scholarly journals Unravelling cytosolic delivery of endosomal escape peptides with a quantitative endosomal escape assay (SLEEQ)

2020 ◽  
Author(s):  
Serena L.Y. Teo ◽  
Joshua J. Rennick ◽  
Daniel Yuen ◽  
Hareth Al-Wassiti ◽  
Angus P.R. Johnston ◽  
...  
Keyword(s):  
Endosomal Escape ◽  
Current Understanding ◽  
Specific Cell ◽  
Essential Requirement ◽  
Intracellular Drug Delivery ◽  
Cytosolic Delivery ◽  
Efficient Delivery ◽  
Highly Sensitive ◽  
Therapeutic Peptides ◽  
Low Sensitivity

AbstractEndosomal escape is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of endosomal escape mechanisms remains limited due to significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we developed a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We applied SLEEQ to evaluate the endosomal escape of a range of widely studied putative endosomal escape peptides (EEPs). We demonstrated that positively-charged EEPs enhanced cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how EEPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.

scholarly journals Unravelling cytosolic delivery of cell penetrating peptides with a quantitative endosomal escape assay

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Serena L. Y. Teo ◽  
Joshua J. Rennick ◽  
Daniel Yuen ◽  
Hareth Al-Wassiti ◽  
Angus P. R. Johnston ◽  
...  
Keyword(s):  
Cell Penetrating Peptides ◽  
Endosomal Escape ◽  
Current Understanding ◽  
Specific Cell ◽  
Intracellular Drug Delivery ◽  
Cytosolic Delivery ◽  
Efficient Delivery ◽  
Cell Penetrating ◽  
Model Protein ◽  
Low Sensitivity

AbstractCytosolic transport is an essential requirement but a major obstacle to efficient delivery of therapeutic peptides, proteins and nucleic acids. Current understanding of cytosolic delivery mechanisms remains limited due to a significant number of conflicting reports, which are compounded by low sensitivity and indirect assays. To resolve this, we develop a highly sensitive Split Luciferase Endosomal Escape Quantification (SLEEQ) assay to probe mechanisms of cytosolic delivery. We apply SLEEQ to evaluate the cytosolic delivery of a range of widely studied cell-penetrating peptides (CPPs) fused to a model protein. We demonstrate that positively charged CPPs enhance cytosolic delivery as a result of increased non-specific cell membrane association, rather than increased endosomal escape efficiency. These findings transform our current understanding of how CPPs increase cytosolic delivery. SLEEQ is a powerful tool that addresses fundamental questions in intracellular drug delivery and will significantly improve the way materials are engineered to increase therapeutic delivery to the cytosol.

scholarly journals Intracellular Delivery of Virus-Like Particles Using a Sheddable Linker

2021 ◽  
Author(s):  
Arezoo Shahrivarkevishahi ◽  
Laurel Hagge ◽  
Zhuo Chen ◽  
Olivia Brohlin ◽  
Alisia Tumac ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Essential Requirement ◽  
Intracellular Targeting ◽  
Cytosolic Delivery ◽  
Virus Like Particle ◽  
Efficient Delivery ◽  
Green Fluorescent ◽  
Mitochondria Targeting ◽  
A549 Lung

Intracellular targeting is an important aspect of the efficient delivery of drugs and nanotherapeutics. Cytosolic transport of nanomaterials is often an essential requirement for therapeutic delivery into cells but remains a challenge owing to the endosomal trap and eventual lysosomal degradation of cargo. To address this, we designed a functional carrier that escapes the endosome and delivers biological materials into the cell's cytoplasm. For this purpose, we synthesized a glutathione-sensitive linker that connects the well-known mitochondria targeting lipophilic triphenylphosphonium cation (TPP) to the surface of a proteinaceous nanoparticle based on the engineered virus-like particle (VLP) Qβ. Once in the cytosol, the thiol sensitive linker severs the TPP from the nanoparticle, halting its trafficking to the mitochondria, and marooning it in the cytosol. We demonstrate the successful in vitro cytosolic delivery of a VLP loaded with Green Fluorescent Protein, where evenly distributed fluorescence is observed in A549 lung cancer cells after four hours. We further demonstrate successful cytosolic delivery by showing that encapsulating siRNA inside the VLP promotes luminescence silencing in luciferase expressing HeLa cells more efficiently than VLPs that lack our sheddable TPP linker.

