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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.

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.

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.

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.

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.

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.

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.

Lack of a system for genetic manipulation of Chlamydia trachomatis has been a key challenge to advancing understanding the molecular genetic basis of virulence for this bacterial pathogen. We developed a non-viral, dendrimer-enabled system for transformation of this organism and used it to characterize the effects of inserting the common 7.5 kbp chlamydial plasmid into strain L2(25667R), a C. trachomatis isolate lacking it. The plasmid was cloned in pUC19 and the clone complexed to polyamidoamine dendrimers, producing ∼83 nm spherical particles. Nearly confluent McCoy cell cultures were infected with L2(25667R) and reference strain L2(434). At 16 h post-infection, medium was replaced with dendrimer–plasmid complexes in medium lacking additives (L2(25667R)) or with additive-free medium alone (L2(434)). Three h later complexes/buffer were removed, and medium was replaced; cultures were harvested at various times post-transformation for analyses. Real time PCR and RT-PCR of nucleic acids from transformed cultures demonstrated plasmid replication and gene expression. A previous report indicated that one or more plasmid-encoded product govern(s) transcription of the glycogen synthase gene (glgA) in standard strains. In L2(25667R) the gene is not expressed, but transformants of that strain given the cloned chlamydial plasmid increase glgA expression, as does L2(434). The cloned plasmid is retained, replicated, and expressed in transformants over at least 5 passages, and GFP is expressed when transformed into growing L2(25667R). This transformation system will allow study of chlamydial gene function in pathogenesis.

A long-term effect of chronically implanted neural electrodes is the formation of a glial scar made up of reactive astrocytes, microglia and the matrix proteins they generate. Studies have shown glial fibrillary acidic protein (GFAP) and cytokines interleukin-1beta (IL-1β), tumor necrosis factor alpha (TNFα), and transforming growth factor beta 1 (TGFβ1) are involved with the initial and modulation phases of reactive astrogliosis. In the present study, nanopatterning of polydimethylsiloxane (PDMS) was attempted as a method for reducing the inflammatory response of glial cells. A unique feature of this study is the use of in vitro brain slice cultures (organotypic cultures) in order to more accurately depict the native response. The aim of the study was to determine whether nanotopography could reduce inflammatory signals typically resultant from neural electrode implantation. Specifically, observation of cell alignment and surveillance of GFAP, IL-1β, TNFα, and TGFβ1 gene expression around the PDMS pins was performed. Results of this study confirm nanopatterning not only influences cell morphology, but some of the molecular signals as well. These results collectively indicate nanopatterning improves the biocompatibility of PDMS by reducing inflammatory markers such as GFAP, IL-1β, TGFβ1 and TNFα compared to the non-patterned PDMS pins.

Drug delivery to the central nervous system (CNS) is challenging due to the inability of many drugs to cross the blood-brain barrier (BBB). Here, we show that wheat germ agglutinin horse radish peroxidase (WGA-HRP) chemically conjugated to gold nanoparticles (AuNPs) can be transported to the spinal cord and brainstem following intramuscular injection into the diaphragm of rats. We synthesized and determined the size and chemical composition of a three-part nanoconjugate consisting of WGA-HRP, AuNPs and drugs for the treatment of diaphragm paralysis associated with high cervical spinal cord injury (SCI). Upon injection into the diaphragm muscle of rats, we show that the nanoconjugate is capable of delivering the drug at a much lower dose than the unconjugated drug injected systemically to effectively induce respiratory recovery in rats following SCI. This study not only demonstrates a promising strategy to deliver drugs to the CNS bypassing the BBB but also contributes a potential nanotherapy for the treatment of respiratory muscle paralysis resulted from cervical SCI.

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.Protein Rad6 is overexpressed in breast cancer cells and its blockade may provide a new treatment against 3N breast cancer. The authors conjugated a small molecule inhibitor SMI#9 for Rad6 to gold nanoparticles in this study and showed that this new formulation specifically targeted chemo-resistant breast cancer cells and highlighted the importance of nanotechnology in drug carrier development.

