Incorporation of Indole Significantly Improves the Transfection Efficiency of Guanidinium-Containing Poly(methacrylamide)s

2020 ◽  
Author(s):  
Ceren Cokca ◽  
Leon Zartner ◽  
Ilja tabujew ◽  
Dagmar Fischer ◽  
Kalina Peneva
Keyword(s):  
Transgene Expression ◽  
Transfection Efficiency ◽  
Fold Increase ◽  
Cell Penetrating Peptides ◽  
Side Chain ◽  
Hydrodynamic Diameter ◽  
Agent Based ◽  
Hydroxypropyl Methacrylamide ◽  
Arginine Residues ◽  
Transfection Agent

<div><div><div><p>In this study, we report a highly efficient transfection agent based on terpolymer consisting of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl) methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide (IEMA) monomers by analogy to the amphipathic cell penetrating peptides containing tryptophan and arginine residues. The incorporation of the indole bearing monomer led to successful plasmid DNA condensation even at nitrogen to phosphate (N/P) ratio 1. The hydrodynamic diameter of polyplexes was determined to be below 200 nm for all N/P ratios. The transfection studies demonstrated 200- fold increase of the transgene expression in comparison to P(HPMA-co-GPMA) with the same guanidinium content. This study reveals the strong potential of the indole group as side chain pending group that can increase the cellular uptake of polymers and the transfection efficiency of the respective polyplexes.</p></div></div></div>

Incorporation of Indole Significantly Improves the Transfection Efficiency of Guanidinium-Containing Poly(methacrylamide)s

2020 ◽  
Author(s):  
Ceren Cokca ◽  
Leon Zartner ◽  
Ilja tabujew ◽  
Dagmar Fischer ◽  
Kalina Peneva
Keyword(s):  
Transgene Expression ◽  
Transfection Efficiency ◽  
Fold Increase ◽  
Cell Penetrating Peptides ◽  
Side Chain ◽  
Hydrodynamic Diameter ◽  
Agent Based ◽  
Hydroxypropyl Methacrylamide ◽  
Arginine Residues ◽  
Transfection Agent

<div><div><div><p>In this study, we report a highly efficient transfection agent based on terpolymer consisting of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl) methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide (IEMA) monomers by analogy to the amphipathic cell penetrating peptides containing tryptophan and arginine residues. The incorporation of the indole bearing monomer led to successful plasmid DNA condensation even at nitrogen to phosphate (N/P) ratio 1. The hydrodynamic diameter of polyplexes was determined to be below 200 nm for all N/P ratios. The transfection studies demonstrated 200- fold increase of the transgene expression in comparison to P(HPMA-co-GPMA) with the same guanidinium content. This study reveals the strong potential of the indole group as side chain pending group that can increase the cellular uptake of polymers and the transfection efficiency of the respective polyplexes.</p></div></div></div>

In-silico Molecular Interaction of Short Synthetic Lipopeptide/Importin-alpha and In-vitro Evaluation of Transgene Expression Mediated by Liposome- Based Gene Carrier

2020 ◽  
Vol 20 (5) ◽  
pp. 383-394
Author(s):  
Tarwadi Tarwadi ◽  
Jalal A. Jazayeri ◽  
Sabar Pambudi ◽  
Alfan D. Arbianto ◽  
Heni Rachmawati ◽  
...  
Keyword(s):  
Molecular Interaction ◽  
In Silico ◽  
Transgene Expression ◽  
Transfection Efficiency ◽  
Dna Molecules ◽  
In Vitro Evaluation ◽  
Nuclear Uptake ◽  
Importin Α ◽  
Transfection Agent

