scholarly journals DNA with zwitterionic and negatively charged phosphate modifications: Formation of DNA triplexes, duplexes and cell uptake studies

2021 ◽  
Vol 17 ◽  
pp. 749-761
Author(s):  
Yongdong Su ◽  
Maitsetseg Bayarjargal ◽  
Tracy K Hale ◽  
Vyacheslav V Filichev
Keyword(s):  
Mouse Fibroblast ◽  
Cell Uptake ◽  
Dna Duplexes ◽  
Nih3t3 Cells ◽  
Dna Triplexes ◽  
Snake Venom Phosphodiesterase ◽  
Nuclease Digestion ◽  
Dna Backbone ◽  
Uptake Studies

Two phosphate modifications were introduced into the DNA backbone using the Staudinger reaction between the 3’,5’-dinucleoside β-cyanoethyl phosphite triester formed during DNA synthesis and sulfonyl azides, 4-(azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide (N+ azide) or p-toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts modification in the DNA sequence. The incorporation of these N+ and Ts modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts-modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs with modification in the middle of the sequence). Additionally, the incorporation of N+ modifications led to the formation of duplexes with a thermal stability that was less dependent on the ionic strength than native DNA duplexes. The thermodynamic analysis of the melting curves revealed that it is the reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell uptake studies showed that Ts-ONs can enter the nucleus of mouse fibroblast NIH3T3 cells without any transfection reagent, whereas, N+ONs remain concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts-modified ONs are promising for various in vivo applications.

scholarly journals DNA with zwitterionic and negatively charged phosphate modifications: formation of DNA triplexes, duplexes and cell permeability studies

2020 ◽  
Author(s):  
Yongdong Su ◽  
Maitsetseg Bayarjargal ◽  
Tracy K Hale ◽  
Vyacheslav V Filichev
Keyword(s):  
Mouse Fibroblast ◽  
Cell Permeability ◽  
Dna Duplexes ◽  
Nih3t3 Cells ◽  
Dna Triplexes ◽  
Snake Venom Phosphodiesterase ◽  
Nuclease Digestion ◽  
Dna Backbone ◽  
Permeability Studies

Two phosphate modifications were introduced into the DNA backbone using Staudinger reaction between the 3’,5’-dinucleoside β-cyanoethyl phosphite triester formed during DNA synthesis and the sulfonyl azides, 4-(azidosulfonyl)-N,N,N-trimethylbutan-1-aminium iodide (N+ azide) or p-toluenesulfonyl (tosyl or Ts) azide, to provide either a zwitterionic phosphoramidate with N+ modification or a negatively charged phosphoramidate for Ts- modification in the DNA sequence. Incorporation of these N+ and Ts- modifications led to the formation of thermally stable parallel DNA triplexes, regardless of the number of modifications incorporated into the oligodeoxynucleotides (ONs). For both N+ and Ts- modified ONs, the antiparallel duplexes formed with complementary RNA were more stable than those formed with complementary DNA (except for ONs where the modification is in the middle of the sequence). Incorporation of N+ modifications led to the formation of duplexes whose thermal stability was less dependent on ionic strength than native DNA duplexes. Thermodynamic analysis of melting curves revealed that it is a reduction in unfavourable entropy, despite the decrease in favourable enthalpy, which is responsible for the stabilisation of duplexes with N+ modification. N+ ONs also demonstrated greater resistance to nuclease digestion by snake venom phosphodiesterase I than the corresponding Ts-ONs. Cell permeability studies showed that Ts- ONs diffuse into the nucleus of mouse fibroblast NIH3T3 cells without the need for transfection reagents. In contrast, N+ ONs were concentrated in vesicles within the cytoplasm. These results indicate that both N+ and Ts- modified ONs are promising for various in vivo applications.

scholarly journals H-CRRETAWAC-OH, a Lead Structure for the Development of Radiotracer Targeting Integrinα5β1?

