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Effects of the Adenosine A1 blockade using 8-cyclopentyl-1,3-diprophyxanthine (DPCPX) nanoconjugate on inducing recovery of the hemidiaphragm paralyzed by hemisection have been thoroughly examined previously; however, the toxicology of DPCPX nanoconjugate remains unknown. This research study investigates the therapeutic efficacy and toxicology of the nanoconjugate DPCPX in the cervical spinal cord injury (SCI) rat model. We hypothesized that a single injection of nanoconjugate DPCPX in the paralyzed left hemidiaphragm (LDH) of hemisected rats at the 2nd cervical segment (C2Hx) would lead to the long-term recovery of LDH while showing minimal toxicity. Adult male rats underwent left C2Hx surgery and the diaphragms' baseline electromyography (EMG). Subsequently, rats were randomized into a control group and four treated subgroups. Three subgroups received a single intradiaphragmatic dose of either 0.09, 0.15, or 0.27 µg/kg, and one subgroup received 0.1 mg/kg of native DPCPX two times per day intravenously (i.v.) for 3 days (total 0.6 mg/kg). Rats were monitored for a total of 56 days. Compared with control, the treatment with nanoconjugate DPCPX at 0.09 µg/kg, 0.15 µg/kg, and 0.27 µg/kg doses elicited significant recovery of paralyzed LDH (i.e., 67% recovery at 8 wk) (

Paclitaxel (PTX) and 5-fluorouracil (5-FU) are clinically relevant chemotherapeutics, but both suffer a range of biopharmaceutical challenges (e.g., either low solubility or permeability and limited controlled release from nanocarriers), which reduces their effectiveness in new medicines. Anticancer drugs have several major limitations, which include non-specificity, wide biological distribution, a short half-life, and systemic toxicity. Here, we investigate the potential of liposome-micelle-hybrid (LMH) carriers (i.e., drug-loaded micelles encapsulated within drug-loaded liposomes) to enhance the co-formulation and delivery of PTX and 5-FU, facilitating new delivery opportunities with enhanced chemotherapeutic performance. We focus on the combination of liposomes and micelles for co-delivery of PTX and 5_FU to investigate increased drug loading, improved solubility, and transport/permeability to enhance chemotherapeutic potential. Furthermore, combination chemotherapy (i.e., containing two or more drugs in a single formulation) may offer improved pharmacological performance. Compared with individual liposome and micelle formulations, the optimized PTX-5FU-LMH carriers demonstrated increased drug loading and solubility, temperature-sensitive release, enhanced permeability in a Caco-2 cell monolayer model, and cancer cell eradication. LMH has significant potential for cancer drug delivery and as a next-generation chemotherapeutic.

The triple-negative breast cancer (TNBC) subtype, regardless of their BRCA1 status, has the poorest outcome compared with other breast cancer subtypes, and currently there are no approved targeted therapies for TNBC. We have previously demonstrated the importance of RAD6-mediated translesion synthesis pathway in TNBC development/progression and chemoresistance, and the potential therapeutic benefit of targeting RAD6 with a RAD6-selective small-molecule inhibitor, SMI#9. To overcome SMI#9 solubility limitations, we recently developed a gold nanoparticle (GNP)-based platform for conjugation and intracellular release of SMI#9, and demonstrated its in vitro cytotoxic activity toward TNBC cells. Here, we characterized the in vivo pharmacokinetic and therapeutic properties of PEGylated GNP-conjugated SMI#9 in BRCA1 wild-type and BRCA1-mutant TNBC xenograft models, and investigated the impact of RAD6 inhibition on TNBC metabolism by 1H-NMR spectroscopy. GNP conjugation allowed the released SMI#9 to achieve higher systemic exposure and longer retention as compared with the unconjugated drug. Systemically administered SMI#9-GNP inhibited the TNBC growth as effectively as intratumorally injected unconjugated SMI#9. Inductively coupled mass spectrometry analysis showed highest GNP concentrations in tumors and liver of SMI#9-GNP and blank-GNP–treated mice; however, tumor growth inhibition occurred only in the SMI#9-GNP–treated group. SMI#9-GNP was tolerated without overt signs of toxicity. SMI#9-induced sensitization was associated with perturbation of a common set of glycolytic pathways in BRCA1 wild-type and BRCA1-mutant TNBC cells. These data reveal novel SMI#9 sensitive markers of metabolic vulnerability for TNBC management and suggest that nanotherapy-mediated RAD6 inhibition offers a promising strategy for TNBC treatment.

Contains Materials, Method of metabolite extraction and 1H-NMR analysis. Supplementary Table S1 shows size measurements of GNPs. Supplementary Table S2 shows summary of metabolic pathway analysis in SUM1315 triple negative breast cancer cells. Supplementary Table S3 shows summary of metabolic pathway analysis in MDA-MB-231 triple negative breast cancer cells. Supplementary Figure S1 shows PCA loading plots of SUM1315 and MDA-MB-231 cells treated with blank-GNP or SMI#9-GNP. Supplementary Figure S2 shows PLS-DA plots of SUM1315 and MDA-MB-231 cells treated with blank-GNP or SMI#9-GNP.

The development of functional nanocoatings using natural compounds is a hallmark of sustainable strategies in the field of green synthesis. Herein, we report a surface-independent nanocoating strategy using natural polyphenols and gallium-based room temperature liquid metal nanoparticles. The nanocoating matrix is composed of tannic acid, crosslinked with group (IV) transition metal ions. Liquid gallium nanoparticles are incorporated into the coatings as a gallium ion releasing depot. The coating deposition is rapid and can be applied to a range of substrates including glass, plastics, paper, and metal surfaces, owing to the versatile adhesive nature of the catechol/gallol functional groups of tannic acid. The coating thickness can be controlled from 100 to 700 nm and the content of liquid gallium nanoparticles can be modulated. This enables the tunable release behaviour of gallium ions into the surrounding from the composite coatings. The coatings are highly biocompatible and display antioxidant and antibacterial properties that can be useful for diverse applications.

