Modulatable endosomalytic, intracellularly biodegradable vectors for gene delivery

In order to achieve efficient gene delivery, we have designed p$K_a$ modulatable oligopeptides (2COPs) by combining with lysine, histidine and cysteine residues which will bind DNA extracellularly, internalize via endocytosis, provide a tunable endosomal release mechanism, and provide a degradable backbone in order that the DNA can be released once in the cytoplasm. The reducible polycations (RPCs) were synthesized from 2COPs. The sizes, surface charges and the stability of RPC polyplexes under the simulated physiological conditions extra- and intracellularly suggested that these RPCs are promising vectors. In addition, the transfections revealed that the RPCs can facilitate endosomal buffering and intracellular reduction and are non-toxic to cells. The nuclear targeting signal (TAT) was incorporated into these vectors. The reducible copolycations (RcPCs) were synthesized via oxidative polymerisation between 2COPs and TAT. The RcPC polyplexes are ~100 nm, and are positively charged. Gel shift assay revealed that RcPCs have less potential than RPCs to be used as vectors as they are less stable extracellularly than the RPCs. In addition, chloroquine was required to enhance the transfection of RcPCs. Furthermore, there is no improvement in transfection of RcPC compared to RPCs. Therefore, this suggests that the incorporation of TAT does not improve the transfection

Selection and characterization of bacteriophages specific to Salmonella Choleraesuis in swine

Background and Aim: Salmonella Choleraesuis is the most common serotype that causes salmonellosis in swine. Recently, the use of bacteriophages as a potential biocontrol strategy has increased. Therefore, this study aimed to isolate and characterize bacteriophages specific to S. Choleraesuis associated with swine infection and to evaluate the efficacy of individual phages and a phage cocktail against S. Choleraesuis strains in simulated intestinal fluid (SIF). Materials and Methods: Three strains of S. Choleraesuis isolated from pig intestines served as host strains for phage isolation. The other 10 Salmonella serovars were also used for the phage host range test. The antibiotic susceptibility of the bacterial strains was investigated. Water samples from natural sources and drain liquid from slaughterhouses were collected for phage isolation. The isolated phages were characterized by determining the efficiency of plating against all Salmonella strains and the stability at a temperature range (4°C–65°C) and at low pH (2.5–4.0) in simulated gastric fluids (SGFs). Furthermore, morphology and genomic restriction analyses were performed for phage classification phages. Finally, S. Choleraesuis reduction in the SIF by the selected individual phages and a phage cocktail was investigated. Results: The antibiotic susceptibility results revealed that most Salmonella strains were sensitive to all tested drugs. Salmonella Choleraesuis KPS615 was multidrug-resistant, showing resistance to three antibiotics. Nine phages were isolated. Most of them could infect four Salmonella strains. Phages vB_SCh-RP5i3B and vB_SCh-RP61i4 showed high efficiency in infecting S. Choleraesuis and Salmonella Rissen. The phages were stable for 1 h at 4°C–45°C. However, their viability decreased when the temperature increased to 65°C. In addition, most phages remained viable at a low pH (pH 2.5–4.0) for 2 h in SGF. The efficiency of phage treatment against S. Choleraesuis in SIF showed that individual phages and a phage cocktail with three phages effectively reduced S. Choleraesuis in SIF. However, the phage cocktails were more effective than the individual phages. Conclusion: These results suggest that the newly isolated phages could be promising biocontrol agents against S. Choleraesuis infection in pigs and could be orally administered. However, further in vivo studies should be conducted

Structural variations in hyperbranched polymers prepared via thermal polycondensation of lysine and histidine and their effects on DNA delivery