Background: Lipopeptide-based gene carriers have shown low cytotoxicity, are capable of cell membrane penetration, are easy to manufacture and therefore are great potential candidates for gene delivery applications. Objectives: This study aims to explore a range of short synthetic lipopeptides, (Lau: Lauryl; Pal: Palmitoyl) consisting of an alkyl chain, one cysteine (C), 1 to 2 histidine (H), and lysine (K) residues by performing in-silico molecular interaction and in-vitro evaluation. Methods: The molecular interactions between the lipopeptides and Importin-α receptor were performed using AutoDock Vina and Amber14. The lipopeptide/DNA complexes were evaluated in- -vitro for their interactions, particle size, zeta potential and transgene expression. Transfection efficiency of the lipopeptides and Pal-CKKHH-derived liposome was carried out based on luciferase transgene expression. Results: The in-silico interaction showed that Lau-CKKH and Pal-CKKHH hypothetically expedited nuclear uptake. Both lipopeptides had lower binding energy (-6.3 kcal/mol and -6.2 kcal/mol, respectively), compared to the native ligand, viz, nuclear localization sequence (-5.4 kcal/mol). The short lipopeptides were able to condense DNA molecules and efficiently form compacted nanoparticles. Based on the in-vitro evaluation on COS-7, Pal-CKKHH was found to be the best transfection agent amongst the lipopeptides. Its transfection efficiency (ng Luc/mg total protein) increased up to ~3-fold higher (1163 + 55) as it was formulated with helper lipid DOPE (1:2). The lipopeptide- based liposome (Pal-CKKHH: DOPE=1:2) also facilitated luciferase transgene expression on human embryonic kidney cells (293T) and human cervical adenocarcinoma cells (HeLa) with transfection efficiency 1779 +52 and 260 + 22, respectively. Conclusion: Our study for the first time has shown that the fully synthesized short lipopeptide Pal- CKKHH is able to interact firmly with the Importin-α. The lipopeptide is able to condense DNA molecules efficiently, facilitate transgene expression, expedite the nuclear uptake process, and hence has the characteristics of a potential transfection agent.

scholarly journals Glucose-Responsive Gene Delivery at Physiological pH through Tertiary-Amine Stabilized Boronate-PVA Particles Synthesized by One-Pot Reaction

Pharmaceutics ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 62
Author(s):  
Mangesh Morey ◽  
Akshay Srivastava ◽  
Abhay Pandit
Keyword(s):  
Gene Delivery ◽  
Fold Increase ◽  
Nonviral Gene Delivery ◽  
Luciferase Reporter ◽  
Hydrodynamic Diameter ◽  
Luciferase Reporter Gene ◽  
One Pot ◽  
Gaussia Luciferase ◽  
Nih3t3 Cells ◽  
The Stability

We report a physiologically stable and cytocompatible glucose-responsive nonviral gene delivery system made up of boronate functionalized polymeric material. Herein, we utilize boronate cis-diol interactions to develop a glucose-responsive submicron particle (SMP) system. The stability of the boronate interaction at a physiological pH was achieved by copolymerization of dimethyl aminoethyl methacrylate (DMAEMA) with acrylamidophenylboronic acid (AAPBA) and the formation of a complex with polyvinylalcohol (PVA) which is governed by cis-diol interactions. The shift in hydrodynamic diameter of SMPs was observed and correlated with increasing glucose concentrations at a physiological pH. Optimal transfection was observed for a 5 µg dose of the gaussia luciferase reporter gene in NIH3T3 cells without any adverse effect on cellular viability. The destabilization of the AAPBA–PVA complex by interacting with glucose allowed the release of encapsulated bovine serum albumin (BSA) in a glucose-responsive manner. In total, 95% of BSA was released from SMPs at a 50 mM glucose concentration after 72 h. A two-fold increase in transfection was observed in 50 mM glucose compared to that of 10 mM glucose.

scholarly journals Resveratrol Stimulates Cortisol Biosynthesis by Activating SIRT-Dependent Deacetylation of P450scc

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3258-3268 ◽  
Author(s):  
Donghui Li ◽  
Eric B. Dammer ◽  
Marion B. Sewer
Keyword(s):  
Protein Expression ◽  
Fold Increase ◽  
Pyridine Nucleotide ◽  
Nucleotide Metabolism ◽  
Side Chain ◽  
Mitochondrial Enzyme ◽  
Adrenocortical Cells ◽  
Protein Levels ◽  
Chain Cleavage ◽  
Cleavage Enzyme

