Photothermal-Enhanced Modulation of Cellular Membrane Potential Using Long-Wavelength-Activated Gold Nanoflowers.

In mammalian cells, plasma membrane potential plays vital roles in both physiology and pathology and it is controlled by a network of membrane-resident ion channels. There is considerable interest in the use of nanoparticles (NPs) to control biological functions, including the modulation of membrane potential. The photoexcitation of gold NPs (AuNPs) tethered close to the plasma membrane has been shown to induce membrane depolarization via localized heating of the AuNP surface coupled with the opening of voltage-gated sodium channels. Previous work has employed spherical AuNPs (AuNS) with absorption in the 500-600 nm range for this purpose. However, AuNP materials with absorption at longer wavelengths [e.g., near-infrared (NIR)] would enable greater tissue penetration depth in vivo. We show here the use of new anisotropic-shaped AuNPs [gold nanoflowers (AuNFs)] with broad absorption spanning into the NIR part of the spectrum (∼650-1000 nm). The AuNFs are directly synthesized with bidentate thiolate ligands, which preserves the AuNF's shape and colloidal stability, while facilitating conjugation to biomolecules. We describe the characterization of the AuNF particles and demonstrate that they adhere to the plasma membrane when bioconjugated to PEGylated cholesterol (PEG-Chol) moieties. The AuNF-PEG-Chol mediated the depolarization of rat adrenal medulla pheochromocytoma (PC-12) neuron-like cells more effectively than AuNS-PEG-Chol and unconjugated AuNS and AuNF when photoexcited at ∼561 or ∼640 nm. Importantly, AuNF induction of depolarization had no impact on cellular viability. This work demonstrates anisotropic AuNFs as an enabling nanomaterial for use in cellular depolarization and the spatiotemporal control of cellular activity.

Gold-Nanoparticle-Mediated Depolarization of Membrane Potential Is Dependent on Concentration and Tethering Distance from the Plasma Membrane.

The photoactivation of plasma-membrane-tethered gold nanoparticles (AuNPs) for the photothermally driven depolarization of membrane potential has recently emerged as a new platform for the controlled actuation of electrically active cells. In this report, we characterize the relationship between AuNP concentration and AuNP-membrane separation distance with the efficiency of photoactivated plasma membrane depolarization. We show in differentiated rat pheochromocytoma (PC-12) cells that AuNPs capped with poly(ethylene glycol) (PEG)-cholesterol ligands localize to the plasma membrane and remain resident for up to 1 h. The efficiency of AuNP-mediated depolarization is directly dependent on the concentration of the NPs on the cell surface. We further show that the efficiency of AuNP-mediated photothermal depolarization of membrane potential is directly dependent on the tethering distance between the AuNP and the plasma membrane, which we control by iteratively tuning the length of the PEG linker. Importantly, the AuNP conjugates do not adversely affect cell viability under the photoactivation conditions required for membrane depolarization. Our results demonstrate the fine control that can be elicited over AuNP bioconjugates and establishes principles for the rational design of functional nanomaterials for the control of electrically excitable cells.

Active Cellular and Subcellular Targeting of Nanoparticles for Drug Delivery.

Nanoparticle (NP)-mediated drug delivery (NMDD) for active targeting of diseases is a primary goal of nanomedicine. NPs have much to offer in overcoming the limitations of traditional drug delivery approaches, including off-target drug toxicity and the need for the administration of repetitive doses. In the last decade, one of the main foci in NMDD has been the realization of NP-mediated drug formulations for active targeted delivery to diseased tissues, with an emphasis on cellular and subcellular targeting. Advances on this front have included the intricate design of targeted NP-drug constructs to navigate through biological barriers, overcome multidrug resistance (MDR), decrease side effects, and improve overall drug efficacy. In this review, we survey advancements in NP-mediated drug targeting over the last five years, highlighting how various NP-drug constructs have been designed to achieve active targeted delivery and improved therapeutic outcomes for critical diseases including cancer, rheumatoid arthritis, and Alzheimer's disease. We conclude with a survey of the current clinical trial landscape for active targeted NP-drug delivery and how we envision this field will progress in the near future.

Nanoparticle-Peptide-Drug Bioconjugates for Unassisted Defeat of Multidrug Resistance in a Model Cancer Cell Line.

