Stimuli-responsive nanocarriers for intracellular delivery

The emergence of different nanoparticles (NPs) has made a significant revolution in the field of medicine. Different NPs in the form of metallic NPs, dendrimers, polymeric NPs, carbon quantum dots and liposomes have been functionalized and used as platforms for intracellular delivery of biomolecules, drugs, imaging agents and nucleic acids. These NPs are designed to improve the pharmacokinetic properties of the drug, improve their bioavailability and successfully surpass physiological or pathological obstacles in the biological system so that therapeutic efficacy is achieved. In this review I present some of the current approaches used in intracellular delivery systems, with a focus on various stimuli-responsive nanocarriers, including cell-penetrating peptides, to highlight their various biomedical applications.     

Nanogel-quantum dot hybrid nanoparticles for live cell imaging

We report here a novel carrier of quantum dots (QDs) for intracellular labeling. Monodisperse hybrid nanoparticles (38 nm in diameter) of QDs were prepared by simple mixing with nanogels of cholesterol-bearing pullulan (CHP) modified with amino groups (CHPNH2). The CHPNH2-QD nanoparticles were effectively internalized into the various human cells examined. The efficiency of cellular uptake was much higher than that of a conventional carrier, cationic liposome. These hybrid nanoparticles could be a promising fluorescent probe for bioimaging.     

Quantum dot-doped silica nanoparticles as probes for targeting of T-lymphocytes

To enhance diagnostic or therapeutic efficacy, novel nanomaterials must be engineered to function in biologically relevant environments, be visible by conventional fluorescent microscopy, and have multivalent loading capacity for easy detection or effective drug delivery. Here we report the fabrication of silica nanoparticles doped with quantum dots and superficially functionalized with amino and phosphonate groups. The amino groups were acylated with a water-soluble biotin-labeling reagent. The biotinylated nanoparticles were subsequently decorated with neutravidin by exploiting the strong affinity between neutravidin and biotin. The resultant neutravidin-decorated fluorescent silica nanoparticles stably dispersed under physiological conditions, were visible by conventional optical and confocal fluorescent microscopy, and could be further functionalized with macromolecules, nucleic acids, and polymers. We also coated the surface of the nanoparticles with biotinylated mouse anti-human CD3 (alphaCD3). The resultant fluorescent nanoassembly was taken up by Jurkat T cells through receptor-mediated endocytosis and was partially released to lysosomes. Thus, quantum dot-doped silica nanoparticles decorated with neutravidin represent a potentially excellent scaffold for constructing specific intracellular nanoprobes and transporters.     

Neurotransmitter Specific,Cellular-Resolution Functional Brain Mapping Using Receptor Coated Nanoparticles: Assessment of the Possibility

Receptor coated resonant nanoparticles and quantum dots are proposed to provide a cellular-level resolution image of neural activities inside the brain. The functionalized nanoparticles and quantum dots in this approach will selectively bind to different neurotransmitters in the extra-synaptic regions of neurons. This allows us to detect neural activities in real time by monitoring the nanoparticles and quantum dots optically. Gold nanoparticles (GNPs) with two different geometries (sphere and rod) and quantum dots (QDs) with different sizes were studied along with three different neurotransmitters: dopamine, gamma-Aminobutyric acid (GABA), and glycine. The absorption/emission spectra of GNPs and QDs before and after binding of neurotransmitters and their corresponding receptors are reported. The results using QDs and nanorods with diameter 25nm and aspect rations larger than three were promising for the development of the proposed functional brain mapping approach.     

New opportunities: the use of nanotechnologies to manipulate and track stem cells

Nanotechnologies are emerging platforms that could be useful in measuring, understanding, and manipulating stem cells. Examples include magnetic nanoparticles and quantum dots for stem cell labeling and in vivo tracking; nanoparticles, carbon nanotubes, and polyplexes for the intracellular delivery of genes/oligonucleotides and protein/peptides; and engineered nanometer-scale scaffolds for stem cell differentiation and transplantation. This review examines the use of nanotechnologies for stem cell tracking, differentiation, and transplantation. We further discuss their utility and the potential concerns regarding their cytotoxicity.     

