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Targeted quantum dots fluorescence probes functionalized with aptamer and peptide for transferrin receptor on tumor cells
Nanotechnology
Abstract and Figures
Quantum dots (QDs) fluorescent probes based on oligonucleotide aptamers and peptides with specific molecular recognition have attracted much attention. In this paper, CdSe/ZnS QDs probes for targeted delivery to mouse and human cells using aptamer GS24 and peptide T7 specific to mouse/human transferrin receptors were developed. Capillary electrophoresis analyses indicated that the optimal molar ratios of QDs to aptamer or peptide were 1:5. Fluorescence and confocal microscope imaging revealed QD-GS24 and QD-T7 probes were able to specifically recognize B16 cells and HeLa cells respectively. Quantitative flow cytometry analysis indicated the transportation of QD-GS24 or QD-T7 into cells could be promoted by corresponding free transferrin. Transmission electron microscopy confirmed the uptake of probes in cells and the effective intracellular delivery. MTT assay suggested the cytotoxicity of probes was related to the surface ligand, and aptamer GS24 (or peptide T7) could reduce the cytotoxicity of probes to a certain degree. The study has great significance for preparing QDs fluorescent probes using non-antibody target molecules.
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... Aptamer-based optical imaging can be divided into direct targeting and activatable probes [10]. Direct conjugation with an aptamer and a fluorescent molecule has been widely studied [11][12][13][14][15][16]. ...
This study investigated the development of aptamer-based molecular probes to detect Methicillin-Resistant Staphylococcus aureus (MRSA) and evaluated the antibacterial activity. Early detection of MRSA infection will improve patients’ recovery and reduce the cost for treating patients. S. aureus can become resistant to methicillin and other β-lactam antibiotics through the expression of PBP2A protein, which is resistant to the action of methicillin. We have developed two aptamer molecular probes against PBP2A protein and whole bacterial cell (MRSA) under optimized in vitro conditions using SELEX approach. Target aptamer sequences were identified, and chemically synthesized aptamer probes were evaluated using fluorescently-labelled aptamer probes using flow cytometry and confocal imaging. Antibacterial activities of those aptamers were also evaluated using a bacterial killing assay. The results showed that high specific aptamers were developed against purified PBP2A protein. However, these aptamers showed less specificity to detect MRSA under in vitro condition. These aptamers showed no cytotoxic effect on 3T3 cells and no antibacterial activity against MRSA. The results suggested that the specific aptamer development and the in vitro selection methodology require further refinement to improve the diagnostic and therapeutic utility of these aptamers.
... Dynamic light scattering (DLS), also known as quasi-elastic light scattering, photon correlation spectroscopy, can be used to determine the size and zeta potential of scattered PELNVs [35]. DLS is the benchmark approach in the evaluation of size distribution of suspension particles in numerous scientific disciplines since it is non-intrusive and ultra-sensitive, requiring only a small sample volume to calculate accurate and precise size [38][39][40]. Furthermore, researchers are increasingly using nanoparticle tracking analysis (NTA) to quantify the amount of ELNVs in a sample container and characterise their size distribution. ...
These days, with the overall spread of various tumors; there is a pressing need in new treatment strategies and prescriptions. With the appearance of medication conveyance strategies, we have gone to another time of treatment. Here, we go to the way that the Plant Exosome-like Nanovesicles will open new open new ways to the logical tests. Various strategies in nano bioelectronics and the significance of Nano science and Nano designing have been examined. Plant Exosome-like Nanovesicles are utilized in drug conveyance framework at nano bio electronic office. It has polar and nonpolar district. Medications will embed into the Plant Exosome-like Nanovesicles. In this paper, they will determine what Plant Exosome-like Nanovesicles are, various procedures to enter Plant Exosome-like Nanovesicles to the objective cells, how it very well may be made, and cooperation's among Plant Exosome-like Nanovesicles and cell layer. Also, the Preparation of PELNVS and Re-Engineering of PELNVs were discussed.