Multidrug resistance (MDR) is a significant challenge in the treatment of many types of cancers as membrane-associated transporters actively pump drugs out of the cell, limiting therapeutic efficacy. While nanoparticle (NP)-based therapeutics have emerged as a mechanism for overcoming MDR, they often rely on the delivery of multiple anticancer drugs, nucleic acid hybrids, or MDR pump inhibitors. The effectiveness of these strategies, however, can be limited by their off-target toxicity or the need for genetic transfection. In this paper, we describe a NP-peptide-drug bioconjugate that achieves significant cell killing in MDR-positive cancer cells without the need for additional drugs. We use a quantum dot (QD) as a central scaffold to append two species of peptide, a cell-uptake peptide to facilitate endocytic internalization and a peptide-drug conjugate that is susceptible to cleavage by esterases found within the endocytic pathway. This approach relies on spatiotemporal control over drug release, where endosomes traffic drug away from membrane-resident pumps and release it closer to the nucleus. Cellular internalization studies showed high uptake of the NP-drug complex and nuclear localization of the drug after 48 h in MDR-positive cells. Additionally, cellular proliferation assays demonstrated a 40% decrease in cell viability for the NP-drug bioconjugate compared to free drug, confirming the utility of this system in overcoming MDR in cancer cells.

Synthesis of a Reactive Oxygen Species-Responsive Doxorubicin Derivative.

A heterobifunctional reactive oxygen species (ROS)-responsive linker for directed drug assembly onto and delivery from a quantum dot (QD) nanoparticle carrier was synthesized and coupled to doxorubicin using N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC)/sulfo⁻NHS coupling. The doxorubicin conjugate was characterized using ¹H NMR and LC-MS and subsequently reacted under conditions of ROS formation (Cu2+/H2O2) resulting in successful and rapid thioacetal oxidative cleavage, which was monitored using ¹H NMR.

The role of nanoparticles in the improvement of systemic anticancer drug delivery.

The systemic delivery of drugs to the body via circulation after oral administration is a preferred method of drug administration during cancer treatment given its ease of implementation. However, the physicochemical properties of many current anticancer drugs limit their effectiveness when delivered by systemic routes. The use of nanoparticles (NPs) has emerged as an effective means of overcoming the inherent limitations of systemic drug delivery. We provide herein an overview of various NP formulations that facilitate improvements in the efficacy of various anticancer drugs compared with the free drug. This review will be useful to the reader who is interested in the role NP technology is playing in shaping the future of chemotherapeutic drug delivery and disease treatment.

Cellular delivery of doxorubicin mediated by disulfide reduction of a peptide-dendrimer bioconjugate.

In this study, we developed a peptide-dendrimer-drug conjugate system for the pH-triggered direct cytosolic delivery of the cancer chemotherapeutic doxorubicin (DOX) using the pH Low Insertion Peptide (pHLIP). We synthesized a pHLIP-dendrimer-DOX conjugate in which a single copy of pHLIP displayed a generation three dendrimer bearing multiple copies of DOX via disulfide linkages. Biophysical analysis showed that both the dendrimer and a single DOX conjugate inserted into membrane bilayers in a pH-dependent manner. Time-resolved confocal microscopy indicate the single DOX conjugate may undergo a faster rate of membrane translocation, due to greater nuclear localization of DOX at 24 h and 48 h post delivery. At 72 h, however, the levels of DOX nuclear accumulation for both constructs were identical. Cytotoxicity assays revealed that both constructs mediated ∼80% inhibition of cellular proliferation at 10 µM, the dendrimer complex exhibited a 17% greater cytotoxic effect at lower concentrations and greater than three-fold improvement in IC50 over free DOX. Our findings show proof of concept that the dendrimeric display of DOX on the pHLIP carrier (1) facilitates the pH-dependent and temporally-controlled release of DOX to the cytosol, (2) eliminates the endosomal sequestration of the drug cargo, and (3) augments DOX cytotoxicity relative to the free drug.

Intracellularly Actuated Quantum Dot-Peptide-Doxorubicin Nanobioconjugates for Controlled Drug Delivery via the Endocytic Pathway.

