Cadmium-containing quantum dots: properties,applications, and toxicity

The marriage of biology with nanomaterials has significantly accelerated advancement of biological techniques, profoundly facilitating practical applications in biomedical fields. With unique optical properties (e.g., tunable broad excitation, narrow emission spectra, robust photostability, and high quantum yield), fluorescent quantum dots (QDs) have been reasonably functionalized with controllable interfaces and extensively used as a new class of optical probe in biological researches. In this review, we summarize the recent progress in synthesis and properties of QDs. Moreover, we provide an overview of the outstanding potential of QDs for biomedical research and innovative methods of drug delivery. Specifically, the applications of QDs as novel fluorescent nanomaterials for biomedical sensing and imaging have been detailedly highlighted and discussed. In addition, recent concerns on potential toxicity of QDs are also introduced, ranging from cell researches to animal models.

     

Folate receptor-targeted aggregation-enhanced near-IR emitting silica nanoprobe for one-photon in vivo and two-photon ex vivo fluorescence bioimaging

A two-photon absorbing (2PA) and aggregation-enhanced near-infrared (NIR) emitting pyran derivative, encapsulated in and stabilized by silica nanoparticles (SiNPs), is reported as a nanoprobe for two-photon fluorescence microscopy (2PFM) bioimaging that overcomes the fluorescence quenching associated with high chromophore loading. The new SiNP probe exhibited aggregate-enhanced emission producing nearly twice as strong a signal as the unaggregated dye, a 3-fold increase in two-photon absorption relative to the DFP in solution, and approximately 4-fold increase in photostability. The surface of the nanoparticles was functionalized with a folic acid (FA) derivative for folate-mediated delivery of the nanoprobe for 2PFM bioimaging. Surface modification of SiNPs with the FA derivative was supported by zeta potential variation and (1)H NMR spectral characterization of the SiNPs as a function of surface modification. In vitro studies using HeLa cells expressing a folate receptor (FR) indicated specific cellular uptake of the functionalized nanoparticles. The nanoprobe was demonstrated for FR-targeted one-photon in vivo imaging of HeLa tumor xenograft in mice upon intravenous injection of the probe. The FR-targeting nanoprobe not only exhibited highly selective tumor targeting but also readily extravasated from tumor vessels, penetrated into the tumor parenchyma, and was internalized by the tumor cells. Two-photon fluorescence microscopy bioimaging provided three-dimensional (3D) cellular-level resolution imaging up to 350 μm deep in the HeLa tumor.     

Targeting and sensing cancer cells with ZnO nanoprobes in vitro

A new class of zinc oxide quantum dots (ZnO QDs) was investigated as nanoprobes for targeting cancer cells in vitro. ZnO nanoparticles were synthesized using conventional sol–gel method and encapsulated using trimethoxy aminopropyl silane. Transferrin, the ligand targeting the cancer cells, was conjugated to the ZnO QDs. In vitro imaging studies using MDA-MB-231 showed the biocompatible ZnO nanoprobe selectively binding to the cell surface receptor and internalizing through receptor-mediated endocytosis. Time-lapsed photobleaching studies indicate the ZnO QDs to be resistant to photobleaching, making them suitable for long term imaging purpose. Investigation of the ZnO nanoprobe as a platform for sensitive bioassays indicates that it can be used as an alternative fluoroprobe for cancer cell targeting and sensing applications.     

Quantum dots and peptides: a bright future together

Nanocrystalline semi-conductor materials, called quantum dots (QDs), exhibit unique optical and spectroscopic properties, which include, broad absorption, narrow and tunable emission, resistance to photobleaching, strong luminescence, and long luminescent lifetimes. These remarkable properties of QDs have resulted in their use as an alternative to both small-molecule and protein fluorophores in innumerable biological applications. The overlap of QDs with the rich chemistry and biology that is characteristic of the peptide arena is an emerging research area. Peptides engineered with appropriate cysteines or histidines have served as ligands for producing water soluble QDs as well as for tagging protein ligands and biosensors to QD surfaces. Incorporation of cell-penetrating peptides on QD surfaces has allowed for the translocation of functionalized QDs into cells for intracellular imaging applications. QDs containing fluorescently labeled peptide substrates have shown utility in the development of novel protease assays. Moreover, QDs-labeled peptides that serve as ligands for cellular receptors provide an alternative to antibody mediated imaging at the whole-cell and single molecule level to study receptor distribution and trafficking. This review highlights the overlap between QD and peptide chemistry and speculates on future research directions.     