... Peptides have been emerged as an ideal molecule for organic electronics due to their intrinsic ability to form a spectrum of ordered self-assembled supramolecular nanostructures . In recent years, peptide conjugated QDs are studied for cellular imaging (Zhang et al. 2012), enhancement of cellular uptake (Choi et al. 2013), and drug delivery (Aswathy et al. 2012). Peptides engineered with different amino acids have served as ligands for producing water-soluble QDs ) Incorporating cellpenetrating peptides on QD surfaces has allowed the translocation of functionalized QDs into cells for intracellular imaging applications (Hild et al. 2008). ...
Spatial confinement of excitons in the nano-crystalline region of semiconducting nanostructures differ significantly from the optoelectronic properties exhibited by the bulk material. We report spike-like absorption observed in the UV spectrum of a phenylalanine hexamer peptide [(Ff)3-OH] nano-assembly, which may be attributed to the spatial confinement of electrons to the dimension of quantum dots. Interdependency of the UV and PLE spectrum of the peptide confirms the existence of quantum confinement in (Ff)3-OH nano-assemblies.
... 65 Being non-intrusive and ultra-sensitive, DLS needs only little sample volume to compute accurate and precise size, which makes it the benchmark technique in evaluation of size distribution of suspension particulates in multiple scientific disciplines. [68][69][70] In addition to this, nanoparticle tracking analysis (NTA) is also gaining popularity among researchers to quantify the number of ELNVs in a sample volume and determine their size distribution. ...
Plant exosome-like nanovesicles, being innately replete with bioactive lipids, proteins, RNA, and other pharmacologically active molecules, offer unique morphological and compositional characteristics as natural nanocarriers. Furthermore, their compelling physicochemical traits underpin their modulative role in physiological processes, all of which have fostered the concept that these nanovesicles may be highly proficient in
the development of next-generation biotherapeutic and drug delivery nanoplatforms to meet the ever-stringent demands of current clinical challenges. This review systemically deals with various facets of plant exosome-like nanovesicles ranging
from their origin and isolation to identification of morphological composition, biological functions, and cargo-loading mechanisms. Efforts are made to encompass their biotherapeutic roles by elucidating their immunological modulating, anti-tumor, regenerative, and anti-inflammatory roles. We also shed light on re-engineering these nanovesicles into robust, innocuous, and non-immunogenic nanovectors for drug delivery through multiple stringent biological hindrances to various targeted organs such as intestine and brain. Finally, recent advances centered around plant exosome-like nanovesicles along with new insights into transdermal, transmembrane and targeting mechanisms of these vesicles are also elucidated. We expect that the continuing development of plant exosome-like nanovesicle-based therapeutic and delivery nanoplatforms will promote their clinical applications.
... Particularly, the fluorescence imaging can easily capture the activities, expressions, and biochemical interactions of specific biomolecule and cells [7]. Recently, several biomarkers, aptamers, receptors, and peptides have been used by different researchers for targeted imaging of cancerous cells [8][9][10][11]. Among them, folate receptor (FR) has been reported to be exceptionally upregulated in several cancers, including breast, kidney, ovarian, lung, endometrial, colorectal, neuroendocrine, and renal carcinomas while rarely found in healthy cells [12,13]. ...
For the first time is reported a facile in situ synthesis of folic acid-conjugated sulfur-doped graphene quantum dots (FA-SGQDs) through simple pyrolysis of citric acid (CA), 3-mercaptopropionic acid (MPA), and FA. The as-prepared FA-SGQDs were extensively characterized to confirm the synthesis and incidence of FA molecule on the surface of SGQDs through advanced characterization techniques. Upon excitation at 370-nm wavelength, FA-SGQDs exhibited blue fluorescence with an emission band at 455 nm. While exhibiting relatively high quantum yield (~ 78%), favorable biocompatibility, excellent photostability, and desirable optical properties, the FA-SGQDs showed suitability as a fluorescent nanoprobe to distinguish the folate receptor (FR)-positive and FR-negative cancer cells. The experimental studies revealed that FA-SGQDs aptly entered into FR-positive cancer cells via a non-immunogenic FR-mediated endocytosis process. Additionally, the FA-SGQDs exhibited excellent free radical scavenging activity. Hence, these FA-SGQDs hold high promise to serve as efficient fluorescent nanoprobes for the pre-diagnosis of cancer through targeted bioimaging and other pertinent biological studies.
