A fluorescent nanosensor for apoptotic cells

A biocompatible surface-functionalized nanoparticle was designed to sense phosphatidylserine exposed on apoptotic cells. We conjugated synthetic artificial phosphatidylserine binding ligands in a multivalent fashion onto magnetofluorescent nanoparticles. Our results show that (1) the synthetic nanoparticles bind to apoptotic cells, (2) there is excellent correlation with annexin V staining by microscopy, and (3) FACS analysis with nanoparticles allows the measurement of therapeutic apoptosis induction. The described nanomaterials should be useful for a variety of biomedical applications including in vivo imaging of apoptosis.     

Targeted Delivery of siRNA‐Generating DNA Nanocassettes Using Multifunctional Nanoparticles

Molecular therapy using a small interfering RNA (siRNA) has shown promise in the development of novel therapeutics. Various formulations have been used for in vivo delivery of siRNAs. However, the stability of short double‐stranded RNA molecules in the blood and efficiency of siRNA delivery into target organs or tissues following systemic administration have been the major issues that limit applications of siRNA in human patients. In this study, multifunctional siRNA delivery nanoparticles are developed that combine imaging capability of nanoparticles with urokinase plasminogen activator receptor‐targeted delivery of siRNA expressing DNA nanocassettes. This theranostic nanoparticle platform consists of a nanoparticle conjugated with targeting ligands and double‐stranded DNA nanocassettes containing a U6 promoter and a shRNA gene for in vivo siRNA expression. Targeted delivery and gene silencing efficiency of firefly luciferase siRNA nanogenerators are demonstrated in tumor cells and in animal tumor models. Delivery of survivin siRNA expressing nanocassettes into tumor cells induces apoptotic cell death and sensitizes cells to chemotherapy drugs. The ability of expression of siRNAs from multiple nanocassettes conjugated to a single nanoparticle following receptor‐mediated internalization should enhance the therapeutic effect of the siRNA‐mediated cancer therapy.     

Real‐Time Label‐Free Monitoring of Nanoparticle Cell Uptake

The surface plasmon resonance technique in combination with whole cell sensing is used for the first time for real‐time label‐free monitoring of nanoparticle cell uptake. The uptake kinetics of several types of nanoparticles relevant to drug delivery applications into HeLa cells is determined. The cell uptake of the nanoparticles is confirmed by confocal microscopy. The cell uptake of silica nanoparticles and polyethylenimine–plasmid DNA polyplexes is studied as a function of temperature, and the uptake energies are determined by Arrhenius plots. The phase transition temperature of the HeLa cell membrane is detected when monitoring cell uptake of silica nanoparticles at different temperatures. The HeLa cell uptake of the mesoporous silica nanoparticles is energy‐independent at temperatures slightly higher than the phase transition temperature of the HeLa cell membrane, while the uptake of polyethylenimine–DNA polyplexes is energy‐dependent and linear as a function of temperature with an activation energy of Ea = 62 ± 7 kJ mol^?1 = 15 ± 2 kcal mol^?1. The HeLa cell uptake of red blood cell derived extracellular vesicles is also studied as a function of the extracellular vesicle concentration. The results show a concentration dependent behavior reaching a saturation level of the extracellular vesicle uptake by HeLa cells.     

The Role of Ligand Density and Size in Mediating Quantum Dot Nuclear Transport

Studying the effects of the physicochemical properties of nanomaterials on cellular uptake, toxicity, and exocytosis can provide the foundation for designing safer and more effective nanoparticles for clinical applications. However, an understanding of the effects of these properties on subcellular transport, accumulation, and distribution remains limited. The present study investigates the effects of surface density and particle size of semiconductor quantum dots on cellular uptake as well as nuclear transport kinetics, retention, and accumulation. The current work illustrates that cellular uptake and nuclear accumulation of nanoparticles depend on surface density of the nuclear localization signal (NLS) peptides with nuclear transport reaching a plateau at 20% surface NLS density in as little as 30 min. These intracellular nanoparticles have no effects on cell viability up to 72 h post treatment. These findings will set a foundation for engineering more sophisticated nanoparticle systems for imaging and manipulating genetic targets in the nucleus.     

