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IMPACT STATEMENT: This article describes a method for producing microRNA (miRNA)-enriched extracellular vesicles in large quantities. It enables in vivo delivery of specific miRNA for therapeutic applications.

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Some RNA species, especially microRNAs, are non-randomly sorted into exosomes, but how selectivity of RNA exosomal sorting is achieved is unknown. We found that all three variants of RNA-binding ubiquitin E3 ligase (MEX3C)-MEX3C-1, MEX3C-2, and MEX3C-3 -interact with adaptor-related protein complex 2 (AP-2), a cargo adaptor in clathrin-mediated endocytosis. MEX3C's C-terminal RING finger domain and the hnRNP K homology (KH) domain shared by the three MEX3C variants are both necessary for MEX3C/AP-2 interaction. MEX3C associates with the endolysosomal compartment through an endocytosis-like process. siRNA-mediated inhibition of the MEX3C or AP-2 complex substantially decreased exosomal but not cellular microRNA miR-451a expression. Exosomal sorting is ceramide-dependent but not ESCRT-dependent in microRNA miR-451a. That RNA-binding protein associates with membrane trafficking machinery, and that its involvement in exosomal microRNA expression, suggest the existence of a mechanism for specific recruiting of RNA molecules to endosomes for subsequent exosomal sorting.

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There is a growing interest in the use of multiwalled carbon nanotubes (MWCNTs) to treat diseases of the brain. Little is known about the effects of MWCNTs on human brain microvascular endothelial cells (HBMECs), which make up the blood vessels in the brain. In our studies, we evaluate the cytotoxicity of MWCNTs and acid oxidized MWNCTs, with or without a phospholipid-polyethylene glycol coating. We determined the cytotoxic effects of MWCNTs on both tissue-mimicking cultures of HBMECs grown on basement membrane and on monolayer cultures of HBMECs grown on plastic. We also evaluated the effects of MWCNT exposure on the capacity of HBMECs to form rings after plating on basement membrane, a commonly used assay to evaluate angiogenesis. We show that tissue-mimicking cultures of HBMECs are less sensitive to all types of MWCNTs than monolayer cultures of HBMECs. Furthermore, we found that MWCNTs have little impact on the capacity of HBMECs to form rings. Our results indicate that relative cytotoxicity of MWCNTs is significantly affected by the type of cell culture model used for testing, and supports further research into the use of tissue-mimicking endothelial cell culture models to help bridge the gap between in vitro and in vivo toxicology.

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We investigated the effects of silver nanoparticle (AgNP) exposure in three ovarian cancer cell lines (A2780, SKOV3, and OVCAR3). We found that AgNPs were highly cytotoxic toward A2780 and SKOV3 cells but OVCAR3 cells were less sensitive to AgNPs. In agreement with the cytotoxicity data, AgNPs caused DNA damage in A2780 and SKOV3 cells, but not in OVCAR3 cells. A2780 and SKOV3 showed higher levels of basal reactive oxygen species (ROS) relative to OVCAR3 cells. AgNP exposure increased ROS levels in both A2780 and SKOV3 cells, but not in OVCAR3 cells. We found that the heterogeneous cytotoxicity was specific to the uptake of intact particles and was not due to differences in sensitivity to silver ions. Furthermore, the combination of AgNPs and standard-of-care platinum therapy, cisplatin (cis-diamminedichloroplatinum(II), CDDP), was synergistic for treatment of A2780 andOVCAR3 cells and the combination of AgNPs and CDDP showed a favorable dose reduction in all cell lines tested. These results provide insight into potential applications of AgNPs for treatment of ovarian cancer.

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Identification of differential sensitivity of cancer cells as compared to normal cells has the potential to reveal a therapeutic window for the use of silver nanoparticles (AgNPs) as a therapeutic agent for cancer therapy. Exposure to AgNPs is known to cause dose-dependent toxicities, including induction of oxidative stress and DNA damage, which can lead to cell death. Triple-negative breast cancer (TNBC) subtypes are more vulnerable to agents that cause oxidative stress and DNA damage than are other breast cancer subtypes. We hypothesized that TNBC may be susceptible to AgNP cytotoxicity, a potential vulnerability that could be exploited for the development of new therapeutic agents. We show that AgNPs are highly cytotoxic toward TNBC cells at doses that have little effect on nontumorigenic breast cells or cells derived from liver, kidney, and monocyte lineages. AgNPs induced more DNA and oxidative damage in TNBC cells than in other breast cells. In vitro and in vivo studies showed that AgNPs reduce TNBC growth and improve radiation therapy. These studies show that unmodified AgNPs act as a self-therapeutic agent with a combination of selective cytotoxicity and radiation dose-enhancement effects in TNBC at doses that are nontoxic to noncancerous breast and other cells.

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Irreversible electroporation (IRE) is a non-thermal focal ablation technique that uses a series of brief but intense electric pulses delivered into a targeted region of tissue, killing the cells by irrecoverably disrupting cellular membrane integrity. This study investigates if there is an improved local anti-tumor response in immunocompetent (IC) BALB/c versus immunodeficient (ID) nude mice, including the potential for a systemic protective effect against rechallenge. Subcutaneous murine renal carcinoma tumors were treated with an IRE pulsing protocol that used 60% of the predicted voltage required to invoke complete regressions in the ID mice. Tumors were followed for 34 days following treatment for 11 treated mice from each strain, and 7 controls from each strain. Mouse survival based on tumor burden and the progression-free disease period was substantially longer in the treated IC mice relative to the treated ID mice and sham controls for both strains. Treated IC mice were rechallenged with the same cell line 18 days after treatment, where growth of the second tumors was shown to be significantly reduced or prevented entirely. There was robust CD3+ cell infiltration in some treated BALB/C mice, with immunocytes focused at the transition between viable and dead tumor. There was no difference in the low immunocyte presence for untreated tumors, nude mice, and matrigel-only injections in both strains. These findings suggest IRE therapy may have greater therapeutic efficacy in immunocompetent patients than what has been suggested by immunodeficient models, and that IRE may invoke a systemic response beyond the targeted ablation region.

