Biocompatible Gold Nanorod Conjugates for Preclinical Biomedical Research

Abstract

Gold nanorods with a peak absorption wavelength of 760 nm were prepared using a seed-mediated method. A novel protocol has been developed to replace hexadecyltrimethylammonium bromide on the surface of the nanorods with 16-mercaptohexadecanoic acid and metoxy-poly(ethylene glycol)-thiol, and the monoclonal antibody HER2. The physical chemistry properties of the conjugates were monitored through optical and zeta-potential measurements to confirm surface chemistry changes. The efficiency of the modifications was quantified through measurement of the average number of antibodies per gold nanorod. The conjugates were investigated for different cells lines: BT-474, MCF7, MCF10, MDCK, and fibroblast. The results show successful cell accumulation of the gold nanorod HER2 conjugates in cells with HER2 overexpression. Incubation of the complexes in heparinized mouse blood demonstrated the low aggregation of the metallic particles through stability of the spectral properties, as verified by UV/VIS spectrometry. Cytotoxicity analysis with LDH release and MTT assay confirms strong targeting and retention of functional activity of the antibody after their conjugation with gold nanorods. Silver staining confirms efficient specific binding to BT-474 cells even in cases where the nanorod complexes were incubated in heparinized mouse blood. This is confirmed through in vivo studies where, following intravenous injection of gold nanorod complexes, silver staining reveals noticeably higher rates of specific binding in mouse tumors than in healthy liver.The conjugates are reproducible, have strong molecular targeting capabilities, have long term stability in vivo and can be used in pre-clinical applications. The conjugates can also be used for molecular and optoacoustic imaging, quantitative sensing of biological substrates, and photothermal therapy.

Figure 1

UV-VIS absorption spectra of GNR. First fraction is from resuspension of the pellet after low speed centrifugation of GNR-CTAB stock solution (not used in this report). The second fraction is from the supernatant which was use in all experiments. Spectra are normalized to match the short wavelength peak of the GNR-CTAB supernatant data.

Figure 2

Different protocols for conjugation of GNR with antibody.

Figure 3

UV-VIS absorption spectra of GNR-Ab (HER2) conjugates with different modifications of GNR surface after incubation at 3 hours in 10% solution of FBS. Control sample is pegylated GNR (PEG). The other three samples are GNR modified with MHDA and PEG (molar ratios are 1, 2 and 4 MHDA for 8 PEG: 1M:8P, 2M:8P and 4M:8P, respectively), activated with cross linkers EDC and sulfo NHS (CL), then conjugated with Ab HER2.

Figure 4

Zeta-Potential (mV) for CTAB coated GNR stock solution (CTAB), GNR after PEGylation (PEG), GNR after removed CTAB with MHDA and PEG (MHDA-PEG) and three different protocols for conjugation and pegylation. GNR with MHDA and PEG, activated with cross linkers EDC and sulfo NHS (CL) and conjugated with Ab: GNR-MHDA/PEG+CL+A (Prot 1); GNR with MHDA, activated with CL, conjugated with Ab and pegylated: GNR-MHDA+CL+Ab+PEG (Prot 2); GNR with MHDA, conjugated with Ab CL complex and pegylated: GNR+MHDA+CL/Ab+PEG (Prot 3) (mean ± SEM, n=10-14 for each conjugate)

Figure 5

Mean number of HER2 molecules on the surface of GNR after conjugation through different protocols (synthesis steps for protocol 1, 2 and 3 are the same as in Figure 4).

Figure 6

Fraction of dead cells for the cell lines (BT 474, MCF 7, MCF 10 and MDCK) after incubation with GNR HER2 conjugates for 48 h, 250 pM (or 1.5 × 10¹¹ GNR/ml). The number of dead cells was counted after staining with Trypan Blue. Control groups of cells received treatment with PBS or GNR after PEGylation (PEG). Synthesis steps for protocols 1, 2 and 3 are the same as in Figure 4 (mean ± SD, n=6 independent measures for each conjugate). Results indicate significantly higher level of cell death for cells with HER2/neu expression, BT 474 and MCF7, and no significant changes without HER2/neu expression: MCF 10 and MDCK.

Figure 7

Metabolically active cells (MTT assay), LDH release, and ratio between LDH release and MTT following incubation of BT 474 cell with GNR conjugates for 48 h, at concentrations up to 500 pM (or 3 × 10¹¹ GNR/ml). Synthesis steps for protocols 1, 2 and 3 are the same as in figure 4 (mean ± SD, n=6 independent measures for each conjugate). All protocols show significant level of cell death, from Prot 1 (highest) to Prot 3 (lowest).

Figure 8

UV-VIS absorption spectra of supernatant solutions of GNR-Ab (HER2) conjugates after incubation for six hours in heparinized mouse blood. Synthesis steps for protocols 1, 2 and 3 are the same as in figure 4. Before and after 1 h incubation all protocols for conjugated and pegylated (PEG and Prot. 1, Prot. 2 and Prot. 3, respectively) have similar spectra. After 6 h incubations a higher level of aggregation is observed in samples from Prot 3, with the best results for Prot.1 (lower aggregation)

Figure 9

Silver staining of fibroblast (no HER2/neu expression) and BT-474 cells following 60 min pre-treatment with pegylated (GNR-PEG) or conjugated through protocol 1 (GNR-PEG-HER2) GNR which were incubated with heparinized mouse blood for four hours. BT 474 shows silver enhancement for GNR-PEG-HER2 conjugates in both conditions: before and after incubation of GNR-conjugates with blood.

Figure 10

Above: Hematoxylin & Eosin (tumor and liver), and Below: silver staining (tumor and liver) of GNR accumulated in mouse tissues following intravenous injection of PBS, GNR-PEG or GNR-PEG-HER2 conjugates. Silver staining shows that GNR peg or Ab conjugates have uniform distribution in liver and noticeably higher number of GNR specific conjugates in mouse tumor.