2014 ◽  
Vol 2014 ◽  
pp. 1-12 ◽  
Author(s):  
Roland Haubner ◽  
Simone Maschauer ◽  
Jürgen Einsiedel ◽  
Iris E. Eder ◽  
Christine Rangger ◽  
...  
Keyword(s):  
Specific Binding ◽  
Radiochemical Yield ◽  
Human Melanoma ◽  
Cell Uptake ◽  
Preclinical Research ◽  
M Cell ◽  
Biodistribution Studies ◽  
Uptake Studies

Imaging of angiogenic processes is of great interest in preclinical research as well as in clinical settings. The most commonly addressed target structure for imaging angiogenesis is the integrinαvβ3. Here we describe the synthesis and evaluation of [18F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH, a radiolabelled peptide designed to selectively target the integrinα5β1. Conjugation of 4-nitrophenyl-(RS)-2-[18F]fluoropropionate provided [18F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH in high radiochemical purity (>95%) and a radiochemical yield of approx. 55%. In vitro evaluation showedα5β1binding affinity in the nanomolar range, whereas affinity toαvβ3andαIIbβ3was >50 μM. Cell uptake studies using human melanoma M21 (αvβ3-positive andα5β1-negative), human melanoma M21-L (αvβ3-negative andα5β1-negative), and human prostate carcinoma DU145 (αvβ3-negative andα5β1-positive) confirmed receptor-specific binding. The radiotracer was stable in human serum and showed low protein binding. Biodistribution studies showed tumour uptake ranging from 2.5 to 3.5% ID/g between 30 and 120 min post-injection. However, blocking studies and studies using mice bearingα5β1-negative M21 tumours did not confirm receptor-specific uptake of [18F]FProp-Cys*-Arg-Arg-Glu-Thr-Ala-Trp-Ala-Cys*-OH, although this radiopeptide revealed high affinity and substantial selectivity toα5β1in vitro. Further experiments are needed to study the in vivo metabolism of this peptide and to develop improved radiopeptide candidates suitable for PET imaging ofα5β1expression in vivo.

scholarly journals Development of a Triazolobenzodiazepine-Based PET Probe for Subtype-Selective Vasopressin 1A Receptor Imaging

2021 ◽  
Author(s):  
Ahmed Haider ◽  
Zhiwei Xiao ◽  
Xiaotian Xia ◽  
Jiahui Chen ◽  
Richard S. Van ◽  
...  
Keyword(s):  
Pet Imaging ◽  
Chinese Hamster ◽  
Autism Spectrum ◽  
Cell Uptake ◽  
Structural Basis ◽  
Receptor Imaging ◽  
V1a Receptor ◽  
Uptake Studies ◽  
Subtype Selective