In the human adrenal cortex, cortisol is synthesized from cholesterol by members of the cytochrome P450 superfamily and hydroxysteroid dehydrogenases. Both the first and last steps of cortisol biosynthesis occur in mitochondria. Based on our previous findings that activation of ACTH signaling changes the ratio of nicotinamide adenine dinucleotide (NAD) phosphate to reduced NAD phosphate in adrenocortical cells, we hypothesized that pyridine nucleotide metabolism may regulate the activity of the mitochondrial NAD+-dependent sirtuin (SIRT) deacetylases. We show that resveratrol increases the protein expression and half-life of P450 side chain cleavage enzyme (P450scc). The effects of resveratrol on P450scc protein levels and acetylation status are dependent on SIRT3 and SIRT5 expression. Stable overexpression of SIRT3 abrogates the cellular content of acetylated P450scc, concomitant with an increase in P450scc protein expression and cortisol secretion. Mutation of K148 and K149 to alanine stabilizes the expression of P450scc and results in a 1.5-fold increase in pregnenolone biosynthesis. Finally, resveratrol also increases the protein expression of P450 11β, another mitochondrial enzyme required for cortisol biosynthesis. Collectively, this study identifies a role for NAD+-dependent SIRT deacetylase activity in regulating the expression of mitochondrial steroidogenic P450.

Multi-modal transfection agent based on monodisperse magnetic nanoparticles for stem cell gene delivery and tracking

Biomaterials ◽  
2014 ◽  
Vol 35 (25) ◽  
pp. 7239-7247 ◽  
Author(s):  
Wooram Park ◽  
Han Na Yang ◽  
Daishun Ling ◽  
Hyeona Yim ◽  
Kyoung Sub Kim ◽  
...  
Keyword(s):  
Stem Cell ◽  
Magnetic Nanoparticles ◽  
Gene Delivery ◽  
Agent Based ◽  
Cell Gene ◽  
Stem Cell Gene ◽  
Transfection Agent

scholarly journals A Novel Gene Encoding Xanthan Lyase of Paenibacillus alginolyticus Strain XL-1

2000 ◽  
Vol 66 (9) ◽  
pp. 3945-3950 ◽  
Author(s):  
Harald J. Ruijssenaars ◽  
Sybe Hartmans ◽  
Jan C. Verdoes
Keyword(s):  
Signal Sequence ◽  
Fold Increase ◽  
Side Chain ◽  
Cloning And Sequencing ◽  
Gene Encoding ◽  
E Coli ◽  
Mature Enzyme ◽  
Locust Bean ◽  
Encoding Gene ◽  
Amino Acid Signal

ABSTRACT Xanthan-modifying enzymes are powerful tools in studying structure-function relationships of this polysaccharide. One of these modifying enzymes is xanthan lyase, which removes the terminal side chain residue of xanthan. In this paper, the cloning and sequencing of the first xanthan lyase-encoding gene is described, i.e., thexalA gene, encoding pyruvated mannose-specific xanthan lyase of Paenibacillus alginolyticus XL-1. ThexalA gene encoded a 100,823-Da protein, including a 36-amino-acid signal sequence. The 96,887-Da mature enzyme could be expressed functionally in Escherichia coli. Like the native enzyme, the recombinant enzyme showed no activity on depyruvated xanthan. Compared to production by P. alginolyticus, a 30-fold increase in volumetric productivity of soluble xanthan lyase was achieved by heterologous production in E. coli. The recombinant xanthan lyase was used to produce modified xanthan, which showed a dramatic loss of the capacity to form gels with locust bean gum.

scholarly journals WRAP-based nanoparticles for siRNA delivery: a SAR study and a comparison with lipid-based transfection reagents

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Karidia Konate ◽  
Emilie Josse ◽  
Milana Tasic ◽  
Karima Redjatti ◽  
Gudrun Aldrian ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gene Silencing ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Sirna Delivery ◽  
Cell Penetrating Peptides ◽  
Sirna Transfection ◽  
Arginine Residues ◽  
Sar Study

