Investigating the Fate of MP1000-LPX In Vivo by Adding Serum to Transfection Medium

2020 ◽  
Vol 8 (5) ◽  
pp. 399-408
Author(s):  
Siyan He ◽  
Shan Xia ◽  
Xiangrong Song ◽  
Hai Huang ◽  
Xueyan Wang ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Tumor Antigens ◽  
Imaging System ◽  
Previous Method ◽  
Organ Distribution ◽  
Transfection Efficacy ◽  
Intracellular Process ◽  
Cellular Transfection

Background: Cationic liposomes (CLs) based messenger RNA (mRNA) vaccine has been a promising approach for cancer treatment. However, rapid lung accumulation after intraveous injection and significantly decreased transfection efficacy (TE) in serum substantially hamper its application. Objective: In this study, we attempt to investigate the fate of Mannose-PEG1000-lipoplex (MP1000-LPX) in vivo, a previous reported mRNA vaccine, and potential mechanism in it. Methods: MP1000-CLs and different type of MP1000-LPX were produced by previous method and characterized by dynamic light scattering (DLS). Organ distribution and Luc-mRNA expression of DiD loaded luciferase (Luc-mRNA)-MP1000-LPX were evaluated by IVIS Spectrum imaging system. Cellular transfection and uptake under serum-free and serum-containing conditions were analysed by flow cytometry and counted by FlowJo software. Results: MP1000-CLs had an average size of 45.3 ± 0.9 nm, a positive charge of 39.9 ± 0.9 mV. When MP1000-LPX formed, the particle size increased to about 130 nm, and zeta potential decreased to about 30 mV. All formulations were in narrow size distribution with PDI < 0.3. 6 h after intraveous injection, Luc-MP1000-LPX mostly distributed to liver, lung and spleen, while only successfully expressed Luc in lung. DC2.4 cellular transfection assay indicated serum substantially lowered TE of MP1000-LPX. However, the cellular uptake on DC2.4 cells was enhanced in the presence of serum. Conclusion: MP1000-LPX distributed to spleen but failed to transfect. Because serum dramatically decreased TE of MP1000-LPX on DC2.4 cells, but not by impeding its interaction to cell membrane. Serum resistance and avoidance of lung accumulation might be prerequisites for CLs based intravenous mRNA vaccines. Lay Summary: mRNA vaccine has been promising immunotherapy to treat cancer by delivering mRNA encoding tumor antigens to APCs and activating immune system against tumor cells. We are investigating the in vivo fate of MP1000-LPX, a CLs based mRNA vaccine. To see if serum causes the fate, we’ll be looking at the influence of serum on transfection and uptake efficacy of MP1000-LPX by DC2.4 cells experiments in vitro. Our findings will imply that serum inhibits transfection but not by decreasing uptake. Thus, we can ultilize serum to enhance transfection if we make intracellular process of MP1000-LPX successful.

scholarly journals Targeting Beclin1 as an Adjunctive Therapy against HIV Using Mannosylated Polyethylenimine Nanoparticles

Pharmaceutics ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 223
Author(s):  
Myosotys Rodriguez ◽  
Yemmy Soler ◽  
Mohan Kumar Muthu Karuppan ◽  
Yuling Zhao ◽  
Elena V. Batrakova ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Imaging System ◽  
Organ Distribution ◽  
Nissl Staining ◽  
Necrosis Factor Alpha ◽  
Human Microglia ◽  
Human Astrocytes ◽  
The Brain

