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Labeling and Intracellular Tracking of Functionally Active Plasmid DNA with Semiconductor Quantum Dots
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doi:10.1016/j.ymthe.2006.03.010
Labeling and Intracellular Tracking of Functionally Active Plasmid DNA with Semiconductor Quantum Dots Charudharshini Srinivasan,1 Jeunghoon Lee,2 Fotios Papadimitrakopoulos,2 Lawrence K. Silbart,3 Minhua Zhao,2 Diane J. Burgess,1,* 1 Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road, Unit 3092, Storrs, CT 06269, USA Nanomaterials Optoelectronics Lab, Polymer Program, Department of Chemistry, Institute of Materials Science, University of Connecticut, 97 North Eagleville Road, Unit 3136, Storrs, CT 06269, USA 3 Department of Animal Science, Center of Excellence for Vaccine Research, University of Connecticut, 1390 Storrs Road, Unit 4163, Storrs, CT 06269, USA 2
*To whom correspondence and reprint requests should be addressed. Fax: +1 860 486 0538. E-mail: [email protected].
Available online 12 May 2006
Semiconductor nanocrystal quantum dots (QDs) allow long-term imaging in the cellular environment with high photostability. QD biolabeling techniques have previously been developed for tagging proteins and peptides as well as oligonucleotides. In this contribution, QD-decorated plasmid DNA was utilized for the first time for long-term intracellular and intranuclear tracking studies. Conjugation of plasmid DNA with phospholipid-coated QDs was accomplished using a peptide nucleic acid (PNA)–N-succinimidyl-3-(2-pyridylthio) propionate linker. Gel electrophoresis and confocal and atomic force microscopy (AFM) were used to confirm the structure of QD–DNA conjugates. AFM imaging also revealed that multiple QDs were attached in a cluster at the PNAreactive site of the plasmid DNA. These QD–DNA conjugates were capable of expressing the reporter protein, enhanced green fluorescent protein, following transfection in Chinese hamster ovary (CHO-K1) cells with an efficiency of ca. 62%, which was comparable to the control (unconjugated) plasmid DNA. Key Words: plasmid DNA, transfection, quantum dots, labeling, QD–DNA conjugates, DNA tracking, nuclear staining, gene expression
INTRODUCTION Nonviral gene delivery systems have gained immense importance, primarily due to their considerable in vivo safety compared to viral vectors [1–4]. In particular, cationic [5–7] and recently anionic liposomes [8–10], as well as cationic polymers [11,12], and nanoparticles [13] are under investigation as nonviral vectors for DNA therapeutics. However, the poor transfection efficiency of these systems in vivo has impeded their development as therapeutics [3,5,14]. To realize the full potential of such delivery systems, a better understanding of the critical steps in the transfection process is needed [15–17]. Methods commonly employed in studying intracellular plasmid trafficking involve labeling of the delivery vehicles (liposomes, polymers, etc.) with commercially available organic dyes [18,19]. The intracellular distribution of poly(dl-lactide-co-glycolide) nanoparticles has been investigated via confocal and fluorescence microscopy using nanoparticles loaded with the lipophilic fluorescent dye 6-coumarin [20,21]. Small molecules, such as oligonucleotides and
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cell-specific antibodies, may also be labeled with organic dyes; however, it is difficult to label large molecules such as plasmid DNA [22]. Attempts to tag plasmid DNA with organic fluorophores have involved cumbersome procedures and the possibility of irreversible changes to the DNA with consequent loss of functional activity [22–24]. Such organic fluorophores are not ideal labels since they rapidly undergo photobleaching (within seconds to a few hours), which renders them unsuitable for long-term imaging studies. Colloidal gold has also been utilized to track plasmid DNA using high-resolution electron microscopy. The colloidal gold and the plasmid DNA are physically associated by charge interactions and are not suitable for live cell fluorescence imaging. Moreover, the plasmid DNA is rendered nonfunctional for gene expression [22–26]. In recent years, semiconductor quantum dots (QDs) have attracted the attention of many research groups due to their scientific and technological significance in microelectronics, optoelectronics, and cellular imaging
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[27]. The broad absorption and narrow emission characteristics of the QDs make it possible to perform multicolor imaging with a single excitation source [28]. As a result of the quantum confinement effect that QDs experience as the particle size becomes smaller than the Bohr exciton radius, the absorption and emission profiles, and hence the color, can be tuned by changing the particle size of the QDs [29–32]. The high fluorescence quantum yield of the QDs and their resistance to photobleaching make them good candidates for fluorescent tagging in biological applications [27]. There have been many reports using QDs for labeling cells, live embryos, tumor cells, antibodies, proteins, and single-stranded oligonucleotides [33–35]. Until now QD labeling of plasmid DNA has not been attempted due to its large size and the perception that any covalent modification of the DNA template might alter the integrity of the plasmid [22]. An ideal probe for imaging the intracellular trafficking of DNA or any other molecule should include the following features: compatibility with cellular substrates, absence of toxicity, high stability, and capacity to emit a high-intensity signal upon illumination over long periods of time. In addition, the btaggedQ plasmid DNA should remain functional and thereby serve as a template for gene transcription upon arrival in the nucleus. To address these issues, we have developed a novel procedure for labeling plasmid DNA using quantum dots. This method involves covalent conjugation of plasmid DNA to phospholipid/ polyethylene oxide-encapsulated cadmium selenide/zinc sulfide core/shell QDs using a peptide nucleic acid–Nsuccinimidyl-3-(2-pyridylthio)propionate (PNA–SPDP) linker [36,37], which facilitates tagging of the plasmid without interfering with its function. After defining the conditions for reproducible conjugation of QDs to plasmid DNA (confirmed by gel electrophoresis and scanning probe and confocal microscopy), we show that QD-tagged DNA can transfect cells with high efficiency and that plasmid DNA intracellular trafficking can be followed through time.
RESULTS Conjugation of Plasmid DNA with Quantum Dots To impart water solubility and reactivity toward thiol groups (used for conjugation with the SPDP–PNA label), we encapsulated the CdSe/ZnS (core/shell) hydrophobic QDs in a 80:20 mixture of polyethylene glycol (PEG)-functionalized phospholipids, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (PEGPE2000) and its maleimide-functionalized analogue 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG(2000)maleimide). These micellar QD dispersions were stable over the 4-month study period
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and may remain stable for considerably longer. We selected the SPDP–PNA linker since it has reactive sites for both the plasmid DNA and the QDs. There are –seven to nine PNA binding sites on each plasmid and we hybridized the SPDP–PNA with the plasmid DNA via triplex formation as presented in Fig. 1. This was followed by reduction of the disulfide bond in the SPDP and reaction with the maleimide group of lipid-encapsulated QDs. For this conjugation reaction, we used a 20-fold excess of QDs and removed the unreacted free QDs using a 1000-kDa membrane and washing three times with Hepes buffer followed by ultracentrifugation. The QD–DNA conjugates were retained above the membrane while the unreacted QDs were washed out, as confirmed by gel electrophoresis (Figs. 2A and 2B). We used excess QDs to maximize the probability that each plasmid DNA molecule was conjugated to one or more QDs. Confirmation of QD–DNA Conjugation To confirm QD–DNA conjugation, we used gel electrophoresis and atomic force microscopy (AFM) confocal imaging. The gel electrophoresis detection method was based on UV-excited (300 nm) photoluminescence emission of both the QDs and the SYBR gold nucleic acid stain. Figs. 2A and 2B show the gel before and after staining with SYBR gold, respectively. The SYBR gold, used to stain the DNA, quenches the QDs and therefore only the bands corresponding to DNA are visualized in Fig. 2B. On the other hand, only the bands corresponding to QDs are visualized in Fig. 2A. Lanes 1 and 3 are controls showing the movement of the free DNA and free QDs, respectively. As expected the movement of the physical mixture (lane 2) is the same as that of the QDs and DNA alone. Free QDs were present in the first filter wash and a small amount in the second wash; however, QDs were not evident in the final wash (Fig. 2A, lanes 6, 7, and 8, and 2B, lanes 6 and 7). A small amount of free DNA was present in the first filter wash but DNA was not evident in the subsequent washes (Fig. 2A, lanes 6 and 7, and 2B, lanes 6, 7, and 8). In addition, following purification through the 1000-kDa membrane and ultracentrifugation of the QD–DNA conjugates, there was no evidence of free DNA in the supernatant (lane 5), confirming the absence of free DNA in the purified QD–DNA conjugate preparation. The purified QD–DNA conjugate showed two bands, one remaining in the well and one with slightly higher mobility than the QDs alone. Both of these bands contained QDs as well as DNA (Figs. 2A and 2B, lane 4). We calculated the relative proportions of QDs and DNA in the conjugate by measuring the absorbance of the free QDs and the free DNA in the pooled filtrates at 573 and 260 nm, respectively. We converted these values to their respective molar concentrations and subtracted
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FIG. 1. Schematic illustration of conjugation of QDs to plasmid DNA. (A) Attachment of PNA-decorated QDs onto plasmid DNA (gWIZ) using pGeneGrip motif [36]. (B) Water solubilization and maleimide functionalization of CdSe/ZnS core/shell QDs and (C) synthetic route for covalently attaching PNA–SPDP linkages onto the maleimide functionalities of QDs (B).
them from the concentrations initially added. According to this calculation the conjugates contained approximately –seven to nine QDs per DNA. This indicates that a few DNA molecules were not fully substituted due to steric hindrance although there are 10 potential PNA binding sites per DNA. The efficiency of the DNA conjugation was approximately 88%, whereas approximately 56.5% of the QDs were lost during purification. Figs. 2C and 2D show AFM images of the plasmid DNA and the QD–DNA conjugates on a mica substrate. The observed heights of the DNA strands for both the QD– DNA conjugate and the naked DNA were approximately 1.1–1.2 nm. The width of the DNA strand was approximately 2 nm, which is comparable to that reported for the DNA double helix [38–40] (Fig. 2C). These results are based on height sections measured on the AFM images.