Surface functionalization of gold nanoparticles using hetero-bifunctional poly(ethylene glycol) spacer for intracellular tracking and delivery

For the development of surface-functionalized gold nanoparticles as cellular probes and delivery agents, we have synthesized hetero-bifunctional poly(ethylene glycol) (PEG, MW 1500) having a thiol group on one terminus and a reactive functional group on the other for use as a flexible spacer. Coumarin, a model fluorescent dye, was conjugated to one end of the PEG spacer and gold nanoparticles were modified with coumarin-PEG-thiol. Surface attachment of coumarin through the PEG spacer decreased the fluorescence quenching effect of gold nanoparticles. The results of cellular cytotoxicity and fluorescence confocal analyses showed that the PEG spacer-modified nanoparticles were essentially non-toxic and could be efficiently internalized in the cells within 1 hour of incubation. Intracellular particle tracking using a Keck 3-D Fusion Microscope System showed that the functionalized gold nanoparticles were rapidly internalized in the cells and localized in the peri-nuclear region. Using the PEG spacer, the gold nano-platform can be conjugated with a variety of biologically relevant ligands such as fluorescent dyes, antibodies, etc in order to target, probe, and induce a stimulus at the target site.     

Bacteria-mediated in vivo delivery of quantum dots into solid tumor

Semiconductor nanocrystals, so-called quantum dots (QDs), promise potential application in bioimaging and diagnosis in vitro and in vivo owing to their high-quality photoluminescence and excellent photostability as well as size-tunable spectra. Here, we describe a biocompatible, comparatively safe bacteria-based system that can deliver QDs specifically into solid tumor of living animals. In our strategy, anaerobic bacterium Bifidobacterium bifidum (B. bifidum) that colonizes selectively in hypoxic regions of animal body was successfully used as a vehicle to load with QDs and transported into the deep tissue of solid tumors. The internalization of lipid-encapsuled QDs into B. bifidum was conveniently carried by electroporation. To improve the efficacy and specificity of tumor targeting, the QDs-carrying bacterium surface was further conjugated with folic acids (FAs) that can bind to the folic acid receptor overexpressed tumor cells. This new approach opens a pathway for delivering different types of functional cargos such as nanoparticles and drugs into solid tumor of live animals for imaging, diagnosis and therapy.     

Targeting of ovarian cancer cell through functionalized gold nanoparticles by novel glypican-3- binding peptide as a ultrasound contrast agents

Gold nanoparticles (AuNPs) are widely studied nanomaterials for their potential employment in advanced biomedical applications, such as selective molecular imaging and targeted drug delivery. AuNPs are generally low cost and highly biocompatible, can be easily functionalized with a wide variety of functional ligands, and have been demonstrated to be effective in enhancing ultrasound contrast at clinical diagnostic frequencies. Therefore, AuNPs might be used as contrast agents in echographic imaging. In this work, we have developed a AuNPs -based system for the in vitro molecular imaging of ovarian carcinoma cells that express high levels of glypican-3 protein (GPC-3) on their surface. In this regard, a novel GPC-3 targeting peptide was designed and conjugated to fluorescent AuNPs nanoparticles. The physicochemical properties, acoustic behavior, and biocompatibility profile of the functionalized AuNPs were characterized. Then, the binding and uptake of both naked and functionalized AuNPs were analyzed by laser scanning confocal microscopy in human HeLa cells (ovarian carcinoma) cell line. The results obtained showed that GPC-3-functionalized fluorescent AuNPs significantly enhanced the ultrasound contrast and were effectively bound and taken up by HeLa cells without affecting their viability.     