Graphical abstract
Quantum dots (QDs) are semiconductor nanocrystals possessing unique optoelectrical properties in that they can emit light energy of specific tunable wavelengths when excited by photons. They are gaining attention nowadays owing to their all-around ability to allow high-quality bio-imaging along with targeted drug delivery. The most lethal central nervous system (CNS) disorders are brain cancers or malignant brain tumors. CNS is guarded by the blood-brain barrier which poses a selective blockade toward drug delivery into the brain. QDs have displayed strong potential to deliver therapeutic agents into the brain successfully. Their bio-imaging capability due to photoluminescence and specific targeting ability through the attachment of ligand biomolecules make them preferable clinical tools for coming times. Biocompatible QDs are emerging as nanotheranostic tools to identify/diagnose and selectively kill cancer cells. The current review focuses on QDs and associated nanoformulations as potential futuristic clinical aids in the continuous battle against brain cancer.
The key issue in the treatment of solid tumors is the lack of efficient strategies for the targeted delivery and accumulation of therapeutic cargoes in the tumor microenvironment (TME). Targeting approaches are designed for more efficient delivery of therapeutic agents to cancer cells while minimizing drug toxicity to normal cells and off-targeting effects, while maximizing the eradication of cancer cells. The highly complicated interrelationship between the physicochemical properties of nanoparticles, and the physiological and pathological barriers that are required to cross, dictates the need for the success of targeting strategies. Dual targeting is an approach that uses both purely biological strategies and physicochemical responsive smart delivery strategies to increase the accumulation of nanoparticles within the TME and improve targeting efficiency towards cancer cells. In both approaches, either one single ligand is used for targeting a single receptor on different cells, or two different ligands for targeting two different receptors on the same or different cells. Smart delivery strategies are able to respond to triggers that are typical of specific disease sites, such as pH, certain specific enzymes, or redox conditions. These strategies are expected to lead to more precise targeting and better accumulation of nano-therapeutics. This review describes the classification and principles of dual targeting approaches and critically reviews the efficiency of dual targeting strategies, and the rationale behind the choice of ligands. We focus on new approaches for smart drug delivery in which synthetic and/or biological moieties are attached to nanoparticles by TME-specific responsive linkers and advanced camouflaged nanoparticles.
The assessment of cytotoxicity of quantum dots is very essential for environmental and health risk analysis. In the present work we have modelled HeLa cell cytotoxicity of sixty one CdSe quantum dots with ZnS shell as a function of its experimental conditions and molecular construction using quasiSMILES representations. The index of ideality of correlation helps in the building of ten statistically significant models having good fitting ability with value of R2 ranging from 0.8414 to 0.9609 for the training set. The split 5 model is rated as the best model with values of R2, Q2F1, Q2F2 and Q2F3 as 0.8964, 0.8267, 0.8264 and 0.8777 respectively for the calibration set. The extraction of features causing increase and decrease of cytotoxicity of quantum dots indicates importance of neutral surface charge, surface modified with protein, 72 hours exposure time, combination of MTT assay with surface protein in decreasing the cytotoxicity. Amphiphilic polymer, polyol ligand with neutral charge, 0.5 – 0.6 nm quantum dot diameter with lipid ligand and unmodified positively charged surface are grouped in toxicity enhancer features. Further, consensus modelling using split 5 and 8 patterns enhances the prediction quality by increasing the R2val to 0.9361 and 0.9656 respectively.