The successful clinical translation of non-viral gene delivery systems has yet to be achieved due to the biological and technical obstacles to preparing a safe, potent and cost-effective vector. Hyper-branched polymers have emerged as promising candidates to address gene delivery barriers owing to their relatively simple synthesis and ease of modification compared to other polymers, which makes them more feasible for scale up and manufacturing. Here, we compare hyperbranched poly(amino acids) synthesised by co-polymerising histidine and lysine, with hyperbranched polylysine prepared using the well-known ‘ultra-facile’ thermal polycondensation route, to investigate the effects of histidine units on the structure and gene delivery applications of the resultant materials. The conditions of polymerisation were optimised to afford water-soluble hyperbranched polylysine-co-histidine of three different molar ratios with molecular masses varying from 13-30 kDa. Spectroscopic, rheological and thermal analysis indicated that the incorporation of histidine modulated the structure of hyperbranched polylysine to produce a more dendritic polymer with less flexible branches. Experiments to probe gene delivery to A549 cells indicated that all the new hyper-branched polymers were well-tolerated but, surprisingly, the co-polymers containing histidine were not more effective in transfecting a luciferase gene than hyper-branched poly(lysine)s synthesised as established literature comparators. We attribute the variations in gene delivery efficacy to the changes induced in polymer architecture by the branching points at histidine residues, and obtain structure-function information relating histidine content with polymer stiffness, pKa and ability to form stable polyplexes with DNA. The results are of significance to nanomedicine design as they indicate that addition of histidine as a co-monomer in the synthetic route to hyper-branched polymers changes not only the buffering capacity of the polymer but has significant effects on the overall structure, architecture and gene delivery efficacy

Interaction of reducible polypeptide gene delivery vectors with supported lipid bilayers: pore formation and structure-function relationships

Real-time atomic force microscopy (AFM) in aqueous buffer has been used to probe interactions of synthetic disulfide-linked polypeptide gene delivery vectors with supported phospholipid bilayers. Disruption of the membranes was apparent in AFM, and the extent of surface heterogeneity and hole (pore) formation was evaluated by depth and area-profiling image analysis. The overall extent of membrane disruption varied with the reducible polycation peptide sequence and block structure and was found to be correlated with the overall degree of transgene expression in two representative cell lines. © 2010 The Royal Society of Chemistry

Combination dual responsive polypeptide vectors for enhanced gene delivery.

Variation in amino acid sequences on a disulfide-linked polypeptide backbone generates differing pK(a) vectors for DNA delivery, which release nucleic acids under reducing conditions and transfect cells with greater efficacy than non-reducible or non-variable pK(a) analogues

Multicomponent synthetic polymers with viral-mimetic chemistry for nucleic acid delivery.

The ability to deliver genetic material for therapy remains an unsolved challenge in medicine. Natural gene carriers, such as viruses, have evolved sophisticated mechanisms and modular biopolymer architectures to overcome these hurdles. Here we describe synthetic multicomponent materials for gene delivery, designed with features that mimic virus modular components and which transfect specific cell lines with high efficacy. The hierarchical nature of the synthetic carriers allows the incorporation of membrane-disrupting peptides, nucleic acid binding components, a protective coat layer, and an outer targeting ligand all in a single nanoparticle, but with functionality such that each is utilized in a specific sequence during the gene delivery process. The experimentally facile assembly suggests these materials could form a generic class of carrier systems that could be customized for many different therapeutic settings

Multicomponent Synthetic Polymers with Viral-Mimetic Chemistry for Nucleic Acid Delivery

The ability to deliver genetic material for therapy remains an unsolved challenge in medicine. Natural gene carriers, such as viruses, have evolved sophisticated mechanisms and modular biopolymer architectures to overcome these hurdles. Here we describe synthetic multicomponent materials for gene delivery, designed with features that mimic virus modular components and which transfect specific cell lines with high efficacy. The hierarchical nature of the synthetic carriers allows the incorporation of membrane-disrupting peptides, nucleic acid binding components, a protective coat layer, and an outer targeting ligand all in a single nanoparticle, but with functionality such that each is utilized in a specific sequence during the gene delivery process. The experimentally facile assembly suggests these materials could form a generic class of carrier systems that could be customized for many different therapeutic settings