Shell-Free Copper Indium Sulfide Quantum Dots Induce Toxicity in Vitro and in Vivo

Abstract

Semiconductor quantum dots (QDs) are attractive fluorescent contrast agents for in vivo imaging due to their superior photophysical properties, but traditional QDs comprise toxic materials such as cadmium or lead. Copper indium sulfide (CuInS₂, CIS) QDs have been posited as a nontoxic and potentially clinically translatable alternative; however, previous in vivo studies utilized particles with a passivating zinc sulfide (ZnS) shell, limiting direct evidence of the biocompatibility of the underlying CIS. For the first time, we assess the biodistribution and toxicity of unshelled CIS and partially zinc-alloyed CISZ QDs in a murine model. We show that bare CIS QDs breakdown quickly, inducing significant toxicity as seen in organ weight, blood chemistry, and histology. CISZ demonstrates significant, but lower, toxicity compared to bare CIS, while our measurements of core/shell CIS/ZnS are consistent with literature reports of general biocompatibility. In vitro cytotoxicity is dose-dependent on the amount of metal released due to particle degradation, linking degradation to toxicity. These results challenge the assumption that removing heavy metals necessarily reduces toxicity: indeed, we find comparable in vitro cytotoxicity between CIS and CdSe QDs, while CIS caused severe toxicity in vivo compared to CdSe. In addition to highlighting the complexity of nanotoxicity and the differences between the in vitro and in vivo outcomes, these unexpected results serve as a reminder of the importance of assessing the biocompatibility of core QDs absent the protective ZnS shell when making specific claims of compositional biocompatibility.

Keywords: CIS; CuInS2; QDs; biodegradable; fluorescent contrast agent; in vivo imaging; nanomedicine; nanotoxicity.

Figure 1.. Characterization of CIS QDs.

(A) Normalized photoluminescence (PL) spectra of CIS, zinc-treated CIS (CISZ), thin-shelled CIS (CIS/ZnS’), and thick-shelled CIS (CIS/ZnS). (B) X-ray diffraction (XRD) profiles of each QD type, along with reference peaks from the Crystallography Open Database (COD). The slight shift of the CIS/ZnS peaks corresponds to the growth of a solid ZnS shell (see reference peaks), while the decreased peak width indicates larger crystals, correlating with the TEM images. CIS and CISZ exhibit nearly identical peak positions. (C) Representative transmission electron microscopy (TEM) images of each QD. Scale bar is 10 nm. (D) Molar ratio of metal components of each QD, as determined by microwave plasma atomic emission spectroscopy (MP-AES).

Figure 2:. In vitro dissolution of CIS-based QDs.

(A) Absorption spectra (mean ± standard deviation, n = 4) of micelle-encapsulated CIS, CISZ, and CIS/ZnS in simulated biological fluid (SBF). Both CIS and CISZ degrade rapidly, while CIS/ZnS remains relatively stable. (B) Photos of CIS and CIS/ZnS solutions in SBF at days 1, 4, and 10. (C) Normalized absorbance at 400 nm of QDs in water, (D) SBF, and (E) artificial lysosomal fluid (ALF). CIS QDs dissolved in all solutions. CISZ exhibited similar dissolution kinetics to CIS in all solutions except water, while CIS/ZnS QDs were the most stable in all solutions. Mean ± standard deviation, n = 4.

Figure 3.. Biodistribution and clearance of CIS QDs.

(A) Organ-specific distribution of indium over time (as % initial dose). Inset shows kidney and heart data on different scale. (B) The summed indium content for CIS, CISZ, and CIS/ZnS. Asterisks indicate significance level of comparison at day 28, as determined Games-Howell ANOVA post-hoc test: **: p < 0.01, ***: p < 0.005, ****: p < 0.001, n=4. Error bars are one standard deviation.

Figure 4.. Blood biochemistry values.

(A) ALT, (B) AST, and (C) BUN values for each animal. Insets are zoomed in regions from dotted rectangles. (D) Organ index (organ weight/total body weight) plotted as percent of control values for four major organs. Error bars are one standard deviation. Asterisks indicate significance level compared to controls, as determined by Games-Howell ANOVA post-hoc test: *: p < 0.05, **: p < 0.01, ***: p < 0.005, ****: p < 0.001. n = 4 in all but two groups for organ index (CIS/ZnS D1 and CIS D7) and three groups for blood biochemistry (CISZ D1, CIS/ZnS D1 and CIS D7), which were n = 3. Note the different organ index axis scaling for the spleen compared to the other organs.

Figure 5.. Histology of QD-dosed mice.

Left, middle, and right columns show liver, spleen, and kidney, respectively. Yellow arrows indicate inflammatory cells; blue arrows point to multinucleated giant cells. Scale bar is 100 μm for primary photos and 50 μm for inserts.

Figure 6.. Comparative toxicity in vivo.

(A)Spleen index (on log scale) plotted against ALT values at day 7 for commercially sourced and lab-made CIS-based QDs as well as CdSe. (B) Results of Games-Howell post-hoc test for spleen index results. p values of pairwise comparisons are given in the table, with statistically significant values (p < 0.05) in bold type. Statistically significant pairwise comparisons of AST and ALT values are also marked with asterisks (*: p < 0.05) and daggers (†: p < 0.05, ††: p < 0.01), respectively. Plots of ALT versus (C) indium dose (D) copper dose, (E) the sum of indium and copper dose, and (F) total cation dose (In + Cu + Zn) for individual mice, with linear fits included.

Figure 7:. In vitro toxicity assay.

(A) Quantification of HepG2 viability after 24 h treatment with QDs, n=4 wells per dose. (B) Dose response curve derived from viability assays for two levels of degradation fit to a 4-parameter Hill equation; shaded region indicates the 95% confidence interval.