Nanoparticle (NP)-mediated drug delivery (NMDD) has emerged as a novel method to overcome the limitations of traditional systemic delivery of therapeutics, including the controlled release of the NP-associated drug cargo. Currently, our most advanced understanding of how to control NP-associated cargos is in the context of soft nanoparticles (e.g., liposomes), but less is known about controlling the release of cargos from the surface of hard NPs (e.g., gold NPs). Here we employ a semiconductor quantum dot (QD) as a prototypical hard NP platform and use intracellularly triggered actuation to achieve spatiotemporal control of drug release and modulation of drug efficacy. Conjugated to the QD are two peptides: (1) a cell-penetrating peptide (CPP) that facilitates uptake of the conjugate into the endocytic pathway and (2) a display peptide conjugated to doxorubicin (DOX) via three different linkages (ester, disulfide, and hydrazone) that are responsive to enzymatic cleavage, reducing conditions, and low pH, respectively. Formation of the QD-[peptide-DOX]-CPP complex is driven by self-assembly that allows control over both the ratio of each peptide species conjugated to the QD and the eventual drug dose delivered to cells. Förster resonance energy transfer assays confirmed successful assembly of the QD-peptide complexes and functionality of the linkages. Confocal microscopy was employed to visualize residence of the QD-[peptide-DOX]-CPP complexes in the endocytic pathway, and distinct differences in DOX localization were noted for the ester linkage, which showed clear signs of nuclear delivery versus the hydrazone, disulfide, and amide control. Finally, delivery of the QD-[peptide-DOX]-CPP conjugate resulted in cytotoxicity for the ester linkage that was comparable to free DOX. Attachment of DOX via the hydrazone linkage facilitated intermediary toxicity, while the disulfide and amide control linkages showed minimal toxicity. Our data demonstrate the utility of hard NP-peptide bioconjugates to function as multifunctional scaffolds for simultaneous control over cellular drug uptake and toxicity and the vital role played by the nature of the chemical linkage that appends the drug to the NP carrier.

Quantum Dot-Peptide-Fullerene Bioconjugates for Visualization of in Vitro and in Vivo Cellular Membrane Potential.

We report the development of a quantum dot (QD)-peptide-fullerene (C60) electron transfer (ET)-based nanobioconjugate for the visualization of membrane potential in living cells. The bioconjugate is composed of (1) a central QD electron donor, (2) a membrane-inserting peptidyl linker, and (3) a C60 electron acceptor. The photoexcited QD donor engages in ET with the C60 acceptor, resulting in quenching of QD photoluminescence (PL) that tracks positively with the number of C60 moieties arrayed around the QD. The nature of the QD-capping ligand also modulates the quenching efficiency; a neutral ligand coating facilitates greater QD quenching than a negatively charged carboxylated ligand. Steady-state photophysical characterization confirms an ET-driven process between the donor-acceptor pair. When introduced to cells, the amphiphilic QD-peptide-C60 bioconjugate labels the plasma membrane by insertion of the peptide-C60 portion into the hydrophobic bilayer, while the hydrophilic QD sits on the exofacial side of the membrane. Depolarization of cellular membrane potential augments the ET process, which is manifested as further quenching of QD PL. We demonstrate in HeLa cells, PC12 cells, and primary cortical neurons significant QD PL quenching (ΔF/F0 of 2-20% depending on the QD-C60 separation distance) in response to membrane depolarization with KCl. Further, we show the ability to use the QD-peptide-C60 probe in combination with conventional voltage-sensitive dyes (VSDs) for simultaneous two-channel imaging of membrane potential. In in vivo imaging of cortical electrical stimulation, the optical response of the optimal QD-peptide-C60 configuration exhibits temporal responsivity to electrical stimulation similar to that of VSDs. Notably, however, the QD-peptide-C60 construct displays 20- to 40-fold greater ΔF/F0 than VSDs. The tractable nature of the QD-peptide-C60 system offers the advantages of ease of assembly, large ΔF/F0, enhanced photostability, and high throughput without the need for complicated organic synthesis or genetic engineering, respectively, that is required of traditional VSDs and fluorescent protein constructs.

Quantum dot-mediated delivery of siRNA to inhibit sphingomyelinase activities in brain-derived cells.

The use of RNAi to suppress protein synthesis offers a potential way of reducing the level of enzymes or the synthesis of mutant toxic proteins but there are few tools currently available for their delivery. To address this problem, bioconjugated quantum dots (QDs) containing a hydrophobic component (N-palmitate) and a sequence VKIKK designed to traverse across cell membranes and visualize drug delivery were developed and tested on cell lines of brain origin. We used the Zn outer shell of the QD to bind HIS6 in JB577 (W•G•Dap(N-Palmitoyl)•VKIKK•P9 •G2 •H6 ) and by a gel-shift assay showed that siRNAs would bind to the positively charged KIKK sequence. By comparing many peptides and QD coatings, we showed that the QD-JB577-siRNA construct was taken up by cells of nervous system origin, distributed throughout the cytosol, and inhibited protein synthesis, implying that JB577 was also promoting endosome egress. By attaching siRNA for luciferase in a cell line over-expressing luciferase, we showed 70% inhibition of mRNA after 24-48 h. To show more specific effects, we synthesized siRNA for neutral (NSMase2), acid (lysosomal ASMase) sphingomyelinase, and sphingosine kinase 1 (SK1), we demonstrated a dose-dependent inhibition of activity. These data suggest that QDs are a useful siRNA delivery tool and QD-siRNA could be a potential theranostic for a variety of diseases.