Labeling TiO2 Nanoparticles with Dyes for Optical Fluorescence Microscopy and Determination of TiO2–DNA Nanoconjugate Stability

Visualization of nanoparticles without intrinsic optical fluorescence properties is a significant problem when performing intracellular studies. Such is the case with titanium dioxide (TiO₂) nanoparticles. These nanoparticles, when electronically linked to single‐stranded DNA oligonucleotides, have been proposed to be used both as gene knockout devices and as possible tumor imaging agents. By interacting with complementary target sequences in living cells, these photoinducible TiO₂–DNA nanoconjugates have the potential to cleave intracellular genomic DNA in a sequence specific and inducible manner. The nanoconjugates also become detectable by magnetic resonance imaging with the addition of gadolinium Gd(III) contrast agents. Herein two approaches for labeling TiO₂ nanoparticles and TiO₂–DNA nanoconjugates with optically fluorescent agents are described. This permits direct quantification of fluorescently labeled TiO₂ nanoparticle uptake in a large population of living cells (>10⁴ cells). X‐ray fluorescence microscopy (XFM) is combined with fluorescent microscopy to determine the relative intracellular stability of the nanoconjugates and used to quantify intracellular nanoparticles. Imaging the DNA component of the TiO₂–DNA nanoconjugate by fluorescent confocal microscopy within the same cell shows an overlap with the titanium signal as mapped by XFM. This strongly implies the intracellular integrity of the TiO₂–DNA nanoconjugates in malignant cells.     

Development of Polymeric Nanoprobes with Improved Lifetime Dynamic Range and Stability for Intracellular Oxygen Sensing

A class of core‐shell nanoparticles possessing a layer of biocompatible shell and hydrophobic core with embedded oxygen‐sensitive platinum‐porphyrin (PtTFPP) dyes is developed via a radical‐initiated microemulsion co‐polymerization strategy. The influences of host matrices and the PtTFPP incorporation manner on the photophysical properties and the oxygen‐sensing performance of the nanoparticles are investigated. Self‐loading capability with cells and intracellular‐oxygen‐sensing ability of the as‐prepared nanoparticle probes in the range 0%–20% oxygen concentration are confirmed. Polymeric nanoparticles with optimized formats are characterized by their relatively small diameter (<50 nm), core‐shell structures with biocompatible shells, covalent‐attachment‐imparted leak‐free construction, improved lifetime dynamic range (up to 44 μs), excellent storage stability and photostability, and facile cell uptake. The nanoparticles’ small sensor diameter and core‐shell structure with biocompatible shell make them suitable for intracellular detection applications. For intracellular detection applications, the leak‐free feature of the as‐prepared nanoparticle sensor effectively minimizes potential chemical interferences and cytotoxicity. As a salient feature, improved lifetime dynamic range of the sensor is expected to enable precise oxygen detection and control in specific practical applications in stem‐cell biology and medical research. Such a feature‐packed nanoparticle oxygen sensor may find applications in precise oxygen‐level mapping of living cells and tissue.     

Measurement of immunotargeted plasmonic nanoparticles' cellular binding: a key factor in optimizing diagnostic efficacy

In this study, we use polarized light scattering to study immunotargeted plasmonic nanoparticles which bind to live SK-BR-3 human breast carcinoma cells. Gold nanoparticles can be conjugated to various biomolecules in order to target specific molecular signatures of disease. This specific targeting provides enhanced contrast in scattering-based optical imaging techniques. While there are papers which report the number of antibodies that bind per nanoparticle, there are almost no reports of the key factor which influences diagnostic or therapeutic efficacy using nanoparticles: the number of targeted nanoparticles that bind per cell. To achieve this goal, we have developed a 'negative' method of determining the binding concentration of those antibody/nanoparticle bioconjugates which are targeted specifically to breast cancer cells. Unlike previously reported methods, we collected unbound nanoparticle bioconjugates and measured the light scattering from dilute solutions of these particles so that quantitative binding information can be obtained. By following this process, the interaction effects of adjacent bound nanoparticles on the cell membrane can be avoided simply by measuring the light scattering from the unbound nanoparticles. Specifically, using nanoshells of two different sizes, we compared the binding concentrations of anti-HER2/nanoshell and anti-IgG/nanoshell bioconjugates targeted to HER2-positive SK-BR-3 breast cancer cells. The results indicate that, for anti-HER2/nanoshell bioconjugates, there are approximately 800-1600 nanoshells bound per cell; for anti-IgG/nanoshell bioconjugates, the binding concentration is significantly lower at nearly 100 nanoshells bound per cell. These results are also supported by dark-field microscopy images of the cells labeled with anti-HER2/nanoshell and anti-IgG/nanoshell bioconjugates.     

