Two-Photon Intravital Microscopy of Glioblastoma in a Murine Model.

The delivery of intravenously administered cancer therapeutics to brain tumors is limited by the blood-brain barrier. A method to directly image the accumulation and distribution of macromolecules in brain tumors in vivo would greatly enhance our ability to understand and optimize drug delivery in preclinical models. This protocol describes a method for real-time in vivo tracking of intravenously administered fluorescent-labeled nanoparticles with two-photon intravital microscopy (2P-IVM) in a mouse model of glioblastoma (GBM). The protocol contains a multi-step description of the procedure, including anesthesia and analgesia of experimental animals, creating a cranial window, GBM cell implantation, placing a head bar, conducting 2P-IVM studies, and post-surgical care for long-term follow-up studies. We show representative 2P-IVM imaging sessions and image analysis, examine the advantages and disadvantages of this technology, and discuss potential applications. This method can be easily modified and adapted for different research questions in the field of in vivo preclinical brain imaging.

Nanopatterned Monolayers of Bioinspired, Sequence-Defined Polypeptoid Brushes for Semiconductor/Bio Interfaces.

The ability to control and manipulate semiconductor/bio interfaces is essential to enable biological nanofabrication pathways and bioelectronic devices. Traditional surface functionalization methods, such as self-assembled monolayers (SAMs), provide limited customization for these interfaces. Polymer brushes offer a wider range of chemistries, but choices that maintain compatibility with both lithographic patterning and biological systems are scarce. Here, we developed a class of bioinspired, sequence-defined polymers, i.e., polypeptoids, as tailored polymer brushes for surface modification of semiconductor substrates. Polypeptoids featuring a terminal hydroxyl (-OH) group are designed and synthesized for efficient melt grafting onto the native oxide layer of Si substrates, forming ultrathin (∼1 nm) monolayers. By programming monomer chemistry, our polypeptoid brush platform offers versatile surface modification, including adjustments to surface energy, passivation, preferential biomolecule attachment, and specific biomolecule binding. Importantly, the polypeptoid brush monolayers remain compatible with electron-beam lithographic patterning and retain their chemical characteristics even under harsh lithographic conditions. Electron-beam lithography is used over polypeptoid brushes to generate highly precise, binary nanoscale patterns with localized functionality for the selective immobilization (or passivation) of biomacromolecules, such as DNA origami or streptavidin, onto addressable arrays. This surface modification strategy with bioinspired, sequence-defined polypeptoid brushes enables monomer-level control over surface properties with a large parameter space of monomer chemistry and sequence and therefore is a highly versatile platform to precisely engineer semiconductor/bio interfaces for bioelectronics applications.

Author Correction: Information-based autonomous reconfiguration in systems of interacting DNA nanostructures.

The original version of this Article omitted a reference to previous work in 'Stojanovic, M. N. & Stefanovic, D. A deoxyribozyme-based molecular automaton. Nat. Biotechnol. 21, 1069-1074 (2003)'. This has been added as reference 42. The following has been added after the third sentence of the fifth paragraph of the Discussion: 'Integration could also allow more sophisticated information processing, for example as shown by the classic deoxyribozyme-based automaton that plays tic-tac-toe42, to direct structural reconfiguration (Supplementary Discussion)'. This has been corrected in the PDF and HTML versions of the Article.

Triangular DNA Origami Tilings.

DNA origami tilings provide methods for creating complex molecular patterns and shapes using flat DNA origami structures as building blocks. Square tiles have been developed to construct micrometer-scale arrays and to generate patterns using stochastic or deterministic strategies. Here we show triangular tiles as a complementary approach for enriching the design space of DNA tilings and for extending the shape of the self-assembled arrays from 2D to 3D. We introduce a computational approach for maximizing binding specificity in a fully symmetric tile design, with which we construct a 20-tile structure resembling a rhombic triacontahedron. We demonstrate controlled transition between 3D and 2D structures using simple methods including tile concentration, magnesium, and fold symmetry in tile edge design. Using these approaches, we construct 2D arrays with unbounded and designed sizes. The programmability of the edge design and the flexibility of the structure make the triangular DNA origami tile an ideal building block for complex self-assembly and reconfiguration in artificial molecular machines and fabricated nanodevices.

