Purification of GNR through centrifugation and filtration was performed to increase the uniformity of the GNR fraction. The results of centrifugation (needed to discard unwanted pellets) can be seen in . Furthermore, filtration caused negligible alteration of the GNR pectral properties. These purification steps at the end of GNR production resulted in a narrow peak around 760nm.
Absorption spectra of GNR: after low speed centrifugation of GNR-CTAB (Pellet), after PEGylation of GNR (PEG Stock), and after filtration of GNR-PEG (PEG-Filtr). Spectra are normalized to match the short wavelength peak of the PEG Stock data
We investigated the influence of concentrations of GNR-PEG conjugates on the physiological status of cell cultures at different ratios between GNR and PEG. In this set of experiments, two types of cells (SKBR3 and MDCK) were incubated with GNR-PEG ( and ).
Cell proliferation (MTT assay), LDH release (LDHR) into medium, and total quantity of LDH in SKBR3 cells after their membrane are destroyed with Triton X100 (LDHC).
Normalization of the LDH release through total LDH (LDH C + LDH R) and MTT, all abbreviations are the same as in .
For the sake of brevity, only data for SKBR3 cells are reported here as our data for MDCK line showed similar trends. The effect of GNR-PEG was estimated using MTT level, activity of LDHR, and LDHC (). We also calculated the ratios of LDHr to total LDH (LDHR + LDHC) and LDHR/MTT after 24 h treatment with GNR-PEG (molar ratios were 10000, 20000, 50000 and 100000).
We observed only minor differences in all parameters for GNR-PEG conjugates with ratios more than 50000. Low molar ratio, such as 20000 or 10000, resulted in toxic effects suppressing cell proliferation (MTT and their ratio with LDH) and damaging cells membranes. From these experiments we can conclude that the optimum molar ratio between GNR and PEG is near 50000.
To confirm the chemistry modification and stability of GNR-PEG conjugates we investigated the Zeta-potential. It was measured for GNR-CTAB, GNR-PEG and thoroughly washed GNR-PEG after 3 h incubation in 5% bovine serum albumin (PBS, pH 7.4) solution. The GNR-CTAB nanoparticles have a positive charge after synthesis. After PEGylation, a negative zeta-potential confirmed chemistry changes to the GNR surface: the CTAB bi-layer was removed (). Stability of GNR-PEG conjugates was observed as the surface charge did not change in a statistically significant manner after incubation in isosmotic 5% Bovine Serum Albumin (BSA) in PBS with 5 % Glucose at pH 7.4, which simulates blood plasma osmolarity. This therefore indicates BSA does not modify the surface of pegylated GNR, confirming stability.
Zeta-Potential (mV) for CTAB coated GNR before PEGylation (GNR-CTAB), GNR after PEGylation (GNR-PEG) and PEGylated GNR after incubation with 5% BSA in PBS, pH 7.4, 3 hours (GNR-PEG+BSA) (mean ± SD, n = 8)
shows how cell viability depends upon concentration of GNR-PEG for a molar ratio of 1:50000 (0.5 nM GNR-CTAB mixed with 25 μM of PEG). After incubation with different concentration of GNR-PEG, cells stained with trypan blue were counted as a percentage of the total amount of IEC-6, MDCK and HEPG2 cells (). shows results for the same types of cells but after incubation with GNR-PEG or GNR-CTAB conjugates, both in concentration of 0.5 nM (n=5). GNR-PEG did not show damaging effects on cells in low concentrations (1.0 nM and below) with non-significant trend for a concentration of 2 nM. Significantly increased cell death, in comparison to control, was apparent only for GNR-PEG at a concentration of 5 nM, as well as GNR-CTAB incubation at 0.5 nM.
Dose dependence effects of GNR on IEC-6, MDCK and HEPG2 cell lines: A) Percentage of dead cells after staining with Trypan Blue, and B) Percentage of dead cells after incubation with GNR coated with CTAB or PEG
Slices of excised liver tissue shown in were taken from another set of mice to track the accumulation of GNR as well as possible toxicity effects over a span of eight days. Silver staining images demonstrate a visible increase of GNR within the liver from day one up to three days following the injection. However, after eight days, it seems that amount of GNR decreases. The studies with hematoxylin and eosin (HE) staining showed no visible differences between the PBS control and the GNR slices. From the HE staining we cannot confirm any morphological changes in mice liver caused by the GNR.
Silver staining and Hematoxylin & Eosin stain of PEGylated GNR accumulated in mouse liver following intravenous injection
OA images are shown for pre-GNR injection, and one hour after PEG-GNR injection in . We can clearly see an increase in number of distinguishable bright objects (blood vessels and organs) an hour following the GNR injection. Peripheral blood vessels around the back, ribs, and underneath the arms are most enhanced. Vertebrae, interestingly enough, are also quite apparent during this time, with various vertebrae discs of the mouse being visualized.
Optoacoustic images of nude mice before, and 1 hour after, intravenous injection of PEG-GNR (3 × 1012 GNR per mouse)