Plasmid DNA Binding Research Articles

Novel antipyrine substituted 4-thiazolidinones: Synthesis, DNA binding and topoisomerase inhibition activities, and in-silico studies

In this study, we report the synthesis and characterization of novel 4-thiazolidinones containing antipyrine (3a-e) derived from the starting compounds of Schiff bases (2a-e). The Schiff bases were synthesized using aldehydes of natural origin, such as citronellal, cuminaldehyde, 2-thiazolecarboxaldehyde, 5-methylfurfural, and syringaldehyde. Their FTIR spectra revealed the enol- tautomeric solid-state structures of compounds 3b and 3d. The pUC18 plasmid DNA binding specificity and capacity of the compounds to catalytically generate topoisomerase I activity were also examined using agarose gel electrophoresis. The compounds induced changes in DNA mobility and showed a topoisomerase I inhibitory effect at all doses. 4-Thiazolidinones had a higher affinity in DNA and topoisomerase I than the Schiff bases. DNA binding and topoisomerase I activities were most effective at low concentration (6 μM) for the compounds of Schiff bases 2a, d, and 4-thiazolidinones 3a, d. Furthermore, this study presents computational methods for addressing the molecules’ drug-like properties. 2a, d, and 3a, d Compounds were used as representative of the most bioactive among the newly synthesized molecules in in-silico studies. The web tools of ADMETlab, SwissADME, OSIRIS, and the BOILED-Egg method were used to conduct in-silico biological studies involving ADME prediction, BBB penetration, gastrointestinal absorption, and toxicity studies. The ADME profile of the query compounds gets within the range of applicability and is at an appropriate level. In-silico studies revealed that compounds 2a, d, and 3a, d are neither hERG blockers nor AMES toxic. However, compounds 2a and 3a have hepatotoxicity.

Synthesis, antioxidant, DNA interaction, electrochemical, and spectroscopic properties of chromene-based Schiff bases: Experimental and theoretical approach

Two chromene-based Schiff base ligands were synthesized by the condensation of 5-hydroxy-2,2-dimethyl-2H-chromene-6-carbaldehyde with 2-aminodiphenylamine and 1,8-diaminonaphthalene: (E)-2,2-dimethyl-6-(((2-(phenylamino)phenyl)imino)methyl)-2H-chromen-5-ol (2) and (E)-6-(((8-aminonaphthalen-1-yl)imino)methyl)-2,2-dimethyl-2H-chromen-5-ol (3). The compounds were characterized by Fourier transform infrared (FT-IR), absorption (UV-Vis) and nuclear magnetic resonance (NMR) spectroscopies, in addition to mass spectrometry (MS) analyses; the results of which were rationalized using density functional theoretical (DFT) calculations. Antioxidant activity of the Schiff bases was determined using the DPPH free radical scavenging and CUPric Ion Reducing Antioxidant Capacity (CUPRAC) assays. Compound 3 (IC50 = 14 μM) showed greater free radical scavenging ability than compound 2 (35 μM), and was also more effective than ascorbic acid and Trolox. DFT studies indicate that the HAT mechanism is favored over SET for DPPH scavenging. Both compounds exhibited stronger Cu(II) ion reducing capacity than Trolox and ascorbic acid with Trolox equivalent activity coefficient (TEAC) values of 2.0 and 1.6 for compounds 2 and 3, respectively. Furthermore, DNA cleavage and binding properties were determined using gel electrophoresis, UV-Vis spectroscopy, and cyclic voltammetry. The compounds showed copper-mediated cleavage of plasmid DNA and strong binding to DNA, with binding constants on the order of 104 M−1. The strength of DNA binding was verified by molecular docking analysis using two DNA models: a B-DNA dodecamer (-8.4 to -9.3 kcal/mol) and a hexamer (-7.8 to -7.9 kcal/mol). Overall, the chromene Schiff bases show promise as antioxidant and DNA binding agents.

Efficient Transfection via an Unexpected Mechanism by Near Neutral Polypiperazines with Tailored Response to Endosomal pH.