Single Chain Epidermal Growth Factor Receptor Antibody Conjugated Nanoparticles for in vivo Tumor Targeting and Imaging

Epidermal growth factor receptor (EGFR) targeted nanoparticle are developed by conjugating a single‐chain anti‐EGFR antibody (ScFvEGFR) to surface functionalized quantum dots (QDs) or magnetic iron oxide (IO) nanoparticles. The results show that ScFvEGFR can be successfully conjugated to the nanoparticles, resulting in compact ScFvEGFR nanoparticles that specifically bind to and are internalized by EGFR‐expressing cancer cells, thereby producing a fluorescent signal or magnetic resonance imaging (MRI) contrast. In vivo tumor targeting and uptake of the nanoparticles in human cancer cells is demonstrated after systemic delivery of ScFvEGFR‐QDs or ScFvEGFR‐IO nanoparticles into an orthotopic pancreatic cancer model. Therefore, ScFvEGFR nanoparticles have potential to be used as a molecular‐targeted in vivo tumor imaging agent. Efficient internalization of ScFvEGFR nanoparticles into tumor cells after systemic delivery suggests that the EGFR‐targeted nanoparticles can also be used for the targeted delivery of therapeutic agents.     

Elucidating the Influences of Size,Surface Chemistry,and Dynamic Flow on Cellular Association of Nanoparticles Made by Polymerization‐Induced Self‐Assembly

The size and surface chemistry of nanoparticles dictate their interactions with biological systems. However, it remains unclear how these key physicochemical properties affect the cellular association of nanoparticles under dynamic flow conditions encountered in human vascular networks. Here, the facile synthesis of novel fluorescent nanoparticles with tunable sizes and surface chemistries and their association with primary human umbilical vein endothelial cells (HUVECs) is reported. First, a one‐pot polymerization‐induced self‐assembly (PISA) methodology is developed to covalently incorporate a commercially available fluorescent dye into the nanoparticle core and tune nanoparticle size and surface chemistry. To characterize cellular association under flow, HUVECs are cultured onto the surface of a synthetic microvascular network embedded in a microfluidic device (SynVivo, INC). Interestingly, increasing the size of carboxylic acid–functionalized nanoparticles leads to higher cellular association under static conditions but lower cellular association under flow conditions, whereas increasing the size of tertiary amine–decorated nanoparticles results in a higher level of cellular association, under both static and flow conditions. These findings provide new insights into the interactions between polymeric nanomaterials and endothelial cells. Altogether, this work establishes innovative methods for the facile synthesis and biological characterization of polymeric nanomaterials for various potential applications.     

An Intrinsically Fluorescent Recognition Ligand Scaffold Based on Chaperonin Protein and Semiconductor Quantum‐Dot Conjugates

Genetic engineering of a novel protein–nanoparticle hybrid system with great potential for biosensing applications and for patterning of various types of nanoparticles is described. The hybrid system is based on a genetically modified chaperonin protein from the hyperthermophilic archaeon Sulfolobus shibatae. This chaperonin is an 18‐subunit double ring, which self‐assembles in the presence of Mg ions and ATP. Described here is a mutant chaperonin (His‐β‐loopless, HBLL) with increased access to the central cavity and His‐tags on each subunit extending into the central cavity. This mutant binds water‐soluble semiconductor quantum dots, creating a protein‐encapsulated fluorescent nanoparticle. The new bioconjugate has high affinity, in the order of strong antibody–antigen interactions, a one‐to‐one protein–nanoparticle stoichiometry, and high stability. By adding selective binding sites to the solvent‐exposed regions of the chaperonin, this protein–nanoparticle bioconjugate becomes a sensor for specific targets.     

