scholarly journals Effect of mRNA Delivery Modality and Formulation on Cutaneous mRNA Distribution and Downstream eGFP Expression

Pharmaceutics ◽  
2022 ◽  
Vol 14 (1) ◽  
pp. 151
Author(s):  
Aditya R. Darade ◽  
Maria Lapteva ◽  
Thomas Hoffmann ◽  
Markus Mandler ◽  
Achim Schneeberger ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Ex Vivo ◽  
Intradermal Injection ◽  
Laser Scanning Microscopy ◽  
Egfp Expression ◽  
Confocal Laser ◽  
Skin Sample ◽  
Messenger Ribonucleic Acid

In vitro transcribed messenger ribonucleic acid (mRNA) constitutes an emerging therapeutic class with several clinical applications. This study presents a systematic comparison of different technologies—intradermal injection, microneedle injection, jet injection, and fractional laser ablation—for the topical cutaneous delivery of mRNA. Delivery of Cy5 labeled mRNA and non-labeled enhanced green fluorescent protein (eGFP) expressing mRNA was investigated in a viable ex vivo porcine skin model and monitored for 48 h. Forty 10 µm-thick horizontal sections were prepared from each skin sample and Cy5 labeled mRNA or eGFP expression visualized as a function of depth by confocal laser scanning microscopy and immunohistochemistry. A pixel-based method was used to create a semi-quantitative biodistribution profile. Different spatial distributions of Cy5 labeled mRNA and eGFP expression were observed, depending on the delivery modality; localization of eGFP expression pointed to the cells responsible. Delivery efficiencies and knowledge of delivery sites can facilitate development of efficient, targeted mRNA-based therapeutics.

scholarly journals Protein-Protein Interactions between Hepatitis C Virus Nonstructural Proteins

2003 ◽  
Vol 77 (9) ◽  
pp. 5401-5414 ◽  
Author(s):  
Maria Dimitrova ◽  
Isabelle Imbert ◽  
Marie Paule Kieny ◽  
Catherine Schuster
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Interactions ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nonstructural Proteins

ABSTRACT Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.

Dynamics of Cytokine Capture Within Hemoadsorption Beads Used to Treat Sepsis

2009 ◽  
Author(s):  
Jeremy D. Kimmel ◽  
Morgan V. DiLeo ◽  
Isabella E. Valenti ◽  
Gregory A. Gibson ◽  
Simon C. Watkins ◽  
...  
Keyword(s):  
Inflammatory Cytokines ◽  
Laser Scanning ◽  
Medical Condition ◽  
Ex Vivo ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Adsorption Dynamics ◽  
The Us ◽  
Rapid Clearance

Sepsis is a serious medical condition characterized by systemic inflammation caused by infection, and affects more than 750,000 individuals per year in the US, with a mortality rate of approximately 30% [1]. The pathophysiology of sepsis is complex and not entirely understood, but is believed to be related to the dysfunction of multiple interdependent humoral mediator pathways, including redundant release of inflammatory cytokines [2]. Removal of both pro- and anti-inflammatory cytokines from the circulating blood is believed to be a promising therapy for severe sepsis [3]. We are developing an extracorporeal hemoadsorption device to remove cytokines from the blood using a novel, biocompatible, sorbent bead technology. A simple model was developed to characterize cytokine adsorption within hemoadsorption beads [4]. Despite rapid clearance of cytokines with hemoadsorption in an ex vivo murine sepsis model [5], our model analysis predicted that only the outer 20μm of each sorbent bead (avg diam = 450μm) adsorbed cytokine. In this work, we used in vitro column capture experiments and confocal laser scanning microscopy (CLSM) to examine cytokine adsorption dynamics within hemoadsorption beads.

scholarly journals Design and Engineering of “Green” Nanoemulsions for Enhanced Topical Delivery of Bakuchiol Achieved in a Sustainable Manner: A Novel Eco-Friendly Approach to Bioretinol