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In Fig. 2D, two plasmid DNAs are observed overlapping each other (indicated by arrowheads pointing to the relaxed and supercoiled DNA), with several QDs attached to each plasmid DNA (shown as bright white spots of approximately 5–6 nm in height in the two-dimensional image). The QDs are clustered on the plasmid DNA ring, presumably at the PNA binding sites. We confirmed the overlap between the two DNA strands by analysis based on average heights of segments on the strands. We analyzed segments I, II, and III indicated by arrows on Fig. 2D and the average heights were 0.52, 1.16, and 1.50 nm, respectively. These data indicate a relaxed plasmid strand at I, a supercoiled plasmid strand at II (twice the average height of I), and the overlap of the relaxed and supercoiled plasmid strands at III (three times the average height of I).
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FIG. 2. Characterization of QD–DNA conjugates. (A and B) Gel electrophoresis (A) before and (B) after staining with SYBR gold: (lane 1) plasmid DNA alone, (lane 2) physical mixture of QDs and plasmid DNA, (lane 3) QDs alone, (lane 4) purified QD–DNA conjugates, (lane 5) supernatant after ultracentrifugation of purified QD–DNA conjugate, (lane 6) first wash filtrate, (lane 7) second wash filtrate, and (lane 8) third wash filtrate. (C and D) Topographical atomic force micrographs: (C) DNA alone and (D) QD–DNA conjugates on a mica substrate. Arrowheads in (D) indicate the QD clusters on the ring structure of two plasmid DNAs. The arrows indicate the different segments, I, II, and III, on the DNA strands of the conjugate. Based on average height analysis, the segments indicate a relaxed plasmid strand at I, a supercoiled plasmid strand at II (twice the average height of I), and the overlap of the relaxed and supercoiled plasmid strands at III (three times the average height of I).
Long-Term Photostability of QD–DNA Conjugates To determine the relative photostability of QD–DNA conjugates versus DNA complexes with the organic dye, rhodamine (rhodamine–PE/DNA complexes) (Fig. 3), we exposed both fluorophores to continuous laser excitation (543 nm) for 100 min. The rhodamine dye started to photobleach within 10 min and by 50 min the dye was no longer observable. Conversely, there was no apparent photobleaching of QD conjugates throughout the study period. Cellular Uptake of Free QDS, QD–DNA Conjugates, and the Physical Mixture Following 3 h of transfection, we investigated uptake into CHO-K1 cells by staining with the Hoechst nuclear stain, washing to remove material that was not internalized, and observing under epifluorescence microscopy (Figs. 4A– 4D). Due to decreased clarity upon overlapping the images obtained from the red and blue channels with the transmitted image, the transmitted image is not included (only the red and blue channel overlapped images are shown). The QDs alone were relatively unsuccessful in penetrating the cells. As shown in Fig. 4A (arrowheads), only one or two QDs (red dots) were observed in the cytoplasm. In the presence of Lipofectamine2000 the QDs associated with the cationic lipids as a result of charge interactions to form aggregates. We observed these aggregates within the cell cytoplasm as a few red dots (shown as arrowheads in Fig. 4B). The uptake of the physical mixture (QDs/DNA/ Lipofectamine) was similar to that of QDs/Lipofectamine
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alone (shown as arrowheads in Fig. 4C). In the case of the QD–DNA conjugates (Fig. 4D) we observed many more QDs within the cytoplasm (red dots, arrowheads shown) and within the nuclear/perinuclear regions (pink dots due to colocalization of the QDs with the blue nuclear stain indicated by arrows). Expression of the QD–DNA Conjugates in Transfected Cells We performed time-lapse imaging experiments using confocal microscopy to assess the functionality of the conjugates via EGFP gene expression (Fig. 5). The conjugates were restricted to a focal plane located above the cells prior to cellular uptake and were therefore not visualized until internalization into the cytoplasm. At the initial time points (0–3 h), the translocation of the QD–DNA conjugates from the outside to the inside of the cells was clearly shown to increase with time. As early as 1 h postincubation we observed the QD–DNA conjugates either at the cell membrane or in the cytoplasm. By 3 h postincubation, most of the QD–DNA conjugates were internalized within the cytoplasm. Low-level EGFP expression was observed by 6 h postincubation and increased steadily with time. Since the cells were at different stages of the cell cycle and QD–DNA nuclear uptake does not occur at the same time in all cells, higher levels of expression in some cells masked EGFP expression in other areas. Therefore, we recorded a time-lapse sequence of cells with initially very low levels of expression at 6 h (Fig. 5, 6–11 h).
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FIG. 3. Comparison of QD and rhodamine long-term photostability. Consecutive images of (A, B, and C) QD–DNA conjugates and (D, E, and F) rhodamine complexes (Rh–PE/DNA) in the presence of Lipofectamine2000 taken at different time points after 3 h transfection in CHO-K1 cells. (G) Graph representing the change in fluorescence intensity of QDs and rhodamine complexes for a total period of 100 min, during which both the fluorophores were exposed to continuous excitation at 543 nm (Kr/Ne laser) (mean F SD, n = 3). QD–DNA conjugates were added as 1.469 1013 particles per 2.5 106 cells per well. Bars, 25 Am.
The arrows indicate a single cell in which EGFP expression increases steadily with time. The bright red dots of QD– DNA conjugates were clearly visible along with the EGFP expression 6–11 h postincubation, and this sequence demonstrated a correlation between QD–DNA conjugate uptake and EGFP expression. Z sections revealed the
locations of the red QDs clearly within the boundaries of the cell membrane (data not shown). By 11 h postincubation most of the cells (approximately 66%) showed EGFP expression. In approximately 16% of the cells, QDs were observed but EGFP expression had not taken place and this may be due to factors such as cells being at
FIG. 4. Cellular uptake studies of conjugated and unconjugated QDs. Overlapped epifluorescence images of: the nuclear stain, Hoechst 33342 (DAPI excitation and Fura-2 emission filters), and red QDs (Texas red excitation filter and Quad emission filters) after 3 h incubation of materials (described below) in live CHO-K1 cells, followed by washing to remove unbound material. (A) QDs alone, (B) QDs in the presence of Lipofectamine2000 mixture, (C) physical mixture of QDs and plasmid DNA in the presence of Lipofectamine2000, and (D) QD–DNA conjugates in the presence of Lipofectamine2000. The arrowheads (A–D) indicate red dots that are in the cytoplasm or outside the cell membrane and arrows in D indicate colocalization of red dots with the blue nuclear stain (shown as pink dots). QD–DNA conjugates were added as 1.469 1013 particles per 2.5 106 cells per well. Bar, 25 Am.
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FIG. 5. In vitro expression and long-term imaging using QD–DNA conjugates. Time-lapse imaging experiments performed using confocal microscopy to track uptake of QD–DNA conjugates (in the presence of Lipofectamine2000) and subsequent EGFP expression in CHO-K1 cells (0–11 h). The arrows indicate an increase in the intensity of EGFP expression associated with the uptake of red QD–DNA conjugates within a selected cell. QD–DNA conjugates were added as 1.469 1013 particles per 2.5 106 cells per well. Bars, 25 Am.
different stages of the cell cycle, failure of the DNA to translocate to the nucleus, or cell stress. To assess the integrity of QD-conjugated plasmid DNA for gene expression in comparison to unlabeled plasmid DNA, we performed a quantitative estimate of transfection efficiency using a Fluostar Optima fluorescence reader at 24 h postincubation. There was no significant difference between the QD-labeled plasmid DNA (trans-
fection efficiency of 62 F 2%, n = 3) and the control plasmid DNA (transfection efficiency of 65 F 2%, n = 3) (data not shown). This demonstrated that QD conjugation did not have an adverse effect on the functionality of the plasmid DNA. Intracellular Tracking Studies for QD–DNA Conjugates We performed intracellular tracking studies in CHO-K1 FIG. 6. Tracking of QD–DNA conjugates. Confocal images following incubation with QD–DNA conjugates at different time points in CHO-K1 cells in the presence of Lipofectamine2000 are shown. Cells were stained with SYTO-16 nuclear stain. Nuclear uptake of QD–DNA conjugates is demonstrated by red QD–DNA conjugates imaged using a 633 nm He–Ne laser line excitation source, which does not excite the SYTO-16 green nuclear stain (ex 488/ em 518). (A, C, and E) Representative images using the red channel (633 nm) at 6, 10, and 24 h, respectively. (B, D, and F) Overlapped images of red and green channel at 6, 10, and 24 h, respectively. Arrows indicate the presence of QD–DNA conjugates (as yellow/orange dots) at the perinuclear/nuclear region at 6-, 10-, and 24-h time points. QD–DNA conjugates were added as 1.469 1013 particles per 2.5 106 cells per well. Bar, 25 Am.
