Peptide-conjugated Quantum Dots Research Articles

Probing the Intracellular Delivery of Nanoparticles into Hard-to-Transfect Cells.

Hard-to-transfect cells are cells that are known to present special difficulties in intracellular delivery of exogenous entities. However, the special transport behaviors underlying the special delivery problem in these cells have so far not been examined carefully. Here, we combine single-particle motion analysis, cell biology studies, and mathematical modeling to investigate nanoparticle transport in bone marrow-derived mesenchymal stem cells (BMSCs), a technologically important type of hard-to-transfect cells. Tat peptide-conjugated quantum dots (QDs-Tat) were used as the model nanoparticles. Two different yet complementary single-particle methods, namely, pair-correlation function and single-particle tracking, were conducted on the same cell samples and on the same viewing stage of a confocal microscope. Our results reveal significant differences in each individual step of transport of QDs-Tat in BMSCs vs a commonly used model cell line, HeLa cells. Single-particle motion analysis demonstrates that vesicle escape and cytoplasmic diffusion are dramatically more difficult in BMSCs than in HeLa cells. Cell biology studies show that BMSCs use different biological pathways for the cellular uptake, vesicular transport, and exocytosis of QDs-Tat than HeLa cells. A reaction-diffusion-advection model is employed to mathematically integrate the individual steps of cellular transport and can be used to predict and design nanoparticle delivery in BMSCs. This work provides dissective, quantitative, and mechanistic understandings of nanoparticle transport in BMSCs. The investigative methods described in this work can help to guide the tailored design of nanoparticle-based delivery in specific types and subtypes of hard-to-transfect cells.

Targeted Fluorescence Imaging and Biological Effects of Peptide Conjugated Quantum Dots on Pancreatic Cancer Cells.

Arginine-glycine-aspartic acid (RGD) peptide sequences exist in a variety of biological extracellular matrices and can specifically bind the cell-surface integrin αvβ3, which is overexpressed in cancer cells and plays important roles in tumor growth and invasion. Quantum dots (QDs) have been applied in the field of cell biology and can be physically conjugated to the surface of cancer cells for imaging. In this research, we developed QDs-RGD nanoparticles and investigated its application in pancreatic cancer cell imaging and its influence on the biological behavior of pancreatic cancer cells. The results of flow cytometric analysis showed that the αvβ3 receptor was markedly overexpressed on pancreatic cancer cells. In cellular uptake studies, the fluorescence signal of QDs-RGD nanoparticles in pancreatic cancer cells was higher than that of QDs without RGD conjugation, as determined by an inverted fluorescence microscope. Furthermore, the biological behavior of pancreatic cancer cells was affected by QDs-RGD nanoparticles, which inhibited proliferation, migration and invasion and induced G2-phase cell cycle arrest. With integrin αvβ3 as a target, QDs-RGD nanoparticles can generate high-quality images of pancreatic cancer cells and have immense potential for use in the targeted diagnosis and therapy of pancreatic cancer.

A potent, minimally invasive and simple strategy of enhancing intracellular targeted delivery of Tat peptide-conjugated quantum dots: organic solvent-based permeation enhancer.

Targeted delivery of nanomaterials to specific intracellular locations is essential for the development of many nanomaterials-based biological applications. Thus far the targeting performance has been limited due to various intracellular transport barriers, especially intracellular vesicle trapping. Here we report the application of permeation enhancers based on organic solvents in small percentage to enhance the intracellular targeted delivery of nanomaterials. Previously permeation enhancers based on organic solvents and ionic liquids have been used in overcoming biological transport barriers at tissue, organ, and cellular levels, but this strategy has so far rarely been examined for its potential in facilitating transport of nanometer-scale entities across intracellular barriers, particularly intracellular vesicle trapping. Using the cell nucleus as a model intracellular target and Tat peptide-conjugated quantum dots (QDs-Tat) as a model nanomaterial-based probe, we demonstrate that a small percentage (e.g. 1%) of organic solvent greatly enhances nucleus targeting specificity as well as increasing endocytosis-based cellular uptake of QDs. We combine vesicle colocalization (DiO dye staining), vesicle integrity (calcein dye release), and single-particle studies (pair-correlation function microscopy) to investigate the process of organic solvent-enhanced vesicle escape of QDs-Tat. The organic solvent based vesicle escape-enhancing approach is found to be not only very effective but minimally invasive, resulting in high vesicle escape efficiency with no significant disruption to the membrane integrity of either intracellular vesicles or cells. This approach drastically outperforms the commonly used vesicle escape-enhancing agent (i.e., chloroquine, whose enhancement effect is based on disrupting vesicle integrity) in both potency and minimal invasiveness. Finally, we apply organic solvent-based targeting enhancement to improve the intracellular delivery of the anticancer drug doxorubicin (DOX).

