The microRNA miR-22 inhibits the histone deacetylase HDAC4 to promote T(H)17 cell-dependent emphysema.

Smoking-related emphysema is a chronic inflammatory disease driven by the T(H)17 subset of helper T cells through molecular mechanisms that remain obscure. Here we explored the role of the microRNA miR-22 in emphysema. We found that miR-22 was upregulated in lung myeloid dendritic cells (mDCs) of smokers with emphysema and antigen-presenting cells (APCs) of mice exposed to smoke or nanoparticulate carbon black (nCB) through a mechanism that involved the transcription factor NF-κB. Mice deficient in miR-22, but not wild-type mice, showed attenuated T(H)17 responses and failed to develop emphysema after exposure to smoke or nCB. We further found that miR-22 controlled the activation of APCs and T(H)17 responses through the activation of AP-1 transcription factor complexes and the histone deacetylase HDAC4. Thus, miR-22 is a critical regulator of both emphysema and T(H)17 responses.

Highly efficient conversion of superoxide to oxygen using hydrophilic carbon clusters.

Many diseases are associated with oxidative stress, which occurs when the production of reactive oxygen species (ROS) overwhelms the scavenging ability of an organism. Here, we evaluated the carbon nanoparticle antioxidant properties of poly(ethylene glycolated) hydrophilic carbon clusters (PEG-HCCs) by electron paramagnetic resonance (EPR) spectroscopy, oxygen electrode, and spectrophotometric assays. These carbon nanoparticles have 1 equivalent of stable radical and showed superoxide (O2 (•-)) dismutase-like properties yet were inert to nitric oxide (NO(•)) as well as peroxynitrite (ONOO(-)). Thus, PEG-HCCs can act as selective antioxidants that do not require regeneration by enzymes. Our steady-state kinetic assay using KO2 and direct freeze-trap EPR to follow its decay removed the rate-limiting substrate provision, thus enabling determination of the remarkable intrinsic turnover numbers of O2 (•-) to O2 by PEG-HCCs at >20,000 s(-1). The major products of this catalytic turnover are O2 and H2O2, making the PEG-HCCs a biomimetic superoxide dismutase.

Synergistic Cysteamine Delivery Nanowafer as an Efficacious Treatment Modality for Corneal Cystinosis.

A synergy between the polymer biomaterial and drug plays an important role in enhancing the therapeutic efficacy, improving the drug stability, and minimizing the local immune responses in the development of drug delivery systems. Particularly, in the case of ocular drug delivery, the need for the development of synergistic drug delivery system becomes more pronounced because of the wet ocular mucosal surface and highly innervated cornea, which elicit a strong inflammatory response to the instilled drug formulations. This article presents the development of a synergistic cysteamine delivery nanowafer to treat corneal cystinosis. Corneal cystinosis is a rare metabolic disease that causes the accumulation of cystine crystals in the cornea resulting in corneal opacity and loss of vision. It is treated with topical cysteamine (Cys) eye drops that need to be instilled 6-12 times a day throughout the patient's life, which causes side effects such as eye pain, redness, and ocular inflammation. As a result, compliance and treatment outcomes are severely compromised. To surmount these issues, we have developed a clinically translatable Cys nanowafer (Cys-NW) that can be simply applied on the eye with a fingertip. During the course of the drug release, Cys-NW slowly dissolves and fades away. The in vivo studies in cystinosin knockout mice demonstrated twice the therapeutic efficacy of Cys-NW containing 10 µg of Cys administered once a day, compared to 44 µg of Cys as topical eye drops administered twice a day. Furthermore, Cys-NW stabilizes Cys for up to four months at room temperature compared to topical Cys eye drops that need to be frozen or refrigerated and still remain active for only 1 week. The Cys-NW, because of its enhanced therapeutic efficacy, safety profile, and extended drug stability at room temperature, can be rapidly translated to the clinic for human trials.

Chronic exposure to carbon black ultrafine particles reprograms macrophage metabolism and accelerates lung cancer.