2021 ◽  
Vol 22 (18) ◽  
pp. 10091
Author(s):  
Agnieszka Lewińska ◽  
Marta Domżał-Kędzia ◽  
Ewa Maciejczyk ◽  
Marcin Łukaszewicz ◽  
Urszula Bazylińska
Keyword(s):  
Human Skin ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Rapeseed Meal ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Biological Investigation ◽  
Vis Spectroscopy

In the present work, we establish novel “environmentally-friendly” oil-in-water nanoemulsions to enhance the transdermal delivery of bakuchiol, the so-called “bioretinol” obtained from powdered Psoralea corylifolia seeds via a sustainable process, i.e., using a supercritical fluid extraction approach with pure carbon dioxide (SC-CO2). According to Green Chemistry principles, five novel formulations were stabilized by “green” hybrid ionic surfactants such as coco-betaine—surfactin molecules obtained from coconut and fermented rapeseed meal. Preliminary optimization studies involving three dispersion stability tests, i.e., centrifugation, heating, and cooling cycles, indicated the most promising candidates for further physicochemical analysis. Finally, nanoemulsion colloidal characterization provided by scattering (dynamic and electrophoretic light scattering as well as backscattering), microscopic (transmission electron and confocal laser scanning microscopy), and spectroscopic (UV–Vis spectroscopy) methods revealed the most stable nanocarrier for transdermal biological investigation. In vitro, topical experiments provided on human skin cell line HaCaT keratinocytes and normal dermal NHDF fibroblasts indicated high cell viability upon treatment of the tested formulation with a final 0.02–0.2 mg/mL bakuchiol concentration. This excellent biocompatibility was confirmed by ex vivo and in vivo tests on animal and human skin tissue. The improved permeability and antiaging potential of the bakuchiol-encapsulated rich extract were observed, indicating that the obtained ecological nanoemulsions are competitive with commercial retinol formulations.

scholarly journals ATP sensing in living plant cells reveals tissue gradients and stress dynamics of energy physiology

2017 ◽  
Author(s):  
Valentina De Col ◽  
Philippe Fuchs ◽  
Thomas Nietzel ◽  
Marlene Elsässer ◽  
Chia Pao Voon ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Fluorescent Protein ◽  
Research Training ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
In Planta ◽  
Science Center ◽  
Living Plant

AbstractGrowth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here we establish live MgATP2− assessment in plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2− changes in planta. A MgATP2− map of the Arabidopsis seedling highlights different MgATP2− concentrations between tissues and in individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.One-sentence SummarySensing of MgATP2− by fluorimetry and microscopy allows dissection of ATP fluxes of isolated organelles, and dynamics of cytosolic MgATP2−in vivo.Funding AgenciesThis work was supported by the Deutsche Forschungsgemeinschaft (DFG) through the Emmy-Noether programme (SCHW1719/1-1; M.S. and GR4251/1-1; C.G.), the Research Training Group GRK 2064 (M.S.; A.J.M.), the Priority Program SPP1710 (A.J.M.) and a grant (SCHW1719/5-1; M.S.) as part of the package PAK918. The Seed Fund grant CoSens from the Bioeconomy Science Center, NRW (A.J.M.; M.S.) is gratefully acknowledged. The scientific activities of the Bioeconomy Science Center were financially supported by the Ministry of Innovation, Science and Research within the framework of the NRW Strategieprojekt BioSC (No. 313/323-400-002 13). A.Co. received funding by the Ministero dell’Istruzione, dell’Università e della Ricerca through the FIRB 2010 programme (RBFR10S1LJ_001) and Piano di Sviluppo di Ateneo 2015 (Università degli Studi di Milano). M.Z. received funding by the Ministero dell’Istruzione, dell’Università e della Ricerca (Italy) through the PRIN 2010 programme (PRIN2010CSJX4F). S.W. and T.N. received travel support by the Deutscher Akademischer Austauschdienst (DAAD). V.D.C. was supported by the European Social Fund, Operational Programme 2007/2013, and an Erasmus+ Traineeship grant. M.D.F was supported by The Human Frontier Science Program (RPG0053/2012), and the Leverhulme Foundation (RPG-2015-437). I.M.M. was supported by a grant from the Danish Council for Independent Research - Natural Sciences. V.C.P. was supported by the Innovation and Technology Fund (Funding Support to Partner State Key Laboratories in Hong Kong) of the HKSAR.AbbreviationsAAC – ADP/ATP carrier; AK – adenylate kinase; cAT – carboxyatractyloside; CCCP – carbonyl cyanide m-chlorophenyl hydrazone; CFP – cyan fluorescent protein; CLSM – confocal laser scanning microscopy; ETC – electron transport chain; FRET – Förster Resonance Energy Transfer; LSFM – light sheet fluorescence microscopy.