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cells using pGeneGrip (gWIZ-LUC) plasmid conjugated to QDs and the SYTO-16 green nuclear stain. We used the luciferase plasmid since EGFP does not allow visualization of the nucleus due to its bright fluorescence effects that mask any nuclear stain (Figs. 6A–6F). Since the SYTO-16 can be excited at 543 nm, the normal excitation wavelength of the QDs in these experiments, the QDs were excited at 633 nm to minimize interference. It should be noted that the intensity of the QDs is lower in this experiment since excitation at 633 nm is suboptimal. Figs. 6A, 6C, and 6E are photomicrographs of the red channel for the QD–DNA conjugate and Figs. 6B, 6D, and 6F are micrographs showing the overlapped images of the green and the red channels. Comparison of Figs. 6A, 6C, and 6E with 6B, 6D, and 6F facilitates determination of the location of the QD conjugates within the cells. In these photomicrographs the intensity of the SYTO dye fluorescence is lower in some cells compared to others as a result of the photobleaching of the organic dye in cells where the laser is focused. SYTO-16 stains DNA and as such shows the nuclear region as green. The cytoplasm is shown as dull green due to staining of the cytoplasmic organelles. In these photomicrographs the intensity of the SYTO dye fluorescence is lower in some cells compared to others as a result of the photobleaching of the organic dye in cells where the laser is focused. The QD–DNA conjugates are observed as yellow/orange dots due to staining of the DNA part of the QD conjugates by the SYTO dye. There was a gradual increase in the number of QD–DNA conjugates localized within the cells over time (0- to 5-h data not shown). The accumulation of QD–DNA conjugates reached a maximum at about the 6-h time point (approximately 78% are within the cytoplasm and approximately 22% are within the nuclear/perinuclear region, Fig. 6B).
FIG. 7. MTT toxicity assay showing percentage cell survival following incubation with QDs and QD–DNA conjugates in CHO-K1 cells after 24 h transfection. QD–DNA conjugates were added as 2.35 1012 particles per 0.4 106 cells per well. The values represent percentage cell survival (means F SD), n = 4.
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Approximately 61% of the QD-labeled plasmid DNAs are colocalized within the nuclear/perinuclear region by the 10-h time point (indicated by arrows in Fig. 6D). At 24 h the QD–DNA conjugates again appear to be concentrated in the nuclear/perinuclear region (approximately 68%) (indicated by arrows in Fig. 6F). This study is in agreement with the EGFP study detailed above, in which approximately 66% of the cells expressed EGFP by 11 h (Fig. 5). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide (MTT) Toxicity Assay We performed an MTT assay to compare any potential cytotoxicity upon incubation with free QDs vs QD–DNA conjugates in CHO-K1 cells. The assay showed 75% cell survival for QD–DNA conjugates, compared to 89, 83, and 81% cell survival for Hepes buffer alone, QDs alone, and the physical mixture of QD and DNA, respectively (Fig. 7). These observations indicated that the QDs are not toxic to the cells.
DISCUSSION Plasmid DNA was successfully linked to QDs using the highly specific SPDP–PNA linker. The conjugation is clearly shown in the AFM images, in which several QDs appear to be clustered, presumably at the PNA binding sites on the plasmid DNA. As well as confirming the QD–DNA conjugation, AFM imaging proved to be an ideal technique to study plasmid DNA since the DNA structure is not altered during either processing or imaging, and the samples can be imaged directly without any further treatment. The AFM image of plasmid DNA on mica is similar to that reported by other researchers in terms of structure and size [38–40] (Fig. 2C). The plasmid DNA is clearly visible in the AFM images and the dimensions (of the DNA strand) are approximately 2 nm in width, 1.1–1.2 nm in height, and the ring structure is on the order of 200–300 nm. These values are in agreement with those previously reported in the literature determined by TEM as well as AFM and cryo-AFM imaging [37–39]. Although the actual diameter of the whole ring structure shown in Figs. 2C and 2D is on the order of 200– 300 nm, when fully stretched a 5.7-kb circular plasmid DNA would be expected to have a diameter of 500 nm. This implies that the DNA is partially folded, as can be visualized by the nonuniform height observed in the AFM images. The bright spots on Fig. 2C of approximately 1 nm in height are typical of any single- or double-stranded DNA [39,40]. The QD structures on each of the two plasmid DNA conjugates determined by AFM (approximately 5–6 nm in height) indicate that they are attached only at a particular region on the ring structure of the plasmid and are sequence specific. AFM images revealed relaxed and supercoiled forms of the QD–DNA conjugates. The bright spots in Fig. 2D not associated with the DNA conjugate are a result of the buffer salts used in the preparation of the conjugates. This was confirmed by the section analysis of the images
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that shows that the heights of these buffer salts are much lower than those of free QDs (data not shown). The QD–DNA conjugation was also confirmed by gel electrophoresis. From the gel electrophoresis it appears that the purification process removes all free QDs by the end of the third wash and free DNA by the end of second wash (Figs. 2A and 2B, lanes 6, 7, and 8). The purified DNA conjugate showed two bands (Figs. 2A and 2B, lane 4). This is considered to be a consequence of the amount of QDs conjugated to the DNA. Since the QDs are added in excess and each DNA has –seven to nine PNA binding sites, the majority of the DNA is expected to contain a high ratio of QDs; however, a minority will contain few or single QDs (i.e., the ratio of QD to DNA is expected to be skewed). The lower band in Figs. 2A and 2B, lane 4, is considered to represent conjugates with approximately one QD per DNA. This lower band travels at a faster rate than that of the QDs alone as a result of the charge on the DNA. The higher band in Figs. 2A and 2B, lane 4, is thought to be due to conjugates that contain multiple QDs. The relative weight of these conjugates is considered to result in their inability to move under the conditions applied. It is evident from lane 4 in Fig. 2B that conjugates containing multiple QDs are in excess (since the lower band is faint). The bands in lane 4, Fig. 2A, are complicated by the differing quantum efficiencies of conjugates with single QDs compared to those with multiple QDs. The close proximity of the PNA binding sites on the DNA is expected to cause some degree of self-quenching of the QDs where multiple QDs are attached [41]. Therefore, although conjugates with multiple QDs are in greater numbers (upper band in Fig. 2B, lane 4), the lower band is easier to discern in lane 4 in Fig. 2A than in 2B. QD–DNA conjugates represent an ideal method of tracking plasmid DNA since they produce a highly stable fluorescent signal that can be used for long-term studies (such as the 24-h time-lapse confocal imaging and photostability experiments) and the conjugation does not affect the DNA functionality. This technique enabled correlation between DNA distribution and protein expression by simultaneous tracking using time-lapse imaging. In addition, the QD–DNA conjugation facilitated tracking within the cellular compartments of live cells when used in combination with a nuclear stain. Previous reports show that PNA linkers have been labeled with organic fluorophores such as fluorescein and rhodamine to conjugate plasmid DNA to follow DNA delivery [36,37]. These studies involved imaging live cells posttransfection; however, the organic fluorophorelabeled plasmid DNA could not be used for long-term time-lapse imaging [42–45] as these dyes are sensitive to photobleaching during prolonged exposure to UV and O2, which compromises their utility. Another important
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aspect to be considered in bioimaging is cell viability. The QD-labeled plasmid DNA did not affect the CHO-K1 cell viability even up to 24 h posttransfection as shown by MTT assay, in which cell survival was comparable to that of a buffer-alone control [43,46]. Previous studies show that QD micelles conjugated to single-stranded DNA and injected into live Xenopus embryos were stable, biocompatible, and nontoxic when used to observe the cell lineage by following QD fluorescence throughout the embryo development up to the tadpole stage[34]. QD–DNA conjugates may be used in future studies to gain a better understanding of the efficiencies of the various processes involved in the cellular and nuclear uptake of plasmid DNA.