Chronic exposure to airborne carbon black ultrafine (nCB) particles generated from incomplete combustion of organic matter drives IL-17A-dependent emphysema. However, whether and how they alter the immune responses to lung cancer remains unknown. Here, we show that exposure to nCB particles increased PD-L1+ PD-L2+ CD206+ antigen-presenting cells (APCs), exhausted T cells, and Treg cells. Lung macrophages that harbored nCB particles showed selective mitochondrial structure damage and decreased oxidative respiration. Lung macrophages sustained the HIF1α axis that increased glycolysis and lactate production, culminating in an immunosuppressive microenvironment in multiple mouse models of non-small cell lung cancers. Adoptive transfer of lung APCs from nCB-exposed wild type to susceptible mice increased tumor incidence and caused early metastasis. Our findings show that nCB exposure metabolically rewires lung macrophages to promote immunosuppression and accelerates the development of lung cancer.

Necrotizing Enterocolitis-like Pneumatosis Intestinalis in an Infant With COVID-19.

We report an infant with COVID-19 who presented with bloody stools, lethargy and imaging findings significant for pneumatosis intestinalis. The infant was treated with conservative therapy, including resuscitation, bowel rest and intravenous antibiotics, successfully avoiding surgical intervention.

Dextran Sulfate Polymer Wafer Promotes Corneal Wound Healing.

Eye injuries due to corneal abrasions, chemical spills, penetrating wounds, and microbial infections cause corneal scarring and opacification that result in impaired vision or blindness. However, presently available eye drop formulations of anti-inflammatory and antibiotic drugs are not effective due to their rapid clearance from the ocular surface or due to drug-related side effects such as cataract formation or increased intraocular pressure. In this article, we presented the development of a dextran sulfate-based polymer wafer (DS-wafer) for the effective modulation of inflammation and fibrosis and demonstrated its efficacy in two corneal injury models: corneal abrasion mouse model and alkali induced ocular burn mouse model. The DS-wafers were fabricated by the electrospinning method. We assessed the efficacy of the DS-wafer by light microscopy, qPCR, confocal fluorescence imaging, and histopathological analysis. These studies demonstrated that the DS-wafer treatment is significantly effective in modulating corneal inflammation and fibrosis and inhibited corneal scarring and opacification compared to the unsulfated dextran-wafer treated and untreated corneas. Furthermore, these studies have demonstrated the efficacy of dextran sulfate as an anti-inflammatory and antifibrotic polymer therapeutic.

Application of Hydrogel Template Strategy in Ocular Drug Delivery.

The hydrogel template strategy was previously developed to fabricate homogeneous polymeric microparticles. Here, we demonstrate the versatility of the hydrogel template strategy for the development of nanowafer-based ocular drug delivery systems. We describe the fabrication of dexamethasone-loaded nanowafers using polyvinyl alcohol and the instillation of a nanowafer on a mouse eye. The nanowafer, a small circular disk, is placed on the ocular surface, and it releases a drug as it slowly dissolves over time, thus increasing ocular bioavailability and enhancing efficiency to treat eye injuries.

Dexamethasone nanowafer as an effective therapy for dry eye disease.

Dry eye disease is a major public health problem that affects millions of people worldwide. It is presently treated with artificial tear and anti-inflammatory eye drops that are generally administered several times a day and may have limited therapeutic efficacy. To improve convenience and efficacy, a dexamethasone (Dex) loaded nanowafer (Dex-NW) has been developed that can release the drug on the ocular surface for a longer duration of time than drops, during which it slowly dissolves. The Dex-NW was fabricated using carboxymethyl cellulose polymer and contains arrays of 500 nm square drug reservoirs filled with Dex. The in vivo efficacy of the Dex-NW was evaluated using an experimental mouse dry eye model. These studies demonstrated that once a day Dex-NW treatment on alternate days during a five-day treatment period was able to restore a healthy ocular surface and corneal barrier function with comparable efficacy to twice a day topically applied dexamethasone eye drop treatment. The Dex-NW was also very effective in down regulating expression of inflammatory cytokines (TNF-α, and IFN-γ), chemokines (CXCL-10 and CCL-5), and MMP-3, that are stimulated by dry eye. Despite less frequent dosing, the Dex-NW has comparable therapeutic efficacy to topically applied Dex eye drops in experimental mouse dry eye model, and these results provide a strong rationale for translation to human clinical trials for dry eye.