Cationic pH-responsive polymers promise to overcome critical challenges in cellular delivery. Ideally, the polymers become selectively charged along the endosomal pathway disturbing only the local membrane and avoiding unintended interactions or cytotoxic side effects at physiological conditions. Polypiperazines represent a novel, hydrophilic class of pH-responsive polymers whose response can be tuned within the relevant pH range (5-7.4). The authors discovered that the polypiperazines are effectively binding plasmid DNA (pDNA) and demonstrate high efficiency in transfection. By design of experiments (DoE), a wide parameter space (pDNA and polymer concentration) is screened to identify the range of effective concentrations for transfection. An isopropyl modified polypiperazine is highly efficient over a wide range of concentrations outperforming linear polyethylenimine (l-PEI, 25kDa) in regions of low N*/P ratios. A quantitative polymerase chain reaction (qPCR) surprisingly revealed that the pDNA within the piperazine-based polyplexes can be amplified in contrast to polyplexes based on l-PEI. The pDNA must therefore be more accessible and bound differently than for other known transfection polymers. Considering the various opportunities to further optimize their structure, polypiperazines represent a promising platform for designing effective soluble polymeric vectors, which are charge-neutral at physiological conditions.

Separation Properties of Plasmid DNA Using a Two-Stage Particle Adsorption-Microfiltration Process

Plasmid DNA is used as a vector for gene therapy and DNA vaccination; therefore, the establishment of a mass production method is required. Membrane filtration is widely employed as a separation method suitable for the mass production of plasmid DNA. Furthermore, the separation of plasmid DNA using microfiltration and ultrafiltration membranes is being investigated. Because plasmid DNA has a circular structure, it undergoes significant deformation during filtration and easily permeates the membrane, hindering the selection of separation membranes based on molecular weight. In this study, we applied affinity microfiltration to plasmid DNA purification. α-Fe2O3 with an isoelectric point of approximately 8 and a particle size of 0.5 μm was selected as the ligand for two-stage affinity microfiltration of plasmid DNA. In the first stage of microfiltration, the experiment was conducted at a pH of 5, and a cake of α-Fe2O3 with bound plasmid DNA was obtained. Next, liquid permeation (pH 9 and 10) through the cake was performed to elute the bound plasmid DNA. Plasmid DNA was eluted during the early phase of liquid permeation at pH 10. Furthermore, agarose gel analysis confirmed the usefulness of the two-stage affinity microfiltration method with adsorption and desorption for plasmid DNA purification.

Generating a Small Shuttle Vector for Effective Genetic Engineering of Methanosarcina mazei Allowed First Insights in Plasmid Replication Mechanism in the Methanoarchaeon.

Due to their role in methane production, methanoarchaea are of high ecological relevance and genetic systems have been ever more established in the last two decades. The system for protein expression in Methanosarcina using a comprehensive shuttle vector is established; however, details about its replication mechanism in methanoarchaea remain unknown. Here, we report on a significant optimisation of the rather large shuttle vector pWM321 (8.9 kbp) generated by Metcalf through a decrease in its size by about 35% by means of the deletion of several non-coding regions and the ssrA gene. The resulting plasmid (pRS1595) still stably replicates in M. mazei and—most likely due to its reduced size—shows a significantly higher transformation efficiency compared to pWM321. In addition, we investigate the essential gene repA, coding for a rep type protein. RepA was heterologously expressed in Escherichia coli, purified and characterised, demonstrating the significant binding and nicking activity of supercoiled plasmid DNA. Based on our findings we propose that the optimised shuttle vector replicates via a rolling circle mechanism with RepA as the initial replication protein in Methanosarcina. On the basis of bioinformatic comparisons, we propose the presence and location of a double-strand and a single-strand origin, which need to be further verified.

Exploring the ct-DNA and plasmid DNA binding affinity of the biogenic synthesized Chloroxine-conjugated silver nanoflowers: Spectroscopic and gel electrophoresis methods

Resistance to antibiotics is a public health issue, and the identification of new antibacterial agents is one of the most important goals of pharmacological research. The medicinal plants-mediated AgNP-antibiotic or AgNP-antibacterial conjugates show excellent antimicrobial efficiency against viruses, bacteria, and other microorganisms. The Chloroxine-conjugated silver nanoflowers (COX-AgNPs) through Echinophora platyloba DC. Extract mediated synthesis that exhibited significant antibacterial activity was utilized in this research. In this investigation, the interaction of COX-AgNPs with ct-DNA and plasmid DNA was investigated employing different spectroscopic and gel electrophoresis methods. We found that the mode of ct-DNA-COX-AgNPs interaction is partial intercalation. Meanwhile, the gel electrophoresis result implied COX-AgNPs are able to perform cleavage of the plasmid DNA. Consequently, plasmid DNA binding and cleavage might be one of the antibacterial mechanisms of COX-AgNPs.