Using nanoparticle-based RNA interference (RNAi), we have previously shown that silencing the host autophagic protein, Beclin1, in HIV-infected human microglia and astrocytes restricts HIV replication and its viral-associated inflammatory responses. Here, we confirmed the efficacy of Beclin1 small interfering RNA (siBeclin1) as an adjunctive antiviral and anti-inflammatory therapy in myeloid human microglia and primary human astrocytes infected with HIV, both with and without exposure to combined antiretroviral (cART) drugs. To specifically target human microglia and human astrocytes, we used a nanoparticle (NP) comprised of linear cationic polyethylenimine (PEI) conjugated with mannose (Man) and encapsulated with siBeclin1. The target specificity of the PEI-Man NP was confirmed in vitro using human neuronal and glial cells transfected with the NP encapsulated with fluorescein isothiocyanate (FITC). PEI-Man-siBeclin1 NPs were intranasally delivered to healthy C57BL/6 mice in order to report the biodistribution of siBeclin1 in different areas of the brain, measured using stem-loop RT-PCR. Postmortem brains recovered at 1–48 h post-treatment with the PEI-Man-siRNA NP showed no significant changes in the secretion of the chemokines regulated on activation, normal T cell expressed and secreted (RANTES) and monocyte chemotactic protein-1 (MCP-1) and showed significant decreases in the secretion of the cytokines interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) when compared to phosphate-buffered saline (PBS)-treated brains. Nissl staining showed minimal differences between the neuronal structures when compared to PBS-treated brains, which correlated with no adverse behavioral affects. To confirm the brain and peripheral organ distribution of PEI-siBeclin1 in living mice, we used the In vivo Imaging System (IVIS) and demonstrated a significant brain accumulation of siBeclin1 through intranasal administration.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

scholarly journals MSCs-engineered biomimetic PMAA nanomedicines for multiple bioimaging-guided and photothermal-enhanced radiotherapy of NSCLC

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Yipengchen Yin ◽  
Yongjing Li ◽  
Sheng Wang ◽  
Ziliang Dong ◽  
Chao Liang ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Cell Membranes ◽  
Imaging System ◽  
Fluorescence Signal ◽  
Bimodal Imaging ◽  
Red Blood Cell Membranes ◽  
Magnetic Resonance Imaging Mri ◽  
Tumor Accumulation

Abstract Background The recently developed biomimetic strategy is one of the mostly effective strategies for improving the theranostic efficacy of diverse nanomedicines, because nanoparticles coated with cell membranes can disguise as “self”, evade the surveillance of the immune system, and accumulate to the tumor sites actively. Results Herein, we utilized mesenchymal stem cell memabranes (MSCs) to coat polymethacrylic acid (PMAA) nanoparticles loaded with Fe(III) and cypate—an derivative of indocyanine green to fabricate Cyp-PMAA-Fe@MSCs, which featured high stability, desirable tumor-accumulation and intriguing photothermal conversion efficiency both in vitro and in vivo for the treatment of lung cancer. After intravenous administration of Cyp-PMAA-Fe@MSCs and Cyp-PMAA-Fe@RBCs (RBCs, red blood cell membranes) separately into tumor-bearing mice, the fluorescence signal in the MSCs group was 21% stronger than that in the RBCs group at the tumor sites in an in vivo fluorescence imaging system. Correspondingly, the T1-weighted magnetic resonance imaging (MRI) signal at the tumor site decreased 30% after intravenous injection of Cyp-PMAA-Fe@MSCs. Importantly, the constructed Cyp-PMAA-Fe@MSCs exhibited strong photothermal hyperthermia effect both in vitro and in vivo when exposed to 808 nm laser irradiation, thus it could be used for photothermal therapy. Furthermore, tumors on mice treated with phototermal therapy and radiotherapy shrank 32% more than those treated with only radiotherapy. Conclusions These results proved that Cyp-PMAA-Fe@MSCs could realize fluorescence/MRI bimodal imaging, while be used in phototermal-therapy-enhanced radiotherapy, providing desirable nanoplatforms for tumor diagnosis and precise treatment of non-small cell lung cancer.

