Structural and elemental characterization of bare and silica-coated CdS QDs
To prepare CdS and silica-coated QDs, we utilized reverse microemulsion methodology as described. The nanoparticles (bare and silica-coated CdS) were successfully synthesized and were highly water-soluble. This facilitated their use in in vitro studies without the need of any further surface modifications. As shown in the TEM micrograph, the CdS QDs () were highly monodispersed and exhibited good crystal lattice fringes, with average sizes of 4–5 nm. show the TEM image of silica-coated CdS QDs. The silica-coated QDs were also highly monodispersed and each particle consisted of a single CdS QD at its core. The sizes of the silica-coated QDs were approximately 35–40 nm. The high-resolution TEM images of the silica-coated QDs are shown in . The crystal lattice patterns of CdS QDs encapsulated in silica shells were clearly visible. Nearly 90% of the particles were of uniform size (35–40 nm) and uniform spherical shape when the reaction time was 8 hours. In the case of targeted silica-coated QDs, no morphological differences with non-targeted nanomaterial were observed under TEM. We could also control the size of the silica coating when the reaction time was reduced from 8 hours. A thin silica coating was observed at 2–3 hours of reaction time. These thinly silica-coated CdS QDs rendered toxicity to cells, which may be attributed to cadmium ions leaching through the thin coat (data not shown).
Transmission electron microscopy (TEM) images of bare cadmium sulfide quantum dots (CdS QDs; A and B), silica-coated CdS QDs (C and D), and high-resolution TEM images of silica-coated CdS QDs (E and F).
shows the EDS spectra of bare () and silica-coated QDs (). The presence of Cd and S confirmed the formation of CdS QDs, and the presence of only Si and O was noted in the case of silica-coated QDs. shows the XPS analysis of bare CdS QDs. , correspond to the Cd and S peaks, whereas shows the wide XPS spectra of the CdS QDs. The presence of Si and O was from the silica substrate used while performing XPS. corresponds to the XPS analysis of silica-coated and CD31 antibody-targeted silica-coated QDs. The presence of Si and O was clear; the substrate used in this case was carbon tape. When CD31-conjugated silica-coated QDs were analyzed for elemental incidence, we observed an enhanced peak of N compared with the amine-functionalized counterpart, suggesting CD31 had successfully conjugated onto the silica-coated QDs ( [bottom]). In both EDS and XPS, the presence of Cd and S peaks could not be observed in the case of silica-coated QDs. These particles, however, exhibited fluorescence under UV illumination. As bare Si particles are nonfluorescent, the fluorescence visualized is certainly from the CdS QDs encapsulated in the silica shell. TEM images also support the presence of CdS cores inside the silica shell. This clearly indicates that QDs were present deep inside the silica shell and were efficiently encapsulated, with no surface adherence of the QDs. EDS mapping was performed to analyze the presence of CdS deep inside the silica shell (). shows the high-resolution TEM image of the sample area where mapping was performed. B–E of correspond to the mapping of the elements Cd, S, Si, and O, respectively. The presence of all four elements was recorded by EDS mapping, confirming the presence of CdS QDs inside silica spheres. The XRD pattern of bare CdS is shown in Figure S1
. The diffraction peaks were assigned to 2θ = 27.1°, 44.8°, and 53.4° of CdS QDs, suggesting a cubic structure.
Energy-dispersive X-ray spectroscopy analysis of (A) bare cadmium sulfide quantum dots (CdS QDs) and (B) silica-coated CdS QDs.
X-ray photoelectron spectroscopy analysis of cadmium (Cd) sulfide (S) QDs showing Cd (A) and S peaks (B) and of wide (C), respectively.
X-ray photoelectron spectroscopy analysis of silica-coated cadmium sulfide (CdS) quantum dots (top) and CD31 antibody-labeled silica-coated CdS QDs (bottom spectrum).
Energy-dispersive X-ray spectroscopy mapping of cadmium sulfide quantum dots coated with silica. (A) Transmission electron microscopy image. (B) Cadmium mapping. (C) Sulfur mapping. (D) Silicon mapping. (E) Oxygen mapping.
