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Boron neutron capture therapy (BNCT) is an encouraging therapeutic modality for cancer treatment. Prostate-specific membrane antigen (PSMA) is a cell membrane protein that is abundantly overexpressed in prostate cancer and can be targeted with radioligand therapies to stimulate clinical responses in patients. In principle, a spatially targeted neutron beam together with specifically targeted PSMA ligands could enable prostate cancer-targeted BNCT. Thus, we developed and tested PSMA-targeted poly(lactide

Multifunctional magnetic nanoparticles have shown great promise as next-generation imaging and perturbation probes for deciphering molecular and cellular processes. As a consequence of multicomponent integration into a single nanosystem, pre-existing nanoprobes are typically large and show limited access to biological targets present in a crowded microenvironment. Here, we apply organic-phase surface PEGylation, click chemistry, and charge-based valency discrimination principles to develop compact, modular, and monovalent magnetofluorescent nanoparticles (MFNs). We show that MFNs exhibit highly efficient labeling to target receptors present in cells with a dense and thick glycocalyx layer. We use these MFNs to interrogate the E-cadherin-mediated adherens junction formation and F-actin polymerization in a three-dimensional space, demonstrating the utility as modular and versatile mechanogenetic probes in the most demanding single-cell perturbation applications.

The ability to sense and manipulate the state of biological systems has been extensively advanced during the last decade with the help of recent developments in physical tools. Unlike standard genetic and pharmacological perturbation techniques – knockdown, overexpression, small molecule inhibition – that provide a basic on/off switching capability, these physical tools provide the capacity to control the spatial, temporal, and mechanical properties of the biological targets. Among the various physical cues, magnetism offers distinct advantages over light or electricity. Magnetic fields freely penetrate biological tissues and are already used for clinical applications. As one of the unique features, magnetic fields can be transformed into mechanical stimuli which can serve as a cue in regulating biological processes. However, their biological applications have been limited due to a lack of high-performance magnetism-to-mechanical force transducers with advanced spatiotemporal capabilities. In this account, we present recent developments in magnetic nano-tweezers (MNTs) as a high-performance tool for interrogating the spatiotemporal control of cells in living tissue. MNTs are comprised of force-generating magnetic nanoparticles and field generators. Through proper design and the integration of individual components, MNTs deliver controlled mechanical stimulation to targeted biomolecules at any desired space and time. We first discuss about MNT configuration with different force-stimulation modes. By modulating geometry of the magnetic field generator, MNTs exert pulling, dipole-dipole attraction, and rotational forces to the target specifically and quantitatively. We discuss the key physical parameters determining force magnitude, which include magnetic field strength, magnetic field gradient, magnetic moment of the magnetic particle, as well as distance between the field generator and the particle. MNTs also can be used over a wide range of biological time scales — By simply adjusting the amplitude and phase of the applied current, MNTs based on electromagnets allow for dynamic control of the magnetic field from microseconds to hours. Chemical design and the nanoscale effects of magnetic particles are also essential for optimizing MNT performance. We discuss key strategies to develop magnetic nanoparticles with improved force-generation capabilities with a particular focus on the effects of size, shape, and composition of the nanoparticles. We then introduce various strategies and design considerations for target-specific biomechanical stimulations with MNTs. One-to-one particle-receptor engagement for delivering a defined force to the targeted receptor and the small size of the nanoparticles are important. Finally, we demonstrate the utility of MNTs for manipulating biological functions and activities with various spatial (single molecule/cell to organisms) and temporal resolution (microseconds to days). MNTs have the potential to be utilized in many exciting applications across diverse biological systems spanning from fundamental biology investigations of spatial and mechanical signaling dynamics at the single-cell and systems levels to in vivo therapeutic applications.

Spatiotemporal interrogation of signal transduction at the single-cell level is necessary to answer a host of important biological questions. This protocol describes a nanotechnology-based single-cell and single-molecule perturbation tool, termed mechanogenetics, that enables precise spatial and mechanical control over genetically encoded cell-surface receptors in live cells. The key components of this tool are a magnetoplasmonic nanoparticle (MPN) actuator that delivers defined spatial and mechanical cues to receptors through target-specific one-to-one engagement and a micromagnetic tweezers (μMT) that remotely controls the magnitude of force exerted on a single MPN. In our approach, a SNAP-tagged cell-surface receptor of interest is conjugated with a single-stranded DNA oligonucleotide, which hybridizes to its complementary oligonucleotide on the MPN. This protocol consists of four major stages: (i) chemical synthesis of MPNs, (ii) conjugation with DNA and purification of monovalent MPNs, (iii) modular targeting of MPNs to cell-surface receptors, and (iv) control of spatial and mechanical properties of targeted mechanosensitive receptors in live cells by adjusting the μMT-to-MPN distance. Using benzylguanine (BG)-functionalized MPNs and model cell lines expressing either SNAP-tagged Notch or vascular endothelial cadherin (VE-cadherin), we provide stepwise instructions for mechanogenetic control of receptor clustering and for mechanical receptor activation. The ability of this method to differentially control spatial and mechanical inputs to targeted receptors makes it particularly useful for interrogating the differential contributions of each individual cue to cell signaling. The entire procedure takes up to 1 week.