Artificial cell membrane-covered nanoparticles embedding quantum dots as stable and highly sensitive fluorescence bioimaging probes

To obtain a stable and highly sensitive bioimaging fluorescence probe, polymer nanoparticles with embedded quantum dots were covered with an artificial cell membrane. These nanoparticles were designed by assembling phospholipid polar groups as a platform, and oligopeptide was immobilized as a bioaffinity moiety on the surface of the nanoparticles. The polymer nanoparticles showed resistance to cellular uptake from HeLa cells owing to the nature of the phosphorylcholine groups. When arginine octapeptide was immobilized at the surface of the nanoparticles, they were able to penetrate the membrane of HeLa cells effectively. Cytotoxicity of the nanoparticles was not observed even after immobilization of oligopeptide. Thus, we obtained stable fluorescent polymer nanoparticles covered with an artificial cell membrane, which are useful as an excellent bioimaging probe and as a novel evaluation tool for oligopeptide functions in the target cells.     

Single particle tracking confirms that multivalent Tat protein transduction domain-induced heparan sulfate proteoglycan cross-linkage activates Rac1 for internalization

The mechanism by which HIV-1-Tat protein transduction domain (TatP) enters the cell remains unclear because of an insufficient understanding of the initial kinetics of peptide entry. Here, we report the successful visualization and tracking of TatP molecular kinetics on the cell surface with 7-nm spatial precision using quantum dots. Strong cell binding was only observed with a TatP valence of ≥8, whereas monovalent TatP binding was negligible. The requirement of the cell-surface heparan sulfate (HS) chains of HS proteoglycans (HSPGs) for TatP binding and intracellular transport was demonstrated by the enzymatic removal of HS and simultaneous observation of two individual particles. Multivalent TatP induces HSPG cross-linking, recruiting activated Rac1 to adjacent lipid rafts and thereby enhancing the recruitment of TatP/HSPG to actin-associated microdomains and its internalization by macropinocytosis. These findings clarify the initial binding mechanism of TatP to the cell surface and demonstrate the importance of TatP valence for strong surface binding and signal transduction. Our data also shed light on the ability of TatP to exploit the machinery of living cells, using HSPG signaling to activate Rac1 and alter TatP mobility and internalization. This work should guide the future design of TatP-based peptides as therapeutic nanocarriers with efficient transduction.     

Site-specific sonoporation of human melanoma cells at the cellular level using high lateral-resolution ultrasonic micro-transducer arrays

We developed a new instrumental method by which human melanoma cells (LU1205) are sonoporated via radiation pressures exerted by highly-confined ultrasonic waves produced by high lateral-resolution ultrasonic micro-transducer arrays (UMTAs). The method enables cellular-level site-specific sonoporation within the cell monolayer due to UMTAs and can be applicable in the delivery of drugs and gene products in cellular assays. In this method, cells are seeded on the biochip that employs UMTAs for high spatial resolution and specificity. UMTAs are driven by 30-MHz sinusoidal signals and the resulting radiation pressures induce sonoporation in the targeted cells. The sonoporation degree and the effective lateral resolution of UMTAs are determined by performing fluorescent microscopy and analysis of carboxylic-acid-derivatized CdSe/ZnS quantum dots passively transported into the cells. Models representing the transducer-generated ultrasound radiation pressure, the ultrasound-inflicted cell membrane wound, and the transmembrane transport through the wound are developed to determine the ultrasound-pressure-dependent wound size and enhanced cellular uptake of nanoparticles. Model-based calculations show that the effective wound size and cellular uptake of nanoparticles increase linearly with increasing ultrasound pressure (i.e., at applied radiation pressures of 0.21, 0.29, and 0.40 MPa, the ultrasound-induced initial effective wound radii are 150, 460, and 650 nm, respectively, and the post-sonoporation intracellular quantum-dot concentrations are 7.8, 22.8, and 29.9 nM, respectively) and the threshold pressure required to induce sonoporation in LU1205 cells is ～0.12 MPa.     

Quantum dots find their stride in single molecule tracking

Thirteen years after the demonstration of quantum dots as biological imaging agents, and nine years after the initial commercial introduction of bioconjugated quantum dots, the brightness and photostability of the quantum dots has enabled a range of investigations using single molecule tracking. These materials are being routinely utilized by a number of groups to track the dynamics of single molecules in reconstituted biophysical systems and on living cells, and are especially powerful for investigations of single molecules over long timescales with short exposure times and high pointing accuracy. New approaches are emerging where the quantum dots are used as 'hard-sphere' probes for intracellular compartments. Innovations in quantum dot surface modification are poised to substantially expand the utility of these materials.     

