Effective DNA Condensation Research Articles

Intrinsically Disordered Ku Protein-Derived Cell-Penetrating Peptides.

Efficient delivery of bioactive ingredients into cells is a major challenge. Cell-penetrating peptides (CPPs) have emerged as promising vehicles for this purpose. We have developed novel CPPs derived from the flexible and disordered tail extensions of DNA-binding Ku proteins. Ku-P4, the lead CPP identified in this study, is biocompatible and displays high internalization efficacy. Biophysical studies show that the proline residue is crucial for preserving the intrinsically disordered state and biocompatibility. DNA binding studies showed effective DNA condensation to form a positively charged polyplex. The polyplex exhibited effective penetration through the cell membrane and delivered the plasmid DNA inside the cell. These novel CPPs have the potential to enhance the cellular uptake and therapeutic efficacy of peptide-drug or gene conjugates.

Cationic cross-linked polymers containing labile disulfide and boronic ester linkages for effective triple responsive DNA release

Disruption of DNA carriers triggered by intracellular bio-stimulants has been broadly considered as most convenient strategy for efficient DNA delivery. In this direction, we have designed and synthesized pH, redox and ATP responsive cationic cross-linked polymers (CLPs) having disulfide and reversible boronic ester linkages. These CLPs also contain folate groups that are known for their targeting capability towards cancer cells. Biophysical studies showed that these cationic CLPs exhibited more effective DNA condensation in comparison to cationic linear polymers resulting in the formation of nano-sized polyplexes with sufficient positive zeta potentials and good colloidal stability at neutral pH (∼7.4). More interestingly, the polyplexes prepared from these CLPs have the ability to selectively release complexed DNA under conditions similar to those prevalent in cancer cells such as acidic pH, ATP rich surroundings or presence of glutathione, as revealed by ethidium bromide exclusion assay, agarose gel electrophoresis, AFM measurements, etc. Therefore, these cross-linked polymers have high potential of being effective non-viral gene delivery vehicles.

A block copolymer containing PEG and histamine-like segments: well-defined functions for gene delivery

PEG-b-PAMPImB with well-defined functions, including high colloidal stability, effective DNA condensation and buffering capacity, self-assembles with pDNA for lower toxicity and higher efficient gene transfection.

Preparation and evaluation of polyethylenimine-functionalized carbon nanotubes tagged with 5TR1 aptamer for targeted delivery of Bcl-xL shRNA into breast cancer cells

In this study, single-walled carbon nanotubes (SWCNTs) were covalently attached to poly(ethylene glycol) (PEG) and polyethylenimine (PEI) 10kDa, or its derivatives, to fabricate efficient carriers for gene delivery. PEI 10kDa was modified by alkylcarboxylation of its primary amines with a series of ω-bromo-alkylcarboxylic acids to provide a range of vectors with increased lipophilicity. PEI 10kDa or its alkylcarboxylate derivatives were conjugated to SWCNT-PEG to develop vectors possessing effective DNA condensation ability which can interact with cell membrane via both nano-needle mechanism and electrostatic interactions produced by SWCNT and PEI, respectively. The results demonstrated that SWCNT-PEG-PEI and SWCNT-PEG-derivatives of PEI could condense DNA into particle size less than 150nm with positive surface charges between 6.3–30.8mV. To improve the antitumor efficacy, we developed a targeted gene delivery system using a 5TR1 aptamer. The most efficient vector, which was prepared by attachment of SWCNT-PEG to modified PEI 10kDa with 10-bromodecanoic acid (10%), showed 8.5–10 folds enhancement in transfection activity at C/P ratio 6 as compared to the gold standard PEI 25kDa at C/P ratio of 0.8. We also showed that the selected polyplex could efficiently and selectively transfer plasmid shRNA to MUC1 positive cells.

Synthesis of bifunctional molecules containing [12]aneN3 and coumarin moieties as effective DNA condensation agents and new non-viral gene vectors.