Information-based autonomous reconfiguration in systems of interacting DNA nanostructures.

The dynamic interactions between complex molecular structures underlie a wide range of sophisticated behaviors in biological systems. In building artificial molecular machines out of DNA, an outstanding challenge is to develop mechanisms that can control the kinetics of interacting DNA nanostructures and that can compose the interactions together to carry out system-level functions. Here we show a mechanism of DNA tile displacement that follows the principles of toehold binding and branch migration similar to DNA strand displacement, but occurs at a larger scale between interacting DNA origami structures. Utilizing this mechanism, we show controlled reaction kinetics over five orders of magnitude and programmed cascades of reactions in multi-structure systems. Furthermore, we demonstrate the generality of tile displacement for occurring at any location in an array in any order, illustrated as a tic-tac-toe game. Our results suggest that tile displacement is a simple-yet-powerful mechanism that opens up the possibility for complex structural components in artificial molecular machines to undergo information-based reconfiguration in response to their environments.

Fractal assembly of micrometre-scale DNA origami arrays with arbitrary patterns.

Self-assembled DNA nanostructures enable nanometre-precise patterning that can be used to create programmable molecular machines and arrays of functional materials. DNA origami is particularly versatile in this context because each DNA strand in the origami nanostructure occupies a unique position and can serve as a uniquely addressable pixel. However, the scale of such structures has been limited to about 0.05 square micrometres, hindering applications that demand a larger layout and integration with more conventional patterning methods. Hierarchical multistage assembly of simple sets of tiles can in principle overcome this limitation, but so far has not been sufficiently robust to enable successful implementation of larger structures using DNA origami tiles. Here we show that by using simple local assembly rules that are modified and applied recursively throughout a hierarchical, multistage assembly process, a small and constant set of unique DNA strands can be used to create DNA origami arrays of increasing size and with arbitrary patterns. We illustrate this method, which we term 'fractal assembly', by producing DNA origami arrays with sizes of up to 0.5 square micrometres and with up to 8,704 pixels, allowing us to render images such as the Mona Lisa and a rooster. We find that self-assembly of the tiles into arrays is unaffected by changes in surface patterns on the tiles, and that the yield of the fractal assembly process corresponds to about 0.95m - 1 for arrays containing m tiles. When used in conjunction with a software tool that we developed that converts an arbitrary pattern into DNA sequences and experimental protocols, our assembly method is readily accessible and will facilitate the construction of sophisticated materials and devices with sizes similar to that of a bacterium using DNA nanostructures.

Programmable disorder in random DNA tilings.

Scaling up the complexity and diversity of synthetic molecular structures will require strategies that exploit the inherent stochasticity of molecular systems in a controlled fashion. Here we demonstrate a framework for programming random DNA tilings and show how to control the properties of global patterns through simple, local rules. We constructed three general forms of planar network-random loops, mazes and trees-on the surface of self-assembled DNA origami arrays on the micrometre scale with nanometre resolution. Using simple molecular building blocks and robust experimental conditions, we demonstrate control of a wide range of properties of the random networks, including the branching rules, the growth directions, the proximity between adjacent networks and the size distribution. Much as combinatorial approaches for generating random one-dimensional chains of polymers have been used to revolutionize chemical synthesis and the selection of functional nucleic acids, our strategy extends these principles to random two-dimensional networks of molecules and creates new opportunities for fabricating more complex molecular devices that are organized by DNA nanostructures.

High Field Solid-State NMR Spectroscopy Investigation of (15)N-Labeled Rosette Nanotubes: Hydrogen Bond Network and Channel-Bound Water.

(15)N-labeled rosette nanotubes were synthesized and investigated using high-field solid-state NMR spectroscopy, X-ray diffraction, atomic force microscopy, and electron microscopy. The results established the H-bond network involved in the self-assembly of the nanostructure as well as bound water molecules in the nanotube's channel.

Caspase-responsive smart gadolinium-based contrast agent for magnetic resonance imaging of drug-induced apoptosis.