Specific antitumor targetable beta-cyclodextrin-poly(ethylene glycol)-folic acid drug delivery bioconjugate

The tumor targeting properties of a new drug carrier synthesized by bioconjugation of folic acid (FA) to beta-cyclodextrins through a poly(ethylene glycol) (PEG) spacer (CD-PEG-FA) were investigated. Surface plasmon resonance demonstrated that CD-PEG-FA specifically interacts with immobilized folate binding protein (FBP) while the naked beta-cyclodextrins do not display any specific interaction. In vitro studies demonstrated that CD-PEG-FA was devoid of cell toxicity. [(3)H]-folic acid/CD-PEG-FA competition binding investigations performed with folate receptor overexpressing human epidermal carcinoma KB cells showed that CD-PEG-FA had about 14 times lower tumor cell binding capacity than free folic acid. The carrier cell trafficking properties were investigated using rhodamine-B as fluorescent probe, which possesses 3000 and 4580 M(-)(1) inclusion constants for CD-PEG-FA and beta-cyclodextrins, respectively. Cell-associated fluorescence measurements showed that CD-PEG-FA does not promote the rhodamine-B uptake into non-folate receptor expressing human lung carcinoma MCF7 cells while 19% higher accumulation in KB cells was found with respect to rhodamine-B loaded beta-cyclodextrins. Confocal laser scanning microscopy indicated the presence of cytosolic red fluorescent spots after 2 h of incubation of KB cells with rhodamine-B included CD-PEG-FA. The fluorescent dye resided primarily in small spots, namely, endosomes and multivesicular bodies. At 1 h after pulsed incubation, wider red fluorescent cellular structures appeared as a fusion of previous structures.     

Intracellular protein target detection by quantum dots optimized for live cell imaging

Imaging of specific intracellular target proteins in living cells has been of great challenge and importance for understanding intracellular events and elucidating various biological phenomena. Highly photoluminescent and water-soluble semiconductor nanocrystal quantum dots (QDs) have been extensively applied to various cellular imaging applications due to the long-term photostability and the tunable narrow emission spectra with broad excitation. Despite the great success of various bioimaging and diagnostic applications, visualization of intracellular targets in live cells still has been of great challenge. Nonspecific binding, difficulty of intracellular delivery, or endosomal trapping of nanosized QDs are the main reasons to hamper specific target binding in live cells. In this context, we prepared the polymer-coated QDs (pcQD) of which the surface was optimized for specific intracellular targeting in live cells. Efficient intracellular delivery was achieved through PEGylation and subsequent cell penetrating peptide (i.e., TAT) conjugation to the pcQD in order to avoid significant endosomal sequestration and to facilitate internalization of the QDs, respectively. In this study, we employed HEK293 cell line overexpressing endothelin A receptor (ET(A)R), a family of G-protein coupled receptor (GPCR), of which the cytosolic c-terminal site is genetically engineered to possess green fluorescent protein (GFP) as our intracellular protein target. The fluorescence signal of the target protein and the well-defined intracellular behavior of the GPCR help to evaluate the targeting specificity of QDs in living cells. To test the hypothesis that the TAT-QDs conjugated with antibody against intracellular target of interest can find the target, we conjugated anti-GFP antibody to TAT-PEG-pcQD using heterobifunctional linkers. Compared to the TAT-PEG-pcQD, which was distributed throughout the cytoplasm, the antiGFP-functionalized TAT-PEG-pcQD could penetrate the cell membrane and colocalize with the GFP. An agonist (endothelin-1, ET-1) treatment induced GFP-ET(A)R translocation into pericentriolar region, where the GFP also significantly colocalized with antiGFP-TAT-PEG-pcQD. These results demonstrate that stepwise optimization of PEG-pcQD conjugation with both a cell penetrating peptide and an antibody against a target of interest allows specific binding to the intracellular target protein with minimized nonspecific binding.     

