Fluorescent CdSe/ZnS Nanocrystal−Peptide Conjugates for Long-term, Nontoxic Imaging and Nuclear Targeting in Living Cells

Abstract

One of the biggest challenges in cell biology is the imaging of living cells. For this purpose, the most commonly used visualization tool is fluorescent markers. However, conventional labels, such as organic fluorescent dyes or green fluorescent proteins (GFP), lack the photostability to allow the tracking of cellular events that happen over a period from minutes to days. In addition, they are either toxic to cells (dyes) or difficult to construct and manipulate (GFP). We report here the use of a new class of fluorescent labels, silanized CdSe/ZnS nanocrystal−peptide conjugates, for imaging the nuclei of living cells. CdSe/ZnS nanocrystals, or so-called quantum dots (qdots), are extremely photostable, and have been used extensively in cellular imaging of fixed cells. Most of the studies about living cells so far have been concerned only with particle entry into the cytoplasm or the localization of receptors on the cell membrane. Specific targeting of qdots to the nucleus of living cells has not been reported in previous studies, due to the lack of a targeting mechanism and proper particle size. Here we demonstrate for the first time the construction of a CdSe/ZnS nanocrystal−peptide conjugate that carries the SV40 large T antigen nuclear localization signal (NLS) and the transfection of the complex into living cells. By a novel adaptation for qdots of a commonly used cell transfection technique, we were able to introduce and retain the NLS−qdots conjugate in living cells for up to a week without detectable negative cellular effects. Moreover, we can visualize the movement of the CdSe/ZnS nanocrystal−peptide conjugates from the cytoplasm to the nucleus, as well as the accumulation of the complex in the cell nucleus, over a long observation time period. This report opens the door for using qdots to visualize long-term biological events that happen in the cell nucleus and provides a new nontoxic, long-term imaging platform for observing nuclear trafficking mechanisms and cell nuclear processes.

Cellular Effect of High Doses of Silica-Coated Quantum Dot Profiled with High Throughput Gene Expression Analysis and High Content Cellomics Measurements

“…23,30 In contrast to Qdots with a nuclear localization sequence on the surface, PEGsilane-Qdots are unable to cross the nuclear membrane, 23 preventing their direct interaction with the genetic machinery in the cell nucleus. This precludes studies requiring the labeling of nuclear materials, creating a definite disadvantage.…”

In vitro and in vivo toxicity assessment of nanoparticles

“…Treatment of Hela cells with CdSe quantum dots with sizes 1, 10 and 100 nm for 2 h resulted in survival of 90% cells [160]. In another experiment human lymphoblastoid cells were treated with CdSe quantum dots at 0.2 µm size for 12 h; the study resulted in decreased cell activity [161].…”

Quantum Dots in Biomedical Research

“…The electroporation is based on the application of one or more strong electrical pulses, which is thought to create localized pores in the membrane bilayer for enhancing its permeability - Figure 9A. Using electroporation, Chen and Gerion delivered QDs into HeLa cells (Chen & Gerion, 2004). The QDs were able to be recognized by the cellular machinery and enter the cell nucleus in living cells, although in a fraction of the total number of cells and as agglomerated particles.…”

“…However, traffic of molecular dynamics, especially in live cells, are almost exclusively carried out for surface molecules -membrane proteins, accessible by the external side of the cells. This limitation arise mainly due to the incapacity of water dispersed QDs to cross the lipid bilayers by simple diffusion (only few works report tracking of intracellular molecules) (Chen & Gerion, 2004). …”