Non-invasive detection of caspase-3/7 activity in vivo has provided invaluable predictive information regarding tumor therapeutic efficacy and anti-tumor drug selection. Although a number of caspase-3/7 targeted fluorescence and positron emission tomography (PET) imaging probes have been developed, there is still a lack of gadolinium (Gd)-based magnetic resonance imaging (MRI) probes that enable high spatial resolution detection of caspase-3/7 activity in vivo. Here we employ a self-assembly approach and develop a caspase-3/7 activatable Gd-based MRI probe for monitoring tumor apoptosis in mice. Upon reduction and caspase-3/7 activation, the caspase-sensitive nano-aggregation MR probe (C-SNAM: 1) undergoes biocompatible intramolecular cyclization and subsequent self-assembly into Gd-nanoparticles (GdNPs). This results in enhanced r1 relaxivity-19.0 (post-activation) vs. 10.2 mM-1 s-1 (pre-activation) at 1 T in solution-and prolonged accumulation in chemotherapy-induced apoptotic cells and tumors that express active caspase-3/7. We demonstrate that C-SNAM reports caspase-3/7 activity by generating a significantly brighter T1-weighted MR signal compared to non-treated tumors following intravenous administration of C-SNAM, providing great potential for high-resolution imaging of tumor apoptosis in vivo.

Bioorthogonal cyclization-mediated in situ self-assembly of small-molecule probes for imaging caspase activity in vivo.

Directed self-assembly of small molecules in living systems could enable a myriad of applications in biology and medicine, and already this has been used widely to synthesize supramolecules and nano/microstructures in solution and in living cells. However, controlling the self-assembly of synthetic small molecules in living animals is challenging because of the complex and dynamic in vivo physiological environment. Here we employ an optimized first-order bioorthogonal cyclization reaction to control the self-assembly of a fluorescent small molecule, and demonstrate its in vivo applicability by imaging caspase-3/7 activity in human tumour xenograft mouse models of chemotherapy. The fluorescent nanoparticles assembled in situ were imaged successfully in both apoptotic cells and tumour tissues using three-dimensional structured illumination microscopy. This strategy combines the advantages offered by small molecules with those of nanomaterials and should find widespread use for non-invasive imaging of enzyme activity in vivo.

Development of novel tumor-targeted theranostic nanoparticles activated by membrane-type matrix metalloproteinases for combined cancer magnetic resonance imaging and therapy.

A major drawback with current cancer therapy is the prevalence of unrequired dose-limiting toxicity to non-cancerous tissues and organs, which is further compounded by a limited ability to rapidly and easily monitor drug delivery, pharmacodynamics and therapeutic response. In this report, the design and characterization of novel multifunctional "theranostic" nanoparticles (TNPs) is described for enzyme-specific drug activation at tumor sites and simultaneous in vivo magnetic resonance imaging (MRI) of drug delivery. TNPs are synthesized by conjugation of FDA-approved iron oxide nanoparticles ferumoxytol to an MMP-activatable peptide conjugate of azademethylcolchicine (ICT), creating CLIO-ICTs (TNPs). Significant cell death is observed in TNP-treated MMP-14 positive MMTV-PyMT breast cancer cells in vitro, but not MMP-14 negative fibroblasts or cells treated with ferumoxytol alone. Intravenous administration of TNPs to MMTV-PyMT tumor-bearing mice and subsequent MRI demonstrates significant tumor selective accumulation of the TNP, an observation confirmed by histopathology. Treatment with CLIO-ICTs induces a significant antitumor effect and tumor necrosis, a response not observed with ferumoxytol. Furthermore, no toxicity or cell death is observed in normal tissues following treatment with CLIO-ICTs, ICT, or ferumoxytol. These findings demonstrate proof of concept for a new nanotemplate that integrates tumor specificity, drug delivery and in vivo imaging into a single TNP entity through attachment of enzyme-activated prodrugs onto magnetic nanoparticles. This novel approach holds the potential to significantly improve targeted cancer therapies, and ultimately enable personalized therapy regimens.

Iron administration before stem cell harvest enables MR imaging tracking after transplantation.