In cells, N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors called SNAREs are involved in membrane fusion. In neurons, for example, target membrane proteins SNAP-25 and syntaxin called t-SNAREs present at the pre-synaptic membrane, and a synaptic vesicle-associated membrane protein (VAMP) or v-SNARE, is part of the conserved protein complex involved in neurotransmission. Cholesterol and LPC (L-α-lysophosphatidylcholine) are known to contribute to the negative and positive curvature respectively of membranes. In this study, using purified recombinant neuronal membrane-associated SNAREs, we demonstrate for the first time that membrane-curvature-influencing lipids profoundly influence SNARE complex disassembly. Exposure of cholesterol-associated t-SNARE and v-SNARE liposome mixtures to NSF–ATP results in dissociated vesicles. In contrast, exposure of LPC-associated t-SNARE and v-SNARE liposome mixtures to NSF–ATP, results in inhibition of t-/v-SNARE disassembly and the consequent accumulation of clustered vesicles. Similarly, exposure of isolated rat brain slices and pancreas to cholesterol or LPC, also demonstrates LPC-induced inhibition of SNARE complex disassembly. Earlier studies demonstrate a strong correlation between altered plasma LPC levels and cancer. The altered plasma LPC levels observed in various cancers may in part contribute to defects in SNARE assembly–disassembly and membrane fusion, consequently affecting protein maturation and secretion in cancer cells.

Wei Song1, Xiaowei Yu2, Sunxi Wang5, Ralph Blasier4, David C Markel3, Guangzhao Mao5, Tong Shi1, Weiping Ren1,31Department of Biomedical Engineering, Wayne State University, Detroit, MI, USA; 2Department of Orthopedic Surgery, Second Affiliated Hospital of Nanjing Medical University, Nanjing, People's Republic of China; 3Detroit Medical Center and Providence Hospital Orthopedic Residency, Detroit, 4Orthopedic Section, St Francis Hospital, Escanaba, 5Department of Chemical Engineering, Wayne State University, Detroit, MI, USABackground: Erythromycin, a hydrophobic antibiotic used to treat infectious diseases, is now gaining attention because of its anti-inflammatory effects and ability to inhibit osteoclasts formation. The aim of this study was to explore a cyclodextrin-erythromycin (CD-EM) complex for sustained treatment of orthopedic inflammation.Methods and results: Erythromycin was reacted with ß-cyclodextrin to form a nonhost-guest CD-EM complex using both kneading and stirring approaches. Physiochemical measurement data indicated that erythromycin and cyclodextrin formed a packing complex driven by intermolecular forces instead of a host-guest structure due to the limited space in the inner cavity of ß-cyclodextrin. The CD-EM complex improved the stability of erythromycin in aqueous solution and had a longer duration of bactericidal activity than free erythromycin. Cytotoxicity and cell differentiation were evaluated in both murine MC3T3 preosteoblast cells and RAW 264.7 murine macrophage cells. The CD-EM complex was noncytotoxic and showed significant inhibition of osteoclast formation but had little effect on osteoblast viability and differentiation.Conclusion: These attributes are especially important for the delivery of an adequate amount of erythromycin to the site of periprosthetic inflammation and reducing local inflammation in a sustained manner.Keywords: erythromycin, cyclodextrin, drug stability, bactericidal activity, osteoclastogenesis

Nanoparticles (NPs) are 1-100 nm constructs explored for their application in cancer diagnosis and treatment. Exposure of cells to ionizing irradiation leads to DNA damage, with DNA double strand breaks (DSBs) being the most toxic that can lead to apoptosis. The presence of gold in NPs enhances radiation damage and DSBs, the latter measured by the levels of phosphorylated DNA histone protein H2AX (γH2AX). The goal of this study was to decipher biological mechanisms of NP radio-sensitization. We analyzed the effects of two different sizes of gold nanoparticles (AuNP) on DSBs in MCF-7 breast cancer cells by assessing H2AX phosphorylation at three photon energies. Cells were incubated with either 4 nm or 14 nm AuNP and irradiated with 2, 4 or 8 Gy using 2.5 FFF MV (60 MU/min), 6 MV (600 MU/min) or 10 FFF MV (2400 MU/min) X-rays. Percent of cells positive for γH2AX was determined by flow cytometry. Live cells (100000) were gated using forward (FSC; cell size) versus side scatter (SSC; complexity) characteristics (gate P1). Cells positive for γH2AX were further gated to identify subpopulations exhibiting brighter (gate P2) or dimmer (gate P3) fluorescence intensity. Treatment with 4 nm AuNP resulted in significantly more γH2AX positive cells after irradiation at all three energies, compared to their respective controls. When treated at 6 MV energy, increases in the percentage of cells positive for H2AX phosphorylation was detected at all three doses (2, 4 and 8 Gy). Increased phosphorylation was accompanied by an increase of cells in the P2 gate accompanied by an increase in their FCS and SSC characteristics, consistent with activation. In cells treated with 10 MV the effect was most pronounced at 4Gy dose, while 2 and 8 Gy resulted in a slight increase in the percentage of γH2AX positive cells. Nevertheless, these cells still exhibited prominent increases within the P2 gate and FCS vs SSC characteristics, compared to their irradiated controls. In cells treated with 2.5 MV energy similar results were observed using 4 and 8 Gy, while no effect was detected with 2 Gy. Data from the experiments using 14 nm size AuNP were less clear and require further investigation. The difference may be due to the localization of the smaller NPs within the nucleus and therefore cause more DNA damage thereby greater H2AX phosphorylation. We hypothesize that the NP radio-sensitization mechanism involves directing cells towards apoptosis by enhancing DNA damage and interfering with DNA repair. Hence, γH2AX positive cells exhibiting bright fluorescence (P2) and an increase in FCS and SSC identified even in the conditions without significant increase in the percent of total γH2AX positive cells, may reflect cells primed for apoptosis. Future studies are planned to elucidate the exact intracellular mechanisms of NP biological radio-sensitization effect. Citation Format: Branislava Janic, Fangchao Liu, Kevin Bobbitt, Stephen Brown, Guangzhao Mao, Indrin J. Chetty, Benjamin Movsas, Ning Winston Wen. Effect of gold nanoparticle on radiation induced DNA damage in MCF7 breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1376.