Tools capable of imaging and perturbing mechanical signaling pathways with fine spatiotemporal resolution have been elusive, despite their importance in diverse cellular processes. The challenge in developing a mechanogenetic toolkit (i.e., selective and quantitative activation of genetically encoded mechanoreceptors) stems from the fact that many mechanically activated processes are localized in space and time yet additionally require mechanical loading to become activated. To address this challenge, we synthesized magnetoplasmonic nanoparticles that can image, localize, and mechanically load targeted proteins with high spatiotemporal resolution. We demonstrate their utility by investigating the cell-surface activation of two mechanoreceptors: Notch and E-cadherin. By measuring cellular responses to a spectrum of spatial, chemical, temporal, and mechanical inputs at the single-molecule and single-cell levels, we reveal how spatial segregation and mechanical force cooperate to direct receptor activation dynamics. This generalizable technique can be used to control and understand diverse mechanosensitive processes in cell signaling. VIDEO ABSTRACT.

Caspases are proteases involved in cell death, where caspase-3 is the chief executioner that produces an irreversible cutting event in downstream protein substrates and whose activity is desired in the management of cancer. To determine such activity in clinically relevant samples with high signal-to-noise, plasmon rulers are ideal because they are sensitively affected by their interparticle separation without ambiguity from photobleaching or blinking effects. A plasmon ruler is a noble metal nanoparticle pair, tethered in close proximity to one another via a biomolecule, that acts through dipole–dipole interactions and results in the light scattering to increase exponentially. In contrast, a sharp decrease in intensity is observed when the pair is confronted by a large interparticle distance. To align the mechanism of protease activity with building a sensor that can report a binary signal in the presence or absence of caspase-3, we present a caspase-3 selective plasmon ruler (C3SPR) composed of a pair of Zn0.4Fe2.6O4@SiO2@Au core–shell nanoparticles connected by a caspase-3 cleavage sequence. The dielectric core (Zn0.4Fe2.6O4@SiO2)-shell (Au) geometry provided a brighter scattering intensity versus solid Au nanoparticles, and the magnetic core additionally acted as a purification handle during the plasmon ruler assembly. By monitoring the decrease in light scattering intensity per plasmon ruler, we detected caspase-3 activity at single molecule resolution across a broad dynamic range. This was observed to be as low as 100 fM of recombinant material or 10 ng of total protein from cellular lysate. By thorough analyses of single molecule trajectories, we show caspase-3 activation in a drug-treated chronic myeloid leukemia (K562) cancer system as early as 4 and 8 h with greater sensitivity (2- and 4-fold, respectively) than conventional reagents. This study provides future implications for monitoring caspase-3 as a biomarker and efficacy of drugs.

The clinical experience with BCR–ABL tyrosine kinase inhibitors (TKI) for the treatment of chronic myelogenous leukemia (CML) provides compelling evidence for oncogene addiction. Yet, the molecular basis of oncogene addiction remains elusive. Through unbiased quantitative phosphoproteomic analyses of CML cells transiently exposed to BCR–ABL TKI, we identified persistent downregulation of growth factor receptor (GF-R) signaling pathways. We then established and validated a tissue-relevant isogenic model of BCR–ABL-mediated addiction, and found evidence for myeloid GF-R signaling pathway rewiring that profoundly and persistently dampens physiologic pathway activation. We demonstrate that eventual restoration of ligand-mediated GF-R pathway activation is insufficient to fully rescue cells from a competing apoptotic fate. In contrast to previous work with BRAFV600E in melanoma cells, feedback inhibition following BCR–ABL TKI treatment is markedly prolonged, extending beyond the time required to initiate apoptosis. Mechanistically, BCR–ABL-mediated oncogene addiction is facilitated by persistent high levels of MAP–ERK kinase (MEK)-dependent negative feedback. Significance: We found that BCR–ABL can confer addiction in vitro by rewiring myeloid GF-R signaling through establishment of MEK-dependent negative feedback. Our findings predict that deeper, more durable responses to targeted agents across a range of malignancies may be facilitated by maintaining negative feedback concurrently with oncoprotein inhibition. Cancer Discov; 4(2); 200–15. ©2013 AACR. This article is highlighted in the In This Issue feature, p. 131

The use of plasmon coupling in metal nanoparticles has shown great potential for the optical characterization of many biological processes. Recently, we have demonstrated the use of “plasmon rulers” to observe conformational changes of single biomolecules in vitro. Plasmon rulers provide robust signals without photobleaching or blinking. Here, we show the first application of plasmon rulers to in vivo studies to observe very long trajectories of single biomolecules in live cells. We present a unique type of plasmon ruler comprised of peptide-linked gold nanoparticle satellites around a core particle, which was used as a probe to optically follow cell-signaling pathways in vivo at the single-molecule level. These “crown nanoparticle plasmon rulers” allowed us to continuously monitor trajectories of caspase-3 activity in live cells for over 2 h, providing sufficient time to observe early-stage caspase-3 activation, which was not possible by conventional ensemble analyses.

