Quantum dots, lighting up the research and development of nanomedicine

Quantum dots (QDs) have proven themselves as powerful inorganic fluorescent probes, especially for long term, multiplexed imaging and detection. The newly developed QDs labeling techniques have facilitated the study of drug delivery on the level of living cells and small animals. Moreover, based on QDs and fluorescence imaging system, multifunctional nanocomplex integrated targeting, imaging and therapeutic functionalities have become effective materials for synchronous cancer diagnosis and treatment. In this review, we will summarize the recent advances of QDs in the research of drug delivery system from the following aspects: surface modification strategies of QDs for drug delivery, QDs as drug nanocarriers, QD-labeled drug nanocarriers, QD-based fluorescence resonance energy transfer (FRET) technique for drug release study as well as the development of multifunctional nanomedicines. Possible perspective in this field will also be discussed. FROM THE CLINICAL EDITOR: This review discusses the role and significance of quantum dots (QDs) from the following aspects: surface modification strategies of QDs for drug delivery, QDs as drug nanocarriers, QD-labeled drug nanocarriers, QD-based fluorescence resonance energy transfer (FRET) technique for drug release study as well as the development of multifunctional nanomedicines.     

Preliminary study of interactivity between mercury and cells labeled with carboxymethyl chitosan coated quantum dots

This paper describes the development of a simplified and rapid method for the aqueous synthesis of quantum dots (QDs) with CdTe cores and gradient CdS external shells (CdTe/CdS QDs) aided by microwave irradiation. In order to improve the biocompatibility of the CdTe/CdS QDs, these QDs were then interacted with carboxymethyl chitosan (CMC) so as they could be used as fluorescent probes in the aqueous phase. As fluorescent probes, these modified QDs were successfully used for imaging live Madin-Darby canine kidney (MDCK) cells. Then mercury was incubated with the micro-system formed by quantum dots labeled MDCK. Fluorescence quenching was occurred in the micro-system after 24 h. The micro-system’s fluorescence quenching caused by mercury(II) was consistent with the fluorescence quenching equation and displayed a good linearity between the quenched fluorescence intensity of mercury(II). The preliminary results indicated that this micro-system can be used for detection of trace amounts of mercury in vivo and interaction process investigation between mercury and cells.     

New strategy of efficient inhibition of cancer cells by carborane carboxylic acid-CdTe nanocomposites

Nanoconjugates composed of drug molecules encapsulated in quantum dots (QDs) attract enormous attention due to their promising bioimaging and biomedical applications. Here, the anticancer efficiency of potential pharmacophore agents (o-carborane (Cb), o-carborane-C-carboxylic acid (Cbac1), and o-carborane-C(1)C(2)-dicarboxylic acid (Cbac2) coupling with cadmium telluride QDs capped with cysteamine (CA-CdTe QDs)) have been explored. Compared with free CA-CdTe QDs, the composites consisting of Cbac1/Cbac2 and safe-dosage QDs can greatly improve the inhibition efficiency toward SMMC-7721 hepatocellular carcinoma cells with the aid of our real-time cell bioelectronic sensing system and the MTT assay. The enhanced cytotoxicity correlates with increased intracellular reactive oxygen species generation and cell apoptosis. Confocal laser scanning fluorescent microscopy shows improved cellular uptake and drug distribution of the Cbac1/Cbac2-CdTe QDs nanoconjugates. This work raises the possibility that the carborane pharmacophore in combination with QDs or other anticancer drugs may be viable for efficient cancer diagnosis and chemotherapy.     

In vivo real-time confocal microscopy for target-specific delivery of hyaluronic acid-quantum dot conjugates

Hyaluronic acid (HA), which is a biocompatible, biodegradable, and linear polysaccharide in the body, has been widely used for various biomedical applications. In this work, real-time bioimaging for target-specific delivery of HA derivatives was carried out using quantum dots (QDs). In vitro confocal microscopy of HA-QD conjugates confirmed the intracellular delivery of HA derivatives to B16F1 cells with HA receptors by HA-receptor-mediated endocytosis. Furthermore in vivo real-time confocal microscopy of HA-QD conjugates successfully visualized the target specific delivery and accumulation of HA-QD conjugates from the fluorescence-labeled blood vessels to the liver tissues. The authors could confirm the feasibility of HA derivatives as a target-specific intracellular drug-delivery carrier for the treatment of liver diseases and the in vivo real-time confocal microscopy as a new bioimaging tool for various drug-delivery applications.From the Clinical EditorThis study demonstrates the possibility of labeling hyaluronic acid with quantum dots for visualization and for targeted intracellular drug delivery in liver disease models.     