Immune Cell‐Mediated Biodegradable Theranostic Nanoparticles for Melanoma Targeting and Drug Delivery

Although tremendous efforts have been made on targeted drug delivery systems, current therapy outcomes still suffer from low circulating time and limited targeting efficiency. The integration of cell‐mediated drug delivery and theranostic nanomedicine can potentially improve cancer management in both therapeutic and diagnostic applications. By taking advantage of innate immune cell's ability to target tumor cells, the authors develop a novel drug delivery system by using macrophages as both nanoparticle (NP) carriers and navigators to achieve cancer‐specific drug delivery. Theranostic NPs are fabricated from a unique polymer, biodegradable photoluminescent poly (lactic acid) (BPLP‐PLA), which possesses strong fluorescence, biodegradability, and cytocompatibility. In order to minimize the toxicity of cancer drugs to immune cells and other healthy cells, an anti‐BRAF V600E mutant melanoma specific drug (PLX4032) is loaded into BPLP‐PLA nanoparticles. Muramyl tripeptide is also conjugated onto the nanoparticles to improve the nanoparticle loading efficiency. The resulting nanoparticles are internalized within macrophages, which are tracked via the intrinsic fluorescence of BPLP‐PLA. Macrophages carrying nanoparticles deliver drugs to melanoma cells via cell–cell binding. Pharmacological studies also indicate that the PLX4032 loaded nanoparticles effectively kill melanoma cells. The “self‐powered” immune cell‐mediated drug delivery system demonstrates a potentially significant advancement in targeted theranostic cancer nanotechnologies.     

Probing specific sequences on single DNA molecules with bioconjugated fluorescent nanoparticles

Nanometer-sized fluorescent particles (latex nanobeads) have been covalently linked to DNA binding proteins to probe specific sequences on stretched single DNA molecules. In comparison with single organic fluorophores, these nanoparticle probes are brighter, are more stable against photobleaching, and do not suffer from intermittent on/off light emission (blinking). Specifically, we demonstrate that the site-specific restriction enzyme EcoRI can be conjugated to 20-nm fluorescent nanoparticles and that the resulting nanoconjugates display DNA binding and cleavage activities of the native enzyme. In the absence of cofactor magnesium ions, the EcoRI conjugates bind to specific sequences on double-stranded DNA but do not initiate enzymatic cutting. For single DNA molecules that are stretched and immobilized on a solid surface, nanoparticles bound at specific sites can be directly visualized by multicolor fluorescence microscopy. Direct observation of site-specific probes on single DNA molecules opens new possibilities in optical gene mapping and in the fundamental study of DNA-protein interactions.     

Role of Curvature-Sensing Proteins in the Uptake of Nanoparticles with Different Mechanical Properties

Nanoparticles of different properties, such as size, charge, and rigidity, are used for drug delivery. Upon interaction with the cell membrane, because of their curvature, nanoparticles can bend the lipid bilayer. Recent results show that cellular proteins capable of sensing membrane curvature are involved in nanoparticle uptake; however, no information is yet available on whether nanoparticle mechanical properties also affect their activity. Here liposomes and liposome-coated silica are used as a model system to compare uptake and cell behavior of two nanoparticles of similar size and charge, but different mechanical properties. High-sensitivity flow cytometry, cryo-TEM, and fluorescence correlation spectroscopy confirm lipid deposition on the silica. Atomic force microscopy is used to quantify the deformation of individual nanoparticles at increasing imaging forces, confirming that the two nanoparticles display distinct mechanical properties. Uptake studies in HeLa and A549 cells indicate that liposome uptake is higher than for the liposome-coated silica. RNA interference studies to silence their expression show that different curvature-sensing proteins are involved in the uptake of both nanoparticles in both cell types. These results confirm that curvature-sensing proteins have a role in nanoparticle uptake, which is not restricted to harder nanoparticles, but includes softer nanomaterials commonly used for nanomedicine applications.     