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Breast tumors contain a small population of tumor initiating stem-like cells, termed breast cancer stem cells (BCSCs). These cells, which are refractory to chemotherapy and radiotherapy, are thought to persist following treatment and drive tumor recurrence. We examined whether BCSCs are similarly resistant to hyperthermic therapy, and whether nanoparticles could be used to overcome this resistance. Using a model of triple-negative breast cancer stem cells, we show that BCSCs are markedly resistant to traditional hyperthermia and become enriched in the surviving cell population following treatment. In contrast, BCSCs are sensitive to nanotube-mediated thermal treatment and lose their long-term proliferative capacity after nanotube-mediated thermal therapy. Moreover, use of this therapy in vivo promotes complete tumor regression and long-term survival of mice bearing cancer stem cell-driven breast tumors. Mechanistically, nanotube thermal therapy promotes rapid membrane permeabilization and necrosis of BCSCs. These data suggest that nanotube-mediated thermal treatment can simultaneously eliminate both the differentiated cells that constitute the bulk of a tumor and the BCSCs that drive tumor growth and recurrence.

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Emerging evidence suggests that multiple tumor types are sustained by a small population of transformed stem-like cells that have the ability to both self-renew and give rise to non-tumorigenic daughter cells that constitute the bulk of a tumor. These cells, which generally constitute a minority of the overall cancer cell population, are highly resistant to conventional therapies and persist following treatment, leading to disease relapse and the formation of distant metastases. Therapies that disrupt the maintenance and survival of cancer stem cells are the subject of active current investigation. This review discusses recent approaches to the application of nanomedicine to the targeting and elimination of cancer stem cells. Specifically, recent publications in the areas of nanoparticle-enabled drug and nucleic acid delivery and photothermal therapy are addressed.

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Multiwalled carbon nanotubes (MWCNTs) are cylindrical tubes of graphitic carbon with unique physical and electrical properties. MWCNTs are being explored for a variety of diagnostic and therapeutic applications. Successful biomedical application of MWCNTs will require compatibility with normal circulatory components, including constituents of the hemostatic cascades. In this manuscript, we compare the thrombotic activity of MWCNTs in vitro and in vivo. We also assess the influence of functionalization of MWCNTs on thrombotic activity. In vitro, MWCNT activate the intrinsic pathway of coagulation as measured by activated partial thromboplastin time (aPTT) assays. Functionalization by amidation or carboxylation enhances this procoagulant activity. Mechanistic studies demonstrate that MWCNTs enhance propagation of the intrinsic pathway via a non-classical mechanism strongly dependent on factor IX. MWCNTs preferentially associate with factor IXa and may provide a platform that enhances its enzymatic activity. In addition to their effects on the coagulation cascade, MWCNTs activate platelets in vitro, with amidated MWCNTs exhibiting greater platelet activation than carboxylated or pristine MWCNTs. However, contrasting trends are obtained in vivo, where functionalization tends to diminish rather than enhance procoagulant activity. Thus, following systemic injection of MWCNTs in mice, pristine MWCNTs decreased platelet counts, increased vWF, and increased D-dimers. In contrast, carboxylated MWCNTS exhibited little procoagulant tendency in vivo, eliciting only a mild and transient decrease in platelets. Amidated MWCNTs elicited no statistically significant change in platelet count. Further, neither carboxylated nor amidated MWCNTs increased vWF or D-dimers in mouse plasma. We conclude that the procoagulant tendencies of MWCNTs observed in vitro are not necessarily recapitulated in vivo. Further, functionalization can markedly attenuate the procoagulant activity of MWCNTs in vivo. This work will inform the rational development of biocompatible MWCNTs for systemic delivery.

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Iron-chelation therapy has its origins in the treatment of iron-overload syndromes. For many years, the standard for this purpose has been deferoxamine. Recently, considerable progress has been made in identifying synthetic chelators with improved pharmacologic properties relative to deferoxamine. Most notable are deferasirox (Exjade(®)) and deferiprone (Ferriprox(®)), which are now available clinically. In addition to treatment of iron overload, there is an emerging role for iron chelators in the treatment of diseases characterized by oxidative stress, including cardiovascular disease, atherosclerosis, neurodegenerative diseases and cancer. While iron is not regarded as the underlying cause of these diseases, it does play an important role in disease progression, either through promotion of cellular growth and proliferation or through participation in redox reactions that catalyze the formation of reactive oxygen species and increase oxidative stress. Thus, iron chelators may be of therapeutic benefit in many of these conditions. Phytochemicals, many of which bind iron, may also owe some of their beneficial properties to iron chelation. This review will focus on the advances in iron-chelation therapy for the treatment of iron-overload disease and cancer, as well as neurodegenerative and chronic inflammatory diseases. Established and novel iron chelators will be discussed, as well as the emerging role of dietary plant polyphenols that effectively modulate iron biochemistry.

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