PURPOSE: To determine whether intravenous ferumoxytol can be used to effectively label mesenchymal stem cells (MSCs) in vivo and can be used for tracking of stem cell transplants. MATERIALS AND METHODS: This study was approved by the institutional animal care and use committee. Sprague-Dawley rats (6-8 weeks old) were injected with ferumoxytol 48 hours prior to extraction of MSCs from bone marrow. Ferumoxytol uptake by these MSCs was evaluated with fluorescence, confocal, and electron microscopy and compared with results of traditional ex vivo-labeling procedures. The in vivo-labeled cells were subsequently transplanted in osteochondral defects of 14 knees of seven athymic rats and were evaluated with magnetic resonance (MR) imaging up to 4 weeks after transplantation. T2 relaxation times of in vivo-labeled MSC transplants and unlabeled control transplants were compared by using t tests. MR data were correlated with histopathologic results. RESULTS: In vivo-labeled MSCs demonstrated significantly higher ferumoxytol uptake compared with ex vivo-labeled cells. With electron microscopy, iron oxide nanoparticles were localized in secondary lysosomes. In vivo-labeled cells demonstrated significant T2 shortening effects in vitro and in vivo when they were compared with unlabeled control cells (T2 in vivo, 15.4 vs 24.4 msec; P < .05) and could be tracked in osteochondral defects for 4 weeks. Histologic examination confirmed the presence of iron in labeled transplants and defect remodeling. CONCLUSION: Intravenous ferumoxytol can be used to effectively label MSCs in vivo and can be used for tracking of stem cell transplants with MR imaging. This method eliminates risks of contamination and biologic alteration of MSCs associated with ex vivo-labeling procedures.

Activatable oligomerizable imaging agents for photoacoustic imaging of furin-like activity in living subjects.

Photoacoustic (PA) imaging is continuing to be applied for physiological imaging and more recently for molecular imaging of living subjects. Owing to its high spatial resolution in deep tissues, PA imaging holds great potential for biomedical applications and molecular diagnostics. There is however a lack of probes for targeted PA imaging, especially in the area of enzyme-activatable probes. Here we introduce a molecular probe, which upon proteolytic processing is retained at the site of enzyme activity and provides PA contrast. The probe oligomerizes via a condensation reaction and accumulates in cells and tumors that express the protease. We demonstrate that this probe reports furin and furin-like activity in cells and tumor models by generating a significantly higher PA signal relative to furin-deficient and nontarget controls. This probe could report enzyme activity in living subjects at depths significantly greater than fluorescence imaging probes and has potential for molecular imaging in deep tumors.

DNA-based programming of quantum dot valency, self-assembly and luminescence.

The electronic and optical properties of colloidal quantum dots, including the wavelengths of light that they can absorb and emit, depend on the size of the quantum dots. These properties have been exploited in a number of applications including optical detection, solar energy harvesting and biological research. Here, we report the self-assembly of quantum dot complexes using cadmium telluride nanocrystals capped with specific sequences of DNA. Quantum dots with between one and five DNA-based binding sites are synthesized and then used as building blocks to create a variety of rationally designed assemblies, including cross-shaped complexes containing three different types of dots. The structure of the complexes is confirmed with transmission electron microscopy, and photophysical studies are used to quantify energy transfer among the constituent components. Through changes in pH, the conformation of the complexes can also be reversibly switched, turning on and off the transfer of energy between the constituent quantum dots.

MRI of tumor-associated macrophages with clinically applicable iron oxide nanoparticles.

PURPOSE: The presence of tumor-associated macrophages (TAM) in breast cancer correlates strongly with poor outcome. The purpose of this study was to develop a clinically applicable, noninvasive diagnostic assay for selective targeting and visualization of TAMs in breast cancer, based on magnetic resonanceI and clinically applicable iron oxide nanoparticles. EXPERIMENTAL DESIGN: F4/80-negative mammary carcinoma cells and F4/80-positive TAMs were incubated with iron oxide nanoparticles and were compared with respect to magnetic resonance signal changes and iron uptake. MMTV-PyMT transgenic mice harboring mammary carcinomas underwent nanoparticle-enhanced magnetic resonance imaging (MRI) up to 1 hour and 24 hours after injection. The tumor enhancement on MRIs was correlated with the presence and location of TAMs and nanoparticles by confocal microscopy. RESULTS: In vitro studies revealed that iron oxide nanoparticles are preferentially phagocytosed by TAMs but not by malignant tumor cells. In vivo, all tumors showed an initial contrast agent perfusion on immediate postcontrast MRIs with gradual transendothelial leakage into the tumor interstitium. Twenty-four hours after injection, all tumors showed a persistent signal decline on MRIs. TAM depletion via αCSF1 monoclonal antibodies led to significant inhibition of tumor nanoparticle enhancement. Detection of iron using 3,3'-diaminobenzidine-enhanced Prussian Blue staining, combined with immunodetection of CD68, localized iron oxide nanoparticles to TAMs, showing that the signal effects on delayed MRIs were largely due to TAM-mediated uptake of contrast agent. CONCLUSION: These data indicate that tumor enhancement with clinically applicable iron oxide nanoparticles may serve as a new biomarker for long-term prognosis, related treatment decisions, and the evaluation of new immune-targeted therapies.