Depicting Binding-Mediated Translocation of HIV-1 Tat Peptides in Living Cells with Nanoscale Pens of Tat-Conjugated Quantum Dots.

Cell-penetrating peptides (CPPs) can translocate across cell membranes, and thus have great potential for the cellular delivery of macromolecular cargoes. However, the mechanism of this cellular uptake process is not yet fully understood. In this study, a time-lapse single-particle light-sheet microscopy technique was implemented to obtain a parallel visualization of the translocating process of individual human immunodeficiency virus 1 (HIV-1) transactivator of transcription (Tat) peptide conjugated quantum dots (TatP-QDs) in complex cellular terrains. Here, TatP-QDs served as nanoscale dynamic pens, which depict remarkable trajectory aggregates of TatP-QDs on the cell surface. Spectral-embedding analysis of the trajectory aggregates revealed a manifold formed by isotropic diffusion and a fraction of directed movement, possibly caused by interaction between the Tat peptides and heparan sulfate groups on the plasma membrane. Further analysis indicated that the membrane deformation induced by Tat-peptide attachment increased with the disruption of the actin framework in cytochalasin D (cyto D)-treated cells, yielding higher interactions on the TatP-QDs. In native cells, the Tat peptides can remodel the actin framework to reduce their interaction with the local membrane environment. Characteristic hot spots for interaction were detected on the membrane, suggesting that a funnel passage may have formed for the Tat-coated particles. This finding offers valuable insight into the cellular delivery of nanoscale cargo, suggesting an avenue for direct therapeutic delivery.

Peptide-Conjugated Quantum Dots Act as the Target Marker for Human Pancreatic Carcinoma Cells

Background/Aims: In the present study, we describe a novel and straightforward approach to produce a cyclic- arginine-glycine-aspartic (RGD)-peptide-conjugated quantum dot (QD) probe as an ideal target tumor biomarker. Due to its specific structure, the probe can be used for targeted imaging of pancreatic carcinoma cells. Methods: Pancreatic carcinoma cells were routinely cultured and marked with QD-RGD probe. The QD-RGD probe on the fluorescence-labeled cancer cell was observed by fluorescence microscopy and laser confocal microscopy. Cancer cell viability was detected by MTT assay after culturing with QD-RGD probe. Results: Fluorescence microscopy and laser confocal microscopy displayed that 10nmol/L QD-RGD probe was able to effectively mark pancreatic carcinoma cells. In comparison with organic dyes and fluorescent proteins, the quantum dot-RGD probe had unique optical and electronic properties. Conclusion: QD-RGD probe has a low cytotoxicity with an excellent optical property and biocompatibility. These findings support further evaluation of QD-RGD probes for the early detection of pancreatic cancer.

Homing peptide conjugated quantum dots for colorectal cancer imaging

Peptide-labeled quantum dots (QDs) have found wide applications in tumor imaging. Especially, homing peptide labeled QDs can target specific tumor tissues and thus serve as an indicator of the location of the tumor. However, most tumor homing agents target general markers instead of a biomarker specific to a certain type of cancers; for example, no specific imaging agents have been reported for colorectal cancer yet. Herein, we report the conjugation of a colorectal tumor homing peptide TCP-1-H6 with QDs by metal affinity-driven assembly, and also the characterization of the details of the assembly process by capillary electrophoresis. TCP-1-H6 conjugated QDs (TCP-1-H6-QD) were then applied to the imaging of colorectal cancer cells as well as tumor tissues from a mouse model of orthotopic colorectal cancers. TCP-1-H6-QD efficiently targeted colon 26 cells, and were internalized into the intracellular space. On tumor tissue sections, TCP-1-H6-QD specifically labeled the tumor tissues but not health tissues. These results indicate that TCP-1-H6-QD retains the unique homing property of the peptide and thus can be used for specific imaging of colorectal cancer. This method thereby widens the road for specific identification and diagnosis of cancer.