Aptamer is oligonucleotide with high affinity and specificity toward the target which is obtained via systematic evolution of ligands by exponential enrichment (SELEX). However, due to the complexity of environmental factors and the unknown influence on the selection and application of aptamers, the optimization of multiple environmental factors through conventional experiments requires a huge amount of work and samples, so there are few systematic studies. Capillary electrophoresis (CE)-SELEX has become a highly efficient technique for aptamers screening because of the advantages of CE, e.g. microscale, high efficiency and low cost. In this work, human apo-transferrin (A-TF) was used as a model to study the effects of factors based on CE, including length of ssDNA library, incubation temperature, type and pH value of incubation buffer, metal ions etc. The results showed that shorter ssDNA library had higher affinity with the target protein. Incubation temperature, type and pH value of incubation buffer affected the formation of complex. Low concentrations of K⁺, Ca²⁺ and Mg²⁺ metal ions promoted the formation of complexes. The A-TF aptamer Seq A3 obtained by three rounds of selection had a dissociation constant (KD) of 0.476 μmol/L, and its affinity and specificity were verified via CE-laser induced fluorescence in human serum. The results indicated that Seq A3 could specifically recognize A-TF in serum.
Glioblastoma (GBM) is one of the deadliest primary brain malignant tumors with a bleak prognosis. Craniotomy surgical resection followed by radiotherapy and chemotherapy was still the standard therapeutic strategy for GBM. As a target alkylating agent, temozolomide (TMZ) was utilized in the therapy of GBM for decades. However, effective treatment for GBM is stymied by rapid acquired resistance and bone marrow suppression. Here, we synthesize a tetrahedral framework nucleic acid (tFNA) nanoparticle that can carry TMZ to enhance the lethality on four glioblastoma cell lines via activating the cell apoptosis and autophagy pathway. Our nanoparticle, namely tFNA-TMZ, show a more obvious efficacy in killing TMZ-sensitive cells (A172 and U87) than single agent TMZ. Besides, tFNA-TMZ was able to attenuate drug resistance in TMZ-resistant cells (T98G and LN-18) via downregulating the expression of O6-methylguanine-DNA-methyltransferase (MGMT). Furthermore, we modified the tFNA with GS24, a DNA aptamer which can specially bind to transferrin receptor (TRF) in cerebral vascular endothelial cell of mouse and enable tFNA nanoparticle to cross the blood brain barrier. In summary, our results demonstrated that tFNA-TMZ have a promising role as a nanoscale vehicle to deliver TMZ to enhance efficacy for glioblastoma.
Two new techniques, aptamer-based specific recognition and quantum dot (QD)-based fluorescence labeling, are becoming increasingly important in biosensing. In this study, these two techniques have been coupled together to construct a new kind of fluorescent QD-labeled aptamer (QD-Apt) nanoprobe by conjugating GBI-10 aptamer to the QD surface. GBI-10 is a single-stranded DNA (ssDNA) aptamer for tenascin-C, which distributes on the surface of glioma cells as a dominant extracellular matrix protein. The QD-Apt nanoprobe can recognize the tenascin-C on the human glioma cell surface, which will be helpful for the development of new convenient and sensitive in vitro diagnostic assays for glioma. The QD-Apt nanoprobe has particular features such as strong fluorescence, stability, monodispersity and uniformity. In addition, this probe preparation method is universal, so it is expected to provide a new type of stable nanoprobe for high-throughput and fast biosensing detection and bioimaging. New methods for real-time and dynamic tracking and imaging can be accordingly developed.
This paper contributes to the debate on how nanosized objects negotiate membrane barriers inside biological cells. The uptake of peptide-modified gold nanoparticles by HeLa cells has been quantified using atomic emission spectroscopy. The TAT peptide from the HIV virus was singled out as a particularly effective promoter of cellular uptake. The evolution of the intracellular distribution of TAT-modified gold nanoparticles with time has been studied in detail by TEM and systematic image analysis. An unusual trend of particles disappearing from the cytosol and the nucleus and accumulating massively in vesicular bodies was observed. Subsequent release of the particles, both by membrane rupture and by direct transfer across the membrane boundary, was frequently found. Ultimately, near total clearing of particles from the cells occurred. This work provides support for the hypothesis that cell-penetrating peptides can enable small objects to negotiate membrane barriers also in the absence of dedicated transport mechanisms.
