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WIREs Nanomed Nanobiotechnol
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Continuing progress toward controlled intracellular delivery of semiconductor quantum dots

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The biological applications of luminescent semiconductor quantum dots (QDs) continue to grow at a nearly unabated pace. This growth is driven, in part, by their unique photophysical and physicochemical properties which have allowed them to be used in many different roles in cellular biology including: as superior fluorophores for a wide variety of cellular labeling applications; as active platforms for assembly of nanoscale sensors; and, more recently, as a powerful tool to understand the mechanisms of nanoparticle mediated drug delivery. Given that controlled cellular delivery is at the intersection of all these applications, the latest progress in delivering QDs to cells is examined here. A brief discussion of relevant considerations including the importance of materials preparation and bioconjugation along with the continuing issue of endosomal sequestration is initially provided for context. Methods for the cellular delivery of QDs are then highlighted including those based on passive exposure, facilitated strategies that utilize peptides or polymers and fully active modalities such as electroporation and other mechanically based methods. Following on this, the exciting advent of QD cellular delivery using multiple or combined mechanisms is then previewed. Several recent methods reporting endosomal escape of QD materials in cells are also examined in detail with a focus on the mechanisms by which access to the cytosol is achieved. The ongoing debate over QD cytotoxicity is also discussed along with a perspective on how this field will continue to evolve in the future. WIREs Nanomed Nanobiotechnol 2015, 7:131–151. doi: 10.1002/wnan.1281 This article is categorized under: Diagnostic Tools > In Vitro Nanoparticle-Based Sensing Implantable Materials and Surgical Technologies > Nanomaterials and Implants Nanotechnology Approaches to Biology > Nanoscale Systems in Biology
Representative or schematic examples of quantum dots (QDs) use as passive fluorophores, active sensors, and theranostic tools. (a) Five‐color immunohistochemical labeling of a mouse splenic tissue section simultaneously stained with QDs is used as a representative example for the passive fluorophore role. (Reprinted with permission from Ref . Copyright 2013 ACS) (b) For active sensing, a schematic describing the activity of a caspase‐1 sensor is highlighted. This nanosensor is composed of QDs and rhodamine‐B molecules, connected through a short peptide, cleavable by caspase‐1. When the QDs are excited, they transfer their energy to the dye molecules by Förster resonance energy transfer (FRET) and the emission is observed at the wavelength specific for the dye. After enzymatic cleavage of the peptide molecules, the acceptor molecules are detached from the QDs which no longer provide an efficient energy transfer channel to them, and emission spectra changes back to that of QDs. Dissected mouse brain showing an example of fluorescence from this nanosensor. (Reprinted with permission from Ref . Copyright 2013 ACS). (c) Theranostic tools are highlighted on the right with a schematic of a nanoparticle (NP) bioconjugate. Each biological molecule would provide a different potential activity to the final conjugate. For example, the antibody would potentially provide targeting, the peptide – cellular uptake, the protein – sensing, the drug and nucleic acid could act as therapeutics. The central NP (read QD) acts as a central nanoscale platform, provides solubility through its polyethylene glycol or PEG layer, and can still contribute to imaging or sensing with inherent fluorescence or magnetic contrast depending upon its constituents and structure.
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Cellular delivery featuring quantum dots (QDs) in a combinatorial role. (a) QD scaffold for antisense oligonucleotide delivery and tracking. The 625 nm‐emitting QDs (red) appended with antisense oligos for knockdown of folate receptor expression and cell uptake peptides (peptide p160) are released from the endosomes of MCF‐7 cells after internalization. Endosomes were labeled with fluorescein‐conjugated transferrin. Yellow coloring shows QDs colocalized with the green transferrin marker while red signal shows QDs liberated from endosomes to the cytosol. Left panel, bright field; right panel, fluorescence. (Reprinted with permission from Ref . Copyright 2013 Wiley) Scale bar is 5 µm. (b) Drug and small interfering RNA (siRNA) delivery mediated by QDs. Confocal images of HeLa cells ∼6 h after loading with QD‐β‐CD‐CPP complexes carrying MDR1‐directed siRNA and doxorubicin (Dox). Cells loaded with MDR1‐directed siRNA showed higher intracellular levels of Dox compared to #siRNA(−) cells. Scale bar, 40 µm. β‐CD is β‐cyclodextrin. (Reprinted with permission from Ref . Copyright 2012 Elsevier) (c) Combinatorial QD delivery for spatiotemporal cell labeling. Multiple colors of QDs were delivered to the cytosol by (yellow), early endosomes (red), late endosomes (green), and the plasma membrane (magenta) using microinjection, peptide‐ and polymer‐mediated delivery. Nucleus is stained blue (DAPI). (Reprinted with permission from Ref . Copyright 2011 ACS) Scale bar is 5 µm. (d) Supported bilayer system for simultaneous delivery of multiple imaging and therapeutic cargos. Nanoporous silica cores (labeled with AlexaFluor 532, yellow) were loaded with four model cargos: calcein (green), Alexa Fluor 647‐labeled dsDNA oligonucleotide (magenta), RFP (orange), and CdSe/ZnS QDs (teal). Hep3B cells (labeled with CellTracker Violet (cytosol, purple) and Hoechst 33342 (nuclei, purple) 4 h after initial delivery display the distribution of cargos to respective, targeted locations. (Reprinted with permission from Ref . Copyright 2013 Macmillan Publishers Ltd) Scale bar, 20 µm.
