Lactose as a “Trojan Horse” for Quantum Dot Cell Transport

A series of glycan-coated quantum dots were prepared to probe the effect of glycan presentation in intracellular localization in HeLa and SV40 epithelial cells. We show that glycan density mostly impacts on cell toxicity, whereas glycan type affects the cell uptake and intracellular localization. Moreover, we show that lactose can act as a “Trojan horse” on bi-functionalized QDs to help intracellular delivery of other non-internalizable glycan moieties and largely avoid the endosomal/lysosomal degradative pathway.


Lipoic Acid-PEG-OH (2).
A solution of S3 (1.79 g, 5.92 mmol) was neutralised in aqueous NaHCO 3 (1 M, 28 mL) and the volume of the solution was reduced to 10 mL in vacuo. The solution was cooled to 4 °C and to it a solution of S4 (2.66 g, 9.23 mmol) was added dropwise, over 90 minutes. The reaction was then warmed to 20 °C and left to stir for 24 hours. The product was extracted with CHCl 3 (3 x 40 mL) and dried over MgSO 4 . The product was evaporated to dryness and purified by flash silica gel chromatography (CH 2 Cl 2 /MeOH gradient: 1/0 to 9/1, v/v) to give 2 as a yellow oil (1.1 g, 70%

Cell culture
HeLa cells were maintained in Minimal Essential Medium (MEM) with GlutaMAX™ and 10% fetal bovine serum (FBS); AS cells were grown in Dulbecco's MEM with Glutamax and 10 % FBS.
Both media were supplemented with antibiotic-antimycotic (Anti-Anti). Confluent cultures were detached from the surface using trypsin (Tryp LE Express) and plated at 10 4 cells/well in either petri dishes (Mat-Tek 35 mm, with 14mm glass microwell) for imaging, or 96-well plates for toxicity tests.
Tests were carried out the next day as specified below. All cell culture media and additives were purchased from Invitrogen, Life Technologies.

Confocal microscopy
Cell trackers used were purchased from Life Technologies: CellLight® Reagents *BacMam 2.0: early Endosomes (Rab5a), late endosomes (Rab7a), ER-Tracker™ for the endoplasmic reticulum, BODIPY TR ceramide for the Golgi reticulum, mitochondria and LysoTracker TM for lysosomes, carbocyanine-based MitoTracker® (not dependent on oxidation status), and NucBlue™ Live Cell Stain (Hoechst 33342) for nuclei. These markers were used in line with manufacturer's protocols. All images were acquired on a Leica SP5 confocal system equipped with a Leica DMI 6000 inverted microscope. For the excitation of the QDs, either the blue laser or the blue lines of the argon ion laser (405 and 488nm respectively) were used. For the excitation of cellular manufacturers specifications were followed. The images were analysed using Volocity software (PerkinElmer). See full list of images at the end of ESI.

Toxicity assays
The influence of quantum dots on cell metabolism was assessed using AlamarBlue, a cytosolic substrate for reductive metabolism (resazurin to resorufin) whose fluorescence spectrum changes on reduction (Invitrogen Life Technologies). Cell numbers and proliferation were assessed by total protein, bicinchoninic acid assay (BCA, Pierce, ThermoScientific). Cells were exposed to quantum dots for 4, 24 and 48h in medium without foetal bovine serum. Results are expressed as changes in metabolism per µg protein. Figure S4 shows that toxic effects depend on oligosaccharide ligand concentration on QDs. The effects of a 24h exposure are illustrated in Figure S5a for Araki Sasaki cells. Figure S5b shows the effect of a 48h exposure on the metabolic compatibility of HeLa cells.
HeLa cells were exposed to Lactose-functionalised QDs for 24h. Foetal bovine serum was omitted from the medium to avoid masking of toxic effects on metabolism.

Figure S5
Reductive metabolism relatie to cell numbers after exposure to equal doses of differently functionalized Quantum dots. a. AS cells exposed for 24 hours; cells exposed to dextran-, galactose-and lactose-functionalised QDs are not significantly different from the controls, while cells exposed to capped quantum dots have significantly lower metabolic activity than the controls (p<0.001). b. HeLa cells exposed for 28 hours; dextran-and lactose-QDs have no effect on HeLa cell metabolism. Capped QDs and galactose-QDs are more toxic than no treatment (p<0.05). Note that most galactose-QDs were located in the early and Late endosomes in HeLa, while just over half colocalised with early endosomes in AS. The low level of colocalization of other sugar-QDs with intracellular organelles is supported by their presence in the cytosol. Images and regions of interest were analyzed for each experimental condition. The data were analyzed by Student's t test or one-way analysis of variance; P < 0.05 was considered significant.