Bio-distribution and toxicity assessment of intravenously injected anti-HER2 antibody conjugated CdSe/ZnS quantum dots in Wistar rats

Abstract

Anti-HER2 antibody conjugated with quantum dots (anti-HER2ab-QDs) is a very recent fluorescent nanoprobe for HER2+ve breast cancer imaging. In this study we investigated in-vivo toxicity of anti-HER2ab conjugated CdSe/ZnS QDs in Wistar rats. For toxicity evaluation of injected QDs sample, body weight, organ coefficient, complete blood count (CBC), biochemistry panel assay (AST, ALT, ALP, and GGTP), comet assay, reactive oxygen species, histology, and apoptosis were determined. Wistar rat (8-10 weeks old) were randomly divided into 4 treatment groups (n = 6). CBC and biochemistry panel assay showed nonsignificant changes in the anti-HER2ab-QDs treated group but these changes were significant (P < 0.05) in QDs treated group. No tissue damage, inflammation, lesions, and QDs deposition were found in histology and TEM images of the anti-HER2ab-QDs treated group. Apoptosis in liver and kidney was not found in the anti-HER2ab-QDs treated group. Animals treated with nonconjugated QDs showed comet formation and apoptosis. Cadmium deposition was confirmed in the QDs treated group compared with the anti-HER2ab-QDs treated group. The QDs concentration (500 nM) used for this study is suitable for in-vivo imaging. The combine data of this study support the biocompatibility of anti-HER2ab-QDs for breast cancer imaging, suggesting that the antibody coating assists in controlling any possible adverse effect of quantum dots.

Keywords: HER2; anti-HER2 antibody; cancer bioimaging; comet assay; quantum dots.

Figure 1

TEM image of quantum dots (QDs) and anti-HER2ab-QDs. Bar size is 20 nm in both images and analysis was done at 200 kv.

Figure 2

Dynamic light scattering histogram for hydrodynamic diameter detection of quantum dots (QDs) and anti-HER2ab-QDs in 10 mM PBS buffer: a) QDs (525 nm) and b) anti-HER2ab-QDs (525 nm). The hydrodynamic sizes of the QDs and anti-HER2ab-QDs were 11.7 nm and 15.7 nm, respectively.

Figure 3

Body weight of Wistar rats following injection of quantum dots (QDs) and anti-HER2ab-QDs. Mean and standard deviation of body weight of Wistar rats treated with QDs, anti-HER2ab-QD, and phosphate buffered saline control were not significantly different over a 2-month period.

Figure 4

Coefficient of organs (liver, kidney, spleen, and brain) for Wistar rats treated with quantum dots (QDs), anti-HER2ab-QDs and PBS. Coefficient of organs is the ratio of weight of the organs (g) to animal weight (g). No significant difference found at α = 0.05. Statistical analysis was performed with a 2-sample t-test, unknown and unequal variances, comparing each sample group to the related control group. Note: *denotes statistically significant results at α = 0.05.

Figure 5

Hematology analysis for the Wistar rats treated with quantum dots (QDs), anti-HER2-ab-QDs, and phosphate buffered saline. A–F) These results show mean and standard deviation of hemoglobin (A), white blood cells (B), neutrophils (C), lymphocytes (D), red blood cell count (E), and platelets (F). Error bars represent standard deviation. Statistical analysis was performed with a 2-sample t-test, unknown and unequal variances, comparing each sample group to the related control group. Note: *denote statistically significant results at α = 0.05.

Figure 6

Biochemistry panel assays from Wistar rats treated with quantum dots (QDs), anti-HER2ab-QDs, and phosphate buffered saline. A–E) Results illustrate mean and standard deviation of total protein, albumin, globulin (A), AST and ALT (B), ALP (C), GGTP (D), and bilirubin (total, direct, indirect) (E). Error bars represent standard deviation. Statistical analysis was performed with a 2-sample t-test, unknown and unequal variances, comparing each sample group to the related control group. Note: *denotes statistically significant result at α = 0.05.

Figure 6

Biochemistry panel assays from Wistar rats treated with quantum dots (QDs), anti-HER2ab-QDs, and phosphate buffered saline. A–E) Results illustrate mean and standard deviation of total protein, albumin, globulin (A), AST and ALT (B), ALP (C), GGTP (D), and bilirubin (total, direct, indirect) (E). Error bars represent standard deviation. Statistical analysis was performed with a 2-sample t-test, unknown and unequal variances, comparing each sample group to the related control group. Note: *denotes statistically significant result at α = 0.05.

Figure 7

N, N-diethyl-pera-phenylenediamine (DEPPD) assay for reactive oxygen species (ROS) measurement from the blood serum of Wistar rats treated with quantum dots (QDs) and anti-HER2ab-QDs. Note: 1 ROS unit = 1 mg/L H₂O₂.

Figure 8

Concentration of cadmium in liver, kidney and spleen of Wistar rats treated with quantum dots (QDs), anti-HER 2ab-QDs, and phosphate buffered saline. Results show mean and standard deviation of cadmium in liver, kidney, and spleen powder. Note: *denotes statistically significant results at α = 0.05.

Figure 9

Graphical representation of comet assay from blood sample of quantum dots (QDs) and anti-HER2ab-QDs treated Wistar rats. X-axis indicates treatment group of animal and y-axis represent comet values (tail moment, tail migration, and tail length). Statistical analysis was performed with a 2-sample t-test, unknown and unequal variances, comparing each sample group to the related control group. Comet analysis was done by Comet assay IV software (Perspective Instruments, UK). Note: *denotes the level of significance at α = 0.05.

Figure 10

Liver, kidney and spleen histology. Hematoxylin and eosin stains of liver (1–3), kidney (4–6), and spleen (7–9) tissues of Wistar rats injected with phosphate buffered saline, quantum dots (QDs), and anti-HER2ab-QDs. One-headed arrow indicates tissue damage (yellow), mineralization (green), and protein fluid (black).

Figure 11

TEM images of liver and kidney of quantum dots (QDs) and anti-HER2ab-QDs treated Wistar rats: A) control (liver), B) animals treated with QDs (liver), C) animals treated with anti-HER2ab-QDs, D) control kidney, E) animals treated with QDs, F) animals treated with anti-HER 2ab-QDs. Single staining was used for all TEM analysis. Random circle in the image show nucleolus, arrow in white color show mitochondria and red arrow indicate RER (rough endoplasmic reticulum).

Figure 12

Live/dead cell viability assay of the liver and kidney of Wistar rats treated with 100 μL of 500 nM quantum dots (QDs) and anti-HER2ab-QDs. Typical histograms of YO-PRO-1/PI stained liver and kidney cells. A–C) liver cells (control, anti-HER2ab-QDs, and QD respectively); D–F) kidney cells (control, anti-HER2ab-QDs, and QD respectively).