Peptides for specifically targeting nanoparticles to cellular organelles: quo vadis?

The interfacing of nanomaterials and especially nanoparticles within all aspects of biological research continues to grow at a nearly unabated pace with projected applications focusing on powerful new tools for cellular labeling, imaging, and sensing, theranostic materials, and drug delivery. At the most fundamental level, many of these nanoparticles are meant to target not only very specific cell-types, regardless of whether they are in a culture, tissue, an animal model, or ultimately a patient, but also in many cases a specific subcellular organelle. During this process, these materials will undergo a complex journey that must first find the target cell of interest, then be taken up by those cells across the extracellular membrane, and ultimately localize to a desired subcellular organelle, which may include the nucleus, plasma membrane, endolysosomal system, mitochondria, cytosol, or endoplasmic reticulum. To accomplish these complex tasks in the correct sequence, researchers are increasingly interested in selecting for and exploiting targeting peptides that can impart the requisite capabilities to a given nanoparticle construct. There are also a number of related criteria that need careful consideration for this undertaking centering on the nature and properties of the peptide vector itself, the peptide-nanoparticle conjugate characteristics, and the target cell. Here, we highlight some important issues and key research areas related to this burgeoning field. We begin by providing a brief overview of some criteria for optimal attachment of peptides to nanoparticles, the predominant methods by which nanoparticles enter cells, and some of the peptide sequences that have been utilized to facilitate nanoparticle delivery to cells focusing on those that engender the initial targeting and uptake. Because almost all materials delivered to cells by peptides utilize the endosomal system of vesicular transport and in many cases remain sequestered within the vesicles, we critically evaluate the issue of endosomal escape in the context of some recently reported successes in this regard. Following from this, peptides that have been reported to deliver nanoparticles to specific subcellular compartments are examined with a focus on what they delivered and the putative mechanisms by which they were able to accomplish this. The last section focuses on two areas that are critical to realizing this overall approach in the long term. The first is how to select for peptidyl sequences capable of improved or more specific cellular or subcellular targeting based upon principles commonly associated with drug discovery. The second looks at what has been done to create modular peptides that incorporate multiple desirable functionalities within a single, contiguous sequence. This provides a viable alternative to either the almost insurmountable challenge of finding one sequence capable of all functions or, alternatively, attaching different peptides with different functionalities to the same nanoparticle in different ratios when trying to orchestrate their net effects. Finally, we conclude with a brief perspective on the future evolution and broader impact of this growing area of bionanoscience.

Delivery and tracking of quantum dot peptide bioconjugates in an intact developing avian brain.

Luminescent semiconductor ∼9.5 nm nanoparticles (quantum dots: QDs) have intrinsic physiochemical and optical properties which enable us to begin to understand the mechanisms of nanoparticle mediated chemical/drug delivery. Here, we demonstrate the ability of CdSe/ZnS core/shell QDs surface functionalized with a zwitterionic compact ligand to deliver a cell-penetrating lipopeptide to the developing chick embryo brain without any apparent toxicity. Functionalized QDs were conjugated to the palmitoylated peptide WGDap(Palmitoyl)VKIKKP9GGH6, previously shown to uniquely facilitate endosomal escape, and microinjected into the embryonic chick spinal cord canal at embryo day 4 (E4). We were subsequently able to follow the labeling of spinal cord extension into the ventricles, migratory neuroblasts, maturing brain cells, and complex structures such as the choroid plexus. QD intensity extended throughout the brain, and peaked between E8 and E11 when fluorescence was concentrated in the choroid plexus before declining to hatching (E21/P0). We observed no abnormalities in embryonic patterning or embryo survival, and mRNA in situ hybridization confirmed that, at key developmental stages, the expression pattern of genes associated with different brain cell types (brain lipid binding protein, Sox-2, proteolipid protein and Class III-ß-Tubulin) all showed a normal labeling pattern and intensity. Our findings suggest that we can use chemically modified QDs to identify and track neural stem cells as they migrate, that the choroid plexus clears these injected QDs/nanoparticles from the brain after E15, and that they can deliver drugs and peptides to the developing brain.