Bioreducible polyethylenimine core–shell nanostructures as efficient and non-toxic gene and drug delivery vectors

Low molecular weight branched polyethylenimine (LMW bPEIs 1.8 kDa) have received considerable attention for the fabrication of nucleic acid carriers due to their biocompatible and non-toxic nature. However, due to the inadequate nucleic acid complexation ability and transportation across the cell membrane, these show poor transfection efficacy, limiting their clinical applications. Therefore, to overcome these challenges, in this study, we have grafted bPEI 1.8 kDa with a disulfide bond containing hydrophobic moiety, 3-(2-pyridyldithio) propionic acid (PDPA), via amide linkages through EDC/NHS-mediated coupling to obtain N-[3-(2-pyridyldithio)] propionoyl polyethylenimine (PDPP) conjugates. The best formulation for nucleic acid transfection was evaluated after preparing a series of PDPP conjugates by varying the amount of PDPA. In an aqueous environment, these PDPP conjugates self-assembled to form spherical shaped core–shell PDPP nanostructures with size ranging from ∼188–307 nm and zeta-potential from ∼ +3 to +19 mV. The positively charged surface of the core–shell nanocomposites helps in the binding of plasmid DNA (pDNA), its transportation inside the cell, and protection against enzymes. Evaluation of PDPP/pDNA complexes on mammalian cells revealed that all these complexes showed significantly improved transfection efficacy without hampering cytocompatibility. Amongst all, the pDNA complex of PDPP-2 exhibited the best transfection efficiency (i.e. >6-fold) in comparison to pDNA complex of the native bPEI. The nanocomposites exhibited the redox responsive behavior advantageous for therapeutic delivery to the tumor cells. The core of the nanostructures facilitate the encapsulation of a hydrophobic model drug, ornidazole. In vitro drug release analysis showed a faster release rate in response to a reductant mimicking the cellular environment. Altogether, these nanostructures have great potential to co-deliver both drug and gene simultaneously in response to tumor cell reductive microenvironment in vitro and could be used as the next-generation delivery system.

3-Imino derivative-sulfahydantoins: Synthesis, in vitro antibacterial and cytotoxic activities and their DNA interactions

Sulfahydantoins are five-membered rings found in the structure of chemicals that exhibit antibacterial, anti-inflammatory, and anticonvulsant properties. They also activate serine protease enzymes that catalyze the hydrolysis of peptide bonds. Five 3-imino sulfahydantoin compounds were synthesized by using Strecker synthesis reaction with minor modifications. We used reflux of various aldehydes with excess sulfamide in 85% methanol in the presence of sodium cyanide. The spectroscopic properties of these compounds were studied in detail. Antibacterial activities of all synthesized new compounds against four Gram-positive (Staphylococcus aureus, Bacillus cereus, Bacillus subtilis, Streptococcus mutans) and four Gram-negative (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae, Salmonella Enteritidis) bacteria were investigated by disc diffusion and microdilution method. pBR322 plasmid DNA binding abilities of compounds were investigated in vitro by agarose gel electrophoresis. In addition, the cytotoxic activities of the compounds against the human malignant pleural mesothelioma (SPC212) cell line were determined by the MTT method. The remarkable result in this study is that the synthesized compounds, especially 4b, 4d, and 4e, have significant biological activities. It has been demonstrated that these compounds, which cause DNA damage, also have an important antibacterial effect on both Gram-negative and Gram-positive bacteria when results compared with the control group antibiotics. Compound 4e exhibited the highest antibacterial potency against Streptococcus mutans (24.33 ± 0.57) from Gram-positive bacteria and Pseudomonas aeruginosa (24.66 ± 1.15) from Gram-negative bacteria. At the same time, MTT results determined that compounds 4b, 4d, and 4e showed cytotoxic activity against the SPC212 cells. In particular, compound 4b had a high cytotoxic effect, and the IC50 value was determined as 6.25 µM.

Supramolecular Surface Functionalization of Iron Oxide Nanoparticles with α-Cyclodextrin-Based Cationic Star Polymer for Magnetically-Enhanced Gene Delivery.