Recent advances in technology are expected to increase our current understanding of neuroscience. Nanotechnology and nanomaterials can alter and control neural functionality in both

Respiratory dysfunction is a major cause of death in people with spinal cord injury (SCI). A remaining unsolved problem in treating SCI is the intolerable side effects of the drugs to patients. In a significant departure from conventional targeted nanotherapeutics to overcome the blood-brain barrier (BBB), this work pursues a drug-delivery approach that uses neural tracing retrograde transport proteins to bypass the BBB and deliver an adenosine A1 receptor antagonist drug, 1,3-dipropyl-8-cyclopentyl xanthine, exclusively to the respiratory motoneurons in the spinal cord and the brainstem. A single intradiaphragmatic injection at one thousandth of the native drug dosage induces prolonged respiratory recovery in a hemisection animal model. To translate the discovery into new treatments for respiratory dysfunction, we carry out this study to characterize the purity and quality of synthesis, stability, and drug-release properties of the neural tracing protein (wheat germ agglutinin chemically conjugated to horseradish peroxidase)-coupled nanoconjugate. We show that the batch-to-batch particle size and drug dosage variations are less than 10%. We evaluate the nanoconjugate size against the spatial constraints imposed by transsynaptic transport from pre to postsynaptic neurons. We determine that the nanoconjugate formulation is capable of sustained drug release lasting for days at physiologic pH, a prerequisite for long-distance transport of the drug from the diaphragm muscle to the brainstem. We model the drug-release profiles using a first-order reaction model and the Noyes-Whitney diffusion model. We confirm via biological electron microscopy that the nanoconjugate particles do not accumulate in the tissues at the injection site. We define the nanoconjugate storage conditions after monitoring the solution dispersion stability under various conditions for 4 months. This study supports further development of neural tracing protein-enabled nanotherapeutics for treating respiratory problems associated with SCI.

Canonical Wnt signaling is critical for melanocyte lineage commitment and melanoma development. RAD6B, a ubiquitin-conjugating enzyme critical for translesion DNA synthesis, potentiates β-catenin stability/activity by inducing proteasome-insensitive polyubiquitination. RAD6B expression is induced by β-catenin, triggering a positive feedback loop between the two proteins. RAD6B function in melanoma development/progression was investigated by targeting RAD6B using CrispR/Cas9 or an RAD6-selective small-molecule inhibitor #9 (SMI#9). SMI#9 treatment inhibited melanoma cell proliferation but not normal melanocytes. RAD6B knockout or inhibition in metastatic melanoma cells downregulated β-catenin, β-catenin–regulated microphthalmia-associated transcription factor (MITF), sex-determining region Y-box 10, vimentin proteins, and MITF-regulated melan A. RAD6B knockout or inhibition decreased migration/invasion, tumor growth, and lung metastasis. RNA-sequencing and stem cell pathway real-time RT-PCR analysis revealed profound reductions in WNT1 expressions in RAD6B knockout M14 cells compared with control. Expression levels of β-catenin–regulated genes VIM, MITF-M, melan A, and TYRP1 (a tyrosinase family member critical for melanin biosynthesis) were reduced in RAD6B knockout cells. Pathway analysis identified gene networks regulating stem cell pluripotency, Wnt signaling, melanocyte development, pigmentation signaling, and protein ubiquitination, besides DNA damage response signaling, as being impacted by RAD6B gene disruption. These data reveal an important and early role for RAD6B in melanoma development besides its bonafide translesion DNA synthesis function, and suggest that targeting RAD6B may provide a novel strategy to treat melanomas with dysregulated canonical Wnt signaling.

Gold nanoparticles (AuNPs) have been shown to enhance cancer radiotherapy (RT) gain by localizing the absorption of radiation energy in the tumor while sparing surrounding normal tissue from radiation toxicity. Previously, we showed that AuNPs enhanced RT induced DNA damage and cytotoxicity in MCF7 breast cancer cells. Interestingly, we found that cancer cells exhibited a size-dependent AuNPs intracellular localization (4 nm preferentially in the cytoplasm and 14 nm in the nucleus). We extended those studies to an in vivo model and examined the AuNPs effects on RT cytotoxicity, survival and immunomodulation of tumor microenvironment (TME) in human triple negative breast cancer (TNBC) xenograft mouse model. We also explored the significance of nanoparticle size in these AuNPs’ effects. Mice treated with RT and RT plus 4 nm or 14 nm AuNPs showed a significant tumor growth delay, compared to untreated animals, while dual RT plus AuNPs treatment exhibited additive effect compared to either RT or AuNPs treatment alone. Survival log-rank test showed significant RT enhancement with 14 nm AuNP alone; however, 4 nm AuNPs did not exhibit RT enhancement. Both sizes of AuNPs enhanced RT induced immunogenic cell death (ICD) that was coupled with significant macrophage infiltration in mice pretreated with 14 nm AuNPs. These results showing significant AuNP size-dependent RT enhancement, as evident by both tumor growth delay and overall survival, reveal additional underlying immunological mechanisms and provide a platform for studying RT multimodal approaches for TNBC that may be combined with immunotherapies, enhancing their effect.

Molybdenum dioxide (MoO2), considering its near-metallic conductivity and surface plasmonic properties, is a great material for electronics, energy storage devices and biosensing. Yet to this day, room-temperature synthesis of large area MoO2, which allows deposition on arbitrary substrates, has remained a challenge. Due to their reactive interfaces and specific solubility conditions, gallium-based liquid metal alloys offer unique opportunities for synthesizing materials that can meet these challenges. Herein, a substrate-independent liquid metal-based method for the room temperature deposition and patterning of MoO2 is presented. By introducing a molybdate precursor to the surrounding of a eutectic gallium-indium alloy droplet, a uniform layer of hydrated molybdenum oxide (H2MoO3) is formed at the interface. This layer is then exfoliated and transferred onto a desired substrate. Utilizing the transferred H2MoO3 layer, a laser-writing technique is developed which selectively transforms this H2MoO3 into crystalline MoO2 and produces electrically conductive MoO2 patterns at room temperature. The electrical conductivity and plasmonic properties of the MoO2 are analyzed and demonstrated. The presented metal oxide room-temperature deposition and patterning method can find many applications in optoelectronics, sensing, and energy industries.