scholarly journals Terahertz characterization of two-dimensional low-conductive layers enabled by metal gratings

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Prashanth Gopalan ◽  
Yunshan Wang ◽  
Berardi Sensale-Rodriguez
Keyword(s):  
Optical Transmission ◽  
Terahertz Spectroscopy ◽  
Design Guidelines ◽  
Two Dimensional ◽  
Highly Sensitive ◽  
Transmission Measurements ◽  
Metal Gratings ◽  
Low Sensitivity ◽  
Non Destructive

AbstractWhile terahertz spectroscopy can provide valuable information regarding the charge transport properties in semiconductors, its application for the characterization of low-conductive two-dimensional layers, i.e., σs <  < 1 mS, remains elusive. This is primarily due to the low sensitivity of direct transmission measurements to such small sheet conductivity levels. In this work, we discuss harnessing the extraordinary optical transmission through gratings consisting of metallic stripes to characterize such low-conductive two-dimensional layers. We analyze the geometric tradeoffs in these structures and provide physical insights, ultimately leading to general design guidelines for experiments enabling non-contact, non-destructive, highly sensitive characterization of such layers.

Peptide-nucleic acid nanostructures for transfection

2012 ◽  
Vol 3 (3) ◽  
pp. 283-293 ◽  
Author(s):  
Burkhard Bechinger
Keyword(s):  
Nucleic Acid ◽  
Physicochemical Properties ◽  
Cellular Uptake ◽  
Peptide Nucleic Acid ◽  
Endosomal Escape ◽  
Medical Applications ◽  
Specific Cell ◽  
Charge Composition ◽  
Cell Surfaces ◽  
Intracellular Targeting

AbstractTo use nucleic acids in biomedical research and medical applications, these highly hydrophilic macromolecules have to be transported through the organism, targeted to specific cell surfaces, and have to cross cellular barriers. To this end, nanosized transfection complexes have been designed and several of them have been successfully tested. Here, the different steps of the transfection process and the particular optimization protocols are reviewed, including the physicochemical properties of such vectors (size, charge, composition), protection in serum, cellular uptake, endosomal escape, and intracellular targeting. The transfection process has been subdivided into separate steps and here special emphasis is given to peptides that have been designed to optimize these steps individually. Finally, complex devices encompassing a multitude of beneficial functionalities for transfection have been developed.

Boosting dsRNA delivery in plant and insect cells with peptide- and polymer-based carriers: case-based current status and future perspectives.

2021 ◽  
pp. 102-116
Author(s):  
Kristof de Schutter ◽  
Olivier Christiaens ◽  
Clauvis Nji Tizi Taning ◽  
Guy Smagghe
Keyword(s):  
Plant Cell Wall ◽  
Endosomal Escape ◽  
Genomic Research ◽  
Current Status ◽  
Functional Genomic ◽  
Future Perspectives ◽  
Agricultural Industry ◽  
Efficient Delivery ◽  
Rnai Response ◽  
Carrier Systems

Abstract Since the discovery of this naturally occurring endogenous regulatory and defence mechanism, RNA interference (RNAi) has been exploited as a powerful tool for functional genomic research. In addition, it has evolved as a promising candidate for a sustainable, specific and ecofriendly strategy for pest management and plant improvement. A key element in this technology is the efficient delivery of dsRNAs into the pest or plant tissues. While several examples using transgenic plants expressing the dsRNAs have proved the potential of this technology, nontransgenic approaches are investigated as alternatives, allowing flexibility and circumventing technical limitations of the transgenic approach. However, the efficacy of environmental RNAi is affected by several barriers, such as extracellular degradation of the dsRNA, inefficient internalization of the dsRNA in the cell and low endosomal escape into the cytoplasm, resulting in variable or low RNAi responses. In the medical field, carrier systems are commonly used to enhance RNA delivery and these systems are being rapidly adopted by the agricultural industry. Using four case studies, this chapter demonstrates the potential of carriers to improve the RNAi response in pest control for aquatic-living mosquito larvae and RNAi-resilient Lepidoptera and to cross the plant cell wall, allowing efficient environmental RNAi in plants.

