scholarly journals Label-free characterization of organic nanocarriers reveals persistent single molecule cores for hydrocarbon sequestration

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Terry McAfee ◽  
Thomas Ferron ◽  
Isvar A. Cordova ◽  
Phillip D. Pickett ◽  
Charles L. McCormick ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Single Molecule ◽  
New Technologies ◽  
X Rays ◽  
Label Free ◽  
Stimuli Responsive ◽  
Local Environments ◽  
Delivery Platform ◽  
And Function

AbstractSelf-assembled molecular nanostructures embody an enormous potential for new technologies, therapeutics, and understanding of molecular biofunctions. Their structure and function are dependent on local environments, necessitating in-situ/operando investigations for the biggest leaps in discovery and design. However, the most advanced of such investigations involve laborious labeling methods that can disrupt behavior or are not fast enough to capture stimuli-responsive phenomena. We utilize X-rays resonant with molecular bonds to demonstrate an in-situ nanoprobe that eliminates the need for labels and enables data collection times within seconds. Our analytical spectral model quantifies the structure, molecular composition, and dynamics of a copolymer micelle drug delivery platform using resonant soft X-rays. We additionally apply this technique to a hydrocarbon sequestrating polysoap micelle and discover that the critical organic-capturing domain does not coalesce upon aggregation but retains distinct single-molecule cores. This characteristic promotes its efficiency of hydrocarbon sequestration for applications like oil spill remediation and drug delivery. Such a technique enables operando, chemically sensitive investigations of any aqueous molecular nanostructure, label-free.

Fabrication of two-layer nanotubes with the pear-like structure by an in-situ voltage up anodization and the application as a drug delivery platform

2015 ◽  
Vol 647 ◽  
pp. 590-595 ◽  
Author(s):  
Lihong Shi ◽  
Hong Xu ◽  
Xiaoming Liao ◽  
Guangfu Yin ◽  
Yadong Yao ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Delivery Platform

scholarly journals Monoclonal Antibodies Generated against Glycoconjugates Recognize Chemical Linkers

Antibodies ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 48
Author(s):  
Jessica Ramadhin ◽  
Vanessa Silva-Moraes ◽  
Thomas Norberg ◽  
Donald Harn
Keyword(s):  
Drug Delivery ◽  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
Structure And Function ◽  
Enzyme Linked Immunosorbent Assay ◽  
Incomplete Freund’S Adjuvant ◽  
Delivery Platform ◽  
Igg1 Mabs ◽  
Human Milk Oligosaccharide ◽  
And Function

Monoclonal antibodies (mAbs) that recognize glycans are useful tools to assess carbohydrates’ structure and function. We sought to produce IgG mAbs to the human milk oligosaccharide (HMO), lacto-N-fucopentaose III (LNFPIII). LNFPIII contains the Lewisx antigen, which is found on the surface of schistosome parasites. mAbs binding the Lewisx antigen are well-reported in the literature, but mAbs recognizing HMO structures are rare. To generate mAbs, mice were immunized with LNFPIII-DEX (P3DEX) plus CpGs in VacSIM®, a novel vaccine/drug delivery platform. Mice were boosted with LNFPIII-HSA (P3HSA) plus CpGs in Incomplete Freund’s Adjuvant (IFA). Splenocytes from immunized mice were used to generate hybridomas and were screened against LNFPIII conjugates via enzyme-linked immunosorbent assay (ELISA). Three positive hybridomas were expanded, and one hybridoma, producing IgG and IgM antibodies, was cloned via flow cytometry. Clone F1P2H4D8D5 was selected because it produced IgG1 mAbs, but rescreening unexpectedly showed binding to both LNFPIII and lacto-N-neotetraose (LNnT) conjugates. To further assess the specificity of the mAb, we screened it on two glycan microarrays and found no significant binding. This finding suggests that the mAb binds to the acetylphenylenediamine (APD) linker-spacer structure of the conjugate. We present the results herein, suggesting that our new mAb could be a useful probe for conjugates using similar linker spacer structures.