MATERIALS
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METHODS
Synthesis and water solubilization of CdSe/ZnS QDs. CdSe quantum dots (Emax = 600 nm) were synthesized using organometallic precursors according to the method of Murray et al. [32]. In short, dimethylcadmium and selenium were dissolved in trioctylphosphine and this precursor mixture was injected into hot (3008C) trioctylphosphine oxide solvent. CdSe QDs formed instantaneously and were grown until the desired particle size was attained. Subsequent ZnS coating was performed by slowly adding diethyl zinc and hexamethyldisilthiane, which were used as Zn and S precursors, respectively. This was followed by annealing at 1508C for several hours. Encapsulation of CdSe/ZnS QDs. CdSe/ZnS QDs were encapsulated in phospholipid/PEG micelles as reported by Dubertret et al. [34] but a maleimide functionalized PEG lipid was used as described below to provide reactive sites toward conjugation with the plasmid DNA. An 80:20 mixture of PEG-PE2000 and maleimide functionalized PEG lipid, DSPE-PEG(2000)maleimide (Avanti Polar Lipids, Alabaster, AL, USA), was added to the QD dispersion in chloroform. After solvent evaporation, a thin film was formed, to which distilled water or PBS buffer was added followed by vigorous shaking to produce a clear QD micellar dispersion. Conjugation of Plasmid DNA to QDs. pGeneGrip (gWIZ-GFP plasmid) and SPDP–PNA linker were purchased from Gene Therapy Systems (Genlantis, San Diego, CA, USA). The pGeneGrip plasmid contains a PNA binding site and has an EGFP reporter gene. The PNA binding sequence [37] within the plasmid is [AG]20CCATGG[AG]20 and the PNA used has the sequence 5V-TCTCTCTC-O-O-O-JTJTTJTJT-3V, where J is a pseudoisocytosine and O is 8-amino-3,6-dioxaoctanoic acid linker [36]. Reduction of the SPDP–PNA-labeled gene grip vector was conducted using 3 mM tris-(2-carboxyethyl)phosphine, hydrochloride) (Invitrogen, Carlsbad, CA, USA) and the coupling reaction was immediately carried out by mixing with maleimide functionalized QDs prepared in 100 mM Hepes buffer (pH 7.2) (a 20-fold excess of QDs) and incubating overnight at room temperature (schematic illustration of conjugation of QDs to plasmid DNA, Fig. 1). For experiments on intracellular tracking in which nuclear staining was performed, a different pGeneGrip plasmid (gWIZLuc plasmid) (Genlantis) that has a luciferase reporter gene and contains a PNA binding site similar to that of gWIZ-GFP plasmid was used. The luciferase gene was preferred since the GFP plasmid results in bright fluorescence that obscures the location of QDs. Purification of the QD–DNA conjugates. The QD–DNA conjugate was separated from free QDs upon passage through a 100-kDa Centricon (Millipore, Bedford, MA, USA) to remove excess salts and subsequent passage through a 1000-kDa cellulose ester-based membrane (Spectrum Labs, CA, USA) with multiple wash steps to remove any unbound QDs and plasmid DNA (the filtrates were collected separately) followed by ultracentrifugation at 45,000 rpm, 48C, for 2 h (TLA-100 ultracentrifuge; Beckman Coulter, CA, USA (100,000 rpm equals 541,000g)). The amount
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of QDs conjugated with DNA was quantified by subtracting the free QDs in the filtrate from the amount of QDs originally added. Direct quantification of the QD–DNA conjugate was not possible since the DNA would interfere with the UV measurement of the QDs (260 nm). The amount of DNA conjugated to QDs was similarly quantified by subtracting any residual free DNA in the filtrate from the amount of DNA initially used for conjugation. For this, the filtrate was concentrated and ultracentrifuged for 3 h at 70,000 rpm and 48C. Agarose gel (0.5% w/v) electrophoresis was run at 67 V/cm, for 2 h. The gel was stained with SYBR Gold nucleic acid gel stain (Molecular Probes, Invitrogen, Carlsbad, CA, USA), visualized using a standard 300 nm UV transilluminator, and photographed using Polaroid 667 black and white film. QDs alone and a physical mixture of QDs and plasmid DNA were used as controls. Plus and minus signs show the direction of the gel run. Atomic force microscopy. AFM experiments were performed using a mica substrate that was freshly cleaved at the time of imaging. Five microliters of diluted sample of either QD–DNA conjugates or free QDs was deposited onto the mica substrate. After 5-min incubation, the surface was washed five or six times with distilled water to remove unbound material and salts, air dried for 15 min, and viewed under AFM. AFM observations were carried out in air in a tapping mode with a MFP-3D atomic force microscope (Asylum Research, Santa Barbara, CA, USA). The silicon probe cantilever was used (OMCL-AC160TS; Olympus) with a nominal spring constant of 42 N/m. The height analysis of the DNA strands was performed either by measuring the value at a single point or by averaging the heights within a segment with a focal width of 7 points on the DNA strand. Photostability. The photostability of QD–DNA conjugates was compared to that of DNA complexes with the organic dye, rhodamine (Rh–PE/DNA complexes). In this experiment 2.6 Ag of gWIZ-Luc plasmid was complexed with Rh–PE at a ratio of 0.3/1 w/w (Rh–PE/plasmid). Both the complexes were transfected into CHO-K1 cells in the presence of Lipofectamine2000. This experiment was performed by exposing both the fluorophores to constant laser excitation (543 nm) for 100 min. Consecutive images were taken every 10 min. Cell transfection. A CHO-K1 cell line was purchased from the ATCC (Manassas, VA, USA). Transfection studies were conducted by plating CHO-K1 cells (0.8–1 105 cells/well) in 8-well LabTekII chamber glass slides (Nalge Nunc International, Rochester, NY, USA). Once the cultures reached 70–80% confluence, the cells were washed with Hanks’ balanced salt solution (HBSS), and transfections were carried out in serum-free F12K medium (Invitrogen). Lipofectamine2000 (Invitrogen) was used as the transfection reagent following the manufacturer’s protocol. QD–DNA conjugates, physical mixtures (gWIZ-GFP plasmid DNA + QD), or QDs alone were mixed with Lipofectamine2000 individually and incubated with cells. Unconjugated (gWIZ-GFP) plasmid DNA and QDs alone with and without Lipofectamine2000 were used as controls. After a 3-h incubation period, the transfection mixtures were removed, and the cells were washed with HBSS and incubated in F12-K medium with 10% FBS, under 5% CO2 at 378C, until further use. A similar procedure was followed when gWIZ-Luc plasmid DNA was used. Quantitative reporter gene expression analysis was conducted by harvesting the cells after a 24-h transfection period, adding them to a 96-well plate, and reading in a FLUOstar Optima microplate reader, Model 413-101 (BMG Labtech, Durham, NC, USA). QD–DNA conjugates were added as 1.469 1013 particles per 2.5 106 cells per well during transfection in CHO-K1 cells. Equivalent amounts of QDs were added for samples containing physical mixtures and QDs alone. Confocal microscopy. A confocal laser scanning microscope equipped with Ar/Kr, Kr/Ne, and He/Ne lasers (Leica SP2 spectral confocal microscope, Bannockburn, IL, USA) was used for live/fixed cell imaging. For time-lapse imaging, a Bioptechs DT4 open dish system (Bioptechs, Inc., Butler, PA, USA) was used to maintain the temperature of the CHO-K1 cells during observation in a L-15 carbon dioxide-free medium. Quantitative estimation of the uptake of QD–DNA conjugates at different time points was performed by counting the number of yellow/orange dots within the nucleus or at the perinuclear region (double-blind test).
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Nuclear staining. CHO-K1 cells were grown on sterile eight-well LabTekii chamber slides and transfected as described above. The chamber slides were then washed with HBSS and stained with nuclear staining dye Hoechst 33342 (in the cellular uptake experiment) or green SYTO-16 (in the study involving tracking of QD–DNA conjugates) at 1 or 2 AM, respectively, for 15 min at 378C and washed twice with HBSS to remove excess dye. The cells were imaged immediately using either a Zeiss fluorescence microscope (Hoechst staining) or Leica confocal laser scanning microscope (SYTO-16 staining) as described above. QD toxicity study. To compare the potential toxicity of the QDs and QDconjugated and unconjugated plasmid DNA, an MTT assay was performed in CHO-K1 cells. MTT is a quantitative colorimetric assay that detects living cells based on the action of mitochondrial dehydrogenases of viable cells that cleave the tetrazolium ring, forming purple formazan crystals. Ten thousand cells/well were plated in 96-well plates, grown to 70–80% confluence, washed with HBSS, and transfections carried out in serum-free F12-K medium in the presence of Lipofectamine2000. QD–DNA conjugates were added as 2.35 1012 particles per 0.4 106 cells per well. Equivalent amounts of QDs were added for samples containing physical mixtures and QDs alone. After 22 h of transfection, 25 Al of MTT solution (5 mg/ml) (Invitrogen) was added and incubated for 2 h (CO2 incubator at 378C). Cells were lysed with lysis buffer (20% SDS in 50% dimethyl formamide) and further incubated for 5–6 h to dissolve the formazan crystals. The absorbance of the resulting purple solution was measured spectrophotometrically at k = 580 nm.
ACKNOWLEDGMENTS The authors thank Shafiuddin Shafiuddin of the Department of Animal Science, Center of Excellence for Vaccine Research, University of Connecticut, for his valuable suggestions in QD–DNA conjugation work. This research was supported in part by the Parenteral Drug Association Foundation Schering–Plough Grant, NSF DMI 0303950, ONR N000LY0610016PR and ARO DAAD-02-1-0381. RECEIVED FOR PUBLICATION SEPTEMBER 14, 2005; REVISED MARCH 7, 2006; ACCEPTED MARCH 10, 2006.
