The morphological, optical, and magnetic properties of MMNCPs were extensively characterized. TEM studies confirmed the formation of 20–40 nm size nanocomposites as shown in . The IONP core and satellite Qdots surrounding the core are clearly visible with low resolution TEM () whereas magnified TEM images clearly show Qdots around the IONP ( inset). The IONP can be discerned in the image by its light grey contrast while the Qdots appeared with dark contrast on the IONP surface. Individual satellite Qdots on the IONP surface can be clearly identified by their single crystalline structure while the IONP in the HRTEM image is obscured by the satellite Qdots (Supplementary Materials, Fig. S1
). Inductively coupled plasma analysis of the sample confirmed the presence of Zn (41 wt%), Fe (6.2 wt%) and Cd (10 wt%) with a relative ratio of 6.6 : 1.0 : 1.6 (W/W). Zeta potential (ξ) measurements correlated with particle surface charge. The ξ w −20 mV, −4.0 mV, −17 mV and −21 mV, respectively. As expected, DHLA modification of IONP drastically reduced its surface charge due to reduction of the negative surface charge of IONP. Further surface modification with the STAT3 drug and folic acid resulted in an increase in the overall negative surface charge on the particle. This is due to the presence of carboxyl groups on the folic acid residues on the particle surface. Pegylation with mPEG, however, showed minimal effect on the particle surface charge due to its neutral nature. It was observed that pegylated particles exhibited good phosphate buffer dispersibility. A comparative analysis of FT-IR spectra of IONP, DHLA, lipoic acid, and IONP-DHLA confirmed successful surface modification of IONP with DHLA (Supplementary Materials, Fig. S2
). The broad band at 3200–3600 cm−1
indicates the surface hydroxyl group of the super paramagnetic IONPs (Fig. S2d
). The bands at 3046 (O-H), 2934(–CH2
–), 1697 (C=O), 1252 (O-H), and 935 (OH) cm−1
were observed for dihydrolipoic acid and lipoic acid (Fig. S2a and b
respectively). The presence of these characteristic bands into the spectra of dihydrolipoic acid coated IONPs (Fig. S2c
) confirmed the dihydrolipoic acid coating on the surface of IONPs. Fluorescence spectroscopy in solution was used to investigate the Qdot luminscence properties at different stages of MMCNP development as well as for tracking the drug release event. GSH, a tripeptide biomolecule found in all animal cells at relatively high cytosolic concentration (1–10 mM[38
], reduced form), effectively reduces disulfide bonds and in this process glutathione is converted to glutathione disulfide (GSSG), its oxidized form. The design of the MMCNPs is such that once it is exposed to the intracellular GSH environment, it will disintegrate into its different constituents that make up the composite nanoparticle. This forms the basis of the intracellular tracking of the STAT3 drug release as schematically shown in . Distinct changes in absorption spectra were observed for IONP-Qdot and IONP-Qdot-STAT3 conjugates in comparison to Qdots (Fig. S3a
). The Qdot absorption spectrum broadens significantly and slightly shifts towards longer wavelength when conjugated to IONPs. However, upon further conjugation with NAC-STAT3 drug, NAC-FA and NAC-EDA, a decrease in spectral width along with slight blue shift was observed with respect to the IONP-Qdots conjugates. Such changes in absorption spectral characteristics support successful surface conjugation of Qdots with IONP, STAT3 drug and FA. The emission of MMCNP was slightly blue shifted compared to either Qdots or IONP-Qdot conjugates (Fig. S3b
TEM image of MMCNPs are showing nearly spherical particles with irregular surface morphology indicative of the presence of satellite Qdots on the IONPs.
Fluorescence data acquired by adding GSH to MMCNPs in solution showed that Qdot fluorescence could be restored completely in less than one hour () after which no further increase in Qdot fluorescence intensity was observed. The inset shows a plot of Qdot fluorescence intensity measured at the peak emission wavelength (582 nm) as a function of time. This plot illustrated that the fluorescence intensity plateaus at 60 minutes. Furthermore, a systematic study on the effect of GSH concentration on the time scale of fluorescence restoration showed that there was no significant effect of varying GSH concentrations in the range of 2.8 mM to 7 mM (Fig. S4
). Even at the lowest GSH concentration, the Qdot fluorescence recovered in approximately 1 hour. Furthermore, the timescale of fluorescence recovery appeared to be independent of GSH concentration higher than 1.4 mM. Since the intracellular concentration of GSH ranges from 1 mM to 15 mM[38
], it was expected that MMCNP uptaken by the cancer cells should release its cargo within an hour. The observed spectral features of the “ON” state Qdots (i.e. after release from MMCNP) () were in good agreement with those of Qdots in solution as shown in Fig. S3b
. The STAT3 inhibitor is also a fluorescent molecule (λex
: 300 nm and λem
: 396 nm) of which the fluorescence was quenched in MMCNPs (). These data of show that full restoration of STAT3 fluorescence occurred within one hour, after which no further increase in STAT3 fluorescence intensity was observed. The inset of the shows a plot of STAT3 fluorescence intensity measured at the peak emission wavelength (430 nm) as a function of time. This plot illustrated that the fluorescence intensity plateaus at 60 minutes. Both IONP and Qdots can quench the fluorescence of STAT3 drug via electron and/or energy transfer processes. Restoration of STAT3 drug emission was observed once MMCNPs were treated with GSH in solution () was thus confirming disintegration of MMCNPs and release of STAT3 inhibitors.