Synthesis and application of a targeting diagnosis system via quantum dots coated by amphiphilic polymer for the detection of liver cancer cells

Water‐soluble quantum dots (QDs) for liver cancer diagnosis were prepared using QDs with oleylamine ligand coated with poly(aspartate)–graft–poly(ethylene glycol)–dodecylamine (PASP–Na–g–PEG–DDA). Dynamic light scattering and transmission electron microscopy imaging showed that the novel QDs have an ellipsoidal morphology with a size of ~ 45 nm which could be used for biomedical application. Furthermore, the PASP–Na–g–PEG–DDA was then modified with anti‐(vascular endothelial growth factor) (VEGF antibody), and a 1‐(4,5‐dimethylthiazol‐2‐yl)‐3,5‐diphenylformazan (MTT) assay showed that the novel anti‐VEGF‐targeting QDs in vitro had low toxicity. Confocal laser scanning microscopy observations revealed an intracellular (HepG2) distribution of the novel anti‐VEGF‐targeting QDs and the targeting efficiency of anti‐VEGF. These novel QDs could be used as a probe for liver cancer cell imaging because of anti‐VEGF targeting. Copyright © 2014 John Wiley & Sons, Ltd.     

Conjugating folic acid to gold nanoparticles through glutathione for targeting and detecting cancer cells

Gold nanoparticles (GNPs) were modified with glutathione (GSH) to form GSH-capped GNPs, which have carboxyl groups on the surface of these nanoparticles. Then folic acid (FA) was conjugated with GNPs through the reaction between amino group of FA and carboxyl group of GSH. These folic acid-conjugated nanoparticles (FA-GSH-GNPs) were stable in aqueous solution over a broad range of pH and ionic strength values. The targeting of FA-GSH-GNPs in human cervices carcinoma cells (HeLa cells) with high-level folate receptor expression was confirmed by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). No cellular uptake of these nanoparticles was observed in A549 cells lack of folate receptor. HeLa cells and mouse fibroblasts incubated with FA-GSH-GNPs were assayed by measuring the relative absorbance of the supernatant collected at low-speed centrifugation. Based on this simple spectroscopic method, HeLa cells have been detected with a detection limit of 10² cells/mL.     

Bioconjugation of InGaP quantum dots for molecular sensing

Fluorescence-based molecular sensing and cellular imaging are commonly carried out with the application of organic dyes. Quantum dots (QDs) are now recognized as better tools because they are brighter, size tunable, and more photostable than dyes. Most of the proposed QD-based biosensing systems involve elements of known toxicity. The present work reports the functionalization of biocompatible InGaP/ZnS core-shell QDs with anti-bovine serum albumin (anti-BSA) to exploit them as fluorescent probes for antigen detection. Successful bioconjugation was characterized with the absorption and emission spectra showing blue shifts of around 40 and 30 nm, respectively. Gel electrophoresis and particle size distribution studies further confirmed the mass increment of QDs after their functionalization with anti-BSA. Surface plasmon resonance spectrometry has been used to study the affinity of QD-(anti-BSA) probes for bovine serum albumin (BSA). Photoluminescence quenching of the developed probe is observed in the presence of BSA.     

Uptake of ricinB-quantum dot nanoparticles by a macropinocytosis-like mechanism

ABSTRACT: BACKGROUND: There is a huge effort in developing ligand-mediated targeting of nanoparticles to deceased cells and tissue. The plant toxin ricin has been shown to enter cells by utilizing both dynamin-dependent and -independent endocytic pathways. Thus, it is a representative ligand for addressing the important issue of whether even a relatively small ligand-nanoparticle conjugate can gain access to the same endocytic pathways as the free ligand. RESULTS: Here we present a systematic study concerning the internalization mechanism of ricinB:Quantum dot (QD) nanoparticle conjugates in HeLa cells. Contrary to uptake of ricin itself, we found that internalization of ricinB:QDs was inhibited in HeLa cells expressing dominant-negative dynamin. Both clathrin-, Rho-dependent uptake as well as a specific form of macropinocytosis involve dynamin. However, the ricinB:QD uptake was not affected by siRNA-mediated knockdown of clathrin or inhibition of Rho-dependent uptake caused by treating cells with the Clostridium C3 transferase. RicinB:QD uptake was significantly reduced by cholesterol depletion with methyl-beta-cyclodextrin and by inhibitors of actin polymerization such as cytochalasin D. Finally, we found that uptake of ricinB:QDs was blocked by the amiloride analog EIPA, an inhibitor of macropinocytosis. Upon entry, the ricinB:QDs co-localized with dextran, a marker for fluid-phase uptake. Thus, internalization of ricinB:QDs in HeLa cells critically relies on a dynamin-dependent macropinocytosis-like mechanism. CONCLUSIONS: Our results demonstrate that internalization of a ligand-nanoparticle conjugate can be dependent on other endocytic mechanisms than those used by the free ligand, highlighting the challenges of using ligand-mediated targeting of nanoparticles-based drug delivery vehicles to cells of diseased tissues.     