AbstractObjectivesVasopressin 1A (V1A) receptors have been linked to autism spectrum disorder, heart failure, diabetes and renal disease. Currently, there is a lack of validated probes for clinical V1A-targeted imaging and previous PET studies have primarily focused on the brain. To enable non-invasive real-time quantification of V1A receptors in peripheral organs, we sought to develop a suitable PET radioligand that would allow specific and selective V1A receptor imaging in vivo.MethodsThe previously reported triazolobenzodiazepine-based V1A antagonist, PF-184563, served as a structural basis for the development of a suitable V1A-targeted PET probe. Initially, PF-184563 and the respective desmethyl precursor for radiolabeling were synthesized via multistep organic synthesis. Inhibitory constants of PF-184563 for V1A, V1B, V2 and oxytocin (OT) receptors were assessed by competitive radioligand binding or fluorescent-based assays. Molecular docking of PF-184563 to the V1A receptor binding pocket was performed to corroborate the high binding affinity, while carbon-11 labeling was accomplished via radiomethylation. To assess the utility of the resulting PET radioligand, [11C]17, cell uptake studies were performed in a human V1A receptor Chinese hamster ovary (CHO) cell line under baseline and blockade conditions, using a series of V1A, V1B and V2 antagonists in >100-fold excess. Further, to show in vivo specificity, we conducted PET imaging and biodistribution experiments, thereby co-administering the clinical V1A-antagonist, balovaptan (3mg/kg), as a blocking agent.ResultsPF-184563 and the respective desmethyl precursor were synthesized in an overall yield of 49% (over 7 steps) and 40% (over 8 steps), respectively. A subnanomolar inhibitory constant (Ki) of 0.9 nM towards the V1A receptor was observed for PF-184563, while the triazolobenzodiazepine derivative concurrently exhibited excellent selectivity over the related V1B, V2 and OT receptor (IC50 >10,000 nM). [11C]17 was obtained in high radiochemical purity (> 99%), molar activities ranging from 37 - 46 GBq/μmol and a non-decay-corrected radiochemical yield of 8%. Cell uptake studies revealed considerable tracer binding, which was significantly reduced in the presence of V1A antagonists. Conversely, there was no significant blockade in the presence of V1B and V2 antagonists. PET imaging and biodistribution studies in CD-1 mice indicated specific tracer binding in the thyroid, pancreas, spleen and the heart.ConclusionWe report the development of a V1A-targeted PET radioligand that is suitable for subtype-selective in vitro and in vivo receptor imaging. Indeed, [11C]17 proved to specifically visualize V1A receptors in several organs including the heart, pancreas, spleen and thyroid. These results suggest that [11C]17 can be a valuable tool to study the role of V1A receptors in cardiovascular and immune-mediated pathologies.

scholarly journals Syndecan Transmembrane Domain Specifically Regulates Downstream Signaling Events of the Transmembrane Receptor Cytoplasmic Domain

2021 ◽  
Vol 22 (15) ◽  
pp. 7918
Author(s):  
Jisun Hwang ◽  
Bohee Jang ◽  
Ayoung Kim ◽  
Yejin Lee ◽  
Joonha Lee ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Growth Factor Receptor ◽  
Cytoplasmic Domain ◽  
Nih3t3 Cells ◽  
C Elegans ◽  
Downstream Signaling ◽  
Downstream Effectors ◽  
Signaling Events

Despite the known importance of the transmembrane domain (TMD) of syndecan receptors in cell adhesion and signaling, the molecular basis for syndecan TMD function remains unknown. Using in vivo invertebrate models, we found that mammalian syndecan-2 rescued both the guidance defects in C. elegans hermaphrodite-specific neurons and the impaired development of the midline axons of Drosophila caused by the loss of endogenous syndecan. These compensatory effects, however, were reduced significantly when syndecan-2 dimerization-defective TMD mutants were introduced. To further investigate the role of the TMD, we generated a chimera, 2eTPC, comprising the TMD of syndecan-2 linked to the cytoplasmic domain of platelet-derived growth factor receptor (PDGFR). This chimera exhibited SDS-resistant dimer formation that was lost in the corresponding dimerization-defective syndecan-2 TMD mutant, 2eT(GL)PC. Moreover, 2eTPC specifically enhanced Tyr 579 and Tyr 857 phosphorylation in the PDGFR cytoplasmic domain, while the TMD mutant failed to support such phosphorylation. Finally, 2eTPC, but not 2eT(GL)PC, induced phosphorylation of Src and PI3 kinase (known downstream effectors of Tyr 579 phosphorylation) and promoted Src-mediated migration of NIH3T3 cells. Taken together, these data suggest that the TMD of a syndecan-2 specifically regulates receptor cytoplasmic domain function and subsequent downstream signaling events controlling cell behavior.

scholarly journals Effects of chitosan-collagen dressing on wound healing in vitro and in vivo assays