Nanotoxicology: Advanced Optical Imaging Reveals the Dependence of Particle Geometry on Interactions Between CdSe Quantum Dots and Immune Cells (Small 3/2011)

The biocompatibility and possible toxicological consequences of engineered nanomaterials, including quantum dots (QDs) due to their unique suitability for biomedical applications, remain intense areas of interest. We utilized advanced imaging approaches to characterize the interactions of CdSe QDs of various sizes and shapes with live immune cells. Particle diffusion and partitioning within the plasma membrane, cellular uptake kinetics, and sorting of particles into lysosomes were all independantly characterized. Using high‐speed total internal reflectance fluorescence (TIRF) microscopy, we show that QDs with an average aspect ratio of 2.0 (i.e., rod‐shaped) diffuse nearly an order of magnitude slower in the plasma membrane than more spherical particles with aspect ratios of 1.2 and 1.6, respectively. Moreover, more rod‐shaped QDs were shown to be internalized into the cell 2‐3 fold more slowly. Hyperspectral confocal fluorescence microscopy demonstrates that QDs tend to partition within the cell membrane into regions containing a single particle type. Furthermore, data examining QD sorting mechanisms indicate that endocytosis and lysosomal sorting increases with particle size. Together, these observations suggest that both size and aspect ratio of a nanoparticle are important characteristics that significantly impact interactions with the plasma membrane, uptake into the cell, and localization within intracellular vesicles. Thus, rather than simply characterizing nanoparticle uptake into cells, we show that utilization of advanced imaging approaches permits a more nuanced and complete examination of the multiple aspects of cell‐nanoparticle interactions that can ultimately aid understanding possible mechanisms of toxicity, resulting in safer nanomaterial designs.     

Layer‐by‐Layer Coated Gold Nanoparticles: Size‐Dependent Delivery of DNA into Cells

Because nanoparticles are finding uses in myriad biomedical applications, including the delivery of nucleic acids, a detailed knowledge of their interaction with the biological system is of utmost importance. Here the size‐dependent uptake of gold nanoparticles (AuNPs) (20, 30, 50 and 80 nm), coated with a layer‐by‐layer approach with nucleic acid and poly(ethylene imine) (PEI), into a variety of mammalian cell lines is studied. In contrast to other studies, the optimal particle diameter for cellular uptake is determined but also the number of therapeutic cargo molecules per cell. It is found that 20 nm AuNPs, with diameters of about 32 nm after the coating process and about 88 nm including the protein corona after incubation in cell culture medium, yield the highest number of nanoparticles and therapeutic DNA molecules per cell. Interestingly, PEI, which is known for its toxicity, can be applied at significantly higher concentrations than its IC₅₀ value, most likely because it is tightly bound to the AuNP surface and/or covered by a protein corona. These results are important for the future design of nanomaterials for the delivery of nucleic acids in two ways. They demonstrate that changes in the nanoparticle size can lead to significant differences in the number of therapeutic molecules delivered per cell, and they reveal that the toxicity of polyelectrolytes can be modulated by an appropriate binding to the nanoparticle surface.     

Zinc Oxide Nanoparticles and Voltage‐Gated Human Kv11.1 Potassium Channels Interact through a Novel Mechanism

Membrane–nanoparticle interactions are important in determining the effects of manufactured nanomaterials on cell physiology and pathology. Here, silica, titanium, zinc, and magnesium oxide nanoparticles are screened against human hERG (K_v11.1) voltage‐gated potassium channels under a whole‐cell voltage clamp. 10 µg mL^?1 ZnO uniquely increases the amplitude of the steady‐state current, decreases the rate of hERG current inactivation during steady‐state depolarization, accelerates channel deactivation during resurgent tail currents, and shows no significant alteration of current activation rate or voltage dependence. In contrast, ZnCl₂ causes increased current suppression with increasing concentration and fails to replicate the nanoparticle effect on decreasing inactivation. The results show a novel class of nanoparticle–biomembrane interaction involving channel gating rather than channel block, and have implications for the use of nanoparticles in biomedicine, drug delivery applications, and nanotoxicology.     