PEGylation of nanoparticles improves their cytoplasmic transport

The efficacy of nucleus-targeted drug- or gene-carrying nanoparticles may be limited by slow transport through the molecularly crowded cytoplasm following endosome escape. Cytoskeletal elements and cellular organelles may pose steric and/or adhesive obstacles to the efficient intracellular transport of nanoparticles. To potentially reduce adhesive interactions of colloids with intracellular components, the surface of model nanoparticles was coated with polyethylene glycol (PEG). Subsequently, multiple-particle tracking (MPT) was used to quantify the cytoplasmic transport rates of particles microinjected into the cytoplasm of live cells. PEGylation increased average nanoparticle diffusivities by 100% compared to unPEGylated particles (time scale of 10 s) in live cells. Faster particle transport correlated with a marked decrease in the number of particles that underwent hindered transport, from 79.2% (unmodified) to 48.8% (PEGylated). This result adds to an impressive list of positive benefits associated with PEGylation of drug and gene delivery vectors.     

Reaching out for signals: filopodia sense EGF and respond by directed retrograde transport of activated receptors

ErbB1 receptors situated on cellular filopodia undergo systematic retrograde transport after binding of the epidermal growth factor (EGF) and activation of the receptor tyrosine kinase. Specific inhibitors of the erbB1 receptor tyrosine kinase as well as cytochalasin D, a disruptor of the actin cytoskeleton, abolish transport but not free diffusion of the receptor-ligand complex. Diffusion constants and transport rates were determined with single molecule sensitivity by tracking receptors labeled with EGF conjugated to fluorescent quantum dots. Retrograde transport precedes receptor endocytosis, which occurs at the base of the filopodia. Initiation of transport requires the interaction and concerted activation of at least two liganded receptors and proceeds at a constant rate mediated by association with actin. These findings suggest a mechanism by which filopodia detect the presence and concentration of effector molecules far from the cell body and mediate cellular responses via directed transport of activated receptors.     

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.     

Simultaneous detection of multiple mutations in epidermal growth factor receptor based on fluorescence quenching of quantum dots

We have developed a simultaneous detection method for two common mutations in the epidermal growth factor receptor gene based on the fluorescence quenching phenomenon caused by aggregation of CdSe quantum dots. For detection of the in-frame deletion in exon 19 and the L858R point mutation in exon 21, water-soluble CdSe quantum dots with two sizes were functionalized using four different types of probe oligonucleotides. Addition of target oligonucleotides with the deletion mutation in exon 19 into the suspensions caused crosslinking-induced aggregation of green-emitting quantum dots, followed by the fluorescence quenching while that with the L858R point mutation resulted in aggregation of yellow-emitting quantum dots. In addition, targets with both deletion and point mutations caused aggregation of both green- and yellow-emitting quantum dots. This method allows a simultaneous detection of mutations in exon 19 and 21 of EGFR gene in a single experiment. We found that minimum mutant concentration that could be detected by this method was as low as 2% for deletion mutation, and 5% for point mutation. PCR products of EGFR gene were also used to confirm that our method could be used to detect mutation in amplified DNA fragments.     

Nondestructive quantum dot-based intracellular serotonin imaging in intact cells

In recent years, quantum dots (Qdot), with their unique physical, chemical, and optical properties, have been used extensively as probes to visualize several cell membrane receptors and extracellular biomolecules. However, Qdot-based intracellular imaging has always been associated with vital lacunas. High affinity between quantum dots may induce serious aggregation in the cytoplasm; as a result, quantum dot aggregates are usually misinterpreted as quantum dot-probed intracellular molecules. Moreover, due to the more viscous nature of the cytoplasm versus the extracellular aqueous media, aggregation issues become more severe during intracellular studies. In this work, we suggest direct nondestructive serotonin imaging in an intact cell using the quantum dot-based immunoassay with a rapid tunable multicolor imaging system based on the acousto-optic tunable filter. Any false-positive intracellular serotonin molecules that appeared due to the aggregation of quantum dots could be completely discriminated from the real intracellular serotonin granules through multicolor cellular imaging. The developed method is quick and has wide applicability in targeting various intracellular proteins, coenzymes, and micronutrients.     