Atmospheric NO2 is of great concern due to its adverse effects on human health and the environment, motivating research on NO2 detection and remediation. Existing low-cost room-temperature NO2 sensors often suffer from low sensitivity at the ppb level or long recovery times, reflecting the trade-off between sensor response and recovery time. Here, we report an atomically dispersed metal ion strategy to address it. We discover that bimetallic PbCdSe quantum dot (QD) gels containing atomically dispersed Pb ionic sites achieve the optimal combination of strong sensor response and fast recovery, leading to a high-performance room-temperature p-type semiconductor NO2 sensor as characterized by a combination of ultra–low limit of detection, high sensitivity and stability, fast response and recovery. With the help of theoretical calculations, we reveal the high performance of the PbCdSe QD gel arises from the unique tuning effects of Pb ionic sites on NO2 binding at their neighboring Cd sites. Quantum dot-based NO2 sensors often suffer from low sensitivity or long recovery times. Here, the authors report that bimetallic PbCdSe quantum dot gels containing atomically dispersed Pb ionic sites enable ultra-sensitive and fast NO2 sensing.

Atmospheric NO2 is of great concern due to its adverse effects on human health and the environment, motivating research on NO2 detection and remediation. Existing low-cost room-temperature NO2 sensors often suffer from low sensitivity at the ppb level or long recovery times, reflecting the trade-off between sensor response and recovery time. Here, we report an atomically dispersed metal ion strategy to address it. We discover that bimetallic PbCdSe quantum dot (QD) gels containing atomically dispersed Pb ionic sites achieve the optimal combination of strong sensor response and fast recovery, leading to a high-performance room-temperature p-type semiconductor NO2 sensor as characterized by a combination of ultra–low limit of detection, high sensitivity and stability, fast response and recovery. With the help of theoretical calculations, we reveal the high performance of the PbCdSe QD gel arises from the unique tuning effects of Pb ionic sites on NO2 binding at their neighboring Cd sites., Quantum dot-based NO2 sensors often suffer from low sensitivity or long recovery times. Here, the authors report that bimetallic PbCdSe quantum dot gels containing atomically dispersed Pb ionic sites enable ultra-sensitive and fast NO2 sensing.

The triple-negative breast cancer (TNBC) subtype, regardless of their BRCA1 status, has the poorest outcome compared with other breast cancer subtypes, and currently there are no approved targeted therapies for TNBC. We have previously demonstrated the importance of RAD6-mediated translesion synthesis pathway in TNBC development/progression and chemoresistance, and the potential therapeutic benefit of targeting RAD6 with a RAD6-selective small-molecule inhibitor, SMI#9. To overcome SMI#9 solubility limitations, we recently developed a gold nanoparticle (GNP)-based platform for conjugation and intracellular release of SMI#9, and demonstrated its in vitro cytotoxic activity toward TNBC cells. Here, we characterized the in vivo pharmacokinetic and therapeutic properties of PEGylated GNP-conjugated SMI#9 in BRCA1 wild-type and BRCA1-mutant TNBC xenograft models, and investigated the impact of RAD6 inhibition on TNBC metabolism by 1H-NMR spectroscopy. GNP conjugation allowed the released SMI#9 to achieve higher systemic exposure and longer retention as compared with the unconjugated drug. Systemically administered SMI#9-GNP inhibited the TNBC growth as effectively as intratumorally injected unconjugated SMI#9. Inductively coupled mass spectrometry analysis showed highest GNP concentrations in tumors and liver of SMI#9-GNP and blank-GNP–treated mice; however, tumor growth inhibition occurred only in the SMI#9-GNP–treated group. SMI#9-GNP was tolerated without overt signs of toxicity. SMI#9-induced sensitization was associated with perturbation of a common set of glycolytic pathways in BRCA1 wild-type and BRCA1-mutant TNBC cells. These data reveal novel SMI#9 sensitive markers of metabolic vulnerability for TNBC management and suggest that nanotherapy-mediated RAD6 inhibition offers a promising strategy for TNBC treatment.

Crystallization is at the heart of many industrial processes in pharmaceuticals, dyes and pigments, microelectronics, and emerging wearable sensors. This paper reviews nucleation and early-stage crystal growth activated by an electrical pulse at microelectrodes and nanoelectrodes. We review thermodynamic and kinetic theories of electrochemistry developed around microelectrodes. We describe various methods to make microelectrodes and nanoelectrodes. Fundamental understanding is still needed for predicting and controlling nucleation and early-stage crystal growth. Using nanoelectrodes, nucleation and growth kinetics can be studied on one nucleation site at a time. In contrast, on macroelectrodes, nanoparticles are nucleated at random sites and at different times. This gives rise to overlapping growth zones resulting in inhomogeneous particle deposition and growth. The random size and density distributions prevent electrodeposition from being widely adopted as a manufacturing tool for making nanodevices. We describe advances in electrodeposition of metal nanoparticles and organic charge-transfer complexes on micro/nanoelectrodes. We anticipate increased interests in applying electrochemistry for making nanodevices particularly nanosensors and nanosensor arrays. These electrochemically fabricated nanosensor arrays will in turn fulfill the promise of nanoelectrodes as the most advanced analytical tools for medical diagnostics, environmental monitoring, and renewable energy.

The stripe phase formed by long-chain alkane derivatives on the graphite lattice provides a unique opportunity for the study of molecular adsorption, aggregation, and reaction on patterns. Fatty acids, such as arachidic acid (AA), self-assemble on graphite into a sheet of parallel stripes with a periodicity of twice its molecular chain length. The molecular pattern is thus defined precisely by the size and functionality of the headgroup and tailgroup of the amphiphile. Complexation of metal ions to AA fixes the number and location of the ion, which can serve as a precursor to semiconductor nanocrystal arrays. In order to understand the effect of the ion complexation, we carry out atomic force microscopy (AFM) and Fourier transform infrared spectroscopy (FTIR) investigations of AA self-assembly in the presence of various metal ions. While the stripe orientation is dictated by the graphite lattice and the stripe periodicity is determined by the AA chain length, the size, shape, and degree of order of the stripe crystalline domain are influenced by the metal ion bond strength to the carboxylic ligand. The change of morphology in the self-assembled pattern shows a trend along the Irving-Williams series.