scholarly journals Tanycyte-independent control of hypothalamic leptin signaling

2019 ◽  
Author(s):  
Sooyeon Yoo ◽  
David Cha ◽  
Dong Won Kim ◽  
Thanh V. Hoang ◽  
Seth Blackshaw
Keyword(s):  
Gene Expression ◽  
Single Molecule ◽  
Leptin Receptor ◽  
Cell Types ◽  
Leptin Signaling ◽  
And Function ◽  
Induced Changes ◽  
The Brain ◽  
Specific Deletion

AbstractLeptin is secreted by adipocytes to regulate appetite and body weight. Recent studies have reported that tanycytes actively transport circulating leptin across the brain barrier into the hypothalamus, and are required for normal levels of hypothalamic leptin signaling. However, direct evidence for leptin receptor (LepR) expression is lacking, and the effect of tanycyte-specific deletion of LepR has not been investigated. In this study, we analyze the expression and function of the tanycytic LepR in mice. Using single-molecule fluorescent in situ hybridization (smfISH), RT-qPCR, single-cell RNA sequencing (scRNA-Seq), and selective deletion of the LepR in tanycytes, we are unable to detect expression of LepR in the tanycytes. Tanycyte-specific deletion of LepR likewise did not affect leptin-induced pSTAT3 expression in hypothalamic neurons, regardless of whether leptin was delivered by intraperitoneal or intracerebroventricular injection. Finally, we use activity-regulated scRNA-Seq (act-Seq) to comprehensively profile leptin-induced changes in gene expression in all cell types in mediobasal hypothalamus. Clear evidence for leptin signaling is only seen in endothelial cells and subsets of neurons, although virtually all cell types show leptin-induced changes in gene expression. We thus conclude that LepR expression in tanycytes is either absent or undetectably low, that tanycytes do not directly regulate hypothalamic leptin signaling through a LepR-dependent mechanism, and that leptin regulates gene expression in diverse hypothalamic cell types through both direct and indirect mechanisms.

scholarly journals Bi-fated tendon-to-bone attachment cells are regulated by shared enhancers and KLF transcription factors

2020 ◽  
Author(s):  
Shiri Kult ◽  
Tsviya Olender ◽  
Marco Osterwalder ◽  
Sharon Krief ◽  
Ronnie Blecher-Gonen ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Single Molecule ◽  
Cell Types ◽  
Underlying Mechanism ◽  
Regulatory Elements ◽  
Transcriptional Enhancer ◽  
Molecular Identity ◽  
And Function

AbstractThe connection between different tissues is vital for the development and function of any organs and systems. In the musculoskeletal system, the attachment of elastic tendons to stiff bones poses a mechanical challenge that is solved by the formation of a transitional tissue, which allows the transfer of muscle forces to the skeleton without tearing. Here, we show that tendon-to-bone attachment cells are bi-fated, activating a mixture of chondrocyte and tenocyte transcriptomes, which is regulated by sharing regulatory elements with these cells and by Krüppel-like factors transcription factors (KLF).To uncover the molecular identity of attachment cells, we first applied high-throughput RNA sequencing to murine humeral attachment cells. The results, which were validated by in situ hybridization and single-molecule in situ hybridization, reveal that attachment cells express hundreds of chondrogenic and tenogenic genes. In search for the underlying mechanism allowing these cells to express these genes, we performed ATAC sequencing and found that attachment cells share a significant fraction of accessible intergenic chromatin areas with either tenocytes or chondrocytes. Epigenomic analysis further revealed transcriptional enhancer signatures for the majority of these regions. We then examined a subset of these regions using transgenic mouse enhancer reporter. Results verified the shared activity of some of these enhancers, supporting the possibility that the transcriptome of attachment cells is regulated by enhancers with shared activities in tenocytes or chondrocytes. Finally, integrative chromatin and motif analyses, as well as the transcriptome data, indicated that KLFs are regulators of attachment cells. Indeed, blocking the expression of Klf2 and Klf4 in the developing limb mesenchyme led to abnormal differentiation of attachment cells, establishing these factors as key regulators of the fate of these cells.In summary, our findings show how the molecular identity of bi-fated attachment cells enables the formation of the unique transitional tissue that connect tendon to bone. More broadly, we show how mixing the transcriptomes of two cell types through shared enhancers and a dedicated set of transcription factors can lead to the formation of a new cell fate that connects them.

