2.1.

Materials

$\mathrm{Cd}\mathrm{Se}∕\mathrm{Zn}\mathrm{S}$ QDs were synthesized according to previous reports.^{27, 28} $\mathrm{Fe}{\mathrm{Cl}}_3$ , defetoxamine (DFO), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and transferrin (Tf) were purchased from Sigma-Aldrich Fine Chemicals (St. Louis, Missouri). A transferrin receptor enzyme-linked immunosorbent assay (ELISA) kit was purchased from R&D Systems (Minneapolis, Minnesota). All other chemicals and materials used in the experiments were of analytical grade and water was deionized.

2.2.

Instruments

The following equipment were purchased from the companies indicated in parentheses: a luminescence spectrometer (LS-55, PerkinElmer, Waltham, Massachusetts), a fiber optic spectrometer (QE65000, Ocean Optics, Dunedin, Florida), an inverted fluorescence microscope (IX71, Olympus, Nagano, Japan), a cooled color charge-coupled device (CCD, Pixera Penguin 150CL, San Jose, California), an atomic absorption spectrophotometer (AA-300, Perkin Elmer, Inc., Wellesley, Massachusetts), a microplate reader (ELX808TM, Biotek, Winooski, Vermont), and a UV-VIS spectrophotometer (UV-2550, Shimadzu, Japan).

2.3.

Preparation of QDs Probes

The whole process of the conjugation of $\mathrm{Cd}\mathrm{Se}∕\mathrm{Zn}\mathrm{S}$ QDs and Tf (QDs-Tf) included three steps. First, $10\mu \mathrm{L}$ EDC ( $5\mathrm{mg}∕\mathrm{mL}$ , pH 7.4) were added to a $40\mu \mathrm{L}$ water-soluble $\mathrm{Cd}\mathrm{Se}∕\mathrm{Zn}\mathrm{S}$ QDs solution $(1.5\times {10}^{-4}\mathrm{M})$ . The size of QDs was about $5\mathrm{nm}$ and the carboxylic groups were on the surface. Then the solution was added to $100\mu \mathrm{L}$ phosphate-buffered saline (PBS, pH 7.2), followed by $15\min$ of shaking. The next step was that $75\mu \mathrm{L}$ of transferrin solution $(2\mathrm{mg}∕\mathrm{mL})$ was added into the mixture, and then the whole solution was further stirred for $2\mathrm{h}$ at room temperature. The resulting mixture was filtered and QDs-Tf was achieved. The synthesization and purification of QDs-TF was reported in previous reports. ^{27, 28}

2.4.

Cell Images

HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum, $100\mathrm{mg}∕\mathrm{mL}$ streptomycin, and $100\mathrm{U}∕\mathrm{mL}$ penicillin at $37°\mathrm{C}$ in a humidified atmosphere with 5% $\mathrm{C}{\mathrm{O}}_2$ . For imaging, the cells were plated on a glass slide in a $3.5\text{-}\mathrm{cm}$ dish. To obtained the high-iron status and the iron-deficient condition, $\mathrm{Fe}{\mathrm{Cl}}_3$ and DFO with the same concentration $(50\mu \mathrm{mol}∕\mathrm{L})$ were added to the dish and maintained in the incubator for $6\mathrm{h}$ . At this time, the cells were in high-iron status with $50\mu \mathrm{mol}∕\mathrm{L}$ $\mathrm{Fe}{\mathrm{Cl}}_3$ . Meanwhile, with $50\mu \mathrm{mol}∕\mathrm{L}$ DFO, the cells were in iron-deficient condition. Following that, the cells were washed three times with PBS (pH 7.2), which was preincubated at $37°\mathrm{C}$ to remove the growth medium; $\mathrm{Fe}{\mathrm{Cl}}_3$ ; and DFO. Then fresh DMEM medium with QDs-Tf were added in the dish. Cells were incubated for $15\min$ at $37°\mathrm{C}$ . This was followed with extensive washing (3 times, $5\min$ /time) in PBS ice. Finally the treated cells were detected by an IX71 inverted fluorescence microscope with a cooled color CCD. The spectra were recorded by the spectrometer.