Multifunctional liquid crystal nanoparticles for intracellular fluorescent imaging and drug delivery.

A continuing goal of nanoparticle (NP)-mediated drug delivery (NMDD) is the simultaneous improvement of drug efficacy coupled with tracking of the intracellular fate of the nanoparticle delivery vehicle and its drug cargo. Here, we present a robust multifunctional liquid crystal NP (LCNP)-based delivery system that affords facile intracellular fate tracking coupled with the efficient delivery and modulation of the anticancer therapeutic doxorubicin (Dox), employed here as a model drug cargo. The LCNPs consist of (1) a liquid crystal cross-linking agent, (2) a homologue of the organic chromophore perylene, and (3) a polymerizable surfactant containing a carboxylate headgroup. The NP core provides an environment to both incorporate fluorescent dye for spectrally tuned particle tracking and encapsulation of amphiphilic and/or hydrophobic agents for intracellular delivery. The carboxylate head groups enable conjugation to biologicals to facilitate the cellular uptake of the particles. Upon functionalization of the NPs with transferrin, we show the ability to differentially label the recycling endocytic pathway in HEK 293T/17 cells in a time-resolved manner with minimal cytotoxicity and with superior dye photostability compared to traditional organic fluorophores. Further, when passively loaded with Dox, the NPs mediate the rapid uptake and subsequent sustained release of Dox from within endocytic vesicles. We demonstrate the ability of the LCNPs to simultaneously serve as both an efficient delivery vehicle for Dox as well as a modulator of the drug's cytotoxicity. Specifically, the delivery of Dox as a LCNP conjugate results in a ∼40-fold improvement in its IC50 compared to free Dox in solution. Cumulatively, our results demonstrate the utility of the LCNPs as an effective nanomaterial for simultaneous cellular imaging, tracking, and delivery of drug cargos.

Evaluation of diverse peptidyl motifs for cellular delivery of semiconductor quantum dots.

Cell-penetrating peptides (CPPs) have rapidly become a mainstay technology for facilitating the delivery of a wide variety of nanomaterials to cells and tissues. Currently, the library of CPPs to choose from is still limited, with the HIV TAT-derived motif still being the most used. Among the many materials routinely delivered by CPPs, nanoparticles are of particular interest for a plethora of labeling, imaging, sensing, diagnostic, and therapeutic applications. The development of nanoparticle-based technologies for many of these uses will require access to a much larger number of functional peptide motifs that can both facilitate cellular delivery of different types of nanoparticles to cells and be used interchangeably in the presence of other peptides and proteins on the same surface. Here, we evaluate the utility of four peptidyl motifs for their ability to facilitate delivery of luminescent semiconductor quantum dots (QDs) in a model cell culture system. We find that an LAH4 motif, derived from a membrane-inserting antimicrobial peptide, and a chimeric sequence that combines a sweet arrow peptide with a portion originating from the superoxide dismutase enzyme provide effective cellular delivery of QDs. Interestingly, a derivative of the latter sequence lacking just a methyl group was found to be quite inefficient, suggesting that even small changes can have significant functional outcomes. Delivery was effected using 1 h incubation with cells, and fluorescent counterstaining strongly suggests an endosomal uptake process that requires a critical minimum number or ratio of peptides to be displayed on the QD surface. Concomitant cytoviability testing showed that the QD-peptide conjugates are minimally cytotoxic in the model COS-1 cell line tested. Potential applications of these peptides in the context of cellular delivery of nanoparticles and a variety of other (bio)molecules are discussed.

Site-specific cellular delivery of quantum dots with chemoselectively-assembled modular peptides.

Modular peptides displaying both quantum dot bioconjugation motifs and specific subcellular targeting domains were constructed using a chemoselective aniline-catalyzed hydrazone coupling chemistry. Peptides were ratiometrically assembled onto quantum dots to facilitate their specific delivery to either the plasma membrane, endosomes, the cytosol or the mitochondria of target cells.

Optimizing protein coordination to quantum dots with designer peptidyl linkers.

