
percentage could be observed in cells exposed to free QDs. The background
fluorescence of cells exposed to free QDs obtained by flow cytometry (Fig.7)
should arise from nonspecific binding of QDs during cell fixation and the cell
autofluorescence. In contrast, after the cells were exposed to Tf-QDs, both MFI
and positive cell percentage increased simultaneously as the exposure time
increased. Taken together, these results, again, illustrate that Tf-QD is
highly specific and potentially useful in activating and binding Tf to cell
receptors.
The inherent stability of the biotin-Tf and
streptavidin-QDs makes us able to obtain the desired degree of ligand loading
simply by adjusting the mixing stoichiometry. To further investigate the effect
of loading rate on nanostructure response for endocytosis of ligand-conjugated
QDs, we cultured the cells after exposure with different loading rates of
biotin-Tf and steptavidin-QDs (Fig. 5). Again, cells showed the
characteristic features of an expansive body and contractive edge (Fig. 5).
Furthermore, increases in Tf-QD concentration and exposure duration resulted in
more accumulation of Tf-QD on the cell surface (Fig. 5), demonstrating
that exposure duration enhanced the binding of the ligand-conjugated QDs before
internalization. Moreover, the nanoparticles on the cell membrane increased
while the endocytosis was still very active. Taken together, these evidences
suggested that the QD nanoparticles did not interfere sterically with the
binding and function of receptors. Therefore ligand-conjugated QDs are potentially
useful in biological labeling of cells at the nanometer scale.
Based on the AFM images, the fluorescent
signal change of Tf-QD in cell bodies might be a dynamic process coupled with
binding and internalization of ligand-conjugated QDs. This inspires us to
further examine whether the fluorescent signal might correlate with the
cellular nanostructure evolution. In Fig. 8, three representative
fluorescent images of cells, after 106 min, 149 min and 168 min of exposure to
Tf-QDs are shown. In these samples, the fluorescent intensity is enhanced
dynamically. Furthermore, the fluorescent intensity in the central part of the
cell is significantly stronger than that in the cell edge. This became clearer
as the exposure time was increased. The most likely explanation for this
phenomenon is the internalization of Tf-QDs upon the binding of Tf-QDs to cell
Tf receptor. Consistently, AFM observations also suggested the similar
phenomenon, suggesting the reliability and applicability of AFM-based
nanostructure analysis. We also found that internalization of Tf-QD appears to
be linearly related to time (Fig. 9). Importantly, this observation is
consistent with the results of EGF-QDs [4], whose internalization is also
related to time linearly.
Based on our observations of cellular
characteristics of endocytosis of Tf-QDs, we proposed a model (Fig. 10)
for the endocytosis of QDs. It seems that the endocytosis of Tf-QDs represents
a typical trajectory of ligand-conjugated QDs activating cell receptors,
binding onto cell surface, and internalizing. As illustrated above, internalization
of Tf-QDs, not only Tf but also Tf-QD conjugates were able to activate TfR,
therefore, two different pathways could achieve stage one. For stage two,
ligand-conjugated QDs bind to the cell receptor. Therefore, the nanoclusters on
the cell surface increased dramatically. For stage three, the ligand-conjugated
QDs experienced internalization, resulting in the expansion of the cell body.
While QDs have
been widely used as a novel fluorescence probe for both in vivo
and in vitro studies, the desired cellular nanostructure
responses for the biological label of QDs have been poorly elucidated. The
well-defined cellular nanostructure analysis should certainly be an important
step for the development of better biological labeling of QDs. First,
elucidation of cellular nanostructures by direct AFM visualization of QDs
endocytosis should significantly extend our understanding of cell
characteristics and be useful for designing and interpreting molecular probes
at the nanometer scale. Second, the insights provided by AFM-based
nanostructure characterization at the nanometer scale allow us to be in a
unique position to study the endocytosis and nanoparticle targeted delivery,
which are difficult to be achieved using other methods such as TEM and SEM
because they require complex pretreatments, which usually damage the cell
structure and functional molecules on the cell membrane. Third, at the
nanometer scale, spatial prevention and nonspecific interactions make it
difficult to detect the desired molecules on the complex cell surface directly.
Apparently, QDs fluorescent probes and AFM can be used to study the inside or
surface molecules of cells, receptor-targeted cross membrane transportation,
and the virus infection at the nanometer scale. Together with confocal laser
scanning microscopy and flow cytometry, direct AFM visualization provides the
background and principles of cellular nanostructure analysis, making a case for
the use of such measurements as disease markers at the nanometer level.
While many groups
have studied the basic principle and application of QDs in biological labeling
in in vivo and in vitro studies, little is known
about the nanostructure evolution induced by endocytosis of ligand-conjugated QDs.
Our AFM study shows that ligand-conjugated QDs are highly specific and bind
strongly to the receptor. Also, the TfR on cell surfaces can be activated
either by Tf or Tf-QD. After receptor stimulation, both pathways can achieve
the desired endocytosis. In this study, we found that the internalization of
Tf-QD is linearly related to time. AFM direct visualization of
ligand-conjugated QDs endocytosis provides novel insights into the biological
labeling of QDs, which should be useful for the design and application of QDs
fluorescent probes.
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