Successful development of ultra-sensitive molecular imaging nanoprobes for the detection of targeted biological objects is a challenging task. Although magnetic nanoprobes have the potential to perform such a role, the results from probes that are currently available have been far from optimal. Here we used artificial engineering approaches to develop innovative magnetic nanoprobes, through a process that involved the systematic evaluation of the magnetic spin, size and type of spinel metal ferrites. These magnetism-engineered iron oxide (MEIO) nanoprobes, when conjugated with antibodies, showed enhanced magnetic resonance imaging (MRI) sensitivity for the detection of cancer markers compared with probes currently available. Also, we successfully visualized small tumors implanted in a mouse. Such high-performance, nanotechnology-based molecular probes could enhance the ability to visualize other biological events critical to diagnostics and therapeutics.

The unique properties of magnetic nanocrystals provide them with high potential as key probes and vectors in the next generation of biomedical applications. Although superparamagnetic iron oxide nanocrystals have been extensively studied as excellent magnetic resonance imaging (MRI) probes for various cell trafficking, gene expression, and cancer diagnosis, further development of in vivo MRI applications has been very limited. Here, we describe in vivo diagnosis of cancer, utilizing a well-defined magnetic nanocrystal probe system with multiple capabilities, such as small size, strong magnetism, high biocompatibility, and the possession of active functionality for desired receptors. Our magnetic nanocrystals are conjugated to a cancer-targeting antibody, Herceptin, and subsequent utilization of these conjugates as MRI probes has been successfully demonstrated for the monitoring of in vivo selective targeting events of human cancer cells implanted in live mice. Further conjugation of these nanocrystal probes with fluorescent dye-labeled antibodies enables both in vitro and ex vivo optical detection of cancer as well as in vivo MRI, which are potentially applicable for an advanced multimodal detection system. Our study finds that high performance in vivo MR diagnosis of cancer is achievable by utilizing improved and multifunctional material properties of iron oxide nanocrystal probes.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

Apoptotic caspases execute programmed cell death, where low levels of caspase activity are linked to cancer (KasibhatlaTseng, 2003). Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process; however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. To overcome this, we describe a procedure to develop peptide-linked gold nanoparticles that have unique optical properties and can serve as beacons to visualize the apoptotic drug response in cancer cells at the single-molecule level. By thorough analyses of their trajectories, one can reveal early-stage caspase-3 activation in live cells continuously and with no ambiguity.

Precise control over interfacial chemistry between nanoparticles and other materials remains a significant challenge limiting the broad application of nanotechnology in biology. To address this challenge, we use “Steric Exclusion” to completely convert commercial quantum dots (QDs) into monovalent imaging probes by wrapping the QD with a functionalized oligonucleotide. We demonstrate the utility of these QDs as modular and non-perturbing imaging probes by tracking individual Notch receptors on live cells.

Synthetic magnetic nanoparticles (MNPs) are emerging as versatile probes in biomedical applications, especially in the area of magnetic resonance imaging (MRI). Their size, which is comparable to biological functional units, and their unique magnetic properties allow their utilization as molecular imaging probes. Herein, we present an overview of recent breakthroughs in the development of new synthetic MNP probes with which the sensitive and target-specific observation of biological events at the molecular and cellular levels is possible.

Magnetic nanoparticles exhibit unique nanoscale properties of superparamagnetism and have the potential to be utilized as excellent probes for magnetic resonance imaging (MRI). Especially, clinically benign iron oxide nanoparticles provide good MR probing capability and some of them are currently available for clinical applications. However, limited magnetic property and inability to escape from reticuloendothelial system (RES) of the currently used nanoparticles impede their further advancements and therefore it is necessary to develop advanced magnetic nanoparticle probes for next-generation molecular MR imaging. In this chapter, we overview recent progresses on the development of magnetic nanoparticle probes for molecular MR imaging. Utilization of these nanoparticle probes for both in vitro and in vivo molecular MR imaging will be described.

High-quality biocompatible magnetic iron oxide (Fe3O4) nanocrystals were developed through a ligand exchange process of hydrophobically capped nanocrystals with hydrophilic molecules. By simple modulation of the nanocrystal surface ligand charge properties, we have been able to prepare magnetic nanocrystals with excellent intracellular labeling capabilities that efficiently label a variety of cell types without the need for additional transport facilitating agents. The excellent intracellular labeling capability of the newly developed cationic WSIO has further led to successful MRI monitoring of the migration of neural stem cells in rat spinal cord. The magnetic nanocrystals developed here have great potential in applications for labeling of various cell types and also the monitoring of cell-based medical treatments and cancer metastasis.

Since the use of magnetic nanocrystals as probes for biomedical system is attractive, it is important to develop optimal synthetic protocols for high-quality magnetic nanocrystals and to have the systematic understanding of their nanoscale properties. Here we present the development of a synthetically controlled magnetic nanocrystal model system that correlates the nanoscale tunabilities in terms of size, magnetism, and induced nuclear spin relaxation processes. This system further led to the development of high-performance nanocrystal-antibody probe systems for the diagnosis of breast cancer cells via magnetic resonance imaging.