Multiphoton imaging of tumor biomarkers with conjugates of single-domain antibodies and quantum dots

An ideal multiphoton fluorescent nanoprobe should combine a nanocrystal with the largest possible two-photon absorption cross section (TPACS) and the smallest highly specific recognition molecules bound in an oriented manner. CdSe/ZnS quantum dots (QDs) conjugated to 13-kDa single-domain antibodies (sdAbs) derived from camelid IgG or streptavidin have been used as efficient two-photon excitation (TPE) probes for carcinoembryonic antigen (CEA) imaging on normal human appendix and colon carcinoma tissue. The TPACS for some conjugates was higher than 49,000 GM (Goeppert-Mayer units), considerably exceeding that of organic dyes being close to the theoretical value of 50,000 GM calculated for CdSe QDs. The ratio of sdAb-QD emission to the autofluorescence for 800 nm TPE was 40 times higher than that for 457.9 nm one-photon excitation. TPE ensures a clear discrimination of CEA-overexpressing tumor areas from normal tissue. Oriented sdAb-QD conjugates are bright specific labels for detecting low concentrations of antigens using multiphoton microscopy.From the Clinical EditorThis study demonstrates carcinoembryonic antigen imaging on normal human appendix and colon carcinoma tissue utilizing CdSe/ZnS quantum dots conjugated to streptavidin or to 13-kDa single-domain antibodies as efficient two-photon excitation probes.     

Intracellular Processing of Poly(Ethylene Imine)/Ribozyme Complexes Can Be Observed in Living Cells by Using Confocal Laser Scanning Microscopy and Inhibitor Experiments

Purpose. Critical steps in the subcellular processing of poly(ethylene imine)/nucleic acid complexes, especially endosomal/lysosomal escape, were visualized by using living cell confocal laser scanning microscopy (CSLM) to obtain an insight into their mechanism. Methods. Living cell confocal microscopy was used to examine the intracellular fate of poly(ethylene imine)/ribozyme and poly(L-lysine)/ribozyme complexes over time, in the presence of and without bafilomycin A1, a selective inhibitor of endosomal/lysosomal acidification. The compartment of complex accumulation was identified by confocal microscopy with a fluorescent acidotropic dye. To confirm microscopic data, luciferase reporter gene expression was determined under similar experimental conditions. Results. Poly(ethylene imine)/ribozyme complexes accumulate in acidic vesicles, most probably lysosomes. Release of complexes occurs in a sudden event, very likely due to bursting of these organelles. After release, poly(ethylene imine) and ribozyme spread throughout the cell, during which slight differences in distribution between cytosol and nucleus are visible. No lysosomal escape was observed with poly(L-lysine)/ribozyme complexes or when poly(ethylene imine)/ribozyme complexes were applied together with bafilomycin A1. Poly(ethylene imine)/plasmid complexes exhibited a high luciferase expression, which was reduced approximately 200-fold when lysosomal acidification was suppressed with bafilomycin A1. Conclusions. Our data provide, for the first time, direct experimental evidence for the escape of poly(ethylene imine)/nucleic acid complexes from the endosomal/lysosomal compartment. CLSM, in conjunction with living cell microscopy, is a promising tool for studying the subcellular fate of polyplexes in nucleic acid/gene delivery.     