Labeling of Hematopoietic Stem Cells by Tat Peptide Conjugated Quantum Dots for Cell Tracking in Mouse Body

Fluorescent quantum dots have great potential to act as labeling tools in biological research, especially cellular tracking and imaging. Tat peptide conjugated quantum dots were introduced into living human hematopoietic stem cells (HSCs). The internalized quantum dots were assessed by laser confocal microscope and flow cytometer. The quantum dots labeled HSCs were injected intravenously into the tail veins of NOD/SCID beta2M null mice. The tissue collections in the major organs were examined with fluorescence microscope to assess the distribution of transplanted stem cells. HSCs with internalized quantum dots offer a promising approach for stem cell transplantation, which will hold a significant impact on stem cell based therapy for several disorders, especially to cure leukemia in current China.

Targeting of Embryonic Stem Cells by Peptide-Conjugated Quantum Dots

BackgroundTargeting stem cells holds great potential for studying the embryonic stem cell and development of stem cell-based regenerative medicine. Previous studies demonstrated that nanoparticles can serve as a robust platform for gene delivery, non-invasive cell imaging, and manipulation of stem cell differentiation. However specific targeting of embryonic stem cells by peptide-linked nanoparticles has not been reported.Methodology/Principal FindingsHere, we developed a method for screening peptides that specifically recognize rhesus macaque embryonic stem cells by phage display and used the peptides to facilitate quantum dot targeting of embryonic stem cells. Through a phage display screen, we found phages that displayed an APWHLSSQYSRT peptide showed high affinity and specificity to undifferentiated primate embryonic stem cells in an enzyme-linked immunoabsorbent assay. These results were subsequently confirmed by immunofluoresence microscopy. Additionally, this binding could be completed by the chemically synthesized APWHLSSQYSRT peptide, indicating that the binding capability was specific and conferred by the peptide sequence. Through the ligation of the peptide to CdSe-ZnS core-shell nanocrystals, we were able to, for the first time, target embryonic stem cells through peptide-conjugated quantum dots.Conclusions/SignificanceThese data demonstrate that our established method of screening for embryonic stem cell specific binding peptides by phage display is feasible. Moreover, the peptide-conjugated quantum dots may be applicable for embryonic stem cell study and utilization.

Effect of peptide-conjugated quantum dots on the tumorigenicity and lymph node metastasis of human tongue squamous cell carcinoma cell line Tca8113 and mouse uterine cervix carcinoma U14 in vivo

To observe the effect of peptide-conjugated quantum dots with a maximal emission of 655 nm (QD655) on growth, proliferation, apoptosis and lymphatic metastasis of human tongue squamous cell carcinoma cell line Tca8113 and mouse uterine cervix carcinoma U14 in vivo. Tca8113 and U14 cells were labeled by QD655 (Tca8113-QD655, U14-QD655). Tca8113-QD655 and U14-QD655 were inoculated subcutaneously into nude mice and Kunming mice. The tumor formation of Tca8113-QD655 and Tca8113, U14-QD655 and U14 was observed and compared in vivo. The proliferation and apoptosis of Tca8113-QD655 and Tca8113, U14-QD655 and U14 cells from tumors formed in vivo were analyzed by flow cytometry. U14-QD655 and U14 were inoculated into the buccal mucosa of Kunming mice to establish the cervical lymph node metastasis model of buccal cancer. The cervical lymph node metastatic ability of U14-QD655 and U14 was compared. The tumor weight and volume of Tca8113-QD655 and Tca8113, U14-QD655 and U14 in vivo were not significantly different (P>0.05), the cell proliferation index and apoptosis index of Tca8113-QD655 and Tca8113, U14-QD655 and U14 in vivo were not significantly different (P>0.05). The cervical lymph node metastasis rate of U14-QD655 and U14 buccal cancer were not significantly different (P>0.05). QD showed no effects on tumorigenicity and lymph node metastasis of Tca8113 and U14 cells. These results provide the scientific basis for noninvasive imaging and long-term tracing study.

Study on the Intracellular Fate of Tat Peptide-Conjugated Quantum Dots by Spectroscopic Investigation

The photoluminescence (PL) spectrum of water-soluble thiol-capped CdTe quantum dots (QDs) conjugated with Tat peptide in solution showed a remarkable redshift as compared to that of unconjugated QDs. After cellular uptake of the Tat-QDs conjugates, the micro-PL spectrum of Tat-QDs in lysosomes showed a spectral blueshift, which was most probably due to the fact that Tat peptide was digested by the enzymes, leaving the Tat-detached QDs in lysosomes. The reasons for the spectral changes have been discussed in detail.