Semiconductor quantum dots (QDs) demonstrate select optical properties that make them of particular use in biological imaging and biosensing. Controlled attachment of biomolecules such as proteins to the QD surface is thus critically necessary for development of these functional nanobiomaterials. QD surface coatings such as poly(ethylene glycol) impart colloidal stability to the QDs, making them usable in physiological environments, but can impede attachment of proteins due to steric interactions. While this problem is being partially addressed through the development of more compact QD ligands, here we present an alternative and complementary approach to this issue by engineering rigid peptidyl linkers that can be appended onto almost all expressed proteins. The linkers are specifically designed to extend a terminal polyhistidine sequence out from the globular protein structure and penetrate the QD ligand coating to enhance binding by metal-affinity driven coordination. α-Helical linkers of two lengths terminating in either a single or triple hexahistidine motif were fused onto a single-domain antibody; these were then self-assembled onto QDs to create a model immunosensor system targeted against the biothreat agent ricin. We utilized this system to systematically evaluate the peptidyl linker design in functional assays using QDs stabilized with four different types of coating ligands including poly(ethylene glycol). We show that increased linker length, but surprisingly not added histidines, can improve protein to QD attachment and sensor performance despite the surface ligand size with both custom and commercial QD preparations. Implications for these findings on the development of QD-based biosensors are discussed.

Quantitative LSPR imaging for biosensing with single nanostructure resolution.

Localized surface plasmon resonance (LSPR) imaging has the potential to map complex spatio-temporal variations in analyte concentration, such as those produced by protein secretions from live cells. A fundamental roadblock to the realization of such applications is the challenge of calibrating a nanoscale sensor for quantitative analysis. Here, we introduce a new, to our knowledge, LSPR imaging and analysis technique that enables the calibration of hundreds of individual gold nanostructures in parallel. The calibration allowed us to map the fractional occupancy of surface-bound receptors at individual nanostructures with nanomolar sensitivity and a temporal resolution of 225 ms. As a demonstration of the technique's applicability to molecular and cell biology, the calibrated array was used for the quantitative LSPR imaging of anti-c-myc antibodies harvested from a cultured 9E10 hybridoma cell line without the need for further purification or processing.

Active cellular sensing with quantum dots: transitioning from research tool to reality; a review.

The application of luminescent semiconductor quantum dots (QDs) within a wide range of biological imaging and sensing formats is now approaching its 15th year. The unique photophysical properties of these nanomaterials have long been envisioned as having the potential to revolutionize biosensing within cellular studies that rely on fluorescence. However, it is only now that these materials are making the transition towards accomplishing this goal. With the idea of understanding how to actively incorporate QDs into different types of cellular biosensing, we review the progress in many of the areas relevant to achieving this goal. This includes the synthesis of the QDs themselves, with an emphasis on minimizing potential toxicity, along with the general methods for making these nanocrystalline structures stable in aqueous media. We next survey some methods for conjugating QDs to biomolecules to allow them to participate in active biosensing. Lastly, we extensively review many of the applications where QDs have been demonstrated in an active role in cellular biosensing. These formats cover a wide range of possibilities including where the QDs have contributed to: monitoring the cell's interaction with its extracellular environment; elucidating the complex molecular interplay that characterizes the plasma membrane; understanding how cells continuously endocytose and exocytose materials across the cellular membrane; visualizing organelle trafficking; and, perhaps most importantly, monitoring the intracellular presence of target molecules such as nucleic acids, nutrients, cofactors, and ions or, alternatively, intracellular responses to external changes in the environment. We illustrate these processes with examples from the recent literature and focus on what QDs can uniquely contribute along with discussing the benefits and liabilities of each sensing strategy. A perspective on where this field is expected to develop in both the near and long-term is also provided.

Nanoparticle targeting to neurons in a rat hippocampal slice culture model.

We have previously shown that CdSe/ZnS core/shell luminescent semiconductor nanocrystals or QDs (quantum dots) coated with PEG [poly(ethylene glycol)]-appended DHLA (dihydrolipoic acid) can bind AcWG(Pal)VKIKKP(9)GGH(6) (Palm1) through the histidine residues. The coating on the QD provides colloidal stability and this peptide complex uniquely allows the QDs to be taken up by cultured cells and readily exit the endosome into the soma. We now show that use of a polyampholyte coating [in which the neutral PEG is replaced by the negatively heterocharged CL4 (compact ligand)], results in the specific targeting of the palmitoylated peptide to neurons in mature rat hippocampal slice cultures. There was no noticeable uptake by astrocytes, oligodendrocytes or microglia (identified by immunocytochemistry), demonstrating neuronal specificity to the overall negatively charged CL4 coating. In addition, EM (electron microscopy) images confirm the endosomal egress ability of the Palm1 peptide by showing a much more disperse cytosolic distribution of the CL4 QDs conjugated to Palm1 compared with CL4 QDs alone. This suggests a novel and robust way of delivering neurotherapeutics to neurons.