Ocular drug delivery nanowafer with enhanced therapeutic efficacy.

Presently, eye injuries are treated by topical eye drop therapy. Because of the ocular surface barriers, topical eye drops must be applied several times in a day, causing side effects such as glaucoma, cataract, and poor patient compliance. This article presents the development of a nanowafer drug delivery system in which the polymer and the drug work synergistically to elicit an enhanced therapeutic efficacy with negligible adverse immune responses. The nanowafer is a small transparent circular disc that contains arrays of drug-loaded nanoreservoirs. The slow drug release from the nanowafer increases the drug residence time on the ocular surface and its subsequent absorption into the surrounding ocular tissue. At the end of the stipulated period of drug release, the nanowafer will dissolve and fade away. The in vivo efficacy of the axitinib-loaded nanowafer was demonstrated in treating corneal neovascularization (CNV) in a murine ocular burn model. The laser scanning confocal imaging and RT-PCR study revealed that once a day administered axitinib nanowafer was therapeutically twice as effective, compared to axitinib delivered twice a day by topical eye drop therapy. The axitinib nanowafer is nontoxic and did not affect the wound healing and epithelial recovery of the ocular burn induced corneas. These results confirmed that drug release from the axitinib nanowafer is more effective in inhibiting CNV compared to the topical eye drop treatment even at a lower dosing frequency.

Nanoparticulate carbon black in cigarette smoke induces DNA cleavage and Th17-mediated emphysema.

Chronic inhalation of cigarette smoke is the major cause of sterile inflammation and pulmonary emphysema. The effect of carbon black (CB), a universal constituent of smoke derived from the incomplete combustion of organic material, in smokers and non-smokers is less known. In this study, we show that insoluble nanoparticulate carbon black (nCB) accumulates in human myeloid dendritic cells (mDCs) from emphysematous lung and in CD11c(+) lung antigen presenting cells (APC) of mice exposed to smoke. Likewise, nCB intranasal administration induced emphysema in mouse lungs. Delivered by smoking or intranasally, nCB persisted indefinitely in mouse lung, activated lung APCs, and promoted T helper 17 cell differentiation through double-stranded DNA break (DSB) and ASC-mediated inflammasome assembly in phagocytes. Increasing the polarity or size of CB mitigated many adverse effects. Thus, nCB causes sterile inflammation, DSB, and emphysema and explains adverse health outcomes seen in smokers while implicating the dangers of nCB exposure in non-smokers.

Hydrophilic carbon clusters as therapeutic, high-capacity antioxidants.

Oxidative stress reflects an excessive accumulation of reactive oxygen species (ROS) and is a hallmark of several acute and chronic human pathologies. Although many antioxidants have been investigated, most have demonstrated poor efficacy in clinical trials. Here we discuss the limitations of current antioxidants and describe a new class of nanoparticle antioxidants, poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs). PEG-HCCs show high capacity to annihilate ROS such as superoxide (O2(•-)) and the hydroxyl (HO(•)) radical, show no reactivity toward the nitric oxide radical (NO(•)), and can be functionalized with targeting moieties without loss of activity. Given these properties, we propose that PEG-HCCs offer an exciting new area of study for the treatment of numerous ROS-induced human pathologies.

Biocompatibility of pristine graphene for neuronal interface.