Nucleic acid-passivated semiconductor nanocrystals: biomolecular templating of form and function.

Bright, photostable luminescent labels are powerful tools for the in vitro and in vivo imaging of biological events. Semiconductor nanocrystals have emerged as attractive alternatives to commonly used organic lumophores because of their high quantum yields and the spectral tunability that can be achieved through synthetic control. Although conventional synthetic methods generally yield high-quality nanocrystals with excellent optical properties for biological imaging, ligand exchange and biological conjugation are necessary to make nanocrystals biocompatible and biospecific. These steps can substantially deteriorate the optical characteristics of these nanocrystals. Moreover, the complexity of multistep nanocrystal synthesis, typically requiring inert and anhydrous conditions, prohibits many end users of these lumiphores from generating their own custom materials. We sought to streamline semiconductor nanocrystal synthesis and develop synthetic routes that would be accessible to scientists from all disciplines. In search of such an approach, we turned to nucleic acids as a programmable and versatile ligand set and found that these biomolecules are indeed appropriate for biocompatible semiconductor nanocrystals preparation. In this Account, we summarize our work on nucleic acids-programmed nanocrystal synthesis that has resulted in the successful development of a one-step synthesis of biofunctionalized nanocrystals in aqueous solution. We first discuss results obtained with nucleotide-capped cadmium and lead chalcogenide-based nanocrystals that served to guide further investigation of polynucleotide-assisted synthesis. We investigated the roles of individual nucleobases and their structures in passivation of the surfaces of nanocrystals and modulating morphology and optical characteristics. The nucleic acid structures and sequences and the reaction conditions greatly influence the nanocrystals' optical properties and morphologies. Moreover, studies using live cells reveal low toxicity and rapid uptake of DNA-passivated CdS nanocrystals, demonstrating their suitability for bioimaging. Finally, we describe a new approach that leads to the production of biofunctionalized, DNA-capped nanocrystals in a single step. Chimeric DNA molecules enable this strategy, providing both a domain for nanocrystal passivation and a domain for biomolecule recognition. Nanocrystals synthesized using this approach possess good spectral characteristics as well as high specificity to cognate DNA, protein, and cancer cell targets. Overall, this approach could make nanocrystal lumiphores more readily accessible to researchers working in the biological sciences.

Synthesis of hydrophobic derivatives of the G/\C base for rosette nanotube self-assembly in apolar media.

Eleven self-complementary G/\C derivatives bearing hydrophobic moieties were synthesized and characterized. One representative derivative from this family was shown to self-assemble into rosette nanotubes in hexane and form Langmuir-Blodgett films at the air-water interface.

Hierarchical self-assembly of organic prolate nanospheroids from hydrophobic rosette nanotubes.

Supramolecular synthesis emerged recently as a new formalism to devise complex architectures held through noncovalent forces. Much of the research endeavor has been devoted to the use of H-bonds as the alphabet for chemical information encoding, and the structures expressed have spanned the range of dimensions and shapes, from discrete to infinite networks. Here we describe the synthesis and characterization of a GwedgeC base bearing two C12 alkyl chains, which undergoes a solvent-controlled multistep hierarchical self-assembly process into lamellar prolate nanospheroids. These assemblies were characterized by AFM, SEM, TEM, XRD, and SAXS, and a mechanism for their formation is proposed.