2021 ◽  
Vol 19 ◽  
pp. 228080002198969
Author(s):  
Min-Xia Zhang ◽  
Wan-Yi Zhao ◽  
Qing-Qing Fang ◽  
Xiao-Feng Wang ◽  
Chun-Ye Chen ◽  
...  
Keyword(s):  
Wound Healing ◽  
Wound Dressing ◽  
Type I Collagen ◽  
Inhibition Zone ◽  
Negative Control ◽  
Type I ◽  
Nih3t3 Cells ◽  
Post Operation

The present study was designed to fabricate a new chitosan-collagen sponge (CCS) for potential wound dressing applications. CCS was fabricated by a 3.0% chitosan mixture with a 1.0% type I collagen (7:3(w/w)) through freeze-drying. Then the dressing was prepared to evaluate its properties through a series of tests. The new-made dressing demonstrated its safety toward NIH3T3 cells. Furthermore, the CCS showed the significant surround inhibition zone than empty controls inoculated by E. coli and S. aureus. Moreover, the moisture rates of CCS were increased more rapidly than the collagen and blank sponge groups. The results revealed that the CCS had the characteristics of nontoxicity, biocompatibility, good antibacterial activity, and water retention. We used a full-thickness excisional wound healing model to evaluate the in vivo efficacy of the new dressing. The results showed remarkable healing at 14th day post-operation compared with injuries treated with collagen only as a negative control in addition to chitosan only. Our results suggest that the chitosan-collagen wound dressing were identified as a new promising candidate for further wound application.

scholarly journals The application prospect of metal/metal oxide nanoparticles in the treatment of osteoarthritis

2021 ◽  
Vol 394 (10) ◽  
pp. 1991-2002
Author(s):  
Junchao Luo ◽  
Yin Zhang ◽  
Senbo Zhu ◽  
Yu Tong ◽  
Lichen Ji ◽  
...  
Keyword(s):  
Metal Oxide ◽  
Metal Oxide Nanoparticles ◽  
Biological Response ◽  
Cell Uptake ◽  
Oxide Nanoparticles ◽  
Therapeutic Effects ◽  
Superoxide Anions ◽  
Metal Metal ◽  
Cartilage Wear

AbstractThe current understanding of osteoarthritis is developing from a mechanical disease caused by cartilage wear to a complex biological response involving inflammation, oxidative stress and other aspects. Nanoparticles are widely used in drug delivery due to its good stability in vivo and cell uptake efficiency. In addition to the above advantages, metal/metal oxide NPs, such as cerium oxide and manganese dioxide, can also simulate the activity of antioxidant enzymes and catalyze the degradation of superoxide anions and hydrogen peroxide. Degrading of metal/metal oxide nanoparticles releases metal ions, which may slow down the progression of osteoarthritis by inhibiting inflammation, promoting cartilage repair and inhibiting cartilage ossification. In present review, we focused on recent research works concerning osteoarthritis treating with metal/metal oxide nanoparticles, and introduced some potential nanoparticles that may have therapeutic effects.

scholarly journals Experimental and theoretical explorations of nanocarriers’ multistep delivery performance for rational design and anticancer prediction

2021 ◽  
Vol 7 (6) ◽  
pp. eaba2458
Author(s):  
Weier Bao ◽  
Falin Tian ◽  
Chengliang Lyu ◽  
Bin Liu ◽  
Bin Li ◽  
...  
Keyword(s):  
Physical Properties ◽  
Rational Design ◽  
Theoretical Models ◽  
Cell Uptake ◽  
Anticancer Efficacy ◽  
Delivery Performance ◽  
Simulation Based ◽  
Multistep Process

The poor understanding of the complex multistep process taken by nanocarriers during the delivery process limits the delivery efficiencies and further hinders the translation of these systems into medicine. Here, we describe a series of six self-assembled nanocarrier types with systematically altered physical properties including size, shape, and rigidity, as well as both in vitro and in vivo analyses of their performance in blood circulation, tumor penetration, cancer cell uptake, and anticancer efficacy. We also developed both data and simulation-based models for understanding the influence of physical properties, both individually and considered together, on each delivery step and overall delivery process. Thus, beyond finding that nanocarriers that are simultaneously endowed with tubular shape, short length, and low rigidity outperformed the other types, we now have a suit of theoretical models that can predict how nanocarrier properties will individually and collectively perform in the multistep delivery of anticancer therapies.