Bioconjugated quantum dots for in vivo molecular and cellular imaging

Semiconductor quantum dots (QDs) are tiny light-emitting particles on the nanometer scale, and are emerging as a new class of fluorescent labels for biology and medicine. In comparison with organic dyes and fluorescent proteins, they have unique optical and electronic properties, with size-tunable light emission, superior signal brightness, resistance to photobleaching, and broad absorption spectra for simultaneous excitation of multiple fluorescence colors. QDs also provide a versatile nanoscale scaffold for designing multifunctional nanoparticles with both imaging and therapeutic functions. When linked with targeting ligands such as antibodies, peptides or small molecules, QDs can be used to target tumor biomarkers as well as tumor vasculatures with high affinity and specificity. Here we discuss the synthesis and development of state-of-the-art QD probes and their use for molecular and cellular imaging. We also examine key issues for in vivo imaging and therapy, such as nanoparticle biodistribution, pharmacokinetics, and toxicology.     

Preparation and in vitro synergistic anticancer effect of vitamin K3 and 1,8-diazabicyclo[5,4,0]undec-7-ene in poly(ethylene glycol)-diacyllipid micelles

Polymeric micelles consisting of poly(ethylene glycol)-distearoyl phosphoethanolamine conjugates (PEG-DSPE) loaded with Vitamin K3 (VK3) to 0.2 mg of drug/mg of carrier and with 1,8-diazabicyclo[5,4,0]undec-7-ene (DBU) to 0.06 mg of drug/mg of carrier were prepared. These micelles were stable for as long as 6 months during storage at 4 degrees C and did not change their size or release the incorporated drugs. Co-encapsulation of VK3 and DBU into PEG-DSPE micelles resulted in synergistic anticancer effects against both murine and human cancer cells in vitro. The synergism may be explained by the fact that the presence of DBU promotes the escape of drug-loaded micelles from the endosomes of cancer cells directly into the cytoplasm as demonstrated by fluorescent microscopy.     

Quantum dots - nano-sized probes for the exploration of cellular and intracellular targeting.

Nanoparticles emerged as promising tool in drug targeting, since, after appropriate modification, they are able to deliver their payload to specific sites, like tissues, cells, or even certain cellular organelles. In this context, the delivery of nanoparticles from the circulation into the target cells represents a crucial step. Here, model drug delivery systems such as quantum dots are ideal candidates to elucidate this process in more detail, since they provide outstanding features like a small and uniform size, unique optical properties for most sensitive detection and modifiable surfaces. Recent progress in the surface chemistry of quantum dots expanded their use in biological applications, reduced their cytotoxicity and rendered quantum dots a powerful tool for the investigation of distinct cellular processes, like uptake, receptor trafficking and intracellular delivery. In this review, we will not only describe the ideal attributes of QDs for biological applications and imaging but also their distinct specific and non-specific pathways into the cells as well as their intracellular fate.     

RGD-modified polymeric micelles as potential carriers for targeted delivery to integrin-overexpressing tumor vasculature and tumor cells

Integrins αvβ3 and αvβ5 are overexpressed in angiogenic tumor endothelial cells and malignant tumor cells, making them attractive targets for cancer therapy. In this study, an integrin αvβ3 and αvβ5 binding tripeptide, RGD (Arg-Gly-Asp), was conjugated with the surface of poly(ethylene glycol)–block–poly(d,l-lactide) (PEG–PLA) micelles. A lipophilic fluorescent probe, DiI, was loaded into both the nontargeted methoxy PEG–PLA (mPEG–PLA) micelles and the targeted RGD-modified PEG–PLA micelles. The DiI-loaded targeted micelles had a size of 24.2?nm. The targeted micelles were stable in phosphate buffered saline and exhibited a negligible leakage in culture medium. Transmission electron microscopy analysis showed that targeted micelles were spherical in shape. Cell uptake of DiI-labeled targeted micelles by human umbilical vein endothelial cells and melanoma B16 cells was investigated by spectrophotofluorometry and confocal microscopy techniques. Results revealed that RGD-modified micelles significantly facilitated the intracellular delivery of the encapsulated agents via integrin-mediated endocytosis. This study suggests that RGD-modified PEG–PLA micelles are promising drug carriers for targeted delivery to both angiogenic tumor endothelial cells and tumor cells and that the targeted micelles may be attractive carriers for combination cancer therapy against both targets.     