2.5.

ELISA for Transferrin Receptor

After being plated in 96 wells for $24\mathrm{h}$ , HeLa cells were washed with fresh medium, and treated with $\mathrm{Fe}{\mathrm{Cl}}_3$ $(50\mu \mathrm{mol}∕\mathrm{L})$ and DFO $(50\mu \mathrm{mol}∕\mathrm{L})$ for $6\mathrm{h}$ , respectively. The cells were washed three times with PBS. The cells were fixed for $10\min$ by 0.25% glutaraldehyde at room temperature, washed three times with warm PBS, and closed by 2% bovine serum albumin for $10\min$ . The expression of transferrin receptor (TfR) in the cells was detected by the ELISA kit. Finally the absorbance of each pore was detected at a wavelength of $450\mathrm{nm}$ in the ELISA meter. In this study, the data were from four independent experiments.

2.6.

Determination of Iron Amount after It Was Transferred into the Cell through the Endocytosis

After HeLa cells were plated in 96-well microtiter plates for $24\mathrm{h}$ , the experiment was divided into three groups: the normal group, the $\mathrm{Fe}{\mathrm{Cl}}_3$ group, and the DFO group. There were two wells in each group with three repeats. First, in the $\mathrm{Fe}{\mathrm{Cl}}_3$ group, the cells were exposed to $50\mu \mathrm{mol}∕\mathrm{L}$ $\mathrm{Fe}{\mathrm{Cl}}_3$ for $6\mathrm{h}$ ; $50\mu \mathrm{l}$ Tf-QDs was added to one of the wells after $\mathrm{Fe}{\mathrm{Cl}}_3$ was washed out at an incubation time of $6\mathrm{h}$ , and incubated for another $15\min$ . Second, in the DFO group, $50\mu \mathrm{mol}∕\mathrm{L}$ DFO was added in one well for $6\mathrm{h}$ , while $50\mu \mathrm{l}$ Tf-QDs was added to another well and incubated at $37°\mathrm{C}$ , 5% $\mathrm{C}{\mathrm{O}}_2$ for $15\min$ after DFO was washed out at the incubation time of $6\mathrm{h}$ . Third, in the normal cell group, the wells were incubated for $6\mathrm{h}$ without $\mathrm{Fe}{\mathrm{Cl}}_3$ and DFO, then one of them was exposed to $50\mu \mathrm{l}$ Tf-QDs for more $15\min$ after washing three times with PBS. Finally, all the cells were washed with cold stripping buffer ( $200\mathrm{mM}$ NaCl, $50\mathrm{mM}$ MES, $1\mathrm{mM}$ $\mathrm{Mg}{\mathrm{Cl}}_2$ , $1\mathrm{mM}$ $\mathrm{Ca}{\mathrm{Cl}}_2$ ) for $2\times 2\min$ to wash out Tf-QDs stained on the plasma membrane. Then the cells were crashed by the cold cell lysis buffer and centrifugated to be collected. The iron concentration was detected by an AA-300 atomic absorption spectrometer (wavelength, $248.3\mathrm{nm}$ ; width of slit, $0.4\mathrm{mm}$ ). The content of intracellular iron which was transferred into cells by TfR was the amount in the wells with Tf-QDs minus that in the wells without Tf-QDs.

3.1.

Characterization of QDs Probes

The conjugation of QDs-Tf and their purification have been reported.^{27, 28} In this study, the fluorescence spectra of QDs and QDs-Tf were measured. As shown in Fig. 1 , photoluminescent (PL) quantum yields (QYs) were 26 and 18%, respectively, for QDs and QDs-Tf. A red shift was observed in QDs-Tf. We found that $606\mathrm{nm}$ (before conjugation) moved to $608\mathrm{nm}$ (after conjugation), and the half-peak width broadened (from 31 into $33\mathrm{nm}$ ), which was possibly due to the combination with Tf to make larger QDs, and the number of Tf binding on the QD surfaces might be also inconsistent, so that size distribution of QDs-Tf increased, leading to a red shift and a half-wavelength width extension.