Preparation of peptide-conjugated quantum dots for tumor vasculature-targeted imaging

To take full advantage of the unique optical properties of quantum dots (QDs) and expedite future near-infrared fluorescence (NIRF) imaging applications, QDs need to be effectively, specifically and reliably directed to a specific organ or disease site after systemic administration. Recently, we reported the use of peptide-conjugated QDs for non-invasive NIRF imaging of tumor vasculature markers in small animal models. In this protocol, we describe the detailed procedure for the preparation of such peptide-conjugated QDs using commercially available PEG-coated QDs and arginine-glycine-aspartic acid (RGD) peptides. Conjugation of the thiolated RGD peptide to the QDs was achieved through a heterobifunctional linker, 4-maleimidobutyric acid N-succinimidyl ester. Competitive cell binding assay, using (125)I-echistatin as the radioligand, and live cell staining were carried out to confirm the successful attachment of the RGD peptides to the QD surface before in vivo imaging of tumor-bearing mice. In general, QD conjugation and in vitro validation of the peptide-conjugated QDs can be accomplished within 1-2 d; in vivo imaging will take another 1-2 d depending on the experimental design.

Applications of Mesenchymal Stem Cells Labeled with Tat Peptide Conjugated Quantum Dots to Cell Tracking in Mouse Body

Fluorescent quantum dots have great potential in cellular labeling and tracking. Here, PEG encapsulated CdSe/ZnS quantum dots have been conjugated with Tat peptide, and introduced into living mesenchymal stem cells. The Tat peptide conjugated quantum dots in mesenchymal stem cells were assessed by fluorescent microscopy, laser confocal microscope and. flow cytometry. The result shows that Tat peptide conjugated quantum dots could enter mesenchymal stem cells efficiently. The Tat-quantum dots labeled stem cells were further injected into the tail veins of NOD/SCID beta2 M null mice, and the tissue distribution of these labeled cells in nude mice were examined with fluorescence microscope. The result shows that characteristic fluorescence of quantum dots was observed primarily in the liver, the lung and the spleen, with little or no quantum dots accumulation in the brain, the heart, or the kidney.

Imaging and Tracking of Tat Peptide-Conjugated Quantum Dots in Living Cells: New Insights into Nanoparticle Uptake, Intracellular Transport, and Vesicle Shedding

We report the use of Tat peptide-conjugated quantum dots (Tat-QDs) to examine the complex behavior of nanoparticle probes in live cells, a topic that is of considerable current interest in developing advanced nanoparticle agents for molecular and cellular imaging. Dynamic confocal imaging studies indicate that the peptide-conjugated QDs are internalized by macropinocytosis, a fluid-phase endocytosis process triggered by Tat-QD binding to negatively charged cell membranes. The internalized Tat-QDs are tethered to the inner vesicle surfaces and are trapped in cytoplasmic organelles. The QD loaded vesicles are found to be actively transported by molecular machines (such as dyneins) along microtubule tracks. The destination of this active transport is an asymmetric perinuclear region (outside the cell nucleus) known as the microtubule organizing center (MTOC). We also find that Tat-QDs strongly bind to cellular membrane structures such as filopodia and that large QD-containing vesicles are released from the tips of filopodia by vesicle shedding. These results provide new insights into the mechanisms of Tat peptide-mediated delivery as well as toward the design of functionalized nanoparticles for molecular imaging and targeted therapy.

Peptide-Conjugated Quantum Dots Activate Neuronal Receptors and Initiate Downstream Signaling of Neurite Growth

Quantum dots (QDs) could serve as fluorescent scaffolds for effecting specific physiological and pharmacological responses in cells. Here, we conjugate the peptide ligand betaNGF to QD surfaces, and confirm surface modification and single QD nanostructure using AFM. We show that betaNGF-QDs retain bioactivity, activate TrkA receptors, and initiate neuronal differentiation in PC12 cells. Receptor-evoked activity of QD-immobilized ligands has wide-ranging implications for the development of molecular tools and therapeutics targeted at understanding and regulating cell function.