Charge reversal induced colloidal hydrogel acts as a multi-stimuli responsive drug delivery platform for synergistic cancer therapy

2019 ◽  
Vol 6 (4) ◽  
pp. 711-716 ◽  
Author(s):  
Gang He ◽  
Sheng Chen ◽  
Yunjun Xu ◽  
Zhaohua Miao ◽  
Yan Ma ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Cancer Therapy ◽  
Stimuli Responsive ◽  
Gelatin Nanoparticles ◽  
Charge Reversal ◽  
Delivery Platform ◽  
A Charge

A charge reversal induced colloidal hydrogel composed of amphoteric gelatin nanoparticles (Gela NPs) and melanin-like polydopamine nanoparticles (PDA NPs) was developed for synergistic cancer therapy.

scholarly journals Potential of Stimuli-Responsive In Situ Gel System for Sustained Ocular Drug Delivery: Recent Progress and Contemporary Research

Polymers ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1340
Author(s):  
Manisha Pandey ◽  
Hira Choudhury ◽  
Azila binti Abd Aziz ◽  
Subrat Kumar Bhattamisra ◽  
Bapi Gorain ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Delivery System ◽  
Ocular Drug Delivery ◽  
Eye Drops ◽  
Stimuli Responsive ◽  
In Situ Gel ◽  
In Vivo Models ◽  
The World ◽  
Gel System

Eyesight is one of the most well-deserved blessings, amid all the five senses in the human body. It captures the raw signals from the outside world to create detailed visual images, granting the ability to witness and gain knowledge about the world. Eyes are exposed directly to the external environment; they are susceptible to the vicissitudes of diseases. The World Health Organization has predicted that the number of individuals affected by eye diseases will rise enormously in the next decades. However, the physical barriers of the eyes and the problems associated with conventional ocular formulations are significant challenges in ophthalmic drug development. This has generated the demand for a sustained ocular drug delivery system, which serves to deliver effective drug concentration at a reduced frequency for consistent therapeutic effect and better patient treatment adherence. Recent advancement in pharmaceutical dosage design has demonstrated that a stimuli-responsive in situ gel system exhibits the favorable characteristics for providing sustained ocular drug delivery and enhanced ocular bioavailability. Stimuli-responsive in situ gels undergo a phase transition (solution–gelation) in response to the ocular environmental temperature, pH, and ions. These stimuli transform the formulation into a gel at the cul de sac to overcome the shortcomings of conventional eye drops, such as rapid nasolacrimal drainage and short contact time with the ocular surface This review highlights the recent successful research outcomes of stimuli-responsive in situ gelling systems in treating in vivo models with glaucoma and various ocular infections. Additionally, it also presents the mechanism, recent development, and safety considerations of stimuli-sensitive in situ gel as the potential sustained ocular delivery system for treating common eye disorders.

scholarly journals Single-molecule Dynamic In-Solution Inhibition Assay: A Method for Full Kinetic Profiling of Drug Candidate Binding to GPCRs in Native Membranes

2021 ◽  
Author(s):  
Tim Patrick Kaminski ◽  
Vladimir P Zhdanov ◽  
Fredrik Hook
Keyword(s):  
Single Molecule ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Drug Binding ◽  
Water Soluble ◽  
Drug Candidate ◽  
Surface Immobilization ◽  
Label Free ◽  
And Function ◽  
G Protein Coupled