Fig. 1

Fluorescence spectra of (a) QDs and (b) QDs-Tf.

3.2.

Imaging and the Fluorescence Intensity Changes of Cells in the Normal Physiological Condition

To compare the effects of iron deficiency and an iron-rich environment on Tf endocytosis, the QDs-Tf endocytosis process of tumor cells was observed first. The cells were incubated with QDs-Tf at $37°\mathrm{C}$ for $15\min$ , and then washed three times quickly. Immediately, the whole process of endocytosis was recorded through the Olympus IX71 inverted fluorescent microscope and CCD. The time for the first photograph was set as $t=0$ . As shown in Fig. 2 , at the beginning, only localized fluorescence appeared in QDs-Tf incubated cells. Then some intermittent fluorescence could be seen on the membrane. Gradually, the fluorescence on the cell membrane increased in brightness, while the punctate fluorophores piled up in the cytoplasm of the cells. Fluorescence throughout the cells grew to be the brightest at around $700\text{to}800\mathrm{s}$ followed by gradually weakening. The fluorescence of the cytoplasm clearly dimmed, while the fluorescence of the membrane was still relatively bright at $5100\mathrm{s}$ . The video in Fig. 2 also provides a description of the entire endocytosis process.

Fig. 2

Video showing bright-field imaging, fluorescence imaging, and fluorescence spectroscopy of the process of receptor-mediated endocytosis of QDs-Tf under the normal physiological condition. Scale bar: $5\mu \mathrm{m}$ . (QuickTime, $77.5\mathrm{KB}$ ). .

To study the time-varying characteristics of cell fluorescence intensity, the total fluorescence intensity in cells was calculated. Figure 3 shows that the change of the fluorescence intensity of cells was consistent with the images. It went through a process transmitted from dark to the light and then to dark again. This is so because the surface of all living cells have TfRs. In the neutral pH environment, the TfR can bind protein and go into cells by endocytosis. In an acidic intracellular environment, Tf releases iron, after which it goes back to the plasma membrane together with the receptor. In the extracellular neutral environment, the Tf is dissociated from the receptor and freely combines with ferric ion again. Then a new cycle begins again.³¹ Therefore, the transfer of Tf for iron ion is different from other receptor-mediated endocytosis processes because its ligand (Tf) and receptor are recycled, and only the iron ion is transported and stored. If the QDs are coupled with Tf protein, they are also involved in the cycle. In the experiments, QDs-Tf was added in the cell culture medium for a certain period of time, after three rinses, and the free probes had already been washed off. In this case, QDs-Tf has undergone combination with the cell membrane receptor and endocytosis into the cell. After the release of iron ions, they also returned to the cell membrane. In this cycle process, QDs have also been transferred from the cell membrane into the cytoplasm, and then back to the membrane, so the cell membrane could be observed to have a very strong fluorescence (Fig. 2). In this process, there is the puzzling phenomenon that, after the free probe are washed off, although a certain amount of QDs probes are maintained, changes in fluorescence was observed in the cells. While fluorescence spectra emission peaks are at $610\mathrm{nm}$ (Fig. 2), which is very consistent with QDs-Tf (Fig. 1, curve b), indicating that fluorescence indeed resulted from the QDs. This phenomenon was also observed by Lidke using QDs as a probe to detect epidermal growth factor signal transduction,³² but the reason is not yet clear.

Fig. 3

Fluorescence (FL) intensity changes of cells in the process of endocytosis under the normal physiological condition.

