Guanidinylated bioreducible polymer (GBP) was developed for gene delivery systems utilizing cellular penetrating ability of guanidine groups. GBP could retard pDNA from a weight ratio of 5 completely in agarose gel electrophoresis but pDNA was released from GBP polyplexes in reducing condition (2.5 mM DTT) due to their biodegradation. GBP also could construct 200 nm-sized and positively charged (~30 mV) polyplex nanoparticles with pDNA. The cytotoxicity of GBP was found to be minimal and GBP showed about 8 folds improved transfection efficiency than a scaffold polymer, poly(cystaminebisacrylamide-diaminohexane) (poly(CBA-DAH)) and even higher transfection efficiency than PEI25k in mammalian cell lines. Its high cellular uptake efficiency (96.1 %) and strong nuclear localization ability for pDNA delivery due to the structural advantage of bioreducible polymer and guanidine groups were also identified, suggesting GBP is a promising candidate for efficient gene delivery systems.
The development of biodegradable cationic polymers for use in somatic gene therapy is desirable because degradable polymers have the potential to overcome cellular toxicities that are related to the high charge densities of the polycationic delivery system. Therefore, to produce a biocompatible gene delivery vehicle, we have designed a novel biodegradable, high molecular weight multiblock copolymer (MBC) of the type (AB) n which consists of repeating units of low molecular weight poly(ethylene glycol) (PEG) conjugated to low molecular weight cationic poly(L-lysine) (PLL). PEG was used not only to impart steric stabilization properties onto the polymer/pDNA complexes but also to introduce biodegradable ester bond linkages into the backbone of the MBCs. Also, to improve the endosome-disrupting capabilities of the polymer, N,N-dimethylhistidine (His) was coupled at various mole ratios (5 mol % His, 9 mol % His, 16 mol % His, 22 mol % His) to the -amines of PLL to produce PEG-PLL-grafted-His (PEG-PLL-g-His) MBCs. Polymer screening revealed that MBCs with 16% His grafted (PEG-PLL-g-16% His) (31 kDa) produced the highest transfection efficiency with minimal cytotoxicity in murine smooth muscle cells (A7r5). The MBCs condensed plasmid DNA (pDNA) into nanostructures with an average particle size between 150 and 200 nm with no aggregation and surface charge of ∼4-45 mV. These MBCs also protected pDNA from endonuclease digestion for at least 2 h. The polymers showed exponential decay with a halflife (t 1/2) of ∼5 h in PBS, pH 7.4 at 37 °C. However, complexes incubated in PBS buffer showed complete stability up to 6 days despite the short polymer t1/2. The pK of the conjugated imidazoles was found to be 4.75 which would facilitate buffering at low pH environments of the late endosome/lysosome. Finally, the ability of the imidazoles to protonate and destabilize membrane vesicles was investigated by the use of bafilomycin A 1 which showed that the MBCs produced about five times higher transfection efficiency in vitro in A7r5 cells compared to the treated cells. This supports the function of histidine as an endosomal disrupting moiety. Therefore, these results suggest that biodegradable multiblock copolymers are promising candidates for long-term gene delivery.
Nonarginine (D-R9) has been reported to be one of the most efficacious protein transduction domains (PTDs) for the intracellular cargo delivery such as DNA, RNA, proteins, and particles. Although oligoarginines are capable of forming polyplex with DNA by electrostatic interaction, the length of oligoarginine can affect the toxicity and gene expression. The reducible poly(oligo-D-arginine) (rPOA) composed of the Cys-(D-R9)-Cys repeating unit forming disulfide bonds between terminal cysteinyl-thiol groups of short peptides was hypothesized to show efficient gene transfection without toxicity. The reducible high molecular weight poly(oligo-D-arginine) may fragment into the Cys-(D-R9)-Cys in cellular environments such as cytosol, cell surface, endosomes, and lysosomes, and enhance DNA transfection efficiency. In the present study, in vitro stability, cytotoxicity, and transfection efficiency of DNA/poly(oligo-D-arginine) polyplex were evaluated. In addition, in vivo delivery of DNA into the lung was performed by intratracheal injection of DNA/poly(oligo-D-arginine) polyplex. The in vivo study with rPOA showed higher level of gene expression than PEI, sustaining for 1 week without toxicity. Reducible high molecular weight poly(oligo-D-arginine) based on R9 PTD is a very promising nonviral gene carrier for lung diseases by efficiently condensing, stabilizing, and transfecting DNA.
High mobility group box 1 (HMGB1) was originally identified as ubiquitously expressed nonhistone DNA-binding protein, but recently, it was found to act as an endogenous danger molecule, which signals danger and traumatic cell death. Previously, the authors showed that HMGB1 is massively released immediately after an ischemic insult and that it subsequently activates microglia and induces inflammation in the postischemic brain. Here, we showed the endogenous danger molecule-like function of HMGB1 in primary cortical cultures. HMGB1 was found to be accumulated in NMDA-treated primary cortical culture media, and media collected from these cultures were able to induce neuronal cell death when added to fresh primary cortical cultures. However, HMGB1-depleted NMDA-conditioned media produced by HMGB1 siRNA transfection or by preincubation with anti-HMGB1 antibody or with HMGB1 A box failed to induce neuronal cell death. Furthermore, siRNA-mediated HMGB1 knockdown substantially suppressed NMDA- or Zn(2+)-induced cell death. It was interesting to find that extracellular HMGB1-induced neuronal apoptosis, as evidenced by TUNEL staining and caspase 3 assay in combination with double immunofluorescence staining. A series of RAGE and HMGB1 co-immunoprecipitation experiments in the presence of SB203580 and PD98059 (p38 MAPK and ERK inhibitors, respectively) demonstrated that RAGE-p38 MAPK and RAGE-ERK pathway might underlie extracellular HMGB1-mediated neuronal apoptosis. These results together with our previous reports regarding microglial activation by extracellular HMGB1 indicate that HMGB1 functions as a novel danger signal, which aggravates brain damage via autocrine and paracrine manners.
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