The effect of cross-linking layer-by-layer (LbL) films consisting of bioreducible poly(2-dimethylaminoethyl methacrylate) (rPDMAEMA) and DNA is examined with regard to rigidity, biodegradability, cell adhesion, and transfection activity using 1,5-diiodopentane (DIP) cross-linker. DIP chemically reacts with the tertiary amines of rPDMAEMA, altering the chemical composition of these LbL films. The result is a change in surface morphology, film swelling behavior, and film rigidity, measured with AFM and ellipsometry. It is found that the apparent Young's modulus is increased more than 4 times its original value upon cross-linking. Cross-linking mass is additionally confirmed with a quartz crystal microbalance with dissipation (QCM-D). Comprehensive analyses of these experimental values were investigated to calculate the degree of cross-linking using the rubber elasticity theory and the Flory-Rehner theory. Additionally, the Flory-Huggins parameter, chi, was calculated. Good agreement in the two methods yields a cross-linking density of approximately 0.82 mmol/cm(3). The Flory-Huggins parameter increased upon cross-linking from 1.07 to 1.2, indicating increased hydrophobicity of the network and formation of bulk water droplets within the films. In addition, the effects of cross-linking on film disassembly by 1,4-dithiothreitol (DTT) are found to be insignificant despite the alteration in film rigidity. Mouse fibroblast cells and smooth muscle cells are used to study the effect of cross-linking on cell adhesion and cell transfection activity. In vitro transfection activity up to seven days is quantified using secreted alkaline phosphatase (SEAP) DNA. Film cross-linking is found to enhance cell adhesion and prolong the duration of cellular transfection. These results contribute to the development of bioreducible polymer coatings for localized gene delivery.

Drug resistance limits the success of many anticancer drugs. Reduced accumulation of the drug at its intracellular site of action because of overexpression of efflux transporters such as P-glycoprotein (P-gp) is a major mechanism of drug resistance. In this study, we investigated whether photodynamic therapy (PDT) using methylene blue, also a P-gp inhibitor, can be used to enhance doxorubicin-induced cytotoxicity in drug-resistant tumor cells. Aerosol OT (AOT)-alginate nanoparticles were used as a carrier for the simultaneous cellular delivery of doxorubicin and methylene blue. Methylene blue was photoactivated using light of 665 nm wavelength. Induction of apoptosis and necrosis following treatment with combination chemotherapy and PDT was investigated in drug-resistant NCI/ADR-RES cells using flow cytometry and fluorescence microscopy. Effect of encapsulation in nanoparticles on the intracellular accumulation of doxorubicin and methylene blue was investigated qualitatively using fluorescence microscopy and was quantitated using HPLC. Encapsulation in AOT-alginate nanoparticles significantly enhanced the cytotoxicity of combination therapy in resistant tumor cells. Nanoparticle-mediated combination therapy resulted in a significant induction of both apoptosis and necrosis. Improvement in cytotoxicity could be correlated with enhanced intracellular and nuclear delivery of the two drugs. Further, nanoparticle-mediated combination therapy resulted in significantly elevated reactive oxygen species (ROS) production compared to single drug treatment. In conclusion, nanoparticle-mediated combination chemotherapy and PDT using doxorubicin and methylene blue was able to overcome resistance mechanisms and resulted in improved cytotoxicity in drug-resistant tumor cells.