A wide range of cellular activities including protein folding and cell secretion, such as neurotransmission or insulin release, are all governed by intracellular pH homeostasis, underscoring the importance of pH on critical life processes. Nano- scale pH measurements of cells and biomolecules therefore hold great promise in understanding a plethora of cellular functions, in addition to disease detection and therapy. In the current study, a novel approach using cadmium telluride quantum dots (CdTeQDs) as pH sensors, combined with fluorescent imaging, spectrofluorimetry, atomic force microscopy (AFM), and Western blot analysis, enabled the study of intracellular pH dynamics at 1 milli-pH sensitivity and 80nm pixel resolution, during insulin secretion. Additionally, the pH-dependent interaction between membrane fusion proteins, also called the soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE), was determined. Glucose stimulation of CdTeQD-loaded insulin secreting Min-6 mouse insulinoma cell line demonstrated the initial (5-6min) intracellular acidification reflected as a loss in QD fluorescence, followed by alkalization and a return to resting pH in 10min. Analysis of the SNARE complex in insulin secreting Min-6 cells demonstrated an initial gain followed by loss of complexed SNAREs in 10min. Stabilization of the SNARE complex at low intracellular pH is further supported by results from studies utilizing both native and AFM measurements of liposome-reconstituted recombinant neuronal SNAREs, providing a molecular understanding of the role of pH during cell secretion.

Targeting the development of stimulus-responsive molecular materials with electronic functionality, we have synthesized and studied the redox and electronic properties of a new bimetallic iron hydrophobe [FeIII2(LN4O6)] (1). The new H6LN4O6 ligand displays bicompartmental topology capable of accomodating two five-coordinate HSFeIII ions bridged by tetraaminobenzene at a close distance of ca. 8 A. We show that the metal-based reduction processes in (1) proceed sequentially, as observed for electronically coupled metal centers. This species forms a well-defined Pockels-Langmuir film at the air–water interface, with collapse pressure of 32 mN m−1. Langmuir–Blodgett monolayers were deposited on gold substrates and used to investigate current–voltage (I–V) measurements. This unprecedented bimetallic hydrophobe [FeIII2(LN4O6)] (1) shows unquestionable molecular rectification and displays a rectification ratio RR between 2 and 15.

This paper describes the synthesis of a new type of nanowires, potassium tetracyanoplatinate sesquihydrate Krogmann salt or K(def)TCP. Seed-mediated electrocrystallization nucleates and confines the K(def)TCP crystals into nanowires. Our data show that the width of K(def)TCP scales proportionally with the diameter of the seed, which is consistent with the nanoconfinement effect of the seed. K(def)TCP nanowires are also electrodeposited on lithographic Au patterns as interconnects and tested for impedance-based gas sensing. The results demonstrate the use of electrocrystallization to construct nanowire sensing units directly on patterned substrates to enable scaling up of nanowire sensor technology. IMPACT STATEMENT We show for the first time nanowires directly formed as interconnects on electronic substrates by electrocrystallization are used as sensor materials to detect gases.

In the past 50 years, isolated blood platelets have had restricted use in wound healing, cancer therapy, and organ and tissue transplant, to name a few. The major obstacle for its unrestricted use has been, among others, the presence of ultrahigh concentrations of growth factors and the presence of both pro-angiogenic and anti-angiogenic proteins. To overcome this problem requires the isolation and separation of the membrane bound secretory vesicles containing the different factors. In the current study, high-resolution imaging of isolated secretory vesicles from human platelets using atomic force microscopy (AFM) and mass spectrometry enabled characterization of the remaining vesicles size and composition following their immunoseparation. The remaining vesicles obtained following osmotic lysis, when subjected to immunoseparation employing antibody to different vesicle-associated membrane proteins (VAMPs), demonstrate for the first time that VAMP-3-, VAMP-7-, and VAMP-8-specific vesicles each possesses distinct size range and composition. These results provide a window into our understanding of the heterogeneous population of vesicles in human platelets and their stability following both physical manipulation using AFM and osmotic lysis of the platelet. This study further provides a platform for isolation and the detailed characterization of platelet granules, with promise for their future use in therapy. Additionally, results from the study demonstrate that secretory vesicles of different size found in cells reflect their unique and specialized composition and function.

Respiratory complications in patients with spinal cord injury (SCI) are common and can have a negative impact on the quality of patients' lives. Previously, we found that intradiaphragmatic administration of the nanoconjugate-bound A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) induced recovery of diaphragm function following SCI in rats. When administered immediately following the injury, recovery was observed as early as 3days following SCI and it persisted until the end of the study, 28days after the drug delivery. The recovery was observed using diaphragmatic electromyography (EMG) as well as phrenic nerve recordings; both of which were conducted under anesthetized conditions. Confounding effects of anesthetic may make data interpretation complex in terms of the impact on overall ventilatory function and clinical relevance. The objective of the present study was to test the hypothesis that intradiaphragmatic administration of nanoconjugate-bound DPCPX, enhances recovery of ventilation following SCI in the unanesthetized rat. To that end, Sprague-Dawley rats underwent C2 spinal cord hemisection (C2Hx) on day 0 and received either: (i) 0.15μg/kg of nanoconjugate-bound DPCPX or (ii) vehicle control (50μl distilled water). To assess ventilation, unrestrained whole body plethysmography (WBP) was performed on day 0 (immediately before the surgery) and 3, 7, 14, 21 and 28days following the SCI. Frequency, tidal volume, and minute ventilation data were analyzed in two minute bins while the animal was calm and awake. We found that a single administration of the nanoconjugate-bound A1 adenosine receptor antagonist facilitated recovery of tidal volume and minute ventilation following SCI. Furthermore, the treatment attenuated SCI-associated increases in respiratory frequency. Taken together, this study suggests that the previously observed DPCPX nanoconjugate-induced recovery in diaphragmatic and phrenic motor outputs may translate to a clinically meaningful improvement in ventilatory function in patients with SCI.