scholarly journals Extraction of Bridge Fundamental Frequencies Utilizing a Smartphone MEMS Accelerometer

Sensors ◽  
2019 ◽  
Vol 19 (14) ◽  
pp. 3143 ◽  
Author(s):  
Ahmed Elhattab ◽  
Nasim Uddin ◽  
Eugene OBrien
Keyword(s):  
Stochastic Resonance ◽  
Wireless Sensing ◽  
Mems Accelerometer ◽  
Fundamental Frequencies ◽  
Acceleration Data ◽  
Highly Sensitive ◽  
Output Noise ◽  
Frequency Independent ◽  
Low Sensitivity ◽  
Traffic Operation

Smartphone MEMS (Micro Electrical Mechanical System) accelerometers have relatively low sensitivity and high output noise density. Therefore, it cannot be directly used to track feeble vibrations such as structural vibrations. This article proposes an effective increase in the sensitivity of the smartphone accelerometer utilizing the stochastic resonance (SR) phenomenon. SR is an approach where, counter-intuitively, feeble signals are amplified rather than overwhelmed by the addition of noise. This study introduces the 2D-frequency independent underdamped pinning stochastic resonance (2D-FI-UPSR) technique, which is a customized SR filter that enables identifying the frequencies of weak signals. To validate the feasibility of the proposed SR filter, an iPhone device is used to collect bridge acceleration data during normal traffic operation and the proposed 2D-FI-UPSR filter is used to process these data. The first four fundamental bridge frequencies are successfully identified from the iPhone data. In parallel to the iPhone, a highly sensitive wireless sensing network consists of 15 accelerometers (Silicon Designs accelerometers SDI-2012) is installed to validate the accuracy of the extracted frequencies. The measurement fidelity of the iPhone device is shown to be consistent with the wireless sensing network data with approximately 1% error in the first three bridge frequencies and 3% error in the fourth frequency.

scholarly journals Multivalent dendritic polyglycerolamine with arginine and histidine end groups for efficient siRNA transfection

2015 ◽  
Vol 11 ◽  
pp. 763-772 ◽  
Author(s):  
Fatemeh Sheikhi Mehrabadi ◽  
Hanxiang Zeng ◽  
Mark Johnson ◽  
Cathleen Schlesener ◽  
Zhibin Guan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Transfection Efficiency ◽  
Endosomal Escape ◽  
Primary Amines ◽  
Ph Responsive ◽  
3T3 Cells ◽  
Sirna Transfection ◽  
Efficient Delivery ◽  
End Groups ◽  
Nih 3T3

The success of siRNA-based therapeutics highly depends on a safe and efficient delivery of siRNA into the cytosol. In this study, we post-modified the primary amines on dendritic polyglycerolamine (dPG-NH2) with different ratios of two relevant amino acids, namely, arginine (Arg) and histidine (His). To investigate the effects from introducing Arg and His to dPG, the resulting polyplexes of amino acid functionalized dPG-NH2s (AAdPGs)/siRNA were evaluated regarding cytotoxicity, transfection efficiency, and cellular uptake. Among AAdPGs, an optimal vector with (1:3) Arg to His ratio, showed efficient siRNA transfection with minimal cytotoxicity (cell viability ≥ 90%) in NIH 3T3 cells line. We also demonstrated that the cytotoxicity of dPG-NH2 decreased as a result of amino acid functionalization. While the incorporation of both cationic (Arg) and pH-responsive residues (His) are important for safe and efficient siRNA transfection, this study indicates that AAdPGs containing higher degrees of His display lower cytotoxicity and more efficient endosomal escape.

scholarly journals Highly Sensitive Noninvasive Cardiac Transplant Rejection Monitoring Using Targeted Quantification of Donor-Specific Cell-Free Deoxyribonucleic Acid