Kinetic profiling of drug-target interactions using surface-based label-free technologies is well established for water-soluble pharmaceutical targets but is difficult to execute for membrane proteins in general and G-protein-coupled receptors (GPCRs) in particular. That is because surface immobilization of GPCRs tends to alter their configuration and function, leading to low target coverage and non-specific binding. We here describe a novel assay for kinetic profiling of drug binding to the GPCR human beta 2 adrenergic receptor (β2AR). The assay involves temporally-resolved imaging of the binding of individual β2AR-containing cell membrane-derived liposomes to a surface-immobilized ligand in the presence of screened drugs. This approach allowed to determine association and dissociation constants of β2AR and suspended alprenolol (antagonist) and fenoterol (agonist). The setup combines a 384 well-plate sensor chip with automated liquid handling and the assay takes minutes to complete, making it well adapted for drug screening campaigns.

scholarly journals Neonatal enthesis healing involves non-inflammatory formation of acellular scar through ECM secretion by resident cells

2021 ◽  
Author(s):  
Ron Carmel Vinestock ◽  
Neta Felsenthal ◽  
Eran Assaraf ◽  
Eldad Katz ◽  
Sarah Rubin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Cellular Response ◽  
Life Quality ◽  
Transmission Electron ◽  
Injury Model ◽  
Fibrotic Scar ◽  
Musculoskeletal Function ◽  
And Function ◽  
Histology And Immunohistochemistry

Wound healing is a well-orchestrated process that typically recruits the immune and vascular systems to restore the structure and function of the injured tissue. Injuries to the enthesis, a hypocellular and avascular tissue, often result in fibrotic scar formation and loss of mechanical properties, thereby severely affecting musculoskeletal function and life quality. This raises questions about the healing capabilities of the enthesis. Here, we established an injury model to the Achilles entheses of neonatal mice to study the possibility that at an early age, the enthesis can heal more effectively. Histology and immunohistochemistry analyses revealed an atypical process that did not involve inflammation or angiogenesis. Instead, neonatal enthesis healing was mediated by secretion of collagen types I and II by resident cells, which formed a permanent hypocellular and avascular scar. Transmission electron microscopy showed that the cellular response to injury, including ER stress, autophagy and cell death, varied between the tendon and cartilage ends of the enthesis. Single-molecule in situ hybridization, immunostaining, and TUNEL assays verified these differences. Finally, gait analysis showed that these processes effectively restored function of the injured leg. Collectively, these findings reveal a novel healing mechanism in neonatal entheses, whereby local ECM secretion by resident cells forms an acellular ECM deposit in the absence of inflammation markers, allowing gait restoration. These insights into the healing mechanism of a complex transitional tissue may lead to new therapeutic strategies for adult enthesis injuries.

scholarly journals Mass spectrometry imaging of the in situ drug release from nanocarriers

2018 ◽  
Vol 4 (10) ◽  
pp. eaat9039 ◽  
Author(s):  
Jinjuan Xue ◽  
Huihui Liu ◽  
Suming Chen ◽  
Caiqiao Xiong ◽  
Lingpeng Zhan ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Drug Delivery ◽  
Drug Release ◽  
Drug Carrier ◽  
Ionization Mass ◽  
Label Free ◽  
Normal Tissues ◽  
Liver Tissues

It is crucial but of a great challenge to study in vivo and in situ drug release of nanocarriers when developing a nanomaterial-based drug delivery platform. We developed a new label-free laser desorption/ionization mass spectrometry (MS) imaging strategy that enabled visualization and quantification of the in situ drug release in tissues by monitoring intrinsic MS signal intensity ratio of loaded drug over the nanocarriers. The proof of concept was demonstrated by investigating the doxorubicin (DOX)/polyethylene glycol–MoS2 nanosheets drug delivery system in tumor mouse models. The results revealed a tissue-dependent release behavior of DOX during circulation with the highest dissociation in tumor and lowest dissociation in liver tissues. The drug-loaded MoS2 nanocarriers are predominantly distributed in lung, spleen, and liver tissues, whereas the accumulation in the tumor was unexpectedly lower than in normal tissues. This new strategy could also be extended to other drug-carrier systems, such as carbon nanotubes and black phosphorus nanosheets, and opened a new path to evaluate the drug release of nanocarriers in the suborgan level.

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