Induction of Winter Flounder Antifreeze Protein Messenger RNA at 4 C in vivo and in vitro

1986 ◽  
Vol 59 (6) ◽  
pp. 679-695 ◽  
Author(s):  
Jeffrey L. Price ◽  
Brian B. Gourlie ◽  
Yuan Lin ◽  
Ru Chih C. Huang
Keyword(s):  
Messenger Rna ◽  
Antifreeze Protein ◽  
Winter Flounder

scholarly journals A Tumbling Magnetic Microrobot System for Biomedical Applications

Micromachines ◽  
2020 ◽  
Vol 11 (9) ◽  
pp. 861
Author(s):  
Elizabeth E. Niedert ◽  
Chenghao Bi ◽  
Georges Adam ◽  
Elly Lambert ◽  
Luis Solorio ◽  
...  
Keyword(s):  
Ultrasound Imaging ◽  
Biomedical Applications ◽  
Imaging System ◽  
Ex Vivo ◽  
Negative Control ◽  
Circular Path ◽  
Rotational Axis ◽  
Significant Difference

A microrobot system comprising an untethered tumbling magnetic microrobot, a two-degree-of-freedom rotating permanent magnet, and an ultrasound imaging system has been developed for in vitro and in vivo biomedical applications. The microrobot tumbles end-over-end in a net forward motion due to applied magnetic torque from the rotating magnet. By turning the rotational axis of the magnet, two-dimensional directional control is possible and the microrobot was steered along various trajectories, including a circular path and P-shaped path. The microrobot is capable of moving over the unstructured terrain within a murine colon in in vitro, in situ, and in vivo conditions, as well as a porcine colon in ex vivo conditions. High-frequency ultrasound imaging allows for real-time determination of the microrobot’s position while it is optically occluded by animal tissue. When coated with a fluorescein payload, the microrobot was shown to release the majority of the payload over a 1-h time period in phosphate-buffered saline. Cytotoxicity tests demonstrated that the microrobot’s constituent materials, SU-8 and polydimethylsiloxane (PDMS), did not show a statistically significant difference in toxicity to murine fibroblasts from the negative control, even when the materials were doped with magnetic neodymium microparticles. The microrobot system’s capabilities make it promising for targeted drug delivery and other in vivo biomedical applications.

The Role of microRNAs in Osteoporosis Diagnostics

2021 ◽  
Vol 30 (03) ◽  
pp. 222-229
Author(s):  
Matthias Hackl ◽  
Elisabeth Semmelrock ◽  
Johannes Grillari
Keyword(s):  
Bone Mass ◽  
Bone Metabolism ◽  
Messenger Rna ◽  
Biological Fluids ◽  
Clinical Diagnostics ◽  
Bone Architecture ◽  
Estrogen Metabolism ◽  
Age Related

AbstractMicroRNAs (miRNAs) are short (18–24 nucleotides) non-coding RNA sequences that regulate gene expression via binding of messenger RNA. It is estimated that miRNAs co-regulate the expression of more than 70% of all human genes, many of which fulfil important roles in bone metabolism and muscle function. In-vitro and in-vivo experiments have shown that the targeted loss of miRNAs in distinct bone cell types (osteoblasts and osteoclasts) results in altered bone mass and bone architecture. These results emphasize the biological relevance of miRNAs for bone health.MiRNAs are not only considered as novel bone biomarkers because of their biological importance to bone metabolism, but also on the basis of other favorable properties: 1) Secretion of miRNAs from cells enables “minimally invasive” detection in biological fluids such as serum. 2) High stability of miRNAs in serum enables the retrospective analysis of frozen blood specimens. 3) Quantification of miRNAs in the serum is based on the RT-PCR - a robust method that is considered as the gold standard for the analysis of nucleic acids in clinical diagnostics.With regard to osteoporosis, it has been shown that many of the known risk factors are characterized by distinct miRNA profiles in the affected tissues: i) age-related loss of bone mass, ii) sarcopenia, iii) changes in estrogen metabolism and related changes Loss of bone mass, and iv) diabetes. Therefore, numerous studies in recent years have dealt with the characterization of miRNAs in the serum of osteoporosis patients and healthy controls, and were able to identify recurring miRNA patterns that are characteristic of osteoporosis. These novel biomarkers have great potential for the diagnosis and prognosis of osteoporosis and its clinical outcomes.The aim of this article is to give a summary of the current state of knowledge on the research and application of miRNA biomarkers in osteoporosis.