OBJECT: Graphene possesses unique electrical, physical, and chemical properties that may offer significant potential as a bioscaffold for neuronal regeneration after spinal cord injury. The purpose of this investigation was to establish the in vitro biocompatibility of pristine graphene for interface with primary rat cortical neurons. METHODS: Graphene films were prepared by chemical vapor deposition on a copper foil catalytic substrate and subsequent apposition on bare Permanox plastic polymer dishes. Rat neuronal cell culture was grown on graphene-coated surfaces, and cell growth and attachment were compared with those on uncoated and poly-d-lysine (PDL)-coated controls; the latter surface is highly favorable for neuronal attachment and growth. Live/dead cell analysis was conducted with flow cytometry using ethidium homodimer-1 and calcein AM dyes. Lactate dehydrogenase (LDH) levels-indicative of cytotoxicity-were measured as markers of cell death. Phase contrast microscopy of active cell culture was conducted to assess neuronal attachment and morphology. RESULTS: Statistically significant differences in the percentage of live or dead neurons were noted between graphene and PDL surfaces, as well as between the PDL-coated and bare surfaces, but there was little difference in cell viability between graphene-coated and bare surfaces. There were significantly lower LDH levels in the graphene-coated samples compared with the uncoated ones, indicating that graphene was not more cytotoxic than the bare control surface. According to phase contrast microscopy, neurons attached to the graphene-coated surface and were able to elaborate long, neuritic processes suggestive of normal neuronal metabolism and morphology. CONCLUSIONS: Further use of graphene as a bioscaffold will require surface modification that enhances hydrophilicity to increase cellular attachment and growth. Graphene is a nanomaterial that is biocompatible with neurons and may have significant biomedical applications.

Design of poly(ethylene glycol)-functionalized hydrophilic carbon clusters for targeted therapy of cerebrovascular dysfunction in mild traumatic brain injury.

Traumatic brain injury (TBI) involves the elaboration of oxidative stress that causes cerebrovascular dysfunction, including impairment of autoregulation of cerebral blood flow. Currently, there is no clinically effective antioxidant treatment for these pathologies. Most currently available antioxidants act through mechanisms in which the antioxidant either transfers the radical or requires regeneration, both of which are impaired in the toxic post-TBI environment. We previously reported that single-walled carbon nanotubes (SWCNTs) and ultrashort SWCNTs possess antioxidant activity, and their characteristics suggest that radical annihilation is the major mechanism. We have now developed a biologically compatible class of carbon-based nanovectors, poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs) that can be further functionalized with antibodies, and hence show promise as targeted drug delivery platforms. Here we report that PEG-HCCs possess innate antioxidant activity and can be rapidly targeted via an antibody to the P-selectin antigen in a model of injured cultured brain endothelial cells. One immediate application of this therapy is to vascular dysfunction that accompanies TBI and worsens outcome in the face of systemic hypotension. These in vitro results support the need for further investigation in animal models.

Antibody-targeted nanovectors for the treatment of brain cancers.

Introduced here is the hydrophilic carbon clusters (HCCs) antibody drug enhancement system (HADES), a methodology for cell-specific drug delivery. Antigen-targeted, drug-delivering nanovectors are manufactured by combining specific antibodies with drug-loaded poly(ethylene glycol)-HCCs (PEG-HCCs). We show that HADES is highly modular, as both the drug and antibody component can be varied for selective killing of a range of cultured human primary glioblastoma multiforme. Using three different chemotherapeutics and three different antibodies, without the need for covalent bonding to the nanovector, we demonstrate extreme lethality toward glioma, but minimal toxicity toward human astrocytes and neurons.

Noncovalent assembly of targeted carbon nanovectors enables synergistic drug and radiation cancer therapy in vivo.

Current chemotherapeutics are characterized by efficient tumor cell-killing and severe side effects mostly derived from off-target toxicity. Hence targeted delivery of these drugs to tumor cells is actively sought. In an in vitro system, we previously demonstrated that targeted drug delivery to cancer cells overexpressing epidermal growth factor receptor (EGFR+) can be achieved by poly(ethylene glycol)-functionalized carbon nanovectors simply mixed with a drug, paclitaxel, and an antibody that binds to the epidermal growth factor receptor, cetuximab. This construct is unusual in that all three components are assembled through noncovalent interactions. Here we show that this same construct is effective in vivo, enhancing radiotherapy of EGFR+ tumors. This targeted nanovector system has the potential to be a new therapy for head and neck squamous cell carcinomas, deserving of further preclinical development.