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Labeling and Intracellular Tracking of Functionally Active Plasmid DNA with Semiconductor Quantum Dots Charudharshini Srinivasan,1 Jeunghoon Lee,2 Fotios Papadimitrakopoulos,2 Lawrence K. Silbart,3 Minhua Zhao,2 Diane J. Burgess,1,* 1 Department of Pharmaceutical Sciences, University of Connecticut, 69 North Eagleville Road, Unit 3092, Storrs, CT 06269, USA Nanomaterials Optoelectronics Lab, Polymer Program, Department of Chemistry, Institute of Materials Science, University of Connecticut, 97 North Eagleville Road, Unit 3136, Storrs, CT 06269, USA 3 Department of Animal Science, Center of Excellence for Vaccine Research, University of Connecticut, 1390 Storrs Road, Unit 4163, Storrs, CT 06269, USA 2
*To whom correspondence and reprint requests should be addressed. Fax: +1 860 486 0538. E-mail: [email protected].
Available online 12 May 2006
Semiconductor nanocrystal quantum dots (QDs) allow long-term imaging in the cellular environment with high photostability. QD biolabeling techniques have previously been developed for tagging proteins and peptides as well as oligonucleotides. In this contribution, QD-decorated plasmid DNA was utilized for the first time for long-term intracellular and intranuclear tracking studies. Conjugation of plasmid DNA with phospholipid-coated QDs was accomplished using a peptide nucleic acid (PNA)–N-succinimidyl-3-(2-pyridylthio) propionate linker. Gel electrophoresis and confocal and atomic force microscopy (AFM) were used to confirm the structure of QD–DNA conjugates. AFM imaging also revealed that multiple QDs were attached in a cluster at the PNAreactive site of the plasmid DNA. These QD–DNA conjugates were capable of expressing the reporter protein, enhanced green fluorescent protein, following transfection in Chinese hamster ovary (CHO-K1) cells with an efficiency of ca. 62%, which was comparable to the control (unconjugated) plasmid DNA. Key Words: plasmid DNA, transfection, quantum dots, labeling, QD–DNA conjugates, DNA tracking, nuclear staining, gene expression
INTRODUCTION Nonviral gene delivery systems have gained immense importance, primarily due to their considerable in vivo safety compared to viral vectors [1–4]. In particular, cationic [5–7] and recently anionic liposomes [8–10], as well as cationic polymers [11,12], and nanoparticles [13] are under investigation as nonviral vectors for DNA therapeutics. However, the poor transfection efficiency of these systems in vivo has impeded their development as therapeutics [3,5,14]. To realize the full potential of such delivery systems, a better understanding of the critical steps in the transfection process is needed [15–17]. Methods commonly employed in studying intracellular plasmid trafficking involve labeling of the delivery vehicles (liposomes, polymers, etc.) with commercially available organic dyes [18,19]. The intracellular distribution of poly(dl-lactide-co-glycolide) nanoparticles has been investigated via confocal and fluorescence microscopy using nanoparticles loaded with the lipophilic fluorescent dye 6-coumarin [20,21]. Small molecules, such as oligonucleotides and
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cell-specific antibodies, may also be labeled with organic dyes; however, it is difficult to label large molecules such as plasmid DNA [22]. Attempts to tag plasmid DNA with organic fluorophores have involved cumbersome procedures and the possibility of irreversible changes to the DNA with consequent loss of functional activity [22–24]. Such organic fluorophores are not ideal labels since they rapidly undergo photobleaching (within seconds to a few hours), which renders them unsuitable for long-term imaging studies. Colloidal gold has also been utilized to track plasmid DNA using high-resolution electron microscopy. The colloidal gold and the plasmid DNA are physically associated by charge interactions and are not suitable for live cell fluorescence imaging. Moreover, the plasmid DNA is rendered nonfunctional for gene expression [22–26]. In recent years, semiconductor quantum dots (QDs) have attracted the attention of many research groups due to their scientific and technological significance in microelectronics, optoelectronics, and cellular imaging
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[27]. The broad absorption and narrow emission characteristics of the QDs make it possible to perform multicolor imaging with a single excitation source [28]. As a result of the quantum confinement effect that QDs experience as the particle size becomes smaller than the Bohr exciton radius, the absorption and emission profiles, and hence the color, can be tuned by changing the particle size of the QDs [29–32]. The high fluorescence quantum yield of the QDs and their resistance to photobleaching make them good candidates for fluorescent tagging in biological applications [27]. There have been many reports using QDs for labeling cells, live embryos, tumor cells, antibodies, proteins, and single-stranded oligonucleotides [33–35]. Until now QD labeling of plasmid DNA has not been attempted due to its large size and the perception that any covalent modification of the DNA template might alter the integrity of the plasmid [22]. An ideal probe for imaging the intracellular trafficking of DNA or any other molecule should include the following features: compatibility with cellular substrates, absence of toxicity, high stability, and capacity to emit a high-intensity signal upon illumination over long periods of time. In addition, the btaggedQ plasmid DNA should remain functional and thereby serve as a template for gene transcription upon arrival in the nucleus. To address these issues, we have developed a novel procedure for labeling plasmid DNA using quantum dots. This method involves covalent conjugation of plasmid DNA to phospholipid/ polyethylene oxide-encapsulated cadmium selenide/zinc sulfide core/shell QDs using a peptide nucleic acid–Nsuccinimidyl-3-(2-pyridylthio)propionate (PNA–SPDP) linker [36,37], which facilitates tagging of the plasmid without interfering with its function. After defining the conditions for reproducible conjugation of QDs to plasmid DNA (confirmed by gel electrophoresis and scanning probe and confocal microscopy), we show that QD-tagged DNA can transfect cells with high efficiency and that plasmid DNA intracellular trafficking can be followed through time.
RESULTS Conjugation of Plasmid DNA with Quantum Dots To impart water solubility and reactivity toward thiol groups (used for conjugation with the SPDP–PNA label), we encapsulated the CdSe/ZnS (core/shell) hydrophobic QDs in a 80:20 mixture of polyethylene glycol (PEG)-functionalized phospholipids, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (PEGPE2000) and its maleimide-functionalized analogue 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N[maleimide(polyethylene glycol)-2000] (ammonium salt) (DSPE-PEG(2000)maleimide). These micellar QD dispersions were stable over the 4-month study period
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and may remain stable for considerably longer. We selected the SPDP–PNA linker since it has reactive sites for both the plasmid DNA and the QDs. There are –seven to nine PNA binding sites on each plasmid and we hybridized the SPDP–PNA with the plasmid DNA via triplex formation as presented in Fig. 1. This was followed by reduction of the disulfide bond in the SPDP and reaction with the maleimide group of lipid-encapsulated QDs. For this conjugation reaction, we used a 20-fold excess of QDs and removed the unreacted free QDs using a 1000-kDa membrane and washing three times with Hepes buffer followed by ultracentrifugation. The QD–DNA conjugates were retained above the membrane while the unreacted QDs were washed out, as confirmed by gel electrophoresis (Figs. 2A and 2B). We used excess QDs to maximize the probability that each plasmid DNA molecule was conjugated to one or more QDs. Confirmation of QD–DNA Conjugation To confirm QD–DNA conjugation, we used gel electrophoresis and atomic force microscopy (AFM) confocal imaging. The gel electrophoresis detection method was based on UV-excited (300 nm) photoluminescence emission of both the QDs and the SYBR gold nucleic acid stain. Figs. 2A and 2B show the gel before and after staining with SYBR gold, respectively. The SYBR gold, used to stain the DNA, quenches the QDs and therefore only the bands corresponding to DNA are visualized in Fig. 2B. On the other hand, only the bands corresponding to QDs are visualized in Fig. 2A. Lanes 1 and 3 are controls showing the movement of the free DNA and free QDs, respectively. As expected the movement of the physical mixture (lane 2) is the same as that of the QDs and DNA alone. Free QDs were present in the first filter wash and a small amount in the second wash; however, QDs were not evident in the final wash (Fig. 2A, lanes 6, 7, and 8, and 2B, lanes 6 and 7). A small amount of free DNA was present in the first filter wash but DNA was not evident in the subsequent washes (Fig. 2A, lanes 6 and 7, and 2B, lanes 6, 7, and 8). In addition, following purification through the 1000-kDa membrane and ultracentrifugation of the QD–DNA conjugates, there was no evidence of free DNA in the supernatant (lane 5), confirming the absence of free DNA in the purified QD–DNA conjugate preparation. The purified QD–DNA conjugate showed two bands, one remaining in the well and one with slightly higher mobility than the QDs alone. Both of these bands contained QDs as well as DNA (Figs. 2A and 2B, lane 4). We calculated the relative proportions of QDs and DNA in the conjugate by measuring the absorbance of the free QDs and the free DNA in the pooled filtrates at 573 and 260 nm, respectively. We converted these values to their respective molar concentrations and subtracted
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FIG. 1. Schematic illustration of conjugation of QDs to plasmid DNA. (A) Attachment of PNA-decorated QDs onto plasmid DNA (gWIZ) using pGeneGrip motif [36]. (B) Water solubilization and maleimide functionalization of CdSe/ZnS core/shell QDs and (C) synthetic route for covalently attaching PNA–SPDP linkages onto the maleimide functionalities of QDs (B).
them from the concentrations initially added. According to this calculation the conjugates contained approximately –seven to nine QDs per DNA. This indicates that a few DNA molecules were not fully substituted due to steric hindrance although there are 10 potential PNA binding sites per DNA. The efficiency of the DNA conjugation was approximately 88%, whereas approximately 56.5% of the QDs were lost during purification. Figs. 2C and 2D show AFM images of the plasmid DNA and the QD–DNA conjugates on a mica substrate. The observed heights of the DNA strands for both the QD– DNA conjugate and the naked DNA were approximately 1.1–1.2 nm. The width of the DNA strand was approximately 2 nm, which is comparable to that reported for the DNA double helix [38–40] (Fig. 2C). These results are based on height sections measured on the AFM images.