Improved tumor targeting of polymer-based nanovesicles using polymer-lipid blends

Block copolymer-based vesicles have recently garnered a great deal of interest as nanoplatforms for drug delivery and molecular imaging applications due to their unique structural properties. These nanovesicles have been shown to direct their cargo to disease sites either through enhanced permeability and retention or even more efficiently via active targeting. Here, we show that the efficacy of nanovesicle targeting can be significantly improved when prepared from polymer-lipid blends compared with block copolymer alone. Polymer-lipid hybrid nanovesicles were produced from the aqueous coassembly of the diblock copolymer, poly(ethylene oxide)-block-polybutadiene (PEO-PBD), and the phospholipid, hydrogenated soy phosphatidylcholine (HSPC). The PEG-based vesicles, 117 nm in diameter, were functionalized with either folic acid or anti-HER2/neu affibodies as targeting ligands to confer specificity for cancer cells. Our results revealed that nanovesicles prepared from polymer-lipid blends led to significant improvement in cell binding compared to nanovesicles prepared from block copolymer alone in both in vitro cell studies and murine tumor models. Therefore, it is envisioned that nanovesicles composed of polymer-lipid blends may constitute a preferred embodiment for targeted drug delivery and molecular imaging applications.     

Dual-mode probe based on mesoporous silica coated gold nanorods for targeting cancer cells

A dual-mode imaging probe for targeting cancer cells has been fabricated based on mesoporous silica coated gold nanorods (MS-GNRs) for the first time. In this probe, fluorescence and surface enhanced Raman scattering (SERS) signals can be generated independently by using different excitation wavelengths. To investigate the targeting performance of the probe, folic acid (FA) is conjugated on the outer surfaces of MS-GNRs as a targeting ligand and HeLa cells were used as model cancer cells because they overexpress folate receptors (FRs). The endocytosis mechanism was verified by competing experiments with free FA through both fluorescence images and SERS mappings. Moreover, the cytotoxicity of the probe was remarkably reduced in comparison with the GNRs without the silica shell as proved by the results of MTT assay. Compared with traditional imaging probes, this new type of nanoprobe has great potential for multiplexed imaging in living cells, which can be easily realized by using fluorescence and SERS signals.     

Improved targeting of ligand-modified adenovirus as a new near infrared fluorescence tumor imaging probe

E1/E3-deleted Adenovirus 5 (Ad.5) possesses a great potential in gene therapy because of its high efficacy in gene transfer and low toxicity. Studies have shown that Coxsackie-Adenovirus receptor (CAR) is the determinant factor for the targeting of Adenovirus vectors. To extend the natural targeting of Ad to low CAR expressing tumors, we covalently attached folic acid (FA) to E1/E3-deleted Ad.5 capsids. Near-infrared (NIR) fluorescent dye ICG-Der-02 was subsequently conjugated with FA-Ad particles for in vivo imaging. The cell experiments and acute toxicity studies demonstrated the low toxicity of FA-Ad-ICG02 to normal cell/tissues. The dynamic behavior and targeting ability of FA-Ad-ICG02 to different tumors were investigated by NIR fluorescence imaging. In vitro and in vivo studies demonstrated its high targeting capability to CAR or FR positive tumors. The results support the potential of using ligand-modified Ad probe for tumor diagnosis and targeted therapy.     