// Ivory Dean 1, 2, 3, 6 , Sijana H. Dzinic 1, 3 , M. Margarida Bernardo 1, 3 , Yi Zou 4 , Vickie Kimler 5, 7 , Xiaohua Li 1, 3, 8 , Alexander Kaplun 1, 3, 9 , James Granneman 3, 4 , Guangzhao Mao 3, 5 , Shijie Sheng 1, 2, 3 1 Department of Pathology, Wayne State University School of Medicine, MI, USA 2 Department of Oncology, Wayne State University School of Medicine, MI, USA 3 The Tumor Biology and Microenvironment Program, Karmanos Cancer Institute, MI, USA 4 Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, MI, USA 5 Department of Chemical Engineering and Materials Science, Wayne State University, MI, USA 6 Current address: Center for Bioengineering and Tissue Regeneration, The University of California San Francisco, San Francisco, CA, USA 7 Current address: Ocular Structure and Imaging Facility, Eye Research Institute, Oakland University, Rochester Hills, MI, USA 8 Current address: Zhangjiagang Aoyang Hospital, Nanjing Medical University, Jiangsu, China 9 Current address: Variantyx, Framingham, MA, USA Correspondence to: Shijie Sheng, email: ssheng@med.wayne.edu Keywords: exosome, tumor progression, exosome cargo, tumor microenvironment, electron microscopy Received: August 15, 2016 Accepted: October 22, 2016 Published: November 11, 2016 ABSTRACT Maspin is an epithelial-specific tumor suppressor shown to exert its biological effects as an intracellular, cell membrane-associated, and secreted free molecule. A recent study suggests that upon DNA-damaging g-irradiation, tumor cells can secrete maspin as an exosome-associated protein. To date, the biological significance of exosomal secretion of maspin is unknown. The current study aims at addressing whether maspin is spontaneously secreted as an exosomal protein to regulate tumor/stromal interactions. We prepared exosomes along with cell extracts and vesicle-depleted conditioned media (VDCM) from normal epithelial (CRL2221, MCF-10A and BEAS-2B) and cancer (LNCaP, PC3 and SUM149) cell lines. Atomic force microscopy and dynamic light scattering analysis revealed similar size distribution patterns and surface zeta potentials between the normal cells-derived and tumor cells-derived exosomes. Electron microscopy revealed that maspin was encapsulated by the exosomal membrane as a cargo protein. While western blotting revealed that the level of exosomal maspin from tumor cell lines was disproportionally lower relative to the levels of corresponding intracellular and VDCM maspin, as compared to that from normal cell lines, maspin knockdown in MCF-10A cells led to maspin-devoid exosomes, which exhibited significantly reduced suppressive effects on the chemotaxis activity of recipient NIH3T3 fibroblast cells. These data are the first to demonstrate the potential of maspin delivered by exosomes to block tumor-induced stromal response, and support the clinical application of exosomal maspin in cancer diagnosis and treatment.

Layer-by-layer (LbL) films are assembled with poly(amido amine)s (PAAs), a type of polycations containing bioreducible disulfide bond, and DNA plasmids to enable LbL film degradation in physiologic conditions by reacting with glutathione or redox-active membrane proteins. The interior layer structure of the LbL films during assembly and disassembly is studied by atomic force microscopy (AFM), ellipsometry, dynamic light scattering (DLS), and fluorescence spectroscopy. Insertion of barrier layers in bioreducible LbL films is necessary to stabilize the interior layer structure and slow down the film degradation rate to achieve sequential gene delivery. Localized gene delivery from the LbL films is demonstrated using human embryonic kidney 293 (HEK 293) cells.

Context: Following a spinal cord hemisection at the second cervical segment the ipsilateral hemidiaphragm is paralyzed due to the disruption of the rostral ventral respiratory group (rVRG) axons descending to the ipsilateral phrenic motoneurons (PN). Systemically administered theophylline activates a functionally latent crossed phrenic pathway (CPP) which decussates caudal to the hemisection and activates phrenic motoneurons ipsilateral to the hemisection. The result is return of function to the paralyzed hemidiaphragm. Unfortunately, in humans, systemically administered theophylline at a therapeutic dose produces many unwanted side effects. Design and setting: A tripartite nanoconjugate was synthesized in which theophylline was coupled to a neuronal tracer, wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), using gold nanoparticles as the coupler. Following intradiaphragmatic injection of the nanoconjugate, WGA-HRP selectively targets the theophylline-bound nanoconjugate to phrenic motoneurons initially, followed by neurons in the rVRG by retrograde transsynaptic transport. Participants: (N/A) Interventions: (N/A) Outcome Measures: Immunostaining, Electromyography (EMG). Results: Delivery of the theophylline-coupled nanoconjugate to the nuclei involved in respiration induces a return of respiratory activity as detected by EMG of the diaphragm and a modest return of phrenic nerve activity. Conclusion: In addition to the modest return of phrenic nerve activity, there were many difficulties using the theophylline nanoconjugate because of its chemical instability, which suggests that the theophylline nanoconjugate should not be developed for clinical use as explained herein.

