5.1 GNP and its conjugates with VS
5.1.1 UV-VIS spectroscopy
Figure 1 describes the normalized UV-VIS absorption spectra of GNP (blue line) and its conjugates, GNP-VS1 (green line) and GNP-VS2 (red line). An intense and sharp surface plasmon absorption peak at 517 nm attributed to GNP collective electron oscillations or localized surface plasmon resonance is presented and is a feature of gold nanoclusters. It is clear that the plasmon absorption peak of GNP shifts to 519 nm (GNP-VS1) and 523(GNP-VS2) nm after the conjugation with VS. The resonance wavelength and bandwidth of nanoparticles are dependent on the particle size and shape, the refractive index of the surrounding medium and the temperature.
5.1.2 Dynamic light scattering
Dynamic Light Scattering study shows (Fig. 2a) statistical size distribution of GNP and its drug conjugates GNP-VS1 and GNP-VS2. The hydrodynamic sizes of the nonmaterial are around 25 nm. The comparative distribution (see Fig. S2 in the Electronic supplementary material) shows the broadening of the distribution pattern that can be described due to the formation of the GNP-VS (green and blue) than that of the GNP (red). From the measurement of the zeta potential (ς) data, it has been shown (Fig. 2b) there is a gradual shift of the ς value when the GNP has been conjugated with VS. This evidence is expected because the drug molecules will replace the citrate molecules during conjugation as a result of which there is a gradual drop and shift of ς value. In this case the steric factor of the drug molecule will contribute to stabilize the particle in colloid.
5.1.3 Fourier transform infrared spectroscopy
The Fourier transform infrared (FTIR) spectra of VS, GNP (see Electronic supplementary material) and GNP-VS conjugates with VS are shown in Fig. 3. The peaks are characterized and marked. Direct evidences of conjugation was observed from peaks at 1,910–1,970 cm−1 for C–H alkyl stretching, 2,549 cm−1 due to S–H deformation and 701 cm−1 for thiol stretching. C = O stretch at 1923 cm−1 and 1,666 cm−1 along with the N–C stretch at 1,621 cm−1, 1,615 cm−1 confirms the bond level interaction of VS with GNP. Several other peaks appeared at 758 and 1,620 cm−1, which are associated with the N–H deformation and stretching vibration, respectively. These results suggest the conjugation of VS with GNP via bond between gold and sulfur, gold and nitrogen in the stable drug-gold conjugate. For further analysis and details of FTIR see Fig. S2 in the Electronic supplementary material.
5.2 Anticancer activity
5.2.1 MTT assay
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) has been employed to study the survival rate of the cells in Fig. 4a. Notably, both VS- and GNP-treated cells showed lower percentage of survival. Further reduction in survival was observed in GNP-VS1 and GNP-VS2 cells with lesser survival with GNP_VS2.
In contrast to the cancer cells, PBMC (peripheral blood mononuclear cells) did not show any alteration in percentage survival pattern when challenged with GNP, VS, GNP-VS conjugates (Fig. 4b).
5.2.2 PI uptake
Cell damage was also assayed with propidium iodide (PI) uptake studies (Fig. 5). Plasma membrane integrity was altered in presence of VS, GNP, and GNP-VS conjugates. In each case, there was higher density of points (representing individual cell) in the upper half of the dot plot (Fig. 5a) implying higher PI entry after treatment with VS, GNP, and GNP-VS conjugates, the effect being dramatically higher for GNP-VS2.
Figure 4 describes survival rate on the basis of the metabolic activity of the cells. The reducing nature in GNP-VS conjugates seems to retard the metabolic rate of the population. This is illustrated by the relative bar heights in Fig. 4. Importantly, there is also an associated loss of plasma membrane integrity upon such conjugation causing enhancement of PI uptake. Reverse is the case for intact plasma membranes, where PI’s are excluded even in case of cell death (see Fig. 5).