The uptake and intracellular fate of a series of different surface coated quantum dots in vitro

Quantum dots (QDs) are potentially beneficial semi-conductor nanocrystals for use in diagnostics and therapeutics. The chemical composition of QDs however, has raised concerns as to their potential toxicity. Although a thorough examination using specific biochemical endpoints is necessary to assess QD toxicity, an understanding of the interaction of QDs, specifically their uptake and intracellular fate, with biological systems is also essential in determining their potential hazardous effects. The aim of this study was to investigate the uptake and intracellular fate of a series of different surface coated QDs (organic, carboxylated (COOH) and amino (NH?) polyethylene glycol (PEG)) in J774.A1 'murine macrophage-like' cells. Model 20 nm and 200 nm COOH polystyrene beads (PBs) were also studied. Results showed that COOH and NH? (PEG) QDs, as well as 20 nm and 200 nm PBs were located within lysosomes and the mitochondria of macrophages after 2 h. Additionally, elemental transmission electron microscopy confirmed both COOH and NH? (PEG) QDs to be located within membrane-bound compartments at this time point. The data from this study combined with current knowledge, indicates that the intracellular localisation of QDs could be directly related to their toxicity.     

Luminescent quantum dots: a very attractive and promising tool in biomedicine

Luminescent semiconductor nanocrystals, also known as quantum dots (QDs), are generally composed of II-VI and III-V elements. Due to their quantum confinement of charge carriers in tiny spaces, QDs show some unique and fascinating optical properties, and are characterized as sharp and symmetrical emission spectra, high quantum yields, broad absorption spectra, good chemical and photo-stability and size dependent emission wavelength tunability. Recently, QDs have been successfully used as new fluorescent tags in many biological and biomedical fields, and will become a new promising tool in biomedical studies, clinical diagnostics, drug delivery and photodynamic therapy. In this review, firstly, the methodology of QDs preparation was introduced, which included organic synthesis, aqueous synthesis and microwave assisted aqueous synthesis. Secondly, some procedures for the QDs bio-conjugation with biomarkers were described. And then, some key applications of QDs were summarized, which mainly covered biomedical imaging, immunoassay, DNA hybridization, and photodynamic therapy. Finally, future prospects were discussed.     

Self-assembled polyion complex micelles for sustained release of hydrophilic drug

Graft copolymer polyethylenimine–graft–poly(N-vinylpyrrolidone) (PEI-g-PVP) was prepared by coupling mono carboxyl-terminated PVP (PVP–COOH) with PEI using N,N′-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) as coupling agents. In aqueous medium, PVP–g–PEI can self-assemble into stable polyion complex micelles with an oppositely charged block copolymer, poly(N-vinylpyrrolidone)–block–poly(2-acrylamido-2-methyl-1-propanesulphonic acid) (PVP-b-PAMPS). Transmission electron microscopy images showed that these micelles were regularly spherical in shape. The micelle size determined by size analysis was around 142?nm. To estimate their feasibility as vehicles for drugs, the model drug folic acid (FA) was incorporated into the cores of the micelles via electrostatic interactions. In vitro release test of FA showed that the drug-release rates are dependent on the pH value of the release media. Based on these results, we can conclude that the polyion complex micelles prepared from the PEI-g-PVP/PVP-b-PAMPS copolymers have great potential as drug delivery nanocarriers.     

Self-assembled polyion complex micelles for sustained release of hydrophilic drug

Graft copolymer polyethylenimine-graft-poly(N-vinylpyrrolidone) (PEI-g-PVP) was prepared by coupling mono carboxyl-terminated PVP (PVP-COOH) with PEI using N,N'-dicyclohexylcarbodiimide (DCC) and N-hydroxysuccinimide (NHS) as coupling agents. In aqueous medium, PVP-g-PEI can self-assemble into stable polyion complex micelles with an oppositely charged block copolymer, poly(N-vinylpyrrolidone)-block-poly(2-acrylamido-2-methyl-1-propanesulphonic acid) (PVP-b-PAMPS). Transmission electron microscopy images showed that these micelles were regularly spherical in shape. The micelle size determined by size analysis was around 142 nm. To estimate their feasibility as vehicles for drugs, the model drug folic acid (FA) was incorporated into the cores of the micelles via electrostatic interactions. In vitro release test of FA showed that the drug-release rates are dependent on the pH value of the release media. Based on these results, we can conclude that the polyion complex micelles prepared from the PEI-g-PVP/PVP-b-PAMPS copolymers have great potential as drug delivery nanocarriers.     

