Effect of Nanoparticle Surface Coating on Cell Toxicity and Mitochondria Uptake

Abstract

We report on the effect of surface charge and the ligand coating composition of CdSe/ZnS core/shell quantum dot (QD) nanoparticles on human keratinocyte toxicity using fluorescent microscopy, flow cytometry, transmission electron microscopy. Two commonly reported positive charged (cysteamine, polyethylenimine) and two negative charged (glutathione, dihydrolipoic acid) ligands were studied. The QDs were fully characterized by UV-vis absorption spectroscopy, fluorescence emission spectroscopy, dynamic light scattering and zeta potential. Differences in surface coatings and charges were evaluated against cellular uptake, ROS generation, cytotoxicity, and mitochondrial targeting. Results show that the negative charged QDs coated with GSH exhibit excellent water solubility, high quantum yield and low cytotoxicity. Ligand composition is more important in ROS generation than surface charge whereas surface charge is an important driver of cytotoxicity. Most importantly we observe the selective accumulation of glutathione coated QDs in vesicles in the mitochondria matrix. This observation suggests a new strategy for developing mitochondria-targeted nanomaterials for drug/gene delivery.

Keywords: Quantum Dots; Keratinoyctes; Endocytosis; Inracellular Localization; Mitochondria.

Figure 1

Comparison of the UV/Vis absorption spectra for the solvent soluble ODA capped QDs and the four different water soluble ligand capped QDs. All solutions were 8 μM. Results show very little change in the absorption properties.

Figure 2

Comparison of the photoluminescence (PL) spectra for the solvent soluble ODA capped QDs and the four different water soluble ligand capped QDs. All solutions were 8 μM. Results show very little change in the PL peak wavelength or shape but differences exist in quantum yield.

Figure 3. ROS generation in HaCaT cells exposed to negative and positive charged QD assessed by flow cytometry.

Representative examples of flow cytometry data showing ROS levels in (A) in HaCaT cells exposed to GSH-QDs (dash line) and DHLA-QDs (solid line); dotted line shows untreated cells and (B) HaCaT cells exposed to CYS-QDs (dash line) and PEI-QDs (solid line); dotted line shows untreated cells. (C) Summary comparison of ROS generation for different ligands capped QDs (GSH-, DHLA-, CYS-, PEI-QDs) in HaCaT cells. ROS levels were determined by the DCF-DA as described in Section 2. Cell cultures were treated with different ligands capped QDs and ROS production was measured as fluorescence intensity. Results are expressed as means±SD (n=3). * indicates significant differences between treated and untreated cell culture (p<0.05).

Figure 4.. Negative and positive charged QD association with HaCaT cells assessed by flow cytometry.

Representative examples of flow cytometry data showing QD association (A) with HaCaT cells exposed to GSH-QDs (dash line) and DHLA-QDs (solid line); dotted line shows untreated cells and (B) with HaCaT cells exposed to CYS-QDs (dash line) and PEI-QDs (solid line); dotted line shows untreated cells. (C) Summary comparison of QD cellular association for the different water soluble ligands capped QDs (GSH-, DHLA-, CYS-, PEI-QDs), (n=3). Data are expressed as fluorescence intensity units. ** indicate significant differences between treated and untreated control cell culture (p<0.001).

Figure 5

Assessment of QDs coated with different ligands induce uptake by HaCaT cell using fluorescence microscope after 24 h incubation with HaCaT cells. (Scale bar: 25μm)

Figure 6

HaCaT cell viability data for (10 nM) QDs coated with different positive and negative charge ligands for 24 h using standard MTT colorimetric assay (n=3).

Figure 7a. TEM images illustrating the cellular uptake and intracellular translocation of positively charged CYS-QDs.

(i) Endocytosis of CYS-QD aggregates into a vesicle at plasma membrane is evident. (ii) Maintenance of CYS-QD aggregates in spherical vesical is evident (blue arrows). Inset shows magnified view of the vesical containing CYS-QDs. (iii) Once internalized the CYS-QD aggregates appear to disrupt the vesicle that contained them and they appear to localize in the cytosol as free aggregates (green arrows). (iv) There was no evidence for accumulation of CYS-QDs in the nucleus (N) or uptake into mitochondria (red arrows).

Figure 7b. TEM images illustrating the cellular uptake and intracellular translocation of positively charged PEI-QDs.

(i) HaCaT cells appear to sequester PEI-QD aggregates in cytosolic vesicles. (ii) Enlarged view of vesicle showing distinct membrane (arrow) (iii) Unlike vesicles containing CYS-QDs, quite often the vesicles containing PEI-QDs appear fluid filled. HaCaT cells also appear to produce much more lipid droplets (L); grey circles. (iv) Enlarged view of fluid filled vesicles containing PEI-QDs. There was no evidence for penetration or accumulation of PEI-QDs in nucleus (N) or uptake into mitochondria (red arrows).

Figure 7c. TEM images illustrating the cellular uptake and intracellular location of negatively charged DLHA-QDs.

(i) In comparison to positive charged QDs (CYC, PEI), much fewer instances of negative charged DHLA-QD aggregates were present in HaCaT cells. (ii) Enlarged view of image in (i) showing small clusters of DLHA-QDs in a vesicle with a distinct membrane (black arrows). (iii) Another example of a HaCaT cell with small DHLA-QD clusters. (iv) Enlarged view of image in (iii) showing small DHLA-QD clusters (blue arrows). There was no evidence for accumulation of PEI-QDs in nucleus (N) or uptake into mitochondria (red arrows).

Figure 7d. TEM images illustrating the cellular uptake and intracellular location of negatively charged GSH -QDs.

(i) Similar to the negative charged DHLA-QDs there are much fewer instances of cytosolic clusters of GSH-QDs compared to the positive charged QDs (CYS, PEI) in HaCaT cells. Interestingly, we find that the preponderance of GSH-QD cytosolic clusters localize in the mitochondria. (ii) Enlarged view of image (i) showing small GSH-QDs clusters in mitochondria (red arrows). (iii) Enlarged view of image (i) showing small GSH-QDs clusters in mitochondria. GSH-QDs appear sequestered in a vesicle in the mitochondria matrix. (iv) Some GSH-QD clusters also localize into vesicles. There was no evidence for accumulation of GSH-QDs in the cell nucleus (N).

Figure 8

Subcellular localization of different ligands coated QDs with mitochondria in HaCaT cells after 24h treatment (scale bars 25μm).