A nano chitosan membrane barrier prepared via Nanospider technology with non-toxic solvent for peritoneal adhesions’ prevention

2021 ◽  
pp. 088532822110081
Author(s):  
Shuo Zhang ◽  
Zhuoyue Xu ◽  
Xuejun Wen ◽  
Changzheng Wei
Keyword(s):  
Mouse Fibroblast ◽  
Electrospun Membrane ◽  
Adhesion Barrier ◽  
Peritoneal Adhesions ◽  
Elongation At Break ◽  
In Vivo Experiments ◽  
L929 Mouse Fibroblast ◽  
Membrane Barrier ◽  
L929 Mouse

Peritoneal adhesion is one of the most common postsurgical complications and can cause bowel obstruction, pelvic pain, and infertility. Setting up a physical barrier directly between the injured site and surrounding tissues is an effective solution for preventing this adverse situation. This study investigated a chitosan electrospun membrane (CSEM) as a potent anti-adhesion barrier, which was prepared by a needleless technology called Nanospider. Scanning electron microscopy revealed that CSEM is a laminated nanofiber with good mechanical properties. The fiber is uniform with the diameter distributing in the range of 100–120 nm. The tensile strength can reach 27.45 ± 6.30 MPa with a maximum elongation at break of 18.50 ± 1.44%, which makes it stick easily to damaged parts but not to be easily damaged by tissue friction. The growth of S. aureus on CSEM was 59.18% lower than the control at 10 h, which indicates its better antibacterial property. In addition, CSEM has good coagulant and biocompatibility characteristics. It can perform hemostatic function within 10 min and the L929 mouse fibroblast viability on it was 92.18% ± 1.08% on the seventh day. In vivo experiments indicated that CSEM significantly prevented peritoneal adhesions within four weeks after surgery with wound surface coverage. These results indicate that CSEM is a promising anti-adhesion barrier material.

scholarly journals Combined Effects of Methylated Cytosine and Molecular Crowding on the Thermodynamic Stability of DNA Duplexes

2021 ◽  
Vol 22 (2) ◽  
pp. 947
Author(s):  
Mitsuki Tsuruta ◽  
Yui Sugitani ◽  
Naoki Sugimoto ◽  
Daisuke Miyoshi
Keyword(s):  
Molecular Crowding ◽  
Dna Duplexes ◽  
Crowding Effect ◽  
Backbone Flexibility ◽  
Cpg Dinucleotides ◽  
Key Factor ◽  
Dna Backbone ◽  
A Cell ◽  
Stabilization Effect ◽  
And Function

Methylated cytosine within CpG dinucleotides is a key factor for epigenetic gene regulation. It has been revealed that methylated cytosine decreases DNA backbone flexibility and increases the thermal stability of DNA. Although the molecular environment is an important factor for the structure, thermodynamics, and function of biomolecules, there are few reports on the effects of methylated cytosine under a cell-mimicking molecular environment. Here, we systematically investigated the effects of methylated cytosine on the thermodynamics of DNA duplexes under molecular crowding conditions, which is a critical difference between the molecular environment in cells and test tubes. Thermodynamic parameters quantitatively demonstrated that the methylation effect and molecular crowding effect on DNA duplexes are independent and additive, in which the degree of the stabilization is the sum of the methylation effect and molecular crowding effect. Furthermore, the effects of methylation and molecular crowding correlate with the hydration states of DNA duplexes. The stabilization effect of methylation was due to the favorable enthalpic contribution, suggesting that direct interactions of the methyl group with adjacent bases and adjacent methyl groups play a role in determining the flexibility and thermodynamics of DNA duplexes. These results are useful to predict the properties of DNA duplexes with methylation in cell-mimicking conditions.

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