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Active cellular quantum dot (QD) delivery. (a) Electrochemically controlled deconjugation for QD delivery to the nucleus. A boron nitride nanoneedle bearing streptavidin‐coated QDs attached to a SAM layer are desorbed when a voltage is applied (left panel) which allowed for single QD tracking within the nucleus of a live HeLa cell (split right panel). The nucleus is denoted with the dashed line (left) alongside the brightfield image (right). Scale bar, 1 µm. (Reprinted with permission from Ref . Copyright 2010 Wiley) (b) Reversible permeabilization facilitates cellular QD entry. The 530 nm‐emitting CdSe/ZnS QDs delivered intracellularly using combination of osmotic fluid transport and membrane‐permeabilizing saponin. Image shows DIC (left) and QD (right) signal in H9C2 rat cardiomyocyte cells. (Reprinted with permission from Ref . Copyright 2013 IOP Publishing) Scale bar, 10 µm. (c) Nanochannel electroporation (NEP) transfection of QD‐antisense‐lipoplex assemblies monitored by Förster resonance energy transfer (FRET). NEP transfection of lipoplex NPs containing QDs delivers lipoplexes directly to the cytosol within 10 min in A549 cells. Note the QD (blue) and Cy5‐antisense (red) signals are matched and separate from the endosomal label (green). QD‐Cy5 FRET was used to monitor the dissolution of the QDs and Cy5‐labeled antisense oligonucleotide from the assemblies over time. (Reprinted with permission from Ref . Copyright 2013 Wiley) (d) Nanoblade‐mediated labeling of cytoskeleton with tubulin‐QD conjugates. Laser‐induced surface plasmons from a titanium‐coated capillary induced transient pores in the plasma membrane allowing the intracellular influx of tubulin‐QD conjugates (green, panel 1) that incorporate into the cytoskeletal network. Immuno‐counterstaining of the tubulin network (red, panel 2) and merged images (panel 3) are shown to illustrate the high degree of overlap. Scale bar, 10 µm. (Reprinted with permission from Ref . Copyright 2012 ACS) (e) Microfluidic device‐mediated cytosolic delivery of QD‐dye FRET constructs. A QD‐Rhodamine donor–acceptor pair joined by a glutathione‐sensitive dithiol linkage is delivered to the cytosol via microfluidic‐driven cellular deformation initially shows full energy transfer of the green QD to the red dye (0 h, red color, left panel). After 15 h, the cytosolic glutathione reduces the thiol linkage resulting in reemission of the QD donor (15 h, green color, right panel). Scale bar, 10 µm. (Reprinted with permission from Ref . Copyright 2012 ACS)
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Peptide‐facilitated endosomal escape of quantum dots (QDs). (a) Simulation of JB577 structure as attached to 550 nm‐emitting QDs. The 550 nm QD core/shell diameter (∼56 Å) and the extension of the polyethylene glycol (PEG) ligand on the QD surface (∼30 Å) are shown. The His6 sequence (light blue) is assumed to be in contact with the QD surface and does not contribute to lateral extension. This is followed by the Gly2 flexible linker (gray) and the Pro9 motif (pink) forms a rigid type II helix designed to extend the rest of the peptide away from the surrounding PEG layer. The QD‐assembled conformation and extension of the His6Gly2Pro9 portion has been repeatedly confirmed with Förster resonance energy transfer (FRET). The VKIKK sequence is then depicted in gray outside the PEG layer along with the palmitoyl (orange) suggesting that both are available for interactions with the cell membrane. QDs appended with the multidomain peptide JB577 exhibit robust cytosolic delivery in (b) COS‐1 cells, (c) primary dermal fibroblasts, and (d) the spinal column of a chick embryo. (Reprinted with permission from Ref . Copyright 2013 ACS)