A novel lanreotide-encoded micelle system targets paclitaxel to the tumors with overexpression of somatostatin receptors

Many tumor cells specifically overexpress somatostatin receptors, in particular, subtype 2 (SSTR2). Lanreotide, a somatostatin analogue with high affinity for SSTR2, can be exploited as a ligand for tumor targeted therapy. In this study, lanreotide was first conjugated to poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-b-PCL) copolymer, and the active targeting micelles with paclitaxel (lanreotide-PM-PTX) or fluorescent agent were constructed and characterized with various analytical methods. Lanreotide-PM-PTX micelles were spherical in shape with a hydrodynamic diameter of 43.2 ± 0.4 nm, high drug encapsulation (87.1 ± 2.8%) and slow drug release rate. Two cancer cell lines (human lung cancer H446 and human breast cancer MCF-7 cells) with different expression levels of SSTR2 were used in this study. As observed by flow cytometry, confocal microscopy and cytotoxicity studies, lanreotide-encoded PEG-b-PCL micelles demonstrated more specific cell uptake and cytotoxicity in SSTR2-positive tumor cells via a receptor-mediated mechanism over the passive targeting micelles. The active targeting micelles showed higher accumulation in tumor tissue and tumor cells in tumor-bearing mice in vivo by near-infrared fluorescence (NIRF) imaging, high-performance liquid chromatography and confocal microscopy, respectively. Furthermore, treatment with lanreotide-PM-PTX micelles resulted in stronger tumor inhibition, increased life span and enhanced tumor cell apoptosis in SSTR2-overexpressing tumor model in athymic nude mice. The in vivo efficacy test with both H446 and MCF-7 tumor models further demonstrated the involvement of receptor-mediated interaction. Finally, the active targeting micelles exhibited less body weight loss, lower hemolysis and lower myelosuppression, as compared with the control groups. In conclusion, lanreotide can serve as an effective homing peptide, and the lanreotide-modified PEG-b-PCL micelles hold considerable promise in the treatment of SSTR2-overexpressing solid tumors.     

Emerging application of quantum dots for drug delivery and therapy

Quantum dots have proven themselves as powerful fluorescent probes, especially for long-term, multiplexed, and quantitative imaging and detection. Newly engineered quantum dots with integrated targeting, imaging and therapeutic functionalities have become excellent material to study drug delivery in cells and small animals. This fluorescent 'prototype' will provide important information in the rational design of biocompatible drug carriers and will serve as a superior alternative to magnetic and radioactive imaging contrast agents in preclinical drug screening, validation and delivery research. This Editorial article is not intended to offer a comprehensive review on drug delivery, but to highlight the breakthroughs in the emerging applications of quantum dots in this field and to provide our perspective on future research.     

Oriented conjugates of single-domain antibodies and quantum dots: toward a new generation of ultrasmall diagnostic nanoprobes

Common strategy for diagnostics with quantum dots (QDs) utilizes the specificity of monoclonal antibodies (mAbs) for targeting. However QD-mAbs conjugates are not always well-suited for this purpose because of their large size. Here, we engineered ultrasmall nanoprobes through oriented conjugation of QDs with 13-kDa single-domain antibodies (sdAbs) derived from llama IgG. Monomeric sdAbs are 12 times smaller than mAbs and demonstrate excellent capacity for refolding. sdAbs were tagged with QDs through an additional cysteine residue integrated within the C terminal of the sdAb. This approach allowed us to develop sdAbs-QD nanoprobes comprising four copies of sdAbs coupled with a QD in a highly oriented manner. sdAbs-QD conjugates specific to carcinoembryonic antigen (CEA) demonstrated excellent specificity of flow cytometry quantitative discrimination of CEA-positive and CEA-negative tumor cells. Moreover, the immunohistochemical labeling of biopsy samples was found to be comparable or even superior to the quality obtained with gold standard protocols of anatomopathology practice. sdAbs-QD-oriented conjugates as developed represent a new generation of ultrasmall diagnostic probes for applications in high-throughput diagnostic platforms. FROM THE CLINICAL EDITOR: The authors report the development of sdAbs-QD-oriented conjugates, comprised of single domain antibodies that are 12 times smaller than regular mAb-s and quantum dots. These ultrasmall diagnostic probes represent a new generation of functionalized ODs for applications in high-throughput diagnostic platforms.     