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In Fig. 2D, two plasmid DNAs are observed overlapping each other (indicated by arrowheads pointing to the relaxed and supercoiled DNA), with several QDs attached to each plasmid DNA (shown as bright white spots of approximately 5–6 nm in height in the two-dimensional image). The QDs are clustered on the plasmid DNA ring, presumably at the PNA binding sites. We confirmed the overlap between the two DNA strands by analysis based on average heights of segments on the strands. We analyzed segments I, II, and III indicated by arrows on Fig. 2D and the average heights were 0.52, 1.16, and 1.50 nm, respectively. These data indicate a relaxed plasmid strand at I, a supercoiled plasmid strand at II (twice the average height of I), and the overlap of the relaxed and supercoiled plasmid strands at III (three times the average height of I).
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FIG. 2. Characterization of QD–DNA conjugates. (A and B) Gel electrophoresis (A) before and (B) after staining with SYBR gold: (lane 1) plasmid DNA alone, (lane 2) physical mixture of QDs and plasmid DNA, (lane 3) QDs alone, (lane 4) purified QD–DNA conjugates, (lane 5) supernatant after ultracentrifugation of purified QD–DNA conjugate, (lane 6) first wash filtrate, (lane 7) second wash filtrate, and (lane 8) third wash filtrate. (C and D) Topographical atomic force micrographs: (C) DNA alone and (D) QD–DNA conjugates on a mica substrate. Arrowheads in (D) indicate the QD clusters on the ring structure of two plasmid DNAs. The arrows indicate the different segments, I, II, and III, on the DNA strands of the conjugate. Based on average height analysis, the segments indicate a relaxed plasmid strand at I, a supercoiled plasmid strand at II (twice the average height of I), and the overlap of the relaxed and supercoiled plasmid strands at III (three times the average height of I).
Long-Term Photostability of QD–DNA Conjugates To determine the relative photostability of QD–DNA conjugates versus DNA complexes with the organic dye, rhodamine (rhodamine–PE/DNA complexes) (Fig. 3), we exposed both fluorophores to continuous laser excitation (543 nm) for 100 min. The rhodamine dye started to photobleach within 10 min and by 50 min the dye was no longer observable. Conversely, there was no apparent photobleaching of QD conjugates throughout the study period. Cellular Uptake of Free QDS, QD–DNA Conjugates, and the Physical Mixture Following 3 h of transfection, we investigated uptake into CHO-K1 cells by staining with the Hoechst nuclear stain, washing to remove material that was not internalized, and observing under epifluorescence microscopy (Figs. 4A– 4D). Due to decreased clarity upon overlapping the images obtained from the red and blue channels with the transmitted image, the transmitted image is not included (only the red and blue channel overlapped images are shown). The QDs alone were relatively unsuccessful in penetrating the cells. As shown in Fig. 4A (arrowheads), only one or two QDs (red dots) were observed in the cytoplasm. In the presence of Lipofectamine2000 the QDs associated with the cationic lipids as a result of charge interactions to form aggregates. We observed these aggregates within the cell cytoplasm as a few red dots (shown as arrowheads in Fig. 4B). The uptake of the physical mixture (QDs/DNA/ Lipofectamine) was similar to that of QDs/Lipofectamine
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alone (shown as arrowheads in Fig. 4C). In the case of the QD–DNA conjugates (Fig. 4D) we observed many more QDs within the cytoplasm (red dots, arrowheads shown) and within the nuclear/perinuclear regions (pink dots due to colocalization of the QDs with the blue nuclear stain indicated by arrows). Expression of the QD–DNA Conjugates in Transfected Cells We performed time-lapse imaging experiments using confocal microscopy to assess the functionality of the conjugates via EGFP gene expression (Fig. 5). The conjugates were restricted to a focal plane located above the cells prior to cellular uptake and were therefore not visualized until internalization into the cytoplasm. At the initial time points (0–3 h), the translocation of the QD–DNA conjugates from the outside to the inside of the cells was clearly shown to increase with time. As early as 1 h postincubation we observed the QD–DNA conjugates either at the cell membrane or in the cytoplasm. By 3 h postincubation, most of the QD–DNA conjugates were internalized within the cytoplasm. Low-level EGFP expression was observed by 6 h postincubation and increased steadily with time. Since the cells were at different stages of the cell cycle and QD–DNA nuclear uptake does not occur at the same time in all cells, higher levels of expression in some cells masked EGFP expression in other areas. Therefore, we recorded a time-lapse sequence of cells with initially very low levels of expression at 6 h (Fig. 5, 6–11 h).
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FIG. 3. Comparison of QD and rhodamine long-term photostability. Consecutive images of (A, B, and C) QD–DNA conjugates and (D, E, and F) rhodamine complexes (Rh–PE/DNA) in the presence of Lipofectamine2000 taken at different time points after 3 h transfection in CHO-K1 cells. (G) Graph representing the change in fluorescence intensity of QDs and rhodamine complexes for a total period of 100 min, during which both the fluorophores were exposed to continuous excitation at 543 nm (Kr/Ne laser) (mean F SD, n = 3). QD–DNA conjugates were added as 1.469 1013 particles per 2.5 106 cells per well. Bars, 25 Am.
The arrows indicate a single cell in which EGFP expression increases steadily with time. The bright red dots of QD– DNA conjugates were clearly visible along with the EGFP expression 6–11 h postincubation, and this sequence demonstrated a correlation between QD–DNA conjugate uptake and EGFP expression. Z sections revealed the
locations of the red QDs clearly within the boundaries of the cell membrane (data not shown). By 11 h postincubation most of the cells (approximately 66%) showed EGFP expression. In approximately 16% of the cells, QDs were observed but EGFP expression had not taken place and this may be due to factors such as cells being at
FIG. 4. Cellular uptake studies of conjugated and unconjugated QDs. Overlapped epifluorescence images of: the nuclear stain, Hoechst 33342 (DAPI excitation and Fura-2 emission filters), and red QDs (Texas red excitation filter and Quad emission filters) after 3 h incubation of materials (described below) in live CHO-K1 cells, followed by washing to remove unbound material. (A) QDs alone, (B) QDs in the presence of Lipofectamine2000 mixture, (C) physical mixture of QDs and plasmid DNA in the presence of Lipofectamine2000, and (D) QD–DNA conjugates in the presence of Lipofectamine2000. The arrowheads (A–D) indicate red dots that are in the cytoplasm or outside the cell membrane and arrows in D indicate colocalization of red dots with the blue nuclear stain (shown as pink dots). QD–DNA conjugates were added as 1.469 1013 particles per 2.5 106 cells per well. Bar, 25 Am.
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FIG. 5. In vitro expression and long-term imaging using QD–DNA conjugates. Time-lapse imaging experiments performed using confocal microscopy to track uptake of QD–DNA conjugates (in the presence of Lipofectamine2000) and subsequent EGFP expression in CHO-K1 cells (0–11 h). The arrows indicate an increase in the intensity of EGFP expression associated with the uptake of red QD–DNA conjugates within a selected cell. QD–DNA conjugates were added as 1.469 1013 particles per 2.5 106 cells per well. Bars, 25 Am.
different stages of the cell cycle, failure of the DNA to translocate to the nucleus, or cell stress. To assess the integrity of QD-conjugated plasmid DNA for gene expression in comparison to unlabeled plasmid DNA, we performed a quantitative estimate of transfection efficiency using a Fluostar Optima fluorescence reader at 24 h postincubation. There was no significant difference between the QD-labeled plasmid DNA (trans-
fection efficiency of 62 F 2%, n = 3) and the control plasmid DNA (transfection efficiency of 65 F 2%, n = 3) (data not shown). This demonstrated that QD conjugation did not have an adverse effect on the functionality of the plasmid DNA. Intracellular Tracking Studies for QD–DNA Conjugates We performed intracellular tracking studies in CHO-K1 FIG. 6. Tracking of QD–DNA conjugates. Confocal images following incubation with QD–DNA conjugates at different time points in CHO-K1 cells in the presence of Lipofectamine2000 are shown. Cells were stained with SYTO-16 nuclear stain. Nuclear uptake of QD–DNA conjugates is demonstrated by red QD–DNA conjugates imaged using a 633 nm He–Ne laser line excitation source, which does not excite the SYTO-16 green nuclear stain (ex 488/ em 518). (A, C, and E) Representative images using the red channel (633 nm) at 6, 10, and 24 h, respectively. (B, D, and F) Overlapped images of red and green channel at 6, 10, and 24 h, respectively. Arrows indicate the presence of QD–DNA conjugates (as yellow/orange dots) at the perinuclear/nuclear region at 6-, 10-, and 24-h time points. QD–DNA conjugates were added as 1.469 1013 particles per 2.5 106 cells per well. Bar, 25 Am.