At the same time, pure QDs instead of Tf-QDs were used to label HeLa cells in the same method as a control. After the same incubation time and being washed as before, photos of the treated cells were taken. As shown in Fig. 4 , no obvious QD fluorescence was observed and only few fluorescent dots appeared in cells and their structure was not clear [Fig. 4]. These images show that unlabeled QDs did not enter the cells in the experiment. This indicates that only Tf-QDs were recognized by the TfR and bound on the membrane and delivered into the cytoplasm after incubation by the TfR. The specificity of endocytosis was also proved in our previous reports.²⁷

Fig. 4

(a) Bright-field images and (b) fluorescence images of HeLa cells labeled with pure QDs. Scale bar: $5\mu \mathrm{m}$ .

3.3.

Imaging and the Fluorescence Intensity Changes of Cells in an Iron-Rich Condition

From the fluorescence imaging, we observed that when the system contained an excessive level of iron, the fluorescence of the cells also experienced a change from dark to light then to dark (Fig. 5 ); fluorescence intensity within the plasma membrane reached the maximum in about $2100\mathrm{s}$ . After the rapid decline in fluorescence intensity, the fluorescence at about $5100\mathrm{s}$ was not as good as in the beginning state. The video in Fig. 5 also provides a description of the entire process of endocytosis of QDs-Tf under an iron-rich condition. Calculation of fluorescence intensity (Fig. 6 ) also confirmed this observation. The results showed that when an increase in iron occurs, Tf endocytosis slows down significantly. This may be because the intracellular iron concentration can affect the TfR mRNA stability. In an iron-rich environment, the stability of TfR mRNA was reduced, the synthesis of receptors was blocked, and expression in cells was depressed.^{29, 33} Thus, QDs-Tf could not combine with more receptors and the cycle of the Tf also slowed.

Fig. 5

Video showing bright-field imaging, fluorescence imaging, and fluorescence spectroscopy of the process of receptor-mediated endocytosis of QDs-Tf under an iron-rich condition. Scale bar: $5\mu \mathrm{m}$ . (QuickTime, $214\mathrm{KB}$ ). .

Fig. 6

Fluorescence intensity changes of cells in the process of endocytosis under an iron-rich condition.

3.4.

Imaging and the Fluorescence Intensity Changes of Cells in an Iron-Deficient Condition

Different from the preceding studies, the results showed that, after being washed off, free QDs and unlabeled compounds, at $0\mathrm{s}$ , the cell membrane and the cytoplasm has a strong fluorescence (Fig. 7 ). Although the fluorescence intensity also showed the transfer from weak to strong then weak process, parts of the cytoplasm has a very strong fluorescence; even at $5100\mathrm{s}$ , very strong fluorescence was still observed (Fig. 7). The video in Fig. 7 also provides a description of the entire process of endocytosis of QDs-Tf under an iron deficient condition. We deduced that the inside and outside exchanges on the membrane were quite frequent, and the transport of transferring iron was faster. The fluorescence intensity (Fig. 8 ) also similarly confirmed this observation. This might be due to the increased demand for cells because of the iron ion deficiency, the mRNA stability of the TfR increased, and receptor synthesis was accelerated, leading to an increase of membrane receptors, so that Tf endocytosis process was accelerated.³⁴ This was embodied in the cell imaging results—the cell fluorescence intensity was enhanced.

Fig. 7

Video showing bright-field imaging, fluorescence imaging, and fluorescence spectroscopy of the process of receptor-mediated endocytosis of QDs-Tf under an iron-deficient condition. Scale bar: $5\mu \mathrm{m}$ . (QuickTime, $154\mathrm{KB}$ ). .

Fig. 8

Fluorescence intensity changes of cells in the process of endocytosis under an iron-deficient condition.

3.5.