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Facilitated quantum dot (QD) delivery. (a) CdS QDs decorated with the proline‐rich sweet arrow peptide localize just inside the inner leaflet of the plasma membrane of HeLa cells. Electrochemical analysis was used to quantify the QDs remaining in the cell culture supernatants. (Reprinted with permission from Ref . Copyright 2011 ACS) (b) Viral capsid‐QD hybrids for real‐time tracking of SV40 infection in Vero cells. QDs (red) encapsidated with SV40 major coat proteins colocalize with a marker of caveolae‐mediated endocytosis (green) as evidenced by the yellow merged color. QD‐viral protein hybrids were not localized with a transferrin marker of recycling endosomes (not shown). Scale bar, 20 µm. (Reprinted with permission from Ref . Copyright 2009 Wiley) (c) Cellular QD delivery facilitated by small molecule ligands. Cysteamine/gambogic acid‐functionalized CdTe QDs internalized by HepG2 cells. Scale bar, 20 µm. (Reprinted with permission from Ref . Copyright 2013 Dove Press) (d) Cytosolic delivery of QDs using His‐rich poly Arg peptides. His‐Arg9 peptides self‐assembled to carboxyl‐capped QDs deliver QDs rapidly to the cytosol in A549 cells. QDs (green), actin (red) and nuclei (blue) show distribution of QDs in the cytosol. Magnification, 600×. (Reprinted with permission from Ref . Copyright 2011 Elsevier) (e) Use of herpes simplex virus derived peptide for cytosolic delivery of QDs. An amphiphilic peptide mediates efficient endocytosis and direct membrane translocation to the cytosol of covalently coupled QDs. Scale bar, 50 µm. (Reprinted with permission from Ref . Copyright 2011 Elsevier)
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Passive quantum dot (QD) cellular delivery. (a) Assessing QD effects on Ca2+ channel activity. Carboxylated 585 nm‐emitting CdSe‐ZnS QDs endocytosed by primary mouse adrenal chromaffin cells were used to study the effects of QD internalization on Ca2+ channel activity and Ca2+‐dependent neurotransmitter secretion. Confocal imaging shows QDs present in punctate endocytic vesicles (left, red) and merged with actin counterstaining (right, green) throughout the cytosol. Scale bar, 40 µm. (Reprinted with permission from Ref . Copyright 2011 Elsevier) (b) QDs spanning a range of sizes were incubated with fixed/permeabilized cell lines to determine size restrictions of intracellular barriers. QDs of size 3.3 nm localize to the cytosol and nucleus of THP‐1 cells (A) but only to the cytosol of Hep‐2 cells (E) while 3.7 nm and 3.9 nm QDs localized to the cytosol of THP‐1 cells (B,C) but were restricted to the plasma membrane in HEp‐2 cells (F,G). 4.4 nm QDs are found on the plasma membrane of THP‐1 cells (D) but are entirely absent from Hep‐2 cells (H). Scale bar, 10 µm. (Reprinted with permission from Ref . Copyright 2009 Wiley) (c) Multistage NP delivery for tumor tissue penetration. QDs of size 10 nm [delivered to a tumor model within ∼100 nm gelatin NPs (QDGelNPs, top row] are released and extravasate throughout the tumor in a time‐resolved manner in response to protease degradation of the gelatin matrix (note the increasingly diffuse nature of the green QD fluorescence with time) while nondegradable (control) silica QDs remain punctate and localized at the site of delivery (bottom row). Scale bar, 100 µm. (Reprinted with permission from Ref . Copyright 2011 PNAS)
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Endocytosis of nanoparticles (NPs). Model of endocytic mechanisms and intracellular transport with a focus on NP uptake into cells. NPs (green dots) and other substances taken up by endocytosis are enclosed within the early endosomes (EE), phagosomes, or macropinosomes (MP). These vesicles with particles then mature down the degradative pathway and become multivesicular bodies/late endosomes (MVB) which fuse with lysosomes (Lys). Alternatively, the NPs may be transported back to the cell surface either directly from EE or through the recycling endosomes (RE). The pH drops gradually from the cell surface to lysosomes where the pH is 4.0–5.5. The lysosomes contain proteases and other enzymes that degrade most biological substances. (Reprinted with permission from Ref . Copyright 2011 Elsevier)
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