Qdot fluorescence emission spectra (excitation wavelength = 375 nm) measured as a function of time at 7.0 mM GSH concentration. The red line is a non-linear fit to the data.
STAT3 fluorescence emission spectra (excitation wavelength = 300 nm) measured as a function of time at 7.0 mM GSH concentration. The red line is a non-linear fit to the data.
To validate the proof-of-concept, we challenged the MMCNP against the intracellular GSH environment where the reported GSH concentration was in the millimolar (mM) range, typically between 2 mM and 15 mM[38
]. The human breast cancer (MDA-MB-231) cell line, known to over-express folate receptors, and the mouse thymus stromal epithelial cell line (TE-71) were incubated for up to 24 hrs with MMCNP at a concentration of 50 μg/mL. As expected, a significant uptake of folate conjugated MMCNP by the cancer cells was observed compared to normal cells as shown in . These results also showed that complete restoration of fluorescence occurred within 3 hr incubation (). This confirmed extensive folate receptor mediated uptake of the MMCNPs. MDA-MB-231 cells incubated with MMCNPs for 24 hours, again was showing significant Qdot fluorescence that was similar to the 3 hour incubation experiment (). These data show that uptake of MMCNPs and subsequent restoration of Qdot fluorescence (indicative of STAT3 release) occurred in less than 3 hours. The control experiment performed with MDA-MB-231 cells to which no MMCNPs were added (). As expected only a minor cellular autofluorescence was observed. We performed another control experiment with mouse thymus stromal epithelial cells, TE-71 incubated with MMCNPs for 24 hours (). Similar to the control experiment shown in , only background autofluorescence was observed at locations that correspond to the locations of the cells, with no evidence of uptake of MMCNPs by the TE-71 normal cells. This control experiment validated that the delivery of MMCNPs was highly targeted to MDA-MB-231 cells, which over-expresses folate receptors. Restoration of fluorescence in cancer cells was a direct confirmation of targeted cellular uptake of MMCNPs and subsequent disintegration of MMCNP into the separate components i.e. IONP, Qdots, and release of ligands including drug molecules.
Fig. 4 Phase-contrast (left panel) and corresponding epi-fluorescence (right panel) microscopy images: (a) MDA-MB-231 cells incubated with MMCNPs for 3 hours, showing significant Qdot fluorescence; (b) MDA-MB-231 cells incubated with MMCNPs for 24 hours, again (more ...)
Systematic optical studies were performed to investigate intracellular drug release at the single cell level (). Confocal microscopy images of the MDA-MB-231 cells incubated with MMCNPs for 5 hrs () clearly show that MMCNP were uptaken by the cells. In addition, strong fluorescence signal from only a few locations in the cell can be observed. These data show that Qdots were released from the MMCNP through the cleavage of disulfide bonds by GSH (see ). Besides the images of single cells incubated with MMCNP, emission spectra of different regions in single cells were also collected (, red and cyan lines). The Qdots in the intracellular environment show emission spectra were red-shifted and broadened with respect to uncoated free Qdots in solution (Fig. S3b
). These spectral differences were attributed to aggregation of the Qdots after release from MMCNP in the cytosol. This observation was confirmed with solution experiments on bare Qdots by observing emission spectra before and after aggregation, as well as addition of GSH to each of these samples (data not shown). We found from control experiments that addition of GSH to a suspension of bare Qdots leads to a stable Qdot suspension and has no noticeable effect on the Qdot emission properties (data not shown). These observations may provide preliminary indication that in the intracellular environment GSH does not necessarily exchange with the NAC/cargo-ligand that is initially present on the Qdot surface due to the fact that intracellular Qdot aggregation is observed after cargo release, although it could be argued that binding constants of both molecules could be comparable given that GSH and NAC both contain a single thiol group in their structure (monodentate ligand). However, to conclusively determine the mechanism of removal of the NAC-ligands from the Qdot surface by GSH further investigations need to be performed, which are planned for the near future. The observation of only a few very bright spots in a single cell in the fluorescence images indicates aggregation of multiple Qdots in a single or a few clusters. The Qdot aggregation is reasonable given that while in the MMCNP Qdots are stabilized by PEG coating, after exposure to GSH this coating is removed by cleavage of disulfide bonds, resulting in hydrophobic Qdots that self-aggregate. In addition, the data show that these Qdot aggregates preferentially localize near the cell membrane, again due to their hydrophobic nature. Other locations where Qdot aggregates are not present only show autofluorescence. It should be noted that while the STAT3 drug itself is also fluorescent (), the experiments on intracellular delivery cannot be reliably performed by measuring the STAT3 drug fluorescence due to weak fluorescence and the presence of cellular auto-fluorescence, hence the need for the optical signal of the Qdots. We performed the normalized ensemble fluorescence emission spectra acquired by sample scanning laser confocal microscopy under 375 nm laser excitation (). The ensembles were constructed by averaging fluorescence emission spectra obtained at different locations inside individual cells under illumination with a diffraction limited laser spots (~ 300 nm). Spectra were acquired at the location of the Qdot aggregates (red line) and the cellular regions without Qdots (autofluorescence, dark cyan line). As a control, the same experiment was completed for Qdots in the “OFF state” (black line) and “ON state” (blue line) on glass substrates. Both the intracellular and extra-cellular “ON state” Qdots appear slightly red shifted with respect to the “OFF state” Qdots. In addition, the “ON state” Qdots were significantly broadened at the blue edge as well as the red edge of the spectra, possibly due to the presence of GSH on the Qdot surface. The difference in the appearance of the red shoulders in the intracellular and extra-cellular “ON state” Qdots is most likely due to difference in the environment. The spectral feature around 500 nm in the intracellular “ON state” Qdot fluorescence emission ensemble spectrum is due the contribution of cellular autofluorescence.