Superparamagnetic iron oxide (SPIO) nanoparticles are widely used in magnetic resonance imaging (MRI) as versatile ultra-sensitive nanoprobes for cellular and molecular imaging of cancer. In this study, we report a one-step procedure for the surface functionalization of SPIO nanoparticles with a lung cancer-targeting peptide. The hydrophobic surfactants on the as-synthesized SPIO are displaced by the peptide containing a poly(ethylene glycol)-tethered cysteine residue through ligand exchange. The resulting SPIO particles are biocompatible and demonstrate high T(2) relaxivity. The nanoprobes are specific in targeting α(v)β(6)-expressing lung cancer cells as demonstrated by MR imaging and Prussian blue staining. This facile surface chemistry and the functional design of the proposed SPIO system may provide a powerful nanoplatform for the molecular diagnosis of lung cancer.
This article reported the high tumor targeting efficacy of RGD peptide labeled near-infrared (NIR) non-cadmium quantum dots (QDs). After using poly(ethylene glycol) to encapsulate InAs/InP/ZnSe QDs (emission maximum at about 800 nm), QD800-PEG dispersed well in PBS buffer with the hydrodynamic diameter (HD) of 15.9 nm and the circulation half-life of approximately 29 min. After coupling QD800-PEG with arginine-glycine-aspartic acid (RGD) or arginine-alanine-aspartic acid (RAD) peptides, we used nude mice bearing subcutaneous U87MG tumor as models to test tumor-targeted fluorescence imaging. The results indicated that the tumor uptake of QD800-RGD is much higher than those of QD800-PEG and QD800-RAD. The semiquantitative analysis of the region of interest (ROI) showed a high tumor uptake of 10.7 +/- 1.5%ID/g in mice injected with QD800-RGD, while the tumor uptakes of QD800-PEG and QD800-RAD were 2.9 +/- 0.3%ID/g and 4.0 +/- 0.5%ID/g, respectively, indicating the specific tumor targeting of QD800-RGD. The high reproducibility of bioconjunction between QDs and the RGD peptide and the feasibility of QD-RGD bioconjugates as tumor-targeted fluorescence probes warrant the successful application of QDs for in vivo molecular imaging.
The extinction coefficient per mole of nanocrystals at the first exitonic absorption peak, ε, for high-quality CdTe, CdSe, and CdS nanocrystals was found to be strongly dependent on the size of the nanocrystals, between a square and a cubic dependence. The measurements were carried out using either nanocrystals purified with monitored purification procedures or nanocrystals prepared through controlled etching methods. The nature of the surface ligands, the refractive index of the solvents, the PL quantum yield of the nanocrystals, the methods used for the synthesis of the nanocrystals, and the temperature for the measurements all did not show detectable influence on the extinction coefficient for a given sized nanocrystal within experimental error.
CdSe/ZnS quantum dots (QDs) exhibited fluorescence emission blue shifts when conjugated to antibodies or DNA aptamers that are bound to bacteria. The intensity of the shifted emission peak increased with the number of bound bacteria. Curiously, the emission was consistently shifted to approximately 440-460 nm, which is distinctly different from the major component of the natural fluorescence spectrum of these QDs. This minor emission peak can grow upon conjugation to antibodies or aptamers and subsequent binding to bacterial cell surfaces. We hypothesize that the wavelength shift is due to changes in the chemical environment of the QD conjugates when they encounter the bacterial surface and may be due to physical deformation of the QD that changes the quantum confinement state. Regardless of the mechanism, these remarkable emission wavelength shifts of greater than 140 nm in some cases strongly suggest new applications for QD-receptor conjugates.
In this study, we report the design and delivery of a tumor-targeted, pH-responsive quantum dot-mucin1 aptamer-doxorubicin (QD-MUC1-DOX) conjugate for the chemotherapy of ovarian cancer. To achieve active cancer targeting, QD was conjugated with a DNA aptamer specific for mutated MUC1 mucin overexpressed in many cancer cells including ovarian carcinoma. DOX was attached to QD via a pH-sensitive hydrazone bond in order to provide the stability of the complex in systemic circulation and drug release in acidic environment inside cancer cells. The data show that this bond is stable at neutral and slightly basic pH and undergoes rapid hydrolysis in mildly acidic pH. Confocal microscopy and in vivo imaging studies show that the developed QD-MUC1-DOX conjugate had higher cytotoxicity than free DOX in multidrug resistant cancer cells and preferentially accumulated in ovarian tumor. Data obtained demonstrate a high potential of the proposed conjugate in treatment of multidrug resistant ovarian cancer.