Optical characterization of bare and silica-coated CdS QDs
The UV–visible spectroscopy absorption spectra of plain silica, bare, and silica-coated CdS QDs were analyzed (data not shown). Plain silica did not show any absorbance, whereas the bare QDs showed peak absorption at 400 nm. The silica-coated QDs showed a slight red shift in absorption. The targeted nanoparticles showed similar absorbance to that of the silica-coated QDs (data not shown). shows the photoluminescence spectra for bare QDs and targeted silica-coated QDs. The excitation wavelength was 365 nm and a clear emission peak was noted, centered at 600 nm for both particles. (inset) shows UV illumination of bare and silica-coated CdS QDs. The quantum yields of bare and silica-coated CdS QDs were found to be 0.02% and 0.03%, respectively. The conjugation of CD31 did not alter any of the optical characteristics of silica-coated CdS QDs. To assess and affirm the increased shelf-life stability of the silica-coated CdS QDs, UV luminescent images of the nanomaterials after 3 months of storage (Figure S2
) were recorded. The silica-coated QDs exhibited excellent fluorescence under UV light, even after storage under normal light for such an extended period. In stark contrast, the fluorescence of QDs without a silica coating was observed to have diminished drastically under normal light and no fluorescence was recorded after 1 month. This clearly favors our claim regarding the long-term storage capability of the silica-shielded CdS QDs and proves their increased photostability.
Photo luminescence spectra of cadmium sulfide (CdS) and silica-coated quantum dots (QDs).
Biocompatibility of QDs and silica-coated QDs
To efficiently employ these nanomaterials as cellular imaging probes, imparting biocompatibility to them is of prime importance. Bare CdS QDs are reported to be toxic to cells, and their cytotoxicity becomes significant with increases in concentration. The cytotoxicity of bare QDs is due to the release of cadmium ions inside live cells, leading to ROS production and glutathione depletion. Our aim with silica coating was to minimize the toxicity, as the silica layer can efficiently block the release of cadmium ions from the CdS core. As described, cytotoxicity tests were carried out using alamarBlue. The cytotoxicity profiles of bare and silica-coated QDs were studied on HUVECs and Gl-1 cells. Upon the subsequent addition of the nanoparticles, cell viability decreased as a function of concentration and time. The densities of viable HUVECs observed under different concentrations after 24 hours incubation with the nanoparticles are presented in . The cells showed an uptake of QDs within 2 hours and of silica-coated nanomaterial within 4 hours of incubation, as evidenced from confocal studies; hence, it was concluded that cytotoxic studies could be carried out after 24 hours. Bare QDs had comparatively higher levels of toxicity, even at the lowest concentrations (1 μg), at which only 55% of cells were viable. At the highest concentration (1 mg), only 10% of the cells were viable. On the contrary, in the case of silica-coated QDs, the viability of the cells was greatly enhanced. The cell viability was as high as 60% at the highest concentration of 1 mg/mL of silica-coated QDs, highlighting the prominent biocompatibility acquired by these nanomaterials post-silica encapsulation. At concentrations of 10 and 1 μg/mL, nearly 88% and 96% of cells were viable, respectively. In the case of Gl-1 cells, the cytotoxicity of bare QDs was highly pronounced, with only 3% of cells viable at the highest concentration (1 mg/mL). Even at a concentration of 1 μg/mL, we could observe only around 40% viability. With silica-coated QDs, the viability was assessed to be around 75% and 40% at lower (1 μg/mL) and higher concentrations (1 mg/mL), respectively. Gl-1 cells are shown to effectively take up nanomaterials at higher levels than HUVECs, which was evidenced by confocal microscopic studies. The higher toxicity in Gl-1 cells can be attributed to this increased uptake of nanomaterials within a short period. In the case of the targeted silica-coated CdS QDs, Gl-1 cells showed less toxicity than those cells treated with non-targeted silica-coated CdS QDs. Meanwhile, in the case of HUVECs, viability remained the same as that of the non-targeted nanoparticle treatment. This might be due to the high specificity of antibody-labeled silica-coated QDs toward endothelial cells compared with Gl-1 cells, resulting in a decreased intake of nanomaterials in the latter. Alternatively, this may be attributed to the absence of CD31 markers on the surface of Gl-1 cell lines, corresponding directly to the significance of the high specificity of our targeted nanomaterial.
Effect of cadmium sulfide quantum dots (CdS QDs), silica-coated CdS QDs, and CD31-targeted silica-coated CdS QDs on human umbilical vein endothelial cell (HUVEC) and glioma cell viability.