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cells using pGeneGrip (gWIZ-LUC) plasmid conjugated to QDs and the SYTO-16 green nuclear stain. We used the luciferase plasmid since EGFP does not allow visualization of the nucleus due to its bright fluorescence effects that mask any nuclear stain (Figs. 6A–6F). Since the SYTO-16 can be excited at 543 nm, the normal excitation wavelength of the QDs in these experiments, the QDs were excited at 633 nm to minimize interference. It should be noted that the intensity of the QDs is lower in this experiment since excitation at 633 nm is suboptimal. Figs. 6A, 6C, and 6E are photomicrographs of the red channel for the QD–DNA conjugate and Figs. 6B, 6D, and 6F are micrographs showing the overlapped images of the green and the red channels. Comparison of Figs. 6A, 6C, and 6E with 6B, 6D, and 6F facilitates determination of the location of the QD conjugates within the cells. In these photomicrographs the intensity of the SYTO dye fluorescence is lower in some cells compared to others as a result of the photobleaching of the organic dye in cells where the laser is focused. SYTO-16 stains DNA and as such shows the nuclear region as green. The cytoplasm is shown as dull green due to staining of the cytoplasmic organelles. In these photomicrographs the intensity of the SYTO dye fluorescence is lower in some cells compared to others as a result of the photobleaching of the organic dye in cells where the laser is focused. The QD–DNA conjugates are observed as yellow/orange dots due to staining of the DNA part of the QD conjugates by the SYTO dye. There was a gradual increase in the number of QD–DNA conjugates localized within the cells over time (0- to 5-h data not shown). The accumulation of QD–DNA conjugates reached a maximum at about the 6-h time point (approximately 78% are within the cytoplasm and approximately 22% are within the nuclear/perinuclear region, Fig. 6B).
FIG. 7. MTT toxicity assay showing percentage cell survival following incubation with QDs and QD–DNA conjugates in CHO-K1 cells after 24 h transfection. QD–DNA conjugates were added as 2.35 1012 particles per 0.4 106 cells per well. The values represent percentage cell survival (means F SD), n = 4.
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Approximately 61% of the QD-labeled plasmid DNAs are colocalized within the nuclear/perinuclear region by the 10-h time point (indicated by arrows in Fig. 6D). At 24 h the QD–DNA conjugates again appear to be concentrated in the nuclear/perinuclear region (approximately 68%) (indicated by arrows in Fig. 6F). This study is in agreement with the EGFP study detailed above, in which approximately 66% of the cells expressed EGFP by 11 h (Fig. 5). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl Tetrazolium Bromide (MTT) Toxicity Assay We performed an MTT assay to compare any potential cytotoxicity upon incubation with free QDs vs QD–DNA conjugates in CHO-K1 cells. The assay showed 75% cell survival for QD–DNA conjugates, compared to 89, 83, and 81% cell survival for Hepes buffer alone, QDs alone, and the physical mixture of QD and DNA, respectively (Fig. 7). These observations indicated that the QDs are not toxic to the cells.
DISCUSSION Plasmid DNA was successfully linked to QDs using the highly specific SPDP–PNA linker. The conjugation is clearly shown in the AFM images, in which several QDs appear to be clustered, presumably at the PNA binding sites on the plasmid DNA. As well as confirming the QD–DNA conjugation, AFM imaging proved to be an ideal technique to study plasmid DNA since the DNA structure is not altered during either processing or imaging, and the samples can be imaged directly without any further treatment. The AFM image of plasmid DNA on mica is similar to that reported by other researchers in terms of structure and size [38–40] (Fig. 2C). The plasmid DNA is clearly visible in the AFM images and the dimensions (of the DNA strand) are approximately 2 nm in width, 1.1–1.2 nm in height, and the ring structure is on the order of 200–300 nm. These values are in agreement with those previously reported in the literature determined by TEM as well as AFM and cryo-AFM imaging [37–39]. Although the actual diameter of the whole ring structure shown in Figs. 2C and 2D is on the order of 200– 300 nm, when fully stretched a 5.7-kb circular plasmid DNA would be expected to have a diameter of 500 nm. This implies that the DNA is partially folded, as can be visualized by the nonuniform height observed in the AFM images. The bright spots on Fig. 2C of approximately 1 nm in height are typical of any single- or double-stranded DNA [39,40]. The QD structures on each of the two plasmid DNA conjugates determined by AFM (approximately 5–6 nm in height) indicate that they are attached only at a particular region on the ring structure of the plasmid and are sequence specific. AFM images revealed relaxed and supercoiled forms of the QD–DNA conjugates. The bright spots in Fig. 2D not associated with the DNA conjugate are a result of the buffer salts used in the preparation of the conjugates. This was confirmed by the section analysis of the images
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that shows that the heights of these buffer salts are much lower than those of free QDs (data not shown). The QD–DNA conjugation was also confirmed by gel electrophoresis. From the gel electrophoresis it appears that the purification process removes all free QDs by the end of the third wash and free DNA by the end of second wash (Figs. 2A and 2B, lanes 6, 7, and 8). The purified DNA conjugate showed two bands (Figs. 2A and 2B, lane 4). This is considered to be a consequence of the amount of QDs conjugated to the DNA. Since the QDs are added in excess and each DNA has –seven to nine PNA binding sites, the majority of the DNA is expected to contain a high ratio of QDs; however, a minority will contain few or single QDs (i.e., the ratio of QD to DNA is expected to be skewed). The lower band in Figs. 2A and 2B, lane 4, is considered to represent conjugates with approximately one QD per DNA. This lower band travels at a faster rate than that of the QDs alone as a result of the charge on the DNA. The higher band in Figs. 2A and 2B, lane 4, is thought to be due to conjugates that contain multiple QDs. The relative weight of these conjugates is considered to result in their inability to move under the conditions applied. It is evident from lane 4 in Fig. 2B that conjugates containing multiple QDs are in excess (since the lower band is faint). The bands in lane 4, Fig. 2A, are complicated by the differing quantum efficiencies of conjugates with single QDs compared to those with multiple QDs. The close proximity of the PNA binding sites on the DNA is expected to cause some degree of self-quenching of the QDs where multiple QDs are attached [41]. Therefore, although conjugates with multiple QDs are in greater numbers (upper band in Fig. 2B, lane 4), the lower band is easier to discern in lane 4 in Fig. 2A than in 2B. QD–DNA conjugates represent an ideal method of tracking plasmid DNA since they produce a highly stable fluorescent signal that can be used for long-term studies (such as the 24-h time-lapse confocal imaging and photostability experiments) and the conjugation does not affect the DNA functionality. This technique enabled correlation between DNA distribution and protein expression by simultaneous tracking using time-lapse imaging. In addition, the QD–DNA conjugation facilitated tracking within the cellular compartments of live cells when used in combination with a nuclear stain. Previous reports show that PNA linkers have been labeled with organic fluorophores such as fluorescein and rhodamine to conjugate plasmid DNA to follow DNA delivery [36,37]. These studies involved imaging live cells posttransfection; however, the organic fluorophorelabeled plasmid DNA could not be used for long-term time-lapse imaging [42–45] as these dyes are sensitive to photobleaching during prolonged exposure to UV and O2, which compromises their utility. Another important
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aspect to be considered in bioimaging is cell viability. The QD-labeled plasmid DNA did not affect the CHO-K1 cell viability even up to 24 h posttransfection as shown by MTT assay, in which cell survival was comparable to that of a buffer-alone control [43,46]. Previous studies show that QD micelles conjugated to single-stranded DNA and injected into live Xenopus embryos were stable, biocompatible, and nontoxic when used to observe the cell lineage by following QD fluorescence throughout the embryo development up to the tadpole stage[34]. QD–DNA conjugates may be used in future studies to gain a better understanding of the efficiencies of the various processes involved in the cellular and nuclear uptake of plasmid DNA.