Supramolecular polymers formed through host–guest complexation have inspired many interesting developments of functional materials for biological and biomedical applications. Here, we report a novel design of a non-viral gene delivery system composed of a cationic star polymer forming supramolecular complexes with the surface oleyl groups of superparamagnetic iron oxide nanoparticles (SPIONs), for magnetically enhanced delivery of DNA into mammalian cells. The cationic star polymer was synthesized by grafting multiple oligoethylenimine (OEI) chains onto an α-cyclodextrin (α-CD) core. The SPIONs were synthesized from iron(III) acetylacetonate and stabilized by hydrophobic oleic acid and oleylamine in hexane, which were characterized in terms of their size, structure, morphology, and magnetic properties. The synthesized magnetic particles were found to be superparamagnetic, making them a suitable ferrofluid for biological applications. In order to change the hydrophobic surface of the SPIONs to a hydrophilic surface with functionalities for plasmid DNA (pDNA) binding and gene delivery, a non-traditional but simple supramolecular surface modification process was used. The α-CD-OEI cationic star polymer was dissolved in water and then mixed with the SPIONs stabilized in hexane. The SPIONs were “pulled” into the water phase through the formation of supramolecular host–guest inclusion complexes between the α-CD unit and the oleyl surface of the SPIONs, while the surface of the SPIONs was changed to OEI cationic polymers. The α-CD-OEI-SPION complex could effectively bind and condense pDNA to form α-CD-OEI-SPION/pDNA polyplex nanoparticles at the size of ca. 200 nm suitable for delivery of genes into cells through endocytosis. The cytotoxicity of the α-CD-OEI-SPION complex was also found to be lower than high-molecular-weight polyethylenimine, which was widely studied previously as a standard non-viral gene vector. When gene transfection was carried out in the presence of an external magnetic field, the α-CD-OEI-SPION/pDNA polyplex nanoparticles greatly increased the gene transfection efficiency by nearly tenfold. Therefore, the study has demonstrated a facile two-in-one method to make the SPIONs water-soluble as well as functionalized for enhanced magnetofection.

Asymmetric Silica Nanoparticles with Tailored Spiky Coverage Derived from Silica-Polymer Cooperative Assembly for Enhanced Hemocompatibility and Gene Delivery.

Asymmetric mesoporous silica nanoparticles (AMSNs) with one side featuring a spiky nanotopography, while the other side is smooth and solid, were synthesized via an ethylenediamine (EDA)-directed silica-polymer cooperative assembly approach. By simply varying the EDA amount (x), AMSNs-x samples with adjustable spiky surface coverage were obtained. It is demonstrated that a spiky coverage higher than 50% improved the hemocompatibility of AMSN-x, possibly due to the reduced contact area of the smooth side exposed to the red blood cell (RBC) membrane. Moreover, AMSNs-175 and AMSNs-200 with high spiky coverage enhanced their plasmid DNA (pDNA) loading and binding capability, as well as cellular uptake into HEK-293T cells, thus resulting in high transfection performance. The good hemocompatibility and high performance in pDNA delivery of AMSNs-x with high spiky coverage allow them to serve as promising nonviral vectors for potential applications in gene therapies and DNA vaccines.

Exploration of the Role of the C-Terminal Domain of Human DNA Topoisomerase IIα in Catalytic Activity.

Human topoisomerase IIα (TOP2A) is a vital nuclear enzyme involved in resolving knots and tangles in DNA during replication and cell division. TOP2A is a homodimer with a symmetrical, multidomain structure. While the N-terminal and core regions of the protein are well-studied, the C-terminal domain is poorly understood but is involved in enzyme regulation and is predicted to be intrinsically disordered. In addition, it appears to be a major region of post-translational modification and includes several Ser and Thr residues, many of which have not been studied for biochemical effects. Therefore, we generated a series of human TOP2A mutants where we changed specific Ser and Thr residues in the C-terminal domain to Ala, Gly, or Ile residues. We designed, purified, and examined 11 mutant TOP2A enzymes. The amino acid changes were made between positions 1272 and 1525 with 1–7 residues changed per mutant. Several mutants displayed increased levels of DNA cleavage without displaying any change in plasmid DNA relaxation or DNA binding. For example, mutations in the regions 1272–1279, 1324–1343, 1351–1365, and 1374–1377 produced 2–3 times more DNA cleavage in the presence of etoposide than wild-type TOP2A. Further, several mutants displayed changes in relaxation and/or decatenation activity. Together, these results support previous findings that the C-terminal domain of TOP2A influences catalytic activity and interacts with the substrate DNA. Furthermore, we hypothesize that it may be possible to regulate the enzyme by targeting positions in the C-terminal domain. Because the C-terminal domain differs between the two human TOP2 isoforms, this strategy may provide a means for selectively targeting TOP2A for therapeutic inhibition. Additional studies are warranted to explore these results in more detail.