We recently developed a small molecule inhibitor SMI#9 for Rad6, a protein overexpressed in aggressive breast cancers and involved in DNA damage tolerance. SMI#9 induces cytotoxicity in cancerous cells but spares normal breast cells; however, its therapeutic efficacy is limited by poor solubility. Here we chemically modified SMI#9 to enable its conjugation and hydrolysis from gold nanoparticle (GNP). SMI#9-GNP and parent SMI#9 activities were compared in mesenchymal and basal triple negative breast cancer (TNBC) subtype cells. Whereas SMI#9 is cytotoxic to all TNBC cells, SMI#9-GNP is endocytosed and cytotoxic only in mesenchymal TNBC cells. SMI#9-GNP endocytosis in basal TNBCs is compromised by aggregation. However, when combined with cisplatin, SMI#9-GNP is imported and synergistically increases cisplatin sensitivity. Like SMI#9, SMI#9-GNP spares normal breast cells. The released SMI#9 is active and induces cell death via mitochondrial dysfunction and PARP-1 stabilization/hyperactivation. This work signifies the development of a nanotechnology-based Rad6-targeting therapy for TNBCs.

Layer-by-layer (LbL) films are assembled with poly(amido amine)s (PAAs), a type of polycations containing bioreducible disulfide bond, and DNA plasmids to enable LbL film degradation in physiologic conditions by reacting with glutathione or redox-active membrane proteins. The interior layer structure of the LbL films during assembly and disassembly is studied by atomic force microscopy (AFM), ellipsometry, dynamic light scattering (DLS), and fluorescence spectroscopy. Insertion of barrier layers in bioreducible LbL films is necessary to stabilize the interior layer structure and slow down the film degradation rate to achieve sequential gene delivery. Localized gene delivery from the LbL films is demonstrated using human embryonic kidney 293 (HEK 293) cells.

This is a supplementary data for a manuscript submitted to JAP

Targeting the development of stimulus-responsive molecular materials with electronic functionality, we have synthesized and studied the redox and electronic properties of a new bimetallic iron hydrophobe [FeIII2(LN4O6)] (1). The new H6LN4O6 ligand displays bicompartmental topology capable of accomodating two five-coordinate HSFeIII ions bridged by tetraaminobenzene at a close distance of ca. 8 Å. We show that the metal-based reduction processes in (1) proceed sequentially, as observed for electronically coupled metal centers. This species forms a well-defined Pockels-Langmuir film at the air-water interface, with collapse pressure of 32 mN m-1. Langmuir-Blodgett monolayers were deposited on gold substrates and used to investigate current-voltage (I-V) measurements. This unprecedented bimetallic hydrophobe [FeIII2(LN4O6)] (1) shows unquestionable molecular rectification and displays a rectification ratio RR between 2 and 15.

Respiratory complications in patients with spinal cord injury (SCI) are common and have a negative impact on the quality of patients' lives. Systemic administration of drugs that improve respiratory function often cause deleterious side effects. The present study examines the applicability of a novel nanotechnology-based drug delivery system, which induces recovery of diaphragm function after SCI in the adult rat model. We developed a protein-coupled nanoconjugate to selectively deliver by transsynaptic transport small therapeutic amounts of an A1 adenosine receptor antagonist to the respiratory centers. A single administration of the nanoconjugate restored 75% of the respiratory drive at 0.1% of the systemic therapeutic drug dose. The reduction of the systemic dose may obviate the side effects. The recovery lasted for 4 weeks (the longest period studied). These findings have translational implications for patients with respiratory dysfunction after SCI.SIGNIFICANCE STATEMENTThe leading causes of death in humans following SCI are respiratory complications secondary to paralysis of respiratory muscles. Systemic administration of methylxantines improves respiratory function but also leads to the development of deleterious side effects due to actions of the drug on nonrespiratory sites. The importance of the present study lies in the novel drug delivery approach that uses nanotechnology to selectively deliver recovery-inducing drugs to the respiratory centers exclusively. This strategy allows for a reduction in the therapeutic drug dose, which may reduce harmful side effects and markedly improve the quality of life for SCI patients.

Triple negative breast cancers (TNBCs) lack estrogen and progesterone receptors and Her2/neu amplification, and are hence not treatable with therapies targeting these molecules. TNBCs have upregulated DNA damage response mechanisms, including the Rad6 postreplication repair (PRR) pathway, that potentially contribute to chemoresistance. Rad6 is a major component of the PRR pathway and its ubiquitin conjugating (UBC) activity is critical for its function. Rad6 expression is low in normal breast cells and tissues but the Rad6 homolog Rad6B is overexpressed in invasive, metastatic and chemoresistant BrCas. Constitutive overexpression of Rad6B in MCF10A cells induces resistance to cisplatin and doxorubicin. TCGA analysis of TNBC patient data showed an association between high Rad6B expression (but not Rad6A) and decreased overall survival. We recently reported the development of a novel Rad6-selective small molecule inhibitor (SMI#9) that inhibits Rad6 UBC activity, migration, and induces apoptosis in TNBC cells but has no effect on MCF10A cells. Since SMI#9 has limited aqueous solubility, in this study we synthesized a modified analog of SMI#9 to enable conjugation via a hydrolyzable ester bond to gold nanoparticle (GNP) and to improve delivery. GNP tethered SMI#9 (SMI#9-GNP) was characterized for purity, ligand conjugation and size by thermogravimetric analysis, atomic force microscopy, transmission electron microscopy, UV-Vis spectroscopy and zeta sizer, and for cellular uptake and drug release by FTIR and mass spectrometry. We compared the activities of SMI#9-GNP and free SMI#9 for cytotoxicity and intracellular localization in mesenchymal (MDA-MB-231 and SUM1315) and basal (MDA-MB-468 and HCC1937) subtypes of TNBC, and in MCF10A cells. Whereas free SMI#9 was cytotoxic to all TNBC cells, SMI#9-GNP demonstrated as good or better cytotoxicity than free SMI#9 only in mesenchymal TNBC cells. MCF10A cells were unaffected by both free and SMI#9-GNP. Consistent with cellular sensitivities, SMI#9-GNP is efficiently endocytosed and processed in lysosomes in mesenchymal TNBC cells, while uptake into basal TNBC cells is compromised by cell microenvironment induced SMI#9-GNP aggregation. SMI#9-GNP treatment induces mitochondrial dysfunction, and stabilization and hyperactivation of PARP-1 that was commensurate with autophagy (indicated by LC3-I to LC3-II conversion). Rad6 loss and PARP-1 hyperactivation are associated with mitochondrial dysfunction, and since inhibition of Rad6 induces both mitochondrial dysfunction and PARP-1 activation this implicates a potential novel role for Rad6 in linking these processes. In summary, our data show that SMI#9-GNP is a suitable delivery vehicle and that the SMI#9 released from GNP conjugate functions similarly as free SMI#9. Our data also illustrate how cell microenvironment induced changes in the physical properties of GNP-drug conjugates can have important implications in the application of nanoparticles in cancer therapy. Supported by NIH R21 CA178117. Citation Format: Haynes B, Zhang Y, Li J, Petit S, Westwell A, Mao G, Shekhar M. Evaluation of the therapeutic efficacy of a Rad6 small molecule inhibitor in triple negative breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P3-14-06.