Modeling particle shape-dependent dynamics in nanomedicine

One of the major challenges in nanomedicine is to improve nanoparticle cell selectivity and adhesion efficiency through designing functionalized nanoparticles of controlled sizes, shapes, and material compositions. Recent data on cylindrically shaped filomicelles are beginning to show that non-spherical particles remarkably improved the biological properties over spherical counterpart. Despite these exciting advances, non-spherical particles have not been widely used in nanomedicine applications due to the lack of fundamental understanding of shape effect on targeting efficiency. This paper intends to investigate the shape-dependent adhesion kinetics of non-spherical nanoparticles through computational modeling. The ligand-receptor binding kinetics is coupled with Brownian dynamics to study the dynamic delivery process of nanorods under various vascular flow conditions. The influences of nanoparticle shape, ligand density, and shear rate on adhesion probability are studied. Nanorods are observed to contact and adhere to the wall much easier than their spherical counterparts under the same configuration due to their tumbling motion. The binding probability of a nanorod under a shear rate of 8 s(-1) is found to be three times higher than that of a nanosphere with the same volume. The particle binding probability decreases with increased flow shear rate and channel height. The Brownian motion is found to largely enhance nanoparticle binding. Results from this study contribute to the fundamental understanding and knowledge on how particle shape affects the transport and targeting efficiency of nanocarriers, which will provide mechanistic insights on the design of shape-specific nanomedicine for targeted drug delivery applications.     

Visualizing Human Telomerase Activity with Primer‐Modified Au Nanoparticles

Telomerase is over‐expressed in over 85% of all known human tumors. This renders the enzyme a valuable biomarker for cancer diagnosis and an important therapeutic target. The most widely used telomeric repeat amplification protocol (TRAP) assay has been questioned for telomerase detection. It is reported that human telomerase activity can be visualized by using primer‐modified Au nanoparticles. The working principle is based on the elongated primers conjugated to the gold nanoparticle (AuNP) surface, which can fold into a G‐quadruplex to protect the AuNPs from the aggregation. The developed simple and sensitive colorimetric assay can measure telomerase activity down to 1 HeLa cell µL^?1. More importantly, this assay can be easily extended to high‐throughput and automatic format. The AuNP‐TS method is PCR‐free and therefore avoids the amplification‐related errors and becomes more reliable to evaluate telomerase activity. This assay has also been used for initial screening of telomerase inhibitors as anticancer drug agents.     

Amplification of Resonant Rayleigh Light Scattering Response Using Immunogold Colloids for Detection of Lysozyme

A strategy for attomolar‐level detection of small molecule‐size proteins is reported based on Rayleigh light scattering spectroscopy of individual nanoplasmonic aptasensors by exploiting the outstanding characteristics of gold colloids to amplify the nontransparent resonant signal at ultralow analyte concentrations. The fabrication method utilizes thiol‐mediated adsorption of a DNA aptamer on the immobilized Au nanoparticle surface, the interfacial binding characteristics of the aptamer with its target molecules, and the antibody–antigen interaction through plasmonic resonance coupling of the Au nanoparticles. Using lysozyme as a model analyte for disease detection, the detection limit of the aptasensor is ～7 × 10³ aM, corresponding to the LSPR λ_max shift of ～2.25 nm. Up to a 380% increase in the localized resonant λ_max shift is demonstrated upon antibody binding to the analyte compared to the primary response during signal amplification using immunogold colloids. This enhancement leads to a limit of detection of ～7 aM, which is an improvement of three orders of magnitude. The results demonstrate substantial promise for developing coupled plasmonic nanostructures for ultrasensitive detection of various biological and chemical analytes.     

Erythrocyte–Platelet Hybrid Membrane Coating for Enhanced Nanoparticle Functionalization

Cell‐membrane‐coated nanoparticles have recently been studied extensively for their biological compatibility, retention of cellular properties, and adaptability to a variety of therapeutic and imaging applications. This class of nanoparticles, which has been fabricated with a variety of cell membrane coatings, including those derived from red blood cells (RBCs), platelets, white blood cells, cancer cells, and bacteria, exhibit properties that are characteristic of the source cell. In this study, a new type of biological coating is created by fusing membrane material from two different cells, providing a facile method for further enhancing nanoparticle functionality. As a proof of concept, the development of dual‐membrane‐coated nanoparticles from the fused RBC membrane and platelet membrane is demonstrated. The resulting particles, termed RBC–platelet hybrid membrane‐coated nanoparticles ([RBC‐P]NPs), are thoroughly characterized, and it is shown that they carry properties of both source cells. Further, the [RBC‐P]NP platform exhibits long circulation and suitability for further in vivo exploration. The reported strategy opens the door for the creation of biocompatible, custom‐tailored biomimetic nanoparticles with varying hybrid functionalities, which may be used to overcome the limitations of current nanoparticle‐based therapeutic and imaging platforms.