A series of bifunctional molecules with different combinations of macrocyclic polyamine [12]aneN3 and coumarin moieties, 4a/b and 5a/b, were synthesized by a two-step copper(I)-mediated alkyne–azide click reactions between 1,3,5-tris(azidomethyl)benzene and Boc-protected N-propynyl-[12]aneN3/7-propynyloxycoumarins. Agarose gel electrophoresis experiments indicated that bifunctional molecules 4b and 5b effectively induced complete plasmid DNA condensation at concentrations up to 40 μM. It was found that the structural variation had a major impact on the condensation behavior of these compounds. The electrostatic interaction involving the [12]aneN3 moiety can be compensated by the binding contribution of the coumarin units during the DNA condensation process. These two types of interaction showed different effects on the reversibility of DNA condensation. Results from studies using dynamic laser scattering, atomic force microscopy, and EB replacement assay further supported the above conclusion. Cytotoxicity assays on bifunctional compounds 4a/b and 5a/b indicated their low cytotoxicity. Results from cellular uptake and cell transfection experiments proved that bifunctional compounds 4b and 5b successfully served as non-viral gene vectors. Furthermore, methyl substituents attached to the coumarin unit (4b and 5b) greatly enhanced their DNA condensation capability and gene transfection. These bifunctional molecules, with the advantages of lower cytotoxicity, good water solubility, and potential structural modification, will have great potential for the development of new non-viral gene delivery agents.

Interaction of a fluorescent cationic surfactant bearing a coumarin derivative with DNA

A fluorescent cationic surfactant with a coumarin derivative (Br-Mac-12) has been designed, synthesized and characterized. The critical association concentration (CAC) and critical micelle concentration (CMC) of Br-Mac-12 was 1.0×10−4 and 1.6×10−3mol/L, respectively. The interaction between Br-Mac-12 and DNA has been investigated using UV–vis and fluorescence spectroscopy as well as gel electrophoresis and atomic force microscopy (AFM). Both linear and plasmid DNA were condensed to nanoparticles by Br-Mac-12 at a concentration of 320μM with incubation at 37°C for 4h. Br-Mac-12 has a more efficient binding capacity with DNA than the corresponding DTAB without the fluorophore. The groove binding between the coumarin derivative and DNA plays an important role in addition to the electrostatic and hydrophobic interaction for effective DNA condensation inductions. In addition, the condensation process was reversible, and the residual fluorescence of Br-Mac-12 upon binding with DNA remains at 37.5% of the initial value. The results suggest that Br-Mac-12 combined with a fluorophore has the potential for applications in tracing membrane transport in gene delivery and transfection due to its excellent fluorescence property and DNA binding affinity.

Syntheses of bifunctional molecules containing [12]aneN3 and carbazol moieties as effective DNA condensation agents

Bifunctional molecules containing macrocyclic polyamine [12]aneN3 and carbazol units, 1–4, have been efficiently synthesized and fully characterized. Through gel electrophoresis, atomic force microscopy, and dynamic light scattering experiments, compounds 3 and 4b bearing both [12]aneN3 and carbazol moieties showed effective DNA condensation ability at the concentration of 80 μM. Investigations from EB displacement fluorescence spectra, viscosity titration, and ionic strength effects revealed that the effective DNA condensation comes from the appropriate combination of carbazol and [12]aneN3 units in the bifunctional molecules, and the DNA condensation process is reversible. The incorporation of triazole units in the molecules clearly reduced the cytotoxicity.

A redox-responsive cationic supramolecular polymer constructed from small molecules as a promising gene vector

A novel class of redox-responsive cationic supramolecular polymer with effective DNA condensation ability and H2O2-induced DNA release behavior has been successfully constructed from small molecules. This supramolecular polymer can be used in vitro as a promising nonviral vector for gene therapy.

Syntheses of [12]aneN3–oligopeptide conjugates as effective DNA condensation agents

With the aim to develop effective and low toxicity DNA condensation agents, a series of oligopeptide derived macrocyclic polyamine [12]aneN3 conjugates 7a–h·3HCl have been designed and synthesized through multi-step amidation reactions. Structure–property study through gel electrophoresis proved that the conjugates containing high hydrophobic ending amino acids exhibited effective condensation ability at concentration of 150–250μM, which was further confirmed by dynamic light scattering and atomic force microscopy experiments. EB displacement assay, ionic salt effect, and structure–property relationship in gel electrophoresis indicated that DNA condensation resulted from both the electrostatic interaction of [12]aneN3 unit and hydrogen-bonding/hydrophobic multi-interaction of oligopeptide moiety in the conjugates with DNA. The reversible condensation process and their low cytotoxicity suggest that the new condensing agents are potential for the development of non-viral vectors.

Effective and reversible DNA condensation induced by bifunctional molecules containing macrocyclic polyamines and naphthyl moieties

A series of bifunctional molecules containing macrocyclic polyamines [12]aneN3 and naphthyl moieties 1–3(a, b) have been designed and synthesized through efficient N-alkylation and copper-mediated alkyne–azide click reactions. Experiments on gel electrophoresis, dynamic light scattering and atomic force microscopy confirmed that 2b and 3b with two [12]aneN3 units efficiently induced the DNA condensation at the concentration of 120μM in less than 5min. The condensation mechanism was studied by EB displacement fluorescence spectra, viscosity titration, and ionic strength effects. The condensation process was found to be reversible, and the presence of both naphthyl and [12]aneN3 units in the molecules was proved to be necessary for the effective DNA condensation inductions. Cytotoxicity assay showed that the presence of triazole moieties can result in lower toxicity.