PEGylation of Guanidinium and Indole Bearing Poly(methacrylamide)s - Biocompatible Terpolymers for pDNA Delivery.

This study describes the first example for shielding of a high performing terpolymer that consists of N-(2-hydroxypropyl)methacrylamide (HPMA), N-(3-guanidinopropyl)methacrylamide (GPMA), and N-(2-indolethyl)methacrylamide monomers (IEMA) by block copolymerization of a polyethylene glycol derivative - poly(nona(ethylene glycol)methyl ether methacrylate) (P(MEO9 MA)) via reversible addition-fragmentation chain transfer (RAFT) polymerization. The molecular weight of P(MEO9 MA) is varied from 3 to 40 kg mol-1 while the comonomer content of HPMA, GPMA, and IEMA is kept comparable. The influence of P(MEO9 MA) block with various molecular weights is investigated over cytotoxicity, plasmid DNA (pDNA) binding, and transfection efficiency of the resulting polyplexes. Overall, the increase in molecular weight of P(MEO9 MA) block demonstrates excellent biocompatibility with higher cell viability in L-929 cells and an efficient binding to pDNA at N/P ratio of 2. The significant transfection efficiency in CHO-K1 cells at N/P ratio 20 is obtained for block copolymers with molecular weight of P(MEO9 MA) up to 10 kg mol-1 . Moreover, a fluorescently labeled analogue of P(MEO9 MA), bearing perylene monoimide methacrylamide (PMIM), is introduced as a comonomer in RAFT polymerization. Polyplexes consisting of labeled block copolymer with 20 kg mol-1 of P(MEO9 MA) and pDNA are incubated in Hela cells and investigated through structured illumination microscopy (SIM).

Nse5/6 inhibits the Smc5/6 ATPase and modulates DNA substrate binding

Eukaryotic cells employ three SMC (structural maintenance of chromosomes) complexes to control DNA folding and topology. The Smc5/6 complex plays roles in DNA repair and in preventing the accumulation of deleterious DNA junctions. To elucidate how specific features of Smc5/6 govern these functions, we reconstituted the yeast holo‐complex. We found that the Nse5/6 sub‐complex strongly inhibited the Smc5/6 ATPase by preventing productive ATP binding. This inhibition was relieved by plasmid DNA binding but not by short linear DNA, while opposing effects were observed without Nse5/6. We uncovered two binding sites for Nse5/6 on Smc5/6, based on an Nse5/6 crystal structure and cross‐linking mass spectrometry data. One binding site is located at the Smc5/6 arms and one at the heads, the latter likely exerting inhibitory effects on ATP hydrolysis. Cysteine cross‐linking demonstrated that the interaction with Nse5/6 anchored the ATPase domains in a non‐productive state, which was destabilized by ATP and DNA. Under similar conditions, the Nse4/3/1 module detached from the ATPase. Altogether, we show how DNA substrate selection is modulated by direct inhibition of the Smc5/6 ATPase by Nse5/6.

Tight Binding of Plasmid DNA With Self-Assembled Tetramethylguanidinium Conjugated Polyethylenimine Suppresses Transfection Efficiency

Here, we have demonstrated that on modification of linear polyethylenimine (lPEI, LP) with amphiphilic 3-bromopropyltetramethylguanidinium (PTMG) linker, the transfection efficiency exhibited by the modified polymers decreased while cell viability improved. A series of LP-PTMG polymers was synthesized by the reaction of varying amounts of 3-bromopropyl tetramethylguanidinium linker with lPEI (25 kDa). These modified polymers interacted efficiently with pDNA and formed nanosized complexes as shown by dynamic light scattering analysis. The size of the complexes in the series LP-PTMG/pDNA was observed in the range of ∼178–205 nm. The interaction of modified polymers with plasmid DNA was stronger than linear PEI as evidenced by heparin release assay which showed ∼83% pDNA release from LP-PTMG-3/pDNA complexes in comparison to ∼95% in lPEI/pDNA complexes on treatment with same amount of heparin suggesting the formation of self-assembled structures in modified polymers. The transfection studies in HeLa and Chinese hamster ovary cells showed a decrease in transfection efficiency of LP-PTMG polymers, the reason for this may be strong binding of modified polymers with pDNA due to accumulation of charge on the surface. This finding showed the significance of optimum binding of polymer and DNA to form polyplexes as well as release of DNA from the polyplexes.