This study explores the use of human embryonic stem cells (hESCs) for assessing nanotoxicology, specifically, the effect of gold nanoparticles (AuNPs) of different core sizes (1.5 nm, 4 nm, and 14 nm) on the viability, pluripotency, neuronal differentiation, and DNA methylation of hESCs. The hESCs exposed to 1.5 nm thiolate-capped AuNPs exhibited loss of cohesiveness and detachment suggesting ongoing cell death at concentrations as low as 0.1 µg/mL. The cells exposed to 1.5 nm AuNPs at this concentration did not form embryoid bodies but rather disintegrated into single cells within 48 hours. Cell death caused by 1.5 nm AuNPs also occurred in hESC-derived neural progenitor cells. None of the other nanoparticles exhibited toxic effects on the hESCs at concentrations as high as 10 µg/mL during a 19 day neural differentiation period. Thiolate-capped 4 nm AuNPs at 10 µg/mL caused a dramatic decrease in global DNA methylation (5mC) and a corresponding increase in global DNA hydroxymethylation (5hmC) of the hESC’s DNA in only 24 hours. This work identifies a type of AuNPs highly toxic to hESCs and demonstrates the potential of hESCs in predicting nanotoxicity and characterizing their ability to alter the DNA methylation and hydroxymethylation patterns in the cells.

The current study was aimed to develop a targeted dendrimer formulation of 3, 4-difluorobenzylidene curcumin (CDF) and evaluate its potential in CD44 targeted therapy for pancreatic cancer. Using amine terminated fourth generation poly(amidoamine) (PAMAM) dendrimer nanocarrier and hyaluronic acid (HA) as a targeting ligand, we engineered a CD44-targeted PAMAM dendrimer (HA-PAMAM) formulation of CDF. The resulting dendrimer nanosystem (HA-PAMAM-CDF) had a particle size and surface charge of 9.3 ± 1.5 nm and -7.02 ± 9.53 mV, respectively. When CD44 receptor overexpressing MiaPaCa-2 and AsPC-1 human pancreatic cancer cells were treated with HA-PAMAM-CDF, a dose-dependent cytotoxicity was observed. Furthermore, blocking the CD44 receptors present on the MiaPaCa-2 cells using free excess soluble HA prior to treatment with HA-PAMAM-CDF nano-formulation resulted in 1.71 fold increase in the IC50 value compared to non-targeted formulation (PAMAM-CDF), confirming target specificity of HA-PAMAM-CDF. Additionally, HA-PAMAM-CDF formulation when compared to PAMAM-CDF, displayed higher cellular uptake in MiaPaCa-2 cancer cell lines as shown by fluorescence studies. In summary, the novel CD44 targeted dendrimer based nanocarriers appear to be proficient in mediating site-specific delivery of CDF via CD44 receptors, with an improved therapeutic margin and safety.

Purpose: Bombarding high-Z material with x-ray radiation releases Auger electrons and Coster–Kronig electrons, along with deeper penetrating fluorescent x-rays and photoelectrons. The Auger and Coster–Kronig electron penetration distance is on the order of nanometers to micrometers in water or tissue, creating a large dose enhancement accompanied by a RBE greater than 1 at the cellular level. The authors’ aim is to measure the gold nanofilm dose enhancement factor (DEF) at the cellular level with unlaminated radiochromic film via primary 50 kVp tungsten x-ray spectrum interaction, similar to an electronic brachytherapy spectrum. Methods: Unlaminated Gafchromic{sup ®} EBT2 film and Monte Carlo modeling were combined to derive DEF models. Gold film of thickness 23.1 ± 4.3 nm and surface roughness of 1.2 ± 0.2 nm was placed in contact with unlaminated radiochromic film in a downstream orientation and exposed to a 50 kVp tungsten bremsstrahlung, mean energy 19.2 keV. Film response correction factors were derived by Monte Carlo modeling of electron energy deposition in the film’s active layer, and by measuring film energy dependence from 4.5 keV to 50 kVp. Results: The measured DEF within a 13.6 μm thick water layer was 0.29 with a mean dose of 94 ± 9.4 cGymore » from Au emissions and 324 ± 32.4 cGy from the 50 kVp primary beam. Monte Carlo derived correction factors allowed determination of Au contributed dose in shallower depths at 0.25 μm intervals. Maximum DEF of 18.31 was found in the first 0.25 μm water depth. Conclusions: Dose enhancement from Au nanofilm can be measured at the cellular level using unlaminated radiochromic film. Complementing the measured dose value with Monte Carlo calculations allows estimation of dose enhancement at depth increments within the cellular range.« less

Forces between colloidal particles determine the performances of many industrial processes and products. Colloidal force measurements conducted between a colloidal particle AFM probe and particles immobilized on a flat substrate are valuable in selecting appropriate surfactants for colloidal stabilization. One of the features of inorganic fillers and extenders is the prevalence of rough surfaces-even the polymer latex particles, often used as model colloidal systems including the current study, have rough surfaces albeit at a much smaller scale. Surface roughness is frequently cited as the reason for disparity between experimental observations and theoretical treatment but seldom verified by direct evidence. This work reports the effect of nanoscale surface roughness on colloidal force measurements carried out in the presence of surfactants. We applied a heating method to reduce the mean surface roughness of commercial latex particles from 30 to 1 nm. We conducted force measurements using the two types of particles at various salt and surfactant concentrations. The surfactants used were pentaethylene glycol monododecyl ether, Pluronic F108, and a styrene/acrylic copolymer, Joncryl 60. In the absence of the surfactant, nanometer surface roughness affects colloidal forces only in high salt conditions when the Debye length becomes smaller than the surface roughness. The adhesion is stronger between colloids with higher surface roughness and requires a higher surfactant concentration to be eliminated. The effect of surface roughness on colloidal forces was also investigated as a function of the adsorbed surfactant layer structure characterized by AFM indentation and dynamic light scattering. We found that when the layer thickness exceeds the surface roughness, the colloidal adhesion is less influenced by surfactant concentration variation. This study demonstrates that surface roughness at the nanoscale can influence colloidal forces significantly and should be taken into account in colloidal dispersion formulations.