Fluorescent Intensity Analysis

Comparison of Figs. 3, 6, 8 shows that after rising to the maximum, the average fluorescence intensities of several cells lasted for different platform times, which were $892\text{to}2090\mathrm{s}$ in Fig. 3. At $4092\mathrm{s}$ , the cell’s fluorescence intensity reduced to the maximum 86.4%, but still was 1.2 times that at start point $(t=0)$ . Under the high-valence iron condition, Fig. 6 displayed the platform of a long period from 710 to approximately $2178\mathrm{s}$ , when the time for a further period of $2000\mathrm{s}$ , the cell fluorescence intensity had dropped to the 65.6% of highest intensity, and was only 0.85 times of the initial value $(t=0)$ . However, in the iron-deficiency state in Fig. 8, the fluorescence intensity of cells from platform terminal $\left(2070\mathrm{s}\right)\text{to}4070\mathrm{s}$ , hardly changed. The strength reduced only 4.7% and its fluorescence value approximately was 1.4 times of the initial value; even after $3200\mathrm{s}$ , when time was at $7200\mathrm{s}$ , the fluorescence intensity reduced to only 90.6% of the maximum, and was still 1.3 times the initial value. Therefore, we concluded that the high-iron and iron-deficiency states have different effects on the Tf endocytosis process, which slowed under a high-valence iron condition and accelerated with low iron reserves.

To more clearly understand the effect of iron on receptor-mediated endocytosis of Tf, fluorescence spectra of whole-cells in three cases were obtained. HeLa cells were treated as already discussed. After the cells were labeled with Tf-QDs in an iron-overloading environment, an iron-deficiency environment, and a normal state, they were washed and centrifuged to collect them. The fluorescence intensity was measured immediately. As the result shown in Fig. 9 demonstrates, the highest fluorescence peaks were about $607\mathrm{nm}$ , indicating that the fluorescence was generated by QDs. The fluorescence intensity was maximal under the iron-deficiency condition, while the minimal was observed under an iron-rich condition. The results of fluorescence spectra coincided with our already presented results. In an iron-rich environment, TfR synthesis was blocked, membrane binding to Tf fell off relatively, and thus the fluorescence intensity was lowest. The cells were in a hard iron-flaw condition while DFO acted on the cell; to reduce the harm to themselves, cells speeded the iron ion transport. Therefore, the cytomembrane acceptor synthesis increased, the transferrin binding to the receptor on the cell relatively increased in the number, and the fluorescence intensity strengthened as well.

Fig. 9

Fluorescence intensity of cells in three kinds of situations: curve a, a normal condition; curve b, an iron-rich condition; and curve c, an iron deficiency condition.

3.6.

TfR Amount and Intracellular Iron Content Transferred into Cells by TfR among Different Environments

In our experiment, the whole process of fluorescence changes in cells was detected among different environments. When the fluorescence signal varied, ion concentration had also changed in fact. While the ion concentration changes could be detected by chemical methods. ELISA and atom absorption spectrum analysis were used to study the biological effects of different iron concentrations on the expression of TfR and the content of intracellular iron transferred by TfR through endocytosis. The results revealed that when the cells were incubated with $50\mu \mathrm{mol}∕\mathrm{L}$ DFO, the TfR was about $1.25\mathrm{nmol}∕\mathrm{L}$ , which was almost twice than in medium with $50\mu \mathrm{mol}∕\mathrm{L}$ $\mathrm{Fe}{\mathrm{Cl}}_3$ (Table 1 ). The expression of TfR on HeLa cells becomes lower with an increase of the iron element in the experiment. In an iron-deficiency environment, the amount of iron transferred into cells by TfRs was the highest, which was almost 4 times that in the high-iron status and three times that in a normal status. In contrast, in a high-iron environment, the iron ion uptake by cells through endocytosis was the least (Table 1). The results coincided with previous reports, which showed that the expression of TfR was relevant to the need for iron in cells.³⁵ Moreover, when the cells were in an iron-deficiency condition, the TfR mRNA expression increased.³³ The amount of TfR decreased when iron increased.²⁹

Table 1

TfR amount and iron content transferred into cells by TfR among different environments.