2014 ◽  
Vol 63 (12) ◽  
pp. 1224-1226 ◽  
Author(s):  
Mats Hidestrand ◽  
Aoy Tomita-Mitchell ◽  
Pip M. Hidestrand ◽  
Arnold Oliphant ◽  
Mary Goetsch ◽  
...  
Keyword(s):  
Deoxyribonucleic Acid ◽  
Transplant Rejection ◽  
Cardiac Transplant ◽  
Specific Cell ◽  
Highly Sensitive

scholarly journals Xpert MTB/RIF Ultra is highly sensitive for the diagnosis of tuberculosis lymphadenitis in an HIV-endemic setting

2021 ◽  
Author(s):  
Stephanie Minnies ◽  
Byron W.P. Reeve ◽  
Loren Rockman ◽  
Georgina Nyawo ◽  
Charissa C. Naidoo ◽  
...  
Keyword(s):  
Clinical Investigation ◽  
False Negative ◽  
Needle Aspiration ◽  
World Health ◽  
Number Of Patients ◽  
Highly Sensitive ◽  
Specimen Processing ◽  
Increased Sensitivity ◽  
Health Organization ◽  
Low Sensitivity

Background: Tuberculosis lymphadenitis (TBL) is the most common extrapulmonary TB (EPTB) manifestation. Xpert MTB/RIF Ultra (Ultra) is a World Health Organization-endorsed diagnostic test, but performance data for TBL, including on non-invasive specimens, are limited. Methods: Fine needle aspiration biopsies (FNABs) from outpatients (≥18 years) with presumptive TBL (n=135) underwent: 1) routine Xpert (later Ultra once programmatically available), 2) a MGIT 960 culture (if Xpert- or Ultra-negative, or rifampicin-resistant), and 3) study Ultra. Concentrated paired urine underwent Ultra. Primary analyses used a microbiological reference standard (MRS). Results: In a head-to-head comparison (n=92) of FNAB study Ultra and Xpert, Ultra had increased sensitivity [91% (95% confidence interval 79, 98) vs. 72% (57, 84); p=0.016] and decreased specificity [76% (61, 87) vs. 93% (82, 99); p=0.020], and detected patients not on treatment. HIV nor alternative reference standards affected sensitivity and specificity. In patients with both routine and study Ultras, the latter detected more cases [+20% (0, 42); p=0.034] and, further indicative of potential laboratory-based room-for-improvement (e.g., specimen processing optimisation), false-negative study Ultras were more inhibited than true-positives. Study Ultra false-positives had less mycobacterial DNA than true-positives [trace-positive proportions 59% (13/22) vs. 12% (5/51); p<0.001]. “Trace” exclusion or recategorization removed potential benefits offered over Xpert. Urine Ultra had low sensitivity [18% (7, 35)]. Conclusions: Ultra on FNABs is highly sensitive and detects more TBL than Xpert. Patients with FNAB Ultra-positive “trace” results, most of whom will be culture-negative, may require additional clinical investigation. Urine Ultra could reduce the number of patients needing invasive sampling.

scholarly journals Highly Sensitive Flow Sensor Based on Flexible Dual-Layer Heating Structures

Sensors ◽  
2020 ◽  
Vol 20 (22) ◽  
pp. 6657
Author(s):  
Yu-Chao Yan ◽  
Cheng-Yu Jiang ◽  
Run-Bo Chen ◽  
Bing-He Ma ◽  
Jin-Jun Deng ◽  
...  
Keyword(s):  
Heat Conduction ◽  
Convection Heat Transfer ◽  
Single Layer ◽  
Flow Sensor ◽  
Film Sensor ◽  
Highly Sensitive ◽  
Layer Heating ◽  
Low Sensitivity ◽  
Flow Detection ◽  
Hot Film

Hot film sensors detect the flow shear stress based on the forced convection heat transfer to the fluid. Current hot film sensors have been significantly hindered by the relatively low sensitivity due to the massive heat conduction to the substrate. This paper describes the design, fabrication, simulation, and testing of a novel flow sensor with dual-layer hot film structures. More specifically, the heat conduction was insulated from the sensing heater to the substrate by controlling both sensing and guarding heaters working at the same temperature, resulting in a higher sensitivity. The experiment and simulation results showed that the sensitivity of the dual-layer hot film sensor was significantly improved in comparison to the single-layer sensor. Additionally, the dual-layer sensor was designed and fabricated in an integrated, flexible, and miniaturized manner. Its small size makes it an excellent candidate for flow detection.

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