MATERIALS
AND
METHODS
Synthesis and water solubilization of CdSe/ZnS QDs. CdSe quantum dots (Emax = 600 nm) were synthesized using organometallic precursors according to the method of Murray et al. [32]. In short, dimethylcadmium and selenium were dissolved in trioctylphosphine and this precursor mixture was injected into hot (3008C) trioctylphosphine oxide solvent. CdSe QDs formed instantaneously and were grown until the desired particle size was attained. Subsequent ZnS coating was performed by slowly adding diethyl zinc and hexamethyldisilthiane, which were used as Zn and S precursors, respectively. This was followed by annealing at 1508C for several hours. Encapsulation of CdSe/ZnS QDs. CdSe/ZnS QDs were encapsulated in phospholipid/PEG micelles as reported by Dubertret et al. [34] but a maleimide functionalized PEG lipid was used as described below to provide reactive sites toward conjugation with the plasmid DNA. An 80:20 mixture of PEG-PE2000 and maleimide functionalized PEG lipid, DSPE-PEG(2000)maleimide (Avanti Polar Lipids, Alabaster, AL, USA), was added to the QD dispersion in chloroform. After solvent evaporation, a thin film was formed, to which distilled water or PBS buffer was added followed by vigorous shaking to produce a clear QD micellar dispersion. Conjugation of Plasmid DNA to QDs. pGeneGrip (gWIZ-GFP plasmid) and SPDP–PNA linker were purchased from Gene Therapy Systems (Genlantis, San Diego, CA, USA). The pGeneGrip plasmid contains a PNA binding site and has an EGFP reporter gene. The PNA binding sequence [37] within the plasmid is [AG]20CCATGG[AG]20 and the PNA used has the sequence 5V-TCTCTCTC-O-O-O-JTJTTJTJT-3V, where J is a pseudoisocytosine and O is 8-amino-3,6-dioxaoctanoic acid linker [36]. Reduction of the SPDP–PNA-labeled gene grip vector was conducted using 3 mM tris-(2-carboxyethyl)phosphine, hydrochloride) (Invitrogen, Carlsbad, CA, USA) and the coupling reaction was immediately carried out by mixing with maleimide functionalized QDs prepared in 100 mM Hepes buffer (pH 7.2) (a 20-fold excess of QDs) and incubating overnight at room temperature (schematic illustration of conjugation of QDs to plasmid DNA, Fig. 1). For experiments on intracellular tracking in which nuclear staining was performed, a different pGeneGrip plasmid (gWIZLuc plasmid) (Genlantis) that has a luciferase reporter gene and contains a PNA binding site similar to that of gWIZ-GFP plasmid was used. The luciferase gene was preferred since the GFP plasmid results in bright fluorescence that obscures the location of QDs. Purification of the QD–DNA conjugates. The QD–DNA conjugate was separated from free QDs upon passage through a 100-kDa Centricon (Millipore, Bedford, MA, USA) to remove excess salts and subsequent passage through a 1000-kDa cellulose ester-based membrane (Spectrum Labs, CA, USA) with multiple wash steps to remove any unbound QDs and plasmid DNA (the filtrates were collected separately) followed by ultracentrifugation at 45,000 rpm, 48C, for 2 h (TLA-100 ultracentrifuge; Beckman Coulter, CA, USA (100,000 rpm equals 541,000g)). The amount
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of QDs conjugated with DNA was quantified by subtracting the free QDs in the filtrate from the amount of QDs originally added. Direct quantification of the QD–DNA conjugate was not possible since the DNA would interfere with the UV measurement of the QDs (260 nm). The amount of DNA conjugated to QDs was similarly quantified by subtracting any residual free DNA in the filtrate from the amount of DNA initially used for conjugation. For this, the filtrate was concentrated and ultracentrifuged for 3 h at 70,000 rpm and 48C. Agarose gel (0.5% w/v) electrophoresis was run at 67 V/cm, for 2 h. The gel was stained with SYBR Gold nucleic acid gel stain (Molecular Probes, Invitrogen, Carlsbad, CA, USA), visualized using a standard 300 nm UV transilluminator, and photographed using Polaroid 667 black and white film. QDs alone and a physical mixture of QDs and plasmid DNA were used as controls. Plus and minus signs show the direction of the gel run. Atomic force microscopy. AFM experiments were performed using a mica substrate that was freshly cleaved at the time of imaging. Five microliters of diluted sample of either QD–DNA conjugates or free QDs was deposited onto the mica substrate. After 5-min incubation, the surface was washed five or six times with distilled water to remove unbound material and salts, air dried for 15 min, and viewed under AFM. AFM observations were carried out in air in a tapping mode with a MFP-3D atomic force microscope (Asylum Research, Santa Barbara, CA, USA). The silicon probe cantilever was used (OMCL-AC160TS; Olympus) with a nominal spring constant of 42 N/m. The height analysis of the DNA strands was performed either by measuring the value at a single point or by averaging the heights within a segment with a focal width of 7 points on the DNA strand. Photostability. The photostability of QD–DNA conjugates was compared to that of DNA complexes with the organic dye, rhodamine (Rh–PE/DNA complexes). In this experiment 2.6 Ag of gWIZ-Luc plasmid was complexed with Rh–PE at a ratio of 0.3/1 w/w (Rh–PE/plasmid). Both the complexes were transfected into CHO-K1 cells in the presence of Lipofectamine2000. This experiment was performed by exposing both the fluorophores to constant laser excitation (543 nm) for 100 min. Consecutive images were taken every 10 min. Cell transfection. A CHO-K1 cell line was purchased from the ATCC (Manassas, VA, USA). Transfection studies were conducted by plating CHO-K1 cells (0.8–1 105 cells/well) in 8-well LabTekII chamber glass slides (Nalge Nunc International, Rochester, NY, USA). Once the cultures reached 70–80% confluence, the cells were washed with Hanks’ balanced salt solution (HBSS), and transfections were carried out in serum-free F12K medium (Invitrogen). Lipofectamine2000 (Invitrogen) was used as the transfection reagent following the manufacturer’s protocol. QD–DNA conjugates, physical mixtures (gWIZ-GFP plasmid DNA + QD), or QDs alone were mixed with Lipofectamine2000 individually and incubated with cells. Unconjugated (gWIZ-GFP) plasmid DNA and QDs alone with and without Lipofectamine2000 were used as controls. After a 3-h incubation period, the transfection mixtures were removed, and the cells were washed with HBSS and incubated in F12-K medium with 10% FBS, under 5% CO2 at 378C, until further use. A similar procedure was followed when gWIZ-Luc plasmid DNA was used. Quantitative reporter gene expression analysis was conducted by harvesting the cells after a 24-h transfection period, adding them to a 96-well plate, and reading in a FLUOstar Optima microplate reader, Model 413-101 (BMG Labtech, Durham, NC, USA). QD–DNA conjugates were added as 1.469 1013 particles per 2.5 106 cells per well during transfection in CHO-K1 cells. Equivalent amounts of QDs were added for samples containing physical mixtures and QDs alone. Confocal microscopy. A confocal laser scanning microscope equipped with Ar/Kr, Kr/Ne, and He/Ne lasers (Leica SP2 spectral confocal microscope, Bannockburn, IL, USA) was used for live/fixed cell imaging. For time-lapse imaging, a Bioptechs DT4 open dish system (Bioptechs, Inc., Butler, PA, USA) was used to maintain the temperature of the CHO-K1 cells during observation in a L-15 carbon dioxide-free medium. Quantitative estimation of the uptake of QD–DNA conjugates at different time points was performed by counting the number of yellow/orange dots within the nucleus or at the perinuclear region (double-blind test).
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Nuclear staining. CHO-K1 cells were grown on sterile eight-well LabTekii chamber slides and transfected as described above. The chamber slides were then washed with HBSS and stained with nuclear staining dye Hoechst 33342 (in the cellular uptake experiment) or green SYTO-16 (in the study involving tracking of QD–DNA conjugates) at 1 or 2 AM, respectively, for 15 min at 378C and washed twice with HBSS to remove excess dye. The cells were imaged immediately using either a Zeiss fluorescence microscope (Hoechst staining) or Leica confocal laser scanning microscope (SYTO-16 staining) as described above. QD toxicity study. To compare the potential toxicity of the QDs and QDconjugated and unconjugated plasmid DNA, an MTT assay was performed in CHO-K1 cells. MTT is a quantitative colorimetric assay that detects living cells based on the action of mitochondrial dehydrogenases of viable cells that cleave the tetrazolium ring, forming purple formazan crystals. Ten thousand cells/well were plated in 96-well plates, grown to 70–80% confluence, washed with HBSS, and transfections carried out in serum-free F12-K medium in the presence of Lipofectamine2000. QD–DNA conjugates were added as 2.35 1012 particles per 0.4 106 cells per well. Equivalent amounts of QDs were added for samples containing physical mixtures and QDs alone. After 22 h of transfection, 25 Al of MTT solution (5 mg/ml) (Invitrogen) was added and incubated for 2 h (CO2 incubator at 378C). Cells were lysed with lysis buffer (20% SDS in 50% dimethyl formamide) and further incubated for 5–6 h to dissolve the formazan crystals. The absorbance of the resulting purple solution was measured spectrophotometrically at k = 580 nm.
ACKNOWLEDGMENTS The authors thank Shafiuddin Shafiuddin of the Department of Animal Science, Center of Excellence for Vaccine Research, University of Connecticut, for his valuable suggestions in QD–DNA conjugation work. This research was supported in part by the Parenteral Drug Association Foundation Schering–Plough Grant, NSF DMI 0303950, ONR N000LY0610016PR and ARO DAAD-02-1-0381. RECEIVED FOR PUBLICATION SEPTEMBER 14, 2005; REVISED MARCH 7, 2006; ACCEPTED MARCH 10, 2006.
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