Preparation of a water soluble aminated β-1,3-D-glucan for gene carrier: The in vitro study of the anti-inflammatory activity and transfection efficiency.

β-1,3-D-glucan has been reported to have a series of bioactivities including antitumor, antimicrobial, anti-inflammatory and antioxidative effects; however, its insolubility in neutral aqueous solution significantly restricts the potential application in biological and medicine fields. Herein, a water-soluble aminated β-1,3-D-glucan (AG) was synthesized and the anti-inflammatory effect, cytotoxicity and plasmid DNA (pDNA) binding capacity of AG, serum stability, the particle sizes and zeta potentials of AG/pDNA nanocomposites, and the transfection efficiency and mechanism of action were studied. AG shows no obvious cytotoxicity within the range of working concentration (1-64 μg/ml) and it exerts potent anti-inflammatory effect independent on Dectin-1 and TLR2. AG/pDNA nanocomposites prepared by electrostatic interaction possess an appropriate particle size ranged from 192.8 to 118.4nm and zeta potentials ranged from 20.880 to 27.16 mV with the N/P ratios from 5 to 100. AG/pDNA nanocomposites at the N/P ratios of 10 and 20 were able to show superior transfection efficiencies in RAW 264.7 cells as a result of their suitable particle size, zeta potential, anti-inflammatory effect, and the specific interaction with pattern recognition receptors (Dectin-1 and TLR2). These results indicate that AG is a potential candidate for DNA delivery system due to its potent anti-inflammatory effect and high transfection efficiency.

Probing the Impact of Serine and Threonine Patches in the C‐terminus on the Catalytic of Human Topoisomerase IIα

Topoisomerase IIα is an essential nuclear enzyme involved in resolving knots and tangles in DNA during replication and cell division. The enzyme is a homodimer with a complex, multidomain structure. While the N-terminal and core regions of the protein are well-studied, the C-terminal domain is poorly understood but appears to be involved in enzyme regulation. In addition, the C-terminus varies widely among isoforms of topoisomerase II and appears to be a major region of post-translational modification including several patches of Ser and Thr residues. Many of these Ser and Thr residues have not been studied for biochemical effects. Therefore, we generated a series of mutant versions of human topoisomerase IIα where we selectively changed patches of Ser and Thr residues in the C-terminal domain to Ala and Gly residues. We designed, purified, and examined the activity of eleven mutant enzymes for changes in catalytic activity. The amino acid changes were made in the region of positions 1272 to 1525 with 1-7 residues changed per mutant. Several mutants displayed varying increased levels of DNA cleavage without displaying any change in plasmid DNA relaxation or DNA binding. For example, mutations in the regions 1272-1279, 1324-1343, 1351-1365, and 1374-1377 all had larger increases in DNA cleavage in the presence of etoposide when compared with wild-type topoisomerase IIα. One mutant displayed decreased DNA cleavage below wild-type levels. This mutant had changes between residues 1469-1476 and displayed a decreased ability to both relax supercoiled DNA and to bind to plasmid DNA. Importantly, several residues in this region (Ser1469, Thr1470, Ser1471, and Ser1474) are known to have a role in mitosis indicating these are key regulators of enzyme function. Together, these results indicate that the C-terminal domain of topoisomerase IIα influences catalytic activity and likely interacts with substrate DNA at various positions. Additional studies are warranted to explore these results in more detail.

Luminescent Amphiphilic Aminoglycoside Probes to Study Transfection.

We report the characterization of amphiphilic aminoglycoside conjugates containing luminophores with aggregation‐induced emission properties as transfection reagents. These inherently luminescent transfection vectors are capable of binding plasmid DNA through electrostatic interactions; this binding results in an emission “on” signal due to restriction of intramolecular motion of the luminophore core. The luminescent cationic amphiphiles effectively transferred plasmid DNA into mammalian cells (HeLa, HEK 293T), as proven by expression of a red fluorescent protein marker. The morphologies of the aggregates were investigated by microscopy as well as ζ‐potential and dynamic light‐scattering measurements. The transfection efficiencies using luminescent cationic amphiphiles were similar to that of the gold‐standard transfection reagent Lipofectamine® 2000.