Fig. 5 (a) Bright field; (b) corresponding epi-luminescence laser microscopy images of MDA-MB-231 cells incubated with MMCNPs for 5 hours; (c) Normalized ensemble fluorescence emission spectra acquired by sample scanning laser confocal microscopy under 375 nm (more ...)
To demonstrate the concept of multimodality of the MMCNPs, an agar phantom was prepared using a 10 mm NMR tube () for MRI and optical imaging studies. shows the schematic of agar phantom design. The bottom part of the tube contains only MDA-MB-231 cells (as a control), middle part contains only MMCNPs (as a control) and the top part of the tube contains the same cells loaded with MMCNPs. All of these were dispersed in 3% agarose gel under same condition. A digital image of the tube was recorded under room light () as well as under illumination by a hand-held 366 nm multiband UV lamp (). The unfiltered images clearly show a light brown color where cells are located in room light conditions whereas an intense red color appears due to Qdot fluorescence under UV illumination. The MDA-MB-231 cells loaded with the MMCNPs emit red fluorescence that is clearly visible to the naked eye. Control cells do not show any detectable fluorescence emission. An MRI image of the phantom () clearly shows cell clusters that correlate well with the fluorescence image. A 3D reconstruction of the MR images is provided in the supplementary section (Fig. S5
). This demonstrated the appearance of strong MRI signal from the MDA-MB-231 cells loaded with the MMCNPs (indicated with false red color) in contrast with the control cells.
Fig. 6 (a) Schematic of agar phantom design. The agar phantom consists of four layers. From bottom to top these layers are: MDA-MB-231 cells embedded in agar (control), MMCNPs embedded in agar (control); MDA-MB-231 cells loaded with the MMCNPs embedded in agar (more ...)
The CyQUANT™ cell proliferation assay was used in a comparative cell viability study to determine cytotoxicity of MMCNPs without STAT3 drug (control particle), STAT3 drug itself, and MMCNPs with STAT3 conjugation. Two cancer cell lines, MDA-MB-231 and pancreatic (Panc-1) cancer cells were used along with mouse thymus stromal epithelial TE-71 cells (control). Results () suggest that MMCNPs (with STAT3) treated cancer cells have lower viability than cells treated with free STAT 3 drug when MMCNP and STAT3 were administered to the cell medium at identical concentrations. Compared to untreated (control), the viability of cells treated with MMCNPs to which no STAT3 is attached (RII 61) was attached is not significantly different, indicating that the MMCNP itself do not compromise cell viability. By contrast, the cells treated with 50 μM STAT3 inhibitor only (drug) showed a 15–20% decrease in cell viability, while cells treated with fully functional MMCNPs to which STAT3 inhibitor was attached showed nearly 30% decrease in cell viability, even though the amount of STAT3 inhibitor contained in the 5 μg of MMCNP administered in 100 μL of cell media was expected to release a far less amount of the STAT3 inhibitor than the 50 μM that was directly added to the cells in the other study. This key observation demonstrates the effectiveness of the reported nanoparticle design in highly targeted drug delivery to the cancer cells, while maximizing cancer cell death with reduced amounts of drugs used compared to conventional approaches. Even though the MMCNPs consume much less STAT3 drug, the delivery efficiency is dramatically increased, thus resulting in increased therapeutic efficiency while minimizing the potential for medical side effects due to presence of excess free drug.
Fig. 7 CyQuant® cell viability assay performed on the human breast (MB-MDA-231) and pancreatic (Panc-1) cancer lines, or the mouse thymus stromal epithelial line, TE-71. Cells were untreated (control) or treated for 24 hours with MMCNPs to which no STAT3 (more ...)