Major barriers for commercializing nanowire sensors are complicated synthesis technique and lack of scalable device assembly methods. Charge transfer salts (CTS), like tetrathiafulvalene bromide (TTFBr), have been well known for unique electric and chemical property. This talk will bring a controlled one-step deposition method to fabricate CTS nanowire sensors. By applying certain potentials to designed substrates, TTFBr nanowires can be directly deposited onto the electrodes. Varying deposition conditions and design of substrates are able to control the geometry, density and composition of TTFBr nanowires. TTFBr nanowire sensor shows high sensitivity, selectivity, and fast response towards ammonia gas. The detection limit is less than 1ppm and the response time, at room temperature, is less than 1 minute. Other CTS, like Cu- tetracyanoquinodimethane, can also be fabricated into nanowire sensor using the same method. This research shows a promising scalable fabrication method for various nanowire sensors and is being commercialized.

Lingxiao Xie, Xiong Ding, Rachel Budry, Guangzhao Mao Department of Chemical Engineering and Materials Science, Wayne State University, Detroit, MI, USA Background: The layer-by-layer (LbL) assembly method offers a molecular level control of the amount and spatial distribution of bioactive molecules. However, successful clinical translation of LbL film technology will most certainly require a better understanding and control of not only the film assembly process, but also film disassembly kinetics in physiologic conditions. Purpose: This work focuses on the understanding and control of degradation properties of LbL films for localized gene delivery. Methods: Bioreducible poly(amido amine)s (PAAs) containing cystaminebisacrylamide (CBA), methylenebisacrylamide, and 5-amino-1-pentanol (APOL) were synthesized by Michael addition polymerization for the construction of bioreducible LbL films capable of sequential gene delivery. Results: The synthesized PAAs were screened for desirable buffering capacity, cell transfection, and cytotoxicity characteristics together with 25 kDa branched polyethylenimine (PEI) and cross-linked 800 Da PEI. By screening the various polycations we were able to identify a copolymer of CBA and APOL for the subsequent construction of the LbL films. By incorporating a highly transfecting polycation and a nondiffusing polycation we were able to improve the overall transfection of HEK293 and MC3T3 cells from the bioreducible LbL films. We also demonstrated the dual-stage release and transfection of two different DNAs from the LbL films. Conclusion: The results indicate that LbL films consisting of bioreducible PAAs and non-diffusing polyelectrolytes have excellent degradation properties for the development of LbL coating technology for localized gene delivery applications. Keywords: biodegradable polymers, cell transfection, interlayer diffusion, localized gene delivery, polyelectrolyte multilayers, sequential release

Nanotechnology has demonstrated tremendous clinical utility, with potential applications in cancer immunotherapy. Although nanoparticles with intrinsic cytotoxicity are often considered unsuitable for clinical applications, such toxicity may be harnessed in the fight against cancer. Nanoparticle-associated toxicity can induce acute necrotic cell death, releasing tumor-associated antigens which may be captured by antigen-presenting cells to initiate or amplify tumor immunity. To test this hypothesis, cytotoxic cationic silica nanoparticles (CSiNPs) were directly administered into B16F10 melanoma implanted in C57BL/6 mice. CSiNPs caused plasma membrane rupture and oxidative stress of tumor cells, inducing local inflammation, tumor cell death and the release of tumor-associated antigens. The CSiNPs were further complexed with bis-(3'-5')-cyclic dimeric guanosine monophosphate (c-di-GMP), a molecular adjuvant which activates the stimulator of interferon genes (STING) in antigen-presenting cells. Compared with unformulated c-di-GMP, the delivery of c-di-GMP with CSiNPs markedly prolonged its local retention within the tumor microenvironment and activated tumor-infiltrating antigen-presenting cells. The combination of CSiNPs and a STING agonist showed dramatically increased expansion of antigen-specific CD8+ T cells, and potent tumor growth inhibition in murine melanoma. These results demonstrate that cationic nanoparticles can be used as an effective in situ vaccine platform which simultaneously causes tumor destruction and immune activation.

Langmuir–Blodgett films of metallosurfactants were used in Au|molecule|Au devices to investigate the mechanisms of current rectification.

This paper describes electrochemical synthesis of nanorods of tetrathiafulvalene (TTF) charge-transfer salt on gold nanoparticle (GNP) seeds. The seed-mediated process was monitored by cyclic voltammetry, AFM, and field-emission SEM. The electrodeposition of GNPs (nucleation seeds) on highly oriented pyrolytic graphite (HOPG) electrodes was studied as a function of the electrolytic conditions. The GNP size increases with increasing HAuCl4 concentration and decreasing applied overpotential. A morphological transition from quasi-spherical particles to dendritic aggregates occurs when the HAuCl4 concentration increases from 0.5 to 1 mM. The electrocrystallization of (TTF)Br0.76 on the GNP-decorated HOPG was investigated as a function of TTF concentration and GNP morphology. We observed a preferential nucleation of (TTF)Br0.76 on the GNP seed. The seed-mediated (TTF)Br0.76 crystals display a confined crystal morphology in comparison to those nucleated on bare HOPG. (TTF)Br0.76 nanorods as small as 7 nm in hei...