Silica layer-dependent leakage of cadmium from CdSe/ZnS quantum dots and comparison of cytotoxicity with polymer-coated analogues

Abstract

The cytotoxicity of cadmium-containing quantum dots (QDs) is reportedly caused by released Cd²⁺ as well as other factors such as ligand, size, and surface modification. The tolerated concentration of QDs therefore deviates from ~ 1 to 1000 nM. However, the concentration of Cd²⁺ released from QDs has seldom been correctly and systematically measured. We prepared highly emitting silica capsules with incorporated multiple CdSe/ZnS QDs through a sol-gel-derived wet method. The concentration of released Cd²⁺ in buffer solution was measured as a function of the preparation conditions of the capsules. When the shell thickness of the capsule was 15 nm, the release was effectively suppressed compared with a capsule shell thickness of 10 nm. Heat treatment at 40 °C further suppressed the leakage. When the silicon alkoxide hydrolysis time was increased from 3 to 15 h, and the surface was modified with COOH groups, the leakage reached a minimum of ~ 0.01 ppb for a QD concentration of 10 nM after 15 h of dispersion. This was two orders of magnitude less than polymer-coated analogues with the same surface functional groups. These silica capsules did not show any toxicity to culture cells, whereas the polymer-coated ones showed high toxicity with the same QD concentration. Silica capsules are therefore not only the expected ideal, but show significantly superior performance when correctly prepared for biomedical application with CdSe/ZnS QDs, owing to their strong suppression of Cd²⁺ leakage into solution.

Appendices

Appendices

Cytotoxicity experiment

Cells (A549 and HaCaT) were incubated with 0.5 mg/ml MTT (Nacalai Tesque, Inc.) at 37 °C for 2 h. Isopropyl alcohol containing 40 mM HCl was added to the culture medium (3:2, by volume) and the cells were mixed by pipette until the formazan was completely dissolved. The optical density of the formazan was measured at 570 nm by using a Multiskan Ascent plate reader (Thermo Labsystems, Helsinki, Finland). In the LDH assays, LDH release was measured with a tetrazoliumsalt by using a Cytotoxicity Detection KitPLUS (LDH) (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s protocol. The amount of formazan salt that formed was measured at 492 nm by using the Multiskan Ascent plate reader. The maximum amount of released LDH was determined by incubating the cells with a lysis solution provided in the kit. The cytotoxicity was calculated as follows: cytotoxicity (%) = (experimental value - low control)/(high control - low control) × 100. The low control, which refers to spontaneous LDH release, was determined as the LDH released from untreated normal cells. The high control, which refers to the maximum LDH release, was determined as the LDH released from cells that were lysed by surfactant treatment.

Concentration of released Cd²⁺ and measurement conditions in Xu M et al., Metallomics, 2, 469–473 (2010)

Based on the PL wavelength (slightly shorter than 550 nm) of the TGA-capped CdTe QDs, their size is estimated to be 2.6 nm [A]. These QDs therefore contain 135 Cd atoms each when the number of Cd and Te atoms is the same. Upon dialysis, a 2-mL solution of QDs (Cd, 0.1 g/L) was dialyzed against 400 mL of PBS solution. This volume of QD solution contains 13.2 nmol of QDs.

When the concentration increment of Cd²⁺ in dialysis solution is 1 ng/mL/mg/h in their measurement, we understand that 1 ng of Cd²⁺ is released per hour into 1 mL of dialysis solution from the QD solution containing 1 mg of Cd atoms.

When 1 mg of Cd atoms is in 2 mL of solution, 66 nmol of QDs are dispersed therein (the concentration is 33 μM). When 1 ng of Cd²⁺ ions from this solution are released in 1 mL of receiving solution per hour, 400 ng of Cd²⁺ are actually released from the total QDs investigated because the volume of the dialysis solution is 400 mL. This corresponds to a concentration increment of 200 μg/L/h. This means that the amount of Cd²⁺ released in 10 nM of QDs is 61 ng/1000 g/h, which corresponds to 0.061 ppb/h. Since we removed the solution after 15 h for our experiment, this means that the concentration of Cd²⁺ is 0.91 ppb under our experimental conditions.

However, we know that Cd source, Te source, and thiol molecules form clusters upon aqueous synthesis of CdTe QDs [B]. Therefore, it is plausible that Cd²⁺ is released in the form of clusters from QDs. The very high concentration of QD solution (6.6 μM) upon dialysis, which is close to the concentration just after preparation [C], may further promote the formation of clusters. The size of the clusters can reach ~ 1 nm. Therefore, we suspect that the size of the pore for dialysis (500 MWCO) is too small to precisely evaluate the total amount of Cd²⁺ released. Another reported dialysis of TGA-capped CdTe uses dialysis tubing with 6000- or 8000 MWCO [D]. As described in the text, our selection for measurement was 3000 MWCO for the CdSe-related QDs, which is slightly smaller than the size of the QD itself. Our measurement of Cd²⁺ from red-emitting TGA-capped CdTe was 80 ppb after 15 h in pure water, which is similar to the value (30 ppb) discussed in the following section on HeLa cells.

Concentration of released Cd²⁺ in Su Y et al., Biomaterials, 31, 4829–4834 (2010)

Since the emission of the QD is in the red region, we assumed the diameter of CdTe to be 3.8 nm. In this case, 420 Cd atoms are contained in each QD if the number of Cd and Te atoms is the same. Since the HeLa cell volume is estimated to be 3000 μm³ [E], the concentration of free Cd²⁺ in cells containing 1 ng/10⁵ cells corresponds to 33 ppb.

Because the intracellular Cd of CSS (CdTe/CdS/ZnS) is in the order of 1 ng/10⁵ cells (Fig. 3b), the concentration of QD is roughly 70 nM. Intracellular Cd²⁺ for CSS is in the order of 0.1 ng/10⁵ cells (Fig. 5b). Therefore, its concentration is 330 ppb. The duration of dispersion is 24 h. This therefore corresponds to ~ 30 ppb for a 10-nM dispersion of QDs over 15 h (our conditions).

References in appendices

[A] Murase N, Gaponik N, Weller H (2007) Effect of chemical composition on luminescence of thiol-stabilized CdTe nanocrystals. Nanoscale Res Lett 2:230–234

[B] Shavel A, Gaponik N, Eychmüller A (2006) Factors governing the quality of aqueous CdTe nanocrystals: calculations and experiment. J Phys Chem B 110, 19:280–19,284

[C] Li CL, Murase N (2005) Surfactant-dependent photoluminescence of CdTe nanocrystals in aqueous solution. Chem Lett 34:92–93

[D] Gao MY, Kirstein S, Möhwald H, Rogach AL, Kornowski A, Eychmüller A, Weller H (1998) Strongly photoluminescent CdTe nanocrystals by proper surface modification. J Phys Chem B 102:8360–8363

[E] http://book.bionumbers.org/how-big-is-a-human-cell/