scholarly journals Skin Targeting of An Optimized Caffeine Nanostructured Lipid Carrier With Improved Efficiency Against Chemotherapy Induced Alopecia

2021 ◽  
Author(s):  
Amna Makky ◽  
Eman Sadddar ◽  
Doaa galaa ◽  
Abeer Khattab
Keyword(s):  
Particle Size ◽  
Laser Scanning ◽  
Ex Vivo ◽  
Entrapment Efficiency ◽  
Tween 80 ◽  
Stability Study ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Nanostructured Lipid Carrier

Abstract The current investigation was designed to develop and optimize caffeine-loaded nanostructured lipid carriers (NLCs) for topical alopecia treatment. Screening of drug solubility in various excipients was executed. The 23 full factorial design was employed for NLCs optimization. Lipid type, surfactant type, and drug concentration were the independent variables. Entrapment efficiency (EE), particle size, polydispersity index (PDI) and % drug release were the chosen responses. Physiochemical evaluation, in vitro release, ex-vivo permeation, and stability study were achieved. The solubility of caffeine in stearic acid and glyceryl monostearate (GMS) was 47.11 ± 3.048 and 32.67 ± 2.955 mg/g, respectively. Oleic acid: garlic oil mixture at ratio 1:1 v/v was the oily phase. Tween 80 and Cremophor EL, Transcutol HP, carbonate buffer (pH 10.8 and ionic strength 200Mm) were chosen as a surfactant, co-surfactant, and aqueous phase, respectively. The optimized formula showed particle size, %EE, PDI, zeta potential of 358nm, 72.55 %, 0.912, -24.8, respectively. The % release was 92.9 ± 4.9 % after 4hours. Confocal laser scanning microscopy showed an improved permeation of caffeine-loaded NLCs to the whole skin layers. The histological examination proved the efficiency of caffeine NLCs optimized formula on promoting hair growth compared to the market formula.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

scholarly journals Effect of dissolution rate and subsequent ion release on cytocompatibility properties of borophosphate glasses

2019 ◽  
Vol 5 (1) ◽  
pp. 85-97
Author(s):  
Nusrat Sharmin ◽  
Mohammad S. Hasan ◽  
Md. Towhidul Islam ◽  
Chengheng Pang ◽  
Fu Gu ◽  
...  
Keyword(s):  
Dissolution Rate ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ion Release ◽  
Scanning Calorimetry ◽  
Cell Culture Study ◽  
Borophosphate Glasses ◽  
Mg63 Cells

AbstractPresent work explores the relationship between the composition, dissolution rate, ion release and cytocompatibility of a series of borophosphate glasses. While, the base glass was selected to be 40mol%P2O5-16mol%CaO-24mol%MgO-20mol%Na2O, three B2O3 modified glass compositions were formulated by replacing Na2O with 1, 5 and 10 mol% B2O3. Ion release study was conducted using inductively coupled plasma atomic emission spectroscopy (ICP-AES). The thermal scans of the glasses as determined by differential scanning calorimetry (DSC) revealed an increment in the thermal properties with increasing B2O3 content in the glasses. On the other hand, the dissolution rate of the glasses decreased with increasing B2O3 content. To identify the effect of boron ion release on the cytocompatibility properties of the glasses, MG63 cells were cultured on the surface of the glass discs. The in vitro cell culture study suggested that glasses with 5 mol% B2O3 (P40B5) showed better cell proliferation and metabolic activity as compares to the glasses with 10 mol% (P40B10) or with no B2O3 (P40B0). The confocal laser scanning microscopy (CLSM) images of live/dead stained MG63 cells attached to the surface of the glasses also revealed that the number of dead cells attached to P40B5 glasses were significantly lower than both P40B0 and P40B10 glasses.