Detection of tumor marker CA125 in ovarian carcinoma using quantum dots

The fluorescent labeling of biological materials usingsmall-molecule organic dyes is widely employed in bio-logical imaging and clinical diagnosis. Organic fluoro-phores, however, have certain characteristics that limittheir advantages in some applications. These limitationsinclude narrow excitation bands and broad emissionbands with red spectral tails, which make the simultaneousevaluation of several light-emitting probes difficult due tospectral overlap. Also, many organic dyes exhibit highp…     

Controlling the thickness of polymeric shell on magnetic nanoparticles loaded with doxorubicin for targeted delivery and MRI contrast agent

The polymeric functionalization of superparamagnetic iron oxides nanoparticles is developed for cancer targeting capability and magnetic resonance imaging. Here the nanoparticles (NP) are decorated through the adsorption of a polymeric layer around the particle surface for the formation of core-shell. The synthesized magnetic nanoparticles (MNPs) are conjugated with fluorescent dye, targeting ligand, and drug molecules for improvement of target specific diagnostic and possible therapeutics applications. In this investigation doxorubicin was loaded into the shell of the MNPs and release study was carried out at different pH. The core-shell structure of magnetic NP coated chitosan matrix was visualized by TEM observation. The cytotoxicity of these magnetic NPs is investigated using MTT assay and receptor mediated internalization by HeLa and NIH3T3 cells are studied by fluorescence microscopy. Moreover, compared with T₂-weighted magnetic resonance imaging (MRI) in the above cells, the synthesized nanoparticles are showed stronger contrast enhancements towards cancer cells.     

Preparation and in vitro evaluation of a folate-linked liposomal curcumin formulation

Curcumin (CUR), a plant-derived compound, exhibits versatile antitumor effects. However, its poor hydrophilic property limits its application. To circumvent these drawbacks, we encapsulated CUR in liposomes modified with folic acid for better solubility and enhanced tumor targeting. This novel formulation was prepared by a film-dispersion method and characterized by size, zeta potential, drug-loading efficiency, and physical-condition stability. In vitro, cellular uptake efficiency, cytotoxicity, and apoptosis analysis by flow cytometry were performed to evaluate tumor targeting and killing ability. Results showed that the folate-receptor (FR)-targeted liposomal CUR (F-CUR-L) performed with improved solubility, sufficient stability, and enhanced antitumor activity. Mean diameter, zeta potential, and drug-loading efficiency were 182?nm, -26 mV, and 68%, respectively, and this formulation exhibited stability in storage at 4 °C for 1 month. In vitro, FR-positive cells endocytosed more F-CUR-L than nontargeted liposomal CUR (CUR-L); thus, the former induced more cellular proliferation inhibition and higher apoptosis than the latter, and the enhanced targeting could be hindered by 1?mM of free folic acid. Further, KB cells were more sensitive to F-CUR-L, compared to Hela cells. Finally, the two kinds of tumor cells treated with F-CUR-L also showed dose- and time-dependent apoptosis.     

Preparation and in vitro evaluation of a folate-linked liposomal curcumin formulation

Curcumin (CUR), a plant-derived compound, exhibits versatile antitumor effects. However, its poor hydrophilic property limits its application. To circumvent these drawbacks, we encapsulated CUR in liposomes modified with folic acid for better solubility and enhanced tumor targeting. This novel formulation was prepared by a film-dispersion method and characterized by size, zeta potential, drug-loading efficiency, and physical-condition stability. In vitro, cellular uptake efficiency, cytotoxicity, and apoptosis analysis by flow cytometry were performed to evaluate tumor targeting and killing ability. Results showed that the folate-receptor (FR)-targeted liposomal CUR (F-CUR-L) performed with improved solubility, sufficient stability, and enhanced antitumor activity. Mean diameter, zeta potential, and drug-loading efficiency were 182?nm, ?26 mV, and 68%, respectively, and this formulation exhibited stability in storage at 4°C for 1 month. In vitro, FR-positive cells endocytosed more F-CUR-L than nontargeted liposomal CUR (CUR-L); thus, the former induced more cellular proliferation inhibition and higher apoptosis than the latter, and the enhanced targeting could be hindered by 1?mM of free folic acid. Further, KB cells were more sensitive to F-CUR-L, compared to Hela cells. Finally, the two kinds of tumor cells treated with F-CUR-L also showed dose- and time-dependent apoptosis.