A novel chitosan oligosaccharide–stearic acid micelles for gene delivery: Properties and in vitro transfection studies

Stearic acid (SA) grafted chitosan oligosaccharide (CSO) (CSO–SA), which was synthesized by an 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-mediated coupling reaction, was demonstrated to form micelle like structure by self-aggregation in aqueous solution. The critical micelle concentration (CMC) of CSO–SA with 15.4% amino substituted degree of CSO was about 0.035 mg/ml. The micelles with 1 mg/ml CSO–SA concentration had 70.6 nm volume average hydrodynamic diameter with a narrow size distribution and 46.4 ± 0.1 mV surface potential. Due to the cationic property, the micelles could compact the plasmid DNA to form micelle/DNA complexes nanoparticles, which can efficiently protect the condensed DNA from enzymatic degradation by DNase I. The volume average hydrodynamic diameter of CSO–SA micelle/DNA complex increased from 203 nm to 318 nm and decreased to 102 nm due to the variation of zeta potential when the N/P ratio increased from 0.25 to 3.6 and from 3.6 to 58. The IC 50 value of the CSO–SA micelle against A549 cells was 543.16 μg/ml, while the IC 50 of Lipofectamine™ 2000 was about 6 μg/ml. The in vitro transfection efficiency of CSO–SA micelles was investigated by using plasmid DNA (pEGFP-C1). The transfection efficiency with CSO–SA/DNA (N/P ratio is 29) was increased with the post-transfection time (in 76 h), while the optimal transfection of Lipofectamine™ 2000/DNA was obtained at 24 h. The transfection of CSO–SA was not interfered in the presence of 10% fetal bovine serum, which showed remarkable enhancement effect. The optimal transfection efficiency of CSO–SA micelles in A549 cells was about 15%, which was higher than that of CSO (about 2%) and approach to that of Lipofectamine™ 2000 (about 20%). The low cytotoxic biodegradable CSO–SA micelles could be used as an effective DNA condensation carrier for gene delivery system.

DNA Delivery for Vaccination and Therapeutics Through the Skin

Cutaneous gene therapy and DNA vaccination are potential applications of plasmid delivery methods where a gene for an antigen or a therapeutic protein is inserted in the plasmid and applied to the skin. However, the delivery of the DNA plasmid is a major challenge due to the unusual physicochemical properties of the DNA, the tissue and cellular barriers and expression difficulties. Even though the skin is the most accessible organ of the body and it is an ideal target for gene therapy, the delivery of plasmid DNA across the skin is very difficult due to the specific barrier function of the stratum corneum and the inconsistent transfection rate of keratinocytes and other epidermal cells. To date there is no gene delivery system that was shown to be optimal for cutaneous gene therapy. In order to develop an efficient non-viral delivery vehicle we need to design a system that provides the combined properties of effective DNA condensation, cutaneous permeation, cellular transfection and sufficiently sustained expression. This paper reviews the formulation approaches and delivery methods for DNA through the skin in the context of the barriers both at the tissue and cellular levels for both vaccine and gene therapy applications.

Poly( l-lysine)-g-poly( d,l-lactic-co-glycolic acid) micelles for low cytotoxic biodegradable gene delivery carriers

Poly(lactic-co-glycolic acid) (PLGA)-grafted poly( l-lysine) (PLL) (PLL-g-PLGA) was synthesized to demonstrate its micelle-forming property in an aqueous solution. The micelles were used as a gene delivery carrier. The hydrodynamic diameter of PLL-g-PLGA micelles in an aqueous solution was ca. 149 nm with a narrow size distribution. Critical micelle concentration (cmc) was 9.6 mg/l. The PLL-g-PLGA micelles could be used to produce compact nanoparticulate complexes with plasmid DNA, which could efficiently protect the complexed DNA from enzymatic degradation by DNase I. The micelle/DNA complexes had highly compacted structure sized between 200–300 nm with a positive surface charge value. The PLL-g-PLGA micelles exhibited much higher transfection efficiency with lower cytotoxicity than PLL. Here, we demonstrated that biodegradable and cationic PLL-g-PLGA micelles could be used as an effective DNA condensation carrier for gene delivery system.