The purpose of this work was to evaluate the potential of HAIYPRH (T7) peptide as a ligand for constructing tumor-targeting drug delivery systems. T7 could target to transferrin-receptor (TfR) through a cavity on the surface of TfR and then transport into cells via endocytosis with the help of transferrin (Tf). In this study, T7-conjugated poly(ethylene glycol) (PEG)-modified polyamidoamine dendrimer (PAMAM) (PAMAM-PEG-T7) was successfully synthesized and further loaded with doxorubicin (DOX), formulating PAMAM-PEG-T7/DOX nanoparticles (NPs). In vitro, almost 100% of DOX was released during 2 h in pH 5.5, while only 55% of DOX was released over 48 h in pH 7.4. The cellular uptake of DOX could be significantly enhanced when treated with T7-modified NPs in the presence of Tf. Also, the in vitro antitumor effect was enhanced markedly. The IC(50) of PAMAM-PEG-T7/DOX NPs with Tf was 231.5 nM, while that of NPs without Tf was 676.7 nM. T7-modified NPs could significantly enhance DOX accumulation in the tumor by approximately 1.7-fold compared to that of unmodified ones and by approximately 5.3-fold compared to that of free DOX. For in vivo antitumor studies, tumor growth of mice treated with PAMAM-PEG-T7/DOX NPs was significantly inhibited compared to that of mice treated with PAMAM-PEG/DOX NPs and saline. The study provides evidence that PAMAM-PEG-T7 can be applied as a potential tumor-targeting drug delivery system. T7 may be a promising ligand for targeted drug delivery to the tumor.
Synthesis of biologically active antibody conjugated quantum dots (QDs) has been of great importance in cellular imaging and diagnostics. Cetuximab (or Erbitux) is the first monoclonal antibody drug which targets the epidermal growth factor receptor (EGFR) overexpressed in most cancer cells. In the present work, we investigated three different conjugation strategies to obtain the biologically functional QD-cetuximab conjugates for the tumor-specific imaging. Successful conjugation of cetuximab to QDs was achieved using PEG conjugated polymer-coated QDs and two long-chain heterobifunctional linkers, sulfo-LC-SPDP and sulfo-SMCC. The dissociation constant of the QD-cetuximab conjugates to EGFR was determined to be 0.61 +/- 0.28 nM. The cancer cell-specific binding ability of the QD-cetuximab conjugates was evaluated in vitro, and the cellular internalization of the QD-cetuximab conjugates was clearly demonstrated in live cells by confocal microscopy. The cellular imaging experiments using the QD-cetuximab conjugates showed a clear endocytosis pathway, which was evidenced by the colocalization of the QD-cetuximab conjugates with dye-labeled transferrin. These results suggest that the QD-cetuximab conjugates as an imaging modality for tumor EGFR overexpression can be expected to provide important information on the expression levels of EGFR on the cancer cells.
We developed highly luminescent and cost-effective quantum dot (QD)-neutravidin (NTV) bioconjugates to detect the tyrosine kinase B (TrkB) receptors distributed in the cultured hippocampus neurons. Hippocampal neurons were incubated with biotinylated anti-TrkB antibody, followed by further incubation with QD-NTV bioconjugates. QD-NTV biomarkers on the extracellular domain of TrkB receptors were imaged by the combined atomic force microscope and confocal laser scanning microscope (AFM-CLSM) providing resolved (nanometer-scale) structural and fluorescent images. We found that TrkB receptors were distributed over the neuronal cell bodies (soma) and neurites. TrkB receptors in the somata looked more concentrated, but those in the neurites appeared punctate. Thus, our QD-based immunocytochemistry technique combined with an AFM-CLSM can be used for three-dimensional morphology of neurons on nanometer-scale structural resolution and their fluorescence images with QDs. Furthermore, this technique can be applied for real-time fluorescence imaging or long-term study of live neurons.