AbstractRecently, we designed novel amphipathic cell-penetrating peptides, called WRAP, able to transfer efficiently siRNA molecules into cells. In order to gain more information about the relationship between amino acid composition, nanoparticle formation and cellular internalization of these peptides composed of only three amino acids (leucine, arginine and tryptophan), we performed a structure–activity relationship (SAR) study. First, we compared our WRAP1 and WRAP5 peptides with the C6M1 peptide also composed of the same three amino acids and showing similar behaviors in siRNA transfection. Afterwards, to further define the main determinants in the WRAP activity, we synthesized 13 new WRAP analogues harboring different modifications like the number and location of leucine and arginine residues, the relative location of tryptophan residues, as well as the role of the α-helix formation upon proline insertions within the native WRAP sequence. After having compared the ability of these peptides to form peptide-based nanoparticles (PBNs) using different biophysical methods and to induce a targeted gene silencing in cells, we established the main sequential requirements of the amino acid composition of the WRAP peptide. In addition, upon measuring the WRAP-based siRNA transfection ability into cells compared to several non-peptide transfection agents available on the markets, we confirmed that WRAP peptides induced an equivalent level of targeted gene silencing but in most of the cases with lower cell toxicity as clearly shown in clonogenic assays.

Changes in lipophosphoglycan and gene expression associated with the development ofLeishmania majorinPhlebotomus papatasi

Parasitology ◽  
1995 ◽  
Vol 111 (3) ◽  
pp. 275-287 ◽  
Author(s):  
E. M. B. Saraiva ◽  
P. F. P. Pimenta ◽  
T. N. Brodin ◽  
E. Rowton ◽  
G. B. Modi ◽  
...  
Keyword(s):  
Differential Expression ◽  
Fold Increase ◽  
Surface Expression ◽  
Side Chain ◽  
Surface Coat ◽  
Natural Vector ◽  
Metacyclic Promastigotes ◽  
Kinetics Of

SUMMARYStage-specific molecular and morphogenic markers were used to follow the kinetics of appearance, number, and position of metacyclic promastigotes developing during the course ofL. majorinfection in a natural vector,Phlebotomus papatasi. Expression of surface lipophosphoglycan (LPG) on transformed promastigotes was delayed until the appearance of nectomonad forms on day 3, and continued to be abundantly expressed by all promastigotes thereafter. An epitope associate with arabinose substitution of LPG side-chain oligosaccharides, identified by its differential expression by metacyclics invitro, was detected on the surface of a low proportion of midgut promastigotes beginning on day 5, and on up to 60% of promatigotes on days 10 and 15. In contrast 100% of the parasites egested from the mouthparts during forced feeding of 15 day infected flies stained strongly for this epitope. At each time-point, the surface expression of the modified LPG was restricted to morphologically distinguished metacyclic forms. Ultrastructural study of the metacyclic surface revealed an approximate 2-fold increase in the thickness of the surface coat compared to nectomonad forms, suggesting elongation of LPG as occurs during metacyclogenesisin vitro. A metacyclic-associated transcript (MAT-1), another marker identified by its differential expression invitro, also showed selective expression by promastigotes in the fly, and was used inin situhybridization studies to demonstrate the positioning of metacyclics in the anterior gut.

Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells

2021 ◽  
Author(s):  
Pardis Moradi ◽  
akbar hasanzadeh ◽  
Fatemh Radmanesh ◽  
Saideh Rajai Daryasarei ◽  
Elaheh Sadat Hosseini ◽  
...  
Keyword(s):  
Gene Editing ◽  
Transfection Efficiency ◽  
Target Cells ◽  
Reporter Cell ◽  
Expression Plasmid ◽  
Safe Delivery ◽  
Gfp Reporter ◽  
Local Brain ◽  
Transfection Agent

Abstract An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with LMW PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8k in the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5 to 10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g., plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid and in vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.

Designed filamentous cell penetrating peptides: probing supramolecular structure-dependent membrane activity and transfection efficiency

2015 ◽  
Vol 51 (59) ◽  
pp. 11757-11760 ◽  
Author(s):  
Dawei Xu ◽  
Derek Dustin ◽  
Linhai Jiang ◽  
Damien S. K. Samways ◽  
He Dong
Keyword(s):  
Supramolecular Structure ◽  
Transfection Efficiency ◽  
Supramolecular Assembly ◽  
Cell Penetrating Peptides ◽  
Cationic Peptides ◽  
Single Chain ◽  
Membrane Activity ◽  
Highly Efficient ◽  
Cell Penetrating ◽  
Filamentous Cell

In this work, we will demonstrate the supramolecular assembly of single-chain cationic peptides into stable macromolecular filamentous nanostructures and investigate their supramolecular structure-dependent membrane activity for the development of highly efficient therapeutic carriers.

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