scholarly journals Cephalosporin nitric oxide-donor prodrug DEA-C3D disperses biofilms formed by clinical cystic fibrosis isolates of Pseudomonas aeruginosa

2019 ◽  
Vol 75 (1) ◽  
pp. 117-125 ◽  
Author(s):  
Odel Soren ◽  
Ardeshir Rineh ◽  
Diogo G Silva ◽  
Yuming Cai ◽  
Robert P Howlin ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Clinical Isolates ◽  
Nitric Oxide Donor ◽  
Confocal Laser ◽  
Broth Microdilution Method

Abstract Objectives The cephalosporin nitric oxide (NO)-donor prodrug DEA-C3D (‘DiEthylAmin-Cephalosporin-3′-Diazeniumdiolate’) has been shown to initiate the dispersal of biofilms formed by the Pseudomonas aeruginosa laboratory strain PAO1. In this study, we investigated whether DEA-C3D disperses biofilms formed by clinical cystic fibrosis (CF) isolates of P. aeruginosa and its effect in combination with two antipseudomonal antibiotics, tobramycin and colistin, in vitro. Methods β-Lactamase-triggered release of NO from DEA-C3D was confirmed using a gas-phase chemiluminescence detector. MICs for P. aeruginosa clinical isolates were determined using the broth microdilution method. A crystal violet staining technique and confocal laser scanning microscopy were used to evaluate the effects of DEA-C3D on P. aeruginosa biofilms alone and in combination with tobramycin and colistin. Results DEA-C3D was confirmed to selectively release NO in response to contact with bacterial β-lactamase. Despite lacking direct, cephalosporin/β-lactam-based antibacterial activity, DEA-C3D was able to disperse biofilms formed by three P. aeruginosa clinical isolates. Confocal microscopy revealed that DEA-C3D in combination with tobramycin produces similar reductions in biofilm to DEA-C3D alone, whereas the combination with colistin causes near complete eradication of P. aeruginosa biofilms in vitro. Conclusions DEA-C3D is effective in dispersing biofilms formed by multiple clinical isolates of P. aeruginosa and could hold promise as a new adjunctive therapy to patients with CF.

scholarly journals Use of electromagnetic stimulation on an Enterococcus faecalis biofilm on root canal treated teeth in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Beatriz H. D. Panariello ◽  
Justin K. Kindler ◽  
Kenneth J. Spolnik ◽  
Ygal Ehrlich ◽  
George J. Eckert ◽  
...  
Keyword(s):  
Enterococcus Faecalis ◽  
Root Canal ◽  
Laser Scanning ◽  
Confocal Imaging ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Microscopy Imaging ◽  
Electromagnetic Stimulation ◽  
Root Canal Disinfection

AbstractRoot canal disinfection is of utmost importance in the success of the treatment, thus, a novel method for achieving root canal disinfection by electromagnetic waves, creating a synergistic reaction via electric and thermal energy, was created. To study electromagnetic stimulation (EMS) for the disinfection of root canal in vitro, single rooted teeth were instrumented with a 45.05 Wave One Gold reciprocating file. Specimens were sterilized and inoculated with Enterococcus faecalis ATCC 29,212, which grew for 15 days to form an established biofilm. Samples were treated with 6% sodium hypochlorite (NaOCl), 1.5% NaOCl 1.5% NaOCl with EMS, 0.9% saline with EMS or 0.9% saline. After treatments, the colony forming units (CFU) was determined. Data was analyzed by Wilcoxon Rank Sums Test (α = 0.05). One sample per group was scored and split for confocal laser scanning microscopy imaging. There was a significant effect with the use of NaOCl with or without EMS versus 0.9% saline with or without EMS (p = 0.012 and 0.003, respectively). CFUs were lower when using 0.9% saline with EMS versus 0.9% saline alone (p = 0.002). Confocal imaging confirmed CFU findings. EMS with saline has an antibiofilm effect against E. faecalis and can potentially be applied for endodontic disinfection.

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