Antioxidant carbon particles improve cerebrovascular dysfunction following traumatic brain injury.

Injury to the neurovasculature is a feature of brain injury and must be addressed to maximize opportunity for improvement. Cerebrovascular dysfunction, manifested by reduction in cerebral blood flow (CBF), is a key factor that worsens outcome after traumatic brain injury (TBI), most notably under conditions of hypotension. We report here that a new class of antioxidants, poly(ethylene glycol)-functionalized hydrophilic carbon clusters (PEG-HCCs), which are nontoxic carbon particles, rapidly restore CBF in a mild TBI/hypotension/resuscitation rat model when administered during resuscitation--a clinically relevant time point. Along with restoration of CBF, there is a concomitant normalization of superoxide and nitric oxide levels. Given the role of poor CBF in determining outcome, this finding is of major importance for improving patient health under clinically relevant conditions during resuscitative care, and it has direct implications for the current TBI/hypotension war-fighter victims in the Afghanistan and Middle East theaters. The results also have relevancy in other related acute circumstances such as stroke and organ transplantation.

Noncovalent functionalization of carbon nanovectors with an antibody enables targeted drug delivery.

Current chemotherapeutics are characterized by efficient tumor cell-killing and severe side effects mostly derived from off-target toxicity. Hence targeted delivery of these drugs to tumor cells is actively sought. We previously demonstrated that poly(ethylene glycol)-functionalized carbon nanovectors are able to sequester paclitaxel, a widely used hydrophobic cancer drug, by simple physisorption and thereby deliver the drug for killing of cancer cells. The cell-killing when these drug-loaded carbon nanoparticles were used was equivalent to when a commercial formulation of paclitaxel was used. Here we show that by further mixing the drug-loaded nanoparticles with Cetuximab, a monoclonal antibody that recognizes the epidermal growth factor receptor (EGFR), paclitaxel is preferentially targeted to EGFR+ tumor cells in vitro. This supports progressing to in vivo studies. Moreover, the construct is unusual in that all three components are assembled through noncovalent interactions. Such noncovalent assembly could enable high-throughput screening of drug/antibody combinations.

Towards hybrid superlattices in graphene.

The controllable and reversible modification of graphene by chemical functionalization can modulate its optical and electronic properties. Here we demonstrate the controlled patterning of graphane/graphene superlattices within a single sheet of graphene. By exchanging the sp(3) C-H bonds in graphane with sp(3) C-C bonds through functionalization, sophisticated multifunctional superlattices can be fabricated on both the macroscopic and microscopic scales. These patterns are visualized using fluorescence quenching microscopy techniques and confirmed using Raman spectroscopy. By tuning the extent of hydrogenation, the density of the sp(3) C functional groups on graphene's basal plane can be controlled from 0.4% to 3.5% with this two-step method. Using such a technique, which allows for both spatial and density control of the functional groups, a route to multifunctional electrical circuits and chemical sensors with specifically patterned recognition sites might be realized across a single graphene sheet, facilitating the development of graphene-based devices.

Improved synthesis of graphene oxide.

An improved method for the preparation of graphene oxide (GO) is described. Currently, Hummers' method (KMnO(4), NaNO(3), H(2)SO(4)) is the most common method used for preparing graphene oxide. We have found that excluding the NaNO(3), increasing the amount of KMnO(4), and performing the reaction in a 9:1 mixture of H(2)SO(4)/H(3)PO(4) improves the efficiency of the oxidation process. This improved method provides a greater amount of hydrophilic oxidized graphene material as compared to Hummers' method or Hummers' method with additional KMnO(4). Moreover, even though the GO produced by our method is more oxidized than that prepared by Hummers' method, when both are reduced in the same chamber with hydrazine, chemically converted graphene (CCG) produced from this new method is equivalent in its electrical conductivity. In contrast to Hummers' method, the new method does not generate toxic gas and the temperature is easily controlled. This improved synthesis of GO may be important for large-scale production of GO as well as the construction of devices composed of the subsequent CCG.