The cellular ROS formation, when subjected to nanoparticle (CdS and silica-coated CdS QDs) treatment, was also studied. As per the alamarBlue assay experiment, HUVECs and Gl-1 cells were treated with three concentrations of CdS and silica-coated CdS QDs. We were able to detect higher levels of ROS formation in both cell lines treated with the CdS QDs than in the control group (). This ROS formation indicates released cadmium toxicity leading to cellular apoptosis. Coomassie protein assay showed that the total cellular protein production of both the cell lineages treated with CdS QDs was drastically reduced when compared with the control group (). This was attributed to the lower cellular metabolic activity of the cells treated with bare QDs. However, the ROS and protein production of silica-coated CdS QDs-treated cells remained comparable to the control group except at the concentration of 1 mg/mL. At this concentration, the ROS production increased slightly. The protein production of cells treated with 1 mg/mL of silica-coated QDs showed slightly compromised metabolic activity. This insignificant increase in ROS and decreased protein production did not cause any lethal effects to the cells, reaffirming the biocompatibility attained with a silica coating.
Effect of cadmium sulfide and silica-coated quantum dots on (A) cellular radical formation (reactive oxygen species [ROS]) and (B) on protein synthesis.
In general, it was clearly evidenced that silica-coated QDs exert no significant cytotoxic effects on endothelial cells when compared with bare QDs, thereby suggesting they are highly safe labeling probes.
Intake of nanomaterials by cells and imaging
The cellular uptake and endocytosis of these nanomaterials were studied by means of a confocal microscope to determine the cellular uptake/entry of bare and silica-coated QDs. HUVECs and Gl-1 cells were treated with as few as 100 μg/mL of both nanomaterials and incubated for 1 hour (). In the case of bare QDs, Gl-1 cells showed efficient uptake of nanomaterials after 1 hour of incubation. However, HUVECs were not efficiently labeled with bare QDs at 1 hour. This may be the reason for pronounced Gl-1 cell cytotoxicity, since they showed high uptake of nanomaterials within a short period. To check whether time-dependent uptake of bare QDs exists, we incubated both cell lines with bare QDs for 2 hours (). After 2 hours, both cells showed efficient uptake of bare QDs and were effectively labeled. When the cells were incubated for a longer duration with bare QDs (24 hours), most of the Gl-1 cells died, rounded, and started to float (); the HUVECs also started to undergo cell death. Shrinkage of HUVECs after 24 hours of incubation with bare QDs was observed.
Imaging of (A) human umbilical vein endothelial cells and (B) glioma cells using cadmium sulfide quantum dots after 1 hour of incubation.
Imaging of (A) human umbilical vein endothelial cells and (B) glioma cells using cadmium sulfide quantum dots after 2 hours of incubation.
Human umbilical vein endothelial cells (HUVECs) (A) and glioma cells (B) after 24 hours of incubation with bare quantum dots.
shows the uptake and internalization of silica-coated QDs by HUVECs and Gl-1 cells after 2 hours of incubation with the nanomaterials. After 1 hour of incubation, no uptake of silica-coated nanomaterials was recorded by either the HUVECs or Gl-1 cells. After 2 hours, both cell lines showed uptake of nanomaterials, but not as significantly as in case of bare QDs incubated for 2 hours. This may be because of the size effect of the nanomaterials under study. Bare QDs are 4–5 nm and are taken up efficiently after 2 hours of incubation, whereas silica-coated QDs are 35–40 nm, thereby presenting minimal intake in both cells. Apart from the size factor, this discrepancy in cellular uptake could also be due to the surface properties of the QDs and silica-coated QDs. When the cells were incubated with silica-coated QDs for 4 hours (), comparable efficient uptake was observed. However, silica-coated QDs did not show rich fluorescence compared with bare QDs. Silica coating over QDs efficiently stabilized the fluorescent property of QDs, reducing their photobleaching, with limited reduction in fluorescence intensity. The cells treated with silica-coated QDs were viable even after 48 hours (data not shown), clearly indicating their biocompatibility. Thus, our results suggest that silica-coated QDs are effective cell labels due to their enhanced photostability for extended periods and their biocompatibility inside live cells.
Imaging of (A) human umbilical vein endothelial cells and (B) glioma cells using silica-coated cadmium sulfide quantum dots after 2 hours of incubation.