Photodynamic therapy (PDT) is a promising treatment modality for cancer. PDT is based on the concept that photosensitizers, when exposed to light of specific wavelength, generate cytotoxic reactive oxygen species (ROS) capable of killing tumor cells. The effectiveness of PDT has been limited in part by the lack of photosensitizers that accumulate sufficiently in tumor cells and poor yield of ROS from existing photosensitizers. In this report, we investigated whether aerosol OT-alginate nanoparticles can be used as a carrier to enhance the therapeutic efficacy of a model photosensitizer, methylene blue. Methylene blue loaded nanoparticles were evaluated for PDT effectiveness in two cancer cell lines, MCF-7 and 4T1. Encapsulation of methylene blue in nanoparticles significantly enhanced intracellular ROS production, and the overall cytotoxicity following PDT. It also resulted in higher incidence of necrosis. Greater effectiveness of nanoparticles could be correlated with higher yield of ROS with nanoparticle-encapsulated methylene blue. Further, treatment of tumor cells with nanoparticle-encapsulated methylene blue resulted in significant nuclear localization of methylene blue while free drug treatment resulted in its accumulation mainly in the endolysosomal vesicles. In conclusion, encapsulation of methylene blue in aerosol OT-alginate nanoparticles enhanced its anticancer photodynamic efficacy in vitro. Increased ROS production and favorable alteration in the subcellular distribution contribute to the enhanced PDT efficacy of nanoparticle-encapsulated photosensitizer.

Phages are promising alternatives to antibodies as the biorecognition element in a variety of biosensing applications. In this study, a monolayer of bacteriophage P22 whose tailspike proteins specifically recognize Salmonella serotypes was covalently bound to glass substrates through a bifunctional cross linker 3-aminopropyltrimethoxysilane. The specific binding of Salmonella typhimurium to the phage monolayer was studied by enzyme-linked immunosorbent assay and atomic force microscopy. Escherichia coli and a Gram-positive bacterium Listeria monocytogenes were also studied as control bacteria. The P22 particles show strong binding affinity to Salmonella typhimurium. In addition, the dried P22 monolayer maintained 50% binding capacity to Salmonella typhimurium after a one-week storage time. This is a promising method to prepare phage monolayer coatings on surface plasmon resonance and acoustic biosensor substrates in order to utilize the nascent phage display technology.

Active β-catenin signaling in melanoma is associated with aggressive disease. We have previously shown that Rad6B, an ubiquitin conjugating enzyme, is a positive regulator of β-catenin stability and transcriptional activity, and is itself a transcriptional target of β-catenin. This stabilization is mediated by Rad6B-induced K63-linked polyubiquitination of β-catenin that renders it insensitive to 26S proteasomal degradation. Immunohistochemical analysis of human cutaneous melanoma samples showed the first detectable expression of Rad6B in melanocyte hyperplasia and continued Rad6B and β-catenin overexpression in primary and metastatic melanomas. These data implicate a critical role for Rad6B in melanocyte transformation and a continuing role in melanoma progression. To analyze the role of Rad6B in melanomagenesis, we targeted Rad6B expression/activity in human M14 metastatic melanoma cells by using a Rad6-selective small molecule inhibitor SMI#9 and the CrispR/Cas9 gene editing system. Immunoblot analysis showed that compared to controls, M14 cells treated with SMI#9 showed decreased levels of β-catenin, and β-catenin driven transcriptional targets Rad6, vimentin, Mitf-M, and Melan A. Although Snail levels were not altered, SMI#9 treated cells showed increase in phosphorylated Snail. Immunocytochemical staining verified the western blot data and showed cytoplasmic relocalization of PCNA and Snail in SMI#9 treated cells which is consistent with increase in phosphorylated Snail. Concordant with the decrease in vimentin and unphosphorylated Snail levels, Rad6B-inhibited cells display poor capacity to migrate as compared to controls. Whereas Rad6B knockout cells were severely growth impaired, Rad6B edited clones showing partial expression (potentially from only one allele) similarly showed decreased levels of β-catenin, vimentin, and Mitf-M. Similar to Rad6B-inhibited cells, Rad6B edited cells were migration- and invasion-impaired. Whereas control cells produced robust tumors and overt lung metastases, the Rad6B gene edited cells were poorly tumorigenic. Rad6B gene edited clones showed complete loss of Melan A. Since Mitf-M is a transcriptional regulator of Melan A and a key player in melanocyte lineage specification, our data suggest that targeting Rad6B could potentially inhibit melanomagenesis by reprogramming melanoma cells. Supported by NIH CA178117 and 3Balls Racing. Citation Format: Ashapurna Sarma, Sarah Petit, Fangchao Liu, Pratima Nangia-Makker, Guangzhao Mao, Malathy Shekhar. Targeting Rad6B suppresses melanomagenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1149. doi:10.1158/1538-7445.AM2017-1149