Differential Effects of Halothane and Thiopental on Surfactant Protein C Messenger RNA  In Vivo and  In Vitro in Rats 

2000 ◽  
Vol 93 (3) ◽  
pp. 805-810 ◽  
Author(s):  
Catherine Paugam-Burtz ◽  
Serge Molliex ◽  
Bernard Lardeux ◽  
Corinne Rolland ◽  
Michel Aubier ◽  
...  
Keyword(s):  
Messenger Rna ◽  
Protein C ◽  
Alveolar Type ◽  
Type Ii Cells ◽  
Type Ii ◽  
Alveolar Type Ii Cells ◽  
Mechanically Ventilated ◽  
Mrna Content

Background Pulmonary surfactant is a complex mixture of proteins and phospholipids synthetized by alveolar type II cells. Volatile anesthetics have been shown to reduce surfactant phospholipid biosynthesis by rat alveolar type II cells. Surfactant-associated protein C (SP-C) is critical for the alveolar surfactant functions. Our goal was to evaluate the effects of halothane and thiopental on SP-C messenger RNA (mRNA) expression in vitro in rat alveolar type II cells and in vivo in mechanically ventilated rats. Methods In vitro, freshly isolated alveolar type II cells were exposed to halothane during 4 h (1, 2, 4%) and 8 h (1%), and to thiopental during 4 h (10, 100 micrometer) and 8 h (100 micrometer). In vivo, rats were anesthetized with intraperitoneal thiopental or inhaled 1% halothane and mechanically ventilated for 4 or 8 h. SP-C mRNA expression was evaluated by ribonuclease protection assay. Results In vitro, 4-h exposure of alveolar type II cells to thiopental 10 and 100 micrometer increased their SP-C mRNA content to 145 and 197%, respectively, of the control values. In alveolar type II cells exposed for 4 h to halothane 1, 2, and 4%, the SP-C mRNA content increased dose-dependently to 160, 235, and 275%, respectively, of the control values. In vivo, in mechanically ventilated rats, 4 h of halothane anesthesia decreased the lung SP-C mRNA content to 53% of the value obtained in control (nonanesthetized, nonventilated) animals; thiopental anesthesia increased to 150% the lung SP-C mRNA content. Conclusions These findings indicate that halothane and thiopental used at clinically relevant concentrations modulate the pulmonary SP-C mRNA content in rats. In vivo, the additive role of mechanical ventilation is suggested.

scholarly journals Relative messenger RNA expression profiling of collagenases and aggrecanases in human articular chondrocytes in vivo and in vitro

2002 ◽  
Vol 46 (10) ◽  
pp. 2648-2657 ◽  
Author(s):  
Brigitte Bau ◽  
Pia M. Gebhard ◽  
Jochen Haag ◽  
Thomas Knorr ◽  
Eckart Bartnik ◽  
...  
Keyword(s):  
Expression Profiling ◽  
Messenger Rna ◽  
Articular Chondrocytes ◽  
Human Articular Chondrocytes ◽  
Rna Expression ◽  
Rna Expression Profiling

scholarly journals Visualization of SARS-CoV-2 infection dynamic

2021 ◽  
Author(s):  
Chengjin Ye ◽  
Kevin Chiem ◽  
Jun-Gyu Park ◽  
Jesus Silvas ◽  
Desarey Morales Vasquez ◽  
...  
Keyword(s):  
Viral Infection ◽  
Cultured Cells ◽  
Imaging System ◽  
Reporter Genes ◽  
Therapeutic Interventions ◽  
Wild Type Virus ◽  
Nucleocapsid Gene ◽  
Recombinant Viruses

Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing rSARS-CoV-2 have jeopardized their use to monitor the dynamics of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the viral nucleocapsid gene followed by a 2A cleavage peptide. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and K18 hACE2 transgenic mice. Importantly, real-time viral infection was readily tracked using a non-invasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2 retained wild-type virus like pathogenicity in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

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