Imaging of (A) human umbilical vein endothelial cells and (B) glioma cells using silica-coated cadmium sulfide quantum dots after 4 hours of incubation.
shows the targeted imaging of endothelial cell lines by CD31-labeled silica-coated CdS QDs. Gl-1 cells were used as a negative control, as they lack CD31 markers on their surfaces. Targeted nanoparticles were added to both cell lines and incubated. After 1 hour of incubation, the cells were washed to remove the unbound nanoparticles and were imaged using a confocal microscope. Highly efficient endothelial labeling was observed by the targeted nanoparticles. CD31 was expressed all over the surface of the cells. Further, the cytoplasm – including the nucleus – of the endothelial cells showed uptake of nanomaterials. Increased uptake was witnessed within 1 hour in the case of the endothelial cells, whereas the Gl-1 cells showed greatly decreased uptake of nanoparticles when compared with non-targeted treatment. Weak fluorescent signals from the nuclear region of HUVECs were observed after 1 hour of incubation. Therefore, the cells were incubated with targeted nanoparticles for 2 hours and a confocal microscopic study was carried out. Excellent uptake and internalization of targeted nanomaterials into the nuclear region was visualized, currently a first (). Even though there was increased uptake of nanomaterials into the nuclear region, the 4′,6-diamidino-2-phenylindole staining of the cells showed proper nuclear morphology. As mentioned previously, there was an increased uptake of non-targeted nanoparticles by Gl-1 cells when compared with HUVECs. However, with targeted nanoparticle treatment, Gl-1 cells showed weak and reduced fluorescent signals, suggesting the highly limited intake of targeted nanomaterial, in turn emphasizing the high specificity of the nanofactor toward HUVECs. Flow cytometry analysis was also carried out to determine the efficiency of targeted silica-coated CdS QDs with higher specificity toward HUVECs, sparing Gl-1s any internalization (, right column). The flow cytometry data reveal that HUVECs showed nearly 104 cells with particle internalization, whereas only 101 Gl-1 cells showed internalization of nanoparticles. These results clearly indicate our success in achieving a breakthrough in high-specificity targeting of endothelial cells, which may carve a significant and prominent niche into in vivo vascular lineage imaging.
Imaging of (A) human umbilical vein endothelial cells and (B) glioma cells using CD31-labeled silica-coated cadmium sulfide quantum dots after 1 hour of incubation.
Viability staining and nuclear penetration of targeted silica-coated cadmium sulfide quantum dots.
In vivo applications
To investigate the applicability and feasibility of bare and silica-coated QDs for in vivo imaging, we employed medaka embryos as test models. Embryos were exposed to both nanoparticles at a concentration of 100 μg/mL of ERM for 24 hours. After 24 hours, the embryos were viewed for the intrinsic biocompatibility of these two nanoparticles (). We found that the embryos treated with CdS QDs had all shrunk, with blood clots evidently visible (). None of the embryos survived, proving the toxicity of cadmium-based QDs within 24 hours of incubation. This lethality of the CdS QDs can be attributed to the release of cadmium ions from the nanocrystals, which led to heavy metal toxicity, which correlates with the in vitro assay results. All embryos treated with silica-coated QDs were viable and healthy, without any notable deformities of the embryonic body, as with their control counterparts (). The survival and hatching rates of the embryos were examined to understand the toxic nature of QDs and the cytobio-amiability of silica-coated CdS QDs (). In the case of CdS QDs, all test embryos were dead by Day 2 of nanoparticle exposure. However, with silica-coated QDs, the embryos remained viable and hatched concurrently with the control group, with the fry exhibiting a healthy profile. The heartbeats of the embryos treated with silica-coated QDs were also monitored (Supplementary video 1
). This increased biocompatibility can be attributed to the presence of a silica shell, which prevents the leakage of cadmium ions from the QD core. The 20 nm silica shell around the core potentially plays a role in improvising biocompatibility. The silica-coated CdS QD-treated embryos were checked for particle uptake, which subsequently leads to fluorescence of the embryonic body of the medaka until hatching ( and ). This strong fluorescence of the silica-coated nanomaterials depicted the efficient uptake and internalization of silica-coated QDs into the embryonic body, clearly denoting the exceptional biocompatibility of the nanoparticles, along with their outstanding imaging ability.
(A and B) In vivo optical imaging of medaka embryos and a viability assessment using bare CdS and silica-coated cadmium sulfide quantum dots at a concentration of 1 μg/mL of embryo-rearing medium.
(A) Survival rate and (B) Hatching rate of embryos treated with bare cadmium sulfide (CdS) and silica-coated CdS quantum dots.
Fluorescence imaging of silica-coated cadmium sulfide quantum dot-treated embryos.
Altogether, these silica-coated nanoparticles thus represent one of the new classes of nanocarriers for vascular theragnostics when targeted through specific ligands. This system holds promise for improving pharmacotherapy and easing antagonistic side effects when utilized for diagnostic therapy.