Endocytosis and Interaction of Poly (Amidoamine) Dendrimers with Caco-2 Cells

Purpose To investigate the internalization and subcellular trafficking of fluorescently labeled poly (amidoamine) (PAMAM) dendrimers in intestinal cell monolayers. Materials and methods PAMAM dendrimers with positive or negative surface charge were conjugated to fluorescein isothiocyanate (FITC) and visualized for colocalization with endocytosis markers using confocal microscopy. Effect of concentration, generation and charge on the morphology of microvilli was observed using transmission electron microscopy. Results Both cationic and anionic PAMAM dendrimers internalized within 20 min, and differentially colocalized with endocytosis markers clathrin, EEA-1, and LAMP-1. Transmission electron microscopy analysis showed a concentration-, generation- and surface charge-dependent effect on microvilli morphology. Conclusion These studies provide visual evidence that endocytic mechanism(s) contribute to the internalization and subcellular trafficking of PAMAM dendrimers across the intestinal cells, and that appropriate selection of PAMAM dendrimers based on surface charge, concentration and generation number allows the application of these polymers for oral drug delivery.     

The delivery of doxorubicin of multifunctional β‐cyclodextrin‐modified CdSe/ZnS quantum dots for bioactivity and nano‐probing

The modified quantum dots (QDs) have been used in intracellular probing and drug delivery because of their special chemical and physical properties. In this paper, two β‐cyclodextrin (β‐CD)‐modified CdSe/ZnS QDs with strong optical emission properties were synthesized as drug carriers to induce apoptosis. The positively charged l ‐Arginine (l ‐Arg) and neutral l ‐Tryptophan (l ‐Trp) were selected as ligands to compare the effect of charge on bioactivity of QDs nanoparticles. The in vitro assays revealed that these modified QDs showed good Dox carrier ability and significantly high inhibition rate to cancer cells. Especially, the more positively charged β‐CD‐l ‐Arg‐polyamine‐coated CdSe/ZnS QDs could effectively deliver the doxorubicin (Dox) into cells and exhibit excellent cell selectivity in cancer versus normal cells. The Dox‐loaded QDs could enter intracellular, which showed that the Dox can efficiently go through the membranes at the existence of β‐CD. Several lines of evidence suggest that the Dox‐loaded QDs can efficiently induce apoptosis likely related to the production of ROS. We expect that the modified QDs can enhance the amount of hydrophobic antitumor drugs in cells and can also be used as fluorescent imaging agents.     

Quantum dot-mediated biosensing assays for specific nucleic acid detection

Two new classes of quantum dot (QD)-mediated biosensing methods have been developed to detect specific DNA sequences in a separation-free format. Both methods use 2 target-specific oligonucleotide probes to recognize a specific target. The first method is based on cross-linking of 2 QDs with distinct emission wavelengths caused by probe-target hybridization. The second method uses QDs as both fluorescent tags and nanoscaffolds that capture multiple fluorescently labeled hybridization products, resulting in amplified target signals. The presence of targets is determined according to spatiotemporal coincidence of 2 different wavelength fluorescent signals emitted from the QD/DNA/probe complexes. With a single wavelength-excitation, dual wavelength-emission confocal spectroscopic system, the fluorescent signals can be measured with single-dot/molecule sensitivity. Compared with other nanoparticle-based, separation-free assays, our method shows advantages in simplicity, testing speed, and multiplexed applications.