Solid Lipid Nanoparticle Carrier Platform Containing Synthetic TLR4 Agonist Mediates Non-Viral DNA Vaccine Delivery.

There is a growing demand for better delivery systems to improve the stability and efficacy of DNA vaccines. Here we report the synthesis of a non-viral DNA vaccine delivery system using a novel adjuvanted solid lipid nanoparticle (SLN-A) platform as a carrier for a DNA vaccine candidate encoding the Urease alpha (UreA) antigen from Helicobacter pylori. Cationic SLN-A particles containing monophosphoryl lipid A (adjuvant) were synthesised by a modified solvent-emulsification method and were investigated for their morphology, zeta potential and in vitro transfection capacity. Particles were found to bind plasmid DNA to form lipoplexes, which were characterised by electron microscopy, dynamic light scattering and fluorescence microscopy. Cellular uptake studies confirmed particle uptake within 3 h, and intracellular localisation within endosomal compartments. In vitro studies further confirmed the ability of SLN-A particles to stimulate expression of pro-inflammatory cytokine tumor necrosis factor alpha (TNF-α) in human macrophage-like Tohoku Hospital Pediatrics-1 (THP-1) cells. Lipoplexes were found to be biocompatible and could be efficiently transfected in murine immune cells for expression of recombinant H. pylori antigen Urease A, demonstrating their potential as a DNA vaccine delivery system.

In Vitro Assessment of Core-Shell Micellar Nanostructures of Amphiphilic Cationic Polymer-Peptide Conjugates as Efficient Gene and Drug Carriers

Design and development of biocompatible, biodegradable and stable dual delivery systems for drug and gene is the need of the hour. Here, we have designed a strategy to develop carrier systems consisting of above mentioned properties by (a) incorporating an unnatural amino acid in the peptide backbone, and b) conjugating a low molecular weight cationic polymer (polyethylenimine, PEI) for incorporating cationic charge. Using this strategy, we have synthesized a small series of Boc-FΔF-AH-polyethylenimine conjugates by varying the concentration of Boc-FΔF-aminohexanoic acid, viz., PP-1, PP-2 and PP-3. These conjugates self-assembled in aqueous medium to form micelles in the size range of ~144–205 nm with zeta potential ~ +7.9–14.2 mV bearing core-shell type of conformation. Positive surface of the micelles facilitated the binding of plasmid DNA as well as transportation inside the cells. The hydrophobic core of the nanostructures helped in the encapsulation of the hydrophobic drug molecule, which was then got released in a controlled manner. DNA complexes of the conjugates were not only found non-toxic but also exhibited higher transfection efficacy than the native polymer and Lipofectamine. Altogether, these nanostructures are capable of delivering a drug and a gene simultaneously in vitro and could be used as next-generation delivery agents.

Consensus protein engineering on the thermostable histone-like bacterial protein HUs significantly improves stability and DNA binding affinity.

Consensus-based protein engineering strategy has been applied to various proteins and it can lead to the design of proteins with enhanced biological performance. Histone-like HUs comprise a protein family with sequence variety within a highly conserved 3D-fold. HU function includes compacting and regulating bacterial DNA in a wide range of biological conditions in bacteria. To explore the possible impact of consensus-based design in the thermodynamic stability of HU proteins, the approach was applied using a dataset of sequences derived from a group of 40 mesostable, thermostable, and hyperthermostable HUs. The consensus-derived HU protein was named HUBest, since it is expected to perform best. The synthetic HU gene was overexpressed in E. coli and the recombinant protein was purified. Subsequently, HUBest was characterized concerning its correct folding and thermodynamic stability, as well as its ability to interact with plasmid DNA. A substantial increase in HUBest stability at high temperatures is observed. HUBest has significantly improved biological performance at ambience temperature, presenting very low Kd values for binding plasmid DNA as indicated from the Gibbs energy profile of HUBest. This Kd may be associated to conformational changes leading to decreased thermodynamic stability and, therefore, higher flexibility at ambient temperature.