5.2.3 Annexin-V
We now need to explore whether the observed cell death is mediated by apoptosis or necrosis, the two different mechanisms by which cells die (Lieberthal et al. 1996; Kostrzewa 2000; Anselmi et al. 2002; Catelas et al. 2005; Vairetti et al. 2005). The dynamics in these two processes differ greatly. The plasma membrane damage occurs as a primary event during necrosis whereas a delayed but massive programmed phenomenon occurs during apoptosis. In Fig. 5, we have shown only the population of the dead cells, but the mechanism of the event is unexplored. In apoptotic cells, the membrane phospholipid phosphatidylserine is flipped from the inner to the outer leaflet of the plasma membrane, hence exposed to the extracellular environment (Kekre et al. 2005). Annexin V, Ca2+-dependent phospholipid-binding protein, conjugated with FITC has been used with flow cytometry to measure this event. Annexin V-FITC assay as shown in Fig. 6 has identified apoptosis as the early stage event. Interestingly, this followed the DNA fragmentation in the nucleus which is a late apoptotic event (data not shown). The population density as shown in Fig. 6a gradually shifted to higher fluorescence while conjugated with VS. The qualitative shifting of the population has been shown in Fig. 6b and the respective quantitation has been shown in Fig. 6c. Now Fig. 6 describes that the cells are undergoing apoptosis in presence of either GNP or VS. For GNP conjugated with VS there is a cumulative effect.
Dual Staining of annexin V–FITC with propidium iodide (PI) was then used (Fig. 7) for identification of early apoptotic cells (PI negative, Annexin V-FITC positive) from dead cells (PI positive, annexin V–FITC positive). In Fig. 7, the different kinetics of Propidium iodide incorporation by necrotic and apoptotic cells (annexin V positive) after treatment with different GNP and its VS conjugates have been represented along with the control. Figure 7a represents the basis for the flow cytometric identification (dot plot) of different cellular subsets by defining appropriate quadrant. The dual positivity (both annexin V positive and Propidium Iodide positive) of the GNP-VS-treated cells strongly implies that the cells are undergoing apoptosis.
5.2.4 Cell cycle
As VS-mediated chemotherapy is primarily targeted towards the cell cycle, cell-cycle analysis was performed with GNP, VS, and GNP-VS. The relative cellular DNA content was identified by the distribution of the cells during the various phases of the cell cycle. Four distinct phases were recognizing in a proliferating cell population as represented in Fig. 8: G0/G1, S (DNA synthesis phase), and G2/M-phase (mitosis), indicated as M2, M3, and M4, respectively, in the histogram. In G2 and M-phase there is a single population of cells showing identical DNA content (M4). VS was found to affect cells in interphase, producing a transient G2 block with all drug conjugates (GNP-VS1 and GNP-VS2). Metaphase index in case of VS was increased as expected, vincristine being a well-known G2/M blocker.
The cell-cycle pattern was affected by GNP, and in a way that was significantly different from cell-cycle shift induced by VS. In case of GNP, there seems to be an arrest at the S phase of the cell cycle. As shown in Fig. 8a, the G2/M-phase cells are increasing in case of treatment with VS. From the histogram described in Fig. 8a, the GNP-VS conjugates show the cumulative response of both the cell-cycle effectors namely GNP and VS. Figure 8b illustrates the different response of the cell cycle using a contour plot, in which the VS-, GNP-, and the GNP-VS-induced population shift are clearly classified. The analysis further reveals that there is an increase in less-than-2n population. This provides evidence for an onset of DNA fragmentation at the late apoptotic phase of cells in presence of VS, GNP, or GNP-VS.
G2/M population is increased in response to VS as expected, implying a mitotic block, a well-known signature of VS. On the other hand, in case of GNP-VS an additional feature, namely, a decrease of the G0/G1 cell population is thus observed.
Conjugation of drug (here VS) with gold nanoparticle is done successfully by a novel synthesis method. After conjugation, the newly postulated drug (GNP-VS) has achieved unique cell killing properties. The results indicate that Gold conjugated vincristine nanoparticle kills cancer cell line by blocking at synthesis phase (S phase) of cell cycle.
The use of GNP as a drug delivery agent is already established. This is, to our knowledge, the first report describing cell-cycle perturbation by gold nanoparticles. The cumulative effect of GNP and VS on cell cycle makes the combination prospective in chemotherapy of slow-growing cells like MM. The dual role of GNP as a drug carrier and a drug affecting the cell cycle is also an important outcome of the work.