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

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

Plasmonic Tuning of Photoluminescence from Semiconducting Quantum Dot Assemblies

We report tuning of photoluminescence enhancement and quenching from closed packed monolayers of cadmium selenide quantum dots doped with gold nanoparticles. Plasmon-mediated control of the emission intensity from the monolayers is achieved by varying the size and packing density of the quantum dots as well as the doping concentration of gold nanoparticles. We observe a unique packing density dependent crossover from enhancement to quenching and vice versa for fixed size of quantum dots and doping concentration of gold nanoparticles. We suggest that this behavior is indicative of a crossover from single particle to collective emission from quantum dots mediated by gold nanoparticles.     

Mortalin imaging in normal and cancer cells with quantum dot immuno-conjugates

Quantum dots are the nanoparticles that are recently emerging as an alternative to organic fluorescence probes in cell biology and biomedicine, and have several predictive advantages. These include their ⑴broad absorption spectra allowing visualization with single light source, ⑵exceptional photo-stability allowing long term studies and ⑶narrow and symmetrical emission spectrum that is controlled by their size and material composition. These unique properties allow simultaneous excitation of different size of quantum dots with a single excitation light source, their simultaneous resolution and visualization as different colors. At present there are only a few studies that have tested quantum dots in cellular imaging. We describe here the use of quantum dots in mortalin imaging of normal and cancer cells. Mortalin staining pattern with quantum dots in both normal and cancer cells mimicked those obtained with organic florescence probes and were considerably stable.     

The emergence of multiple particle tracking in intracellular trafficking of nanomedicines

A growing number of nanoparticle systems, termed “nanomedicines”, are being developed for diagnostic and therapeutic applications. Nanoparticles can employ various cellular entry pathways and trafficking mechanisms to effectively deliver drugs, biomolecules, and imaging agents to precise sub-cellular locations. However, the dynamic transport of nanoparticles through the complex intracellular environment is not well understood, having been primarily studied with static or bulk averaged methods in the past. Such techniques do not provide detailed information regarding the transport mechanism and rates of individual nanoparticles, where understanding of the interaction of nanoparticles with the cellular environment remains incomplete. Recent advances in live-cell fluorescence microscopy and real-time multiple particle tracking (MPT) have facilitated an improved understanding of cell trafficking pathways. Understanding the dynamic transport of nanoparticles as they are delivered into complex cellular components may lead to rational improvements in the design of nanomedicines. This review discusses different cellular uptake and trafficking pathways of nanomedicines, briefly highlights current fluorescence microscopy tools, and provides examples from the recent literature on the use of MPT and its applications.     

纳米药物递送系统在前列腺癌治疗中的应用

前列腺癌（PCa）是全球最常见的男性泌尿生殖系统恶性肿瘤。手术、内分泌治疗、放疗和化疗是PCa的主要临床治疗选择。纳米药物递送系统具有良好的可控释放特性和较好的肿瘤靶向能力，并可通过增强的渗透性和保留（EPR）效应被动靶向肿瘤。通过精巧的设计组装和外表修饰赋予纳米递药系统与众不同的肿瘤治疗效果。本文介绍用于PCa治疗的先进纳米药物递送系统以及未来发展。     

Optimization of conditions for cadmium selenide quantum dot biosynthesis in Saccharomyces cerevisiae

The biosynthesis of quantum dots has been explored as an alternative to traditional physicochemical methods; however, relatively few studies have determined optimal synthesis parameters. Saccharomyces cerevisiae sequentially treated with sodium selenite and cadmium chloride synthesized CdSe quantum dots in the cytoplasm. These nanoparticles displayed a prominent yellow fluorescence, with an emission maximum of approximately 540 nm. The requirement for glutathione in the biosynthetic mechanism was explored by depleting its intracellular content through cellular treatments with 1-chloro-2,4-dinitrobenzene and buthionine sulfoximine. Synthesis was significantly inhibited by both of these reagents when they were applied after selenite treatment prior to the addition of cadmium, thereby indicating that glutathione contributes to the biosynthetic process. Determining the optimum conditions for biosynthesis revealed that quantum dots were produced most efficiently at entry into stationary phase followed by direct addition of 1 mM selenite for only 6 h and then immediately incubating these cells in fresh growth medium containing 3 mM Cd (II). Synthesis of quantum dots reached a maximum at 84 h of reaction time. Biosynthesis of 800-μg g⁻¹ fresh weight cells was achieved. For the first time, significant efforts have been undertaken to optimize each aspect of the CdSe biosynthetic procedure in S. cerevisiae, resulting in a 70% increased production.