Amphiphilic peptide carrier for the combined delivery of curcumin and plasmid DNA into the lungs

In this study, the R7L10 peptide, which is composed of a 7-arginine stretch and a 10-leucine stretch, was evaluated as a carrier for the combined delivery of curcumin and plasmid DNA (pDNA) into the lungs. Curcumin is a natural product with anti-inflammatory and anti-tumor effects. Curcumin-loaded R7L10 (R7L10-curucmin) was prepared by an oil-in-water (O/W) emulsion/solvent evaporation method. In vitro transfection showed that R7L10-curcumin had higher transfection efficiency than R7L10. Although R7L10-curcumin had lower transfection efficiency than polyethylenimine (25 kDa, PEI25k) and lipofectamine, R7L10-curcumin had lower cytotoxicity. In gel retardation assays and heparin competition assays, R7L10-curcumin formed a more stable complex with pDNA than R7L10. The intracellular curcumin delivery efficiency of R7L10-curcumin was higher than that of curcumin only. Furthermore, R7L10-curcumin more efficiently decreased TNF-α level in lipopolysaccharide (LPS)-activated Raw264.7 macrophage cells than curcumin only. For in vivo evaluation, pDNA/R7L10-curcumin complexes were administered into mouse lungs by intratracheal instillation. The results revealed that R7L10-curcumin delivered pDNA more efficiently than R7L10, poly-L-lysine (PLL), or PEI25k. In addition, R7L10-curcumin decreased TNF-α level in lung tissues in an acute lung injury mouse model. In contrast to PEI25k, R7L10-curcumin did not show liver toxicity after intravenous injection. These results suggest that R7L10-curcumin is a useful carrier for the combined delivery of curcumin and pDNA into the lungs.     

Long-circulating DNA-complexed biodegradable multiblock copolymers for gene delivery: degradation profiles and evidence of dysopsonization.

Biodegradable cationic polymers have become promising alternatives to traditional polycationic gene delivery systems in which the high charge densities of high molecular weight polymers contribute significantly to cellular toxicities. Previous research has shown that biodegradable, multiblock copolymers (MBC), PEG-PLL-g-16% His, are efficient gene carriers with negligible cellular toxicities. The present research was designed to characterize the polymer degradation as well as to determine the biodistribution of the MBC after systemic administration. Polymer degradation was performed in buffer as a function of pH, in serum and within polymer/pDNA complexes. The MBC exhibited exponential decay with a half-life (t1/2) of approximately 14 min at pH 9.0, approximately 5 h at pH 7.4 and approximately 2 h in serum. However, there was little or no degradation observed at pH 4.0 and the MBC within the complexes degraded between 4 and 8 h in serum. Biodistribution data performed with fluorescently labeled polymer and pDNA revealed that intact complexes remained in the blood up to 3 days, which was also reflected in the organs as a function of time. Therefore, the cumulative data suggest that PEG may be sterically stabilizing complexes in vivo via dysopsonization in which serum proteins mask the complexes from elements of the reticuloendothelial system (RES).     

Prevention of Autoimmune Insulitis by Delivery of Interleukin-4 Plasmid Using a Soluble and Biodegradable Polymeric Carrier

Purpose. We delivered interleukin-4 (IL-4) plasmid (pCAGGS-IL-4) using the biodegradable polymer, poly[-(4-aminobutyl)-L-glycolic acid] (PAGA), to prevent autoimmune insulitis in NOD mice. Methods. The pCAGGS-IL-4/PAGA complex was transfected to 293T cells. The expression level of IL-4 was measured by ELISA. The pCAGGS IL-4/PAGA complex was injected once to NOD mice intravenously at the age of 4 weeks. RT-PCR was performed to evaluate the level of the IL-4 mRNA in the liver. At 6 weeks after the injection, the grade of insulitis of the mice was evaluated by double blind methods. Results. In vitro transfecton assays showed that PAGA enhanced the expression of IL-4 in 293T cells. RT-PCR of the liver showed that IL-4 was expressed highest in the complex injected group. In the plasmid/PAGA complex injected group, the prevalence of severe insulitis in NOD mice was markedly improved, suggesting that PAGA enhanced the delivery of IL-4 plasmid. Conclusion. The pCAGGS-IL-4/PAGA complex is an effective system to prevent autoimmune insulitis in NOD mice and applicable for the prevention of autoimmune diabetes.     

A New Potent hFIX Plasmid for Hemophilia B Gene Therapy

PURPOSE: The purpose of this work was to construct and characterize a new potent hFIX plasmid, p2SV-hFIX, which has two hFIX expression units containing the SV40 promoter/enhancer for hemophilia B gene therapy. METHODS: p1SV-hFIX was constructed by insertion of amplified hFIX cDNA at the ECORI and Xbal sites of pSI expression vector containing simian virus 40 (SV40) promoter/enhancer. To construct p2SV-hFIX, the hFIX expression cassette was isolated from p1SV-hFIX by digestion with restriction enzymes, and the purified expression cassette was inserted at the BglII site of another plSV-hFIX. The gene expression of p1SV-hFIX, p2SV-hFIX, and a plasmid containing a liver-specific apoE enhancer and alpha antitrypsin promoter, pAAV-hAAT-hFIX. were evaluated in various cell lines using polyethylenimine (PEI) as a gene carrier in vitro. RESULTS: The construction of p1SV-hFIX and p2SV-hFIX were confirmed by restriction enzyme studies. The transfection efficiency of p2SV-hFIX was 3.83-fold and 7.16-fold higher than that of pAAV-hAAT-hFIX in C2C12 and NIH3T3 cells, respectively. p2SV-hFIX also showed higher transfection efficiency than p1SV-hFIX in both cells. CONCLUSIONS: In accordance with these results, p2SV-hFIX is a new potent hFIX plasmid that can be transfected in various cells. Systemic delivery of p2SV-hFIX via intravenous or intramuscular injection is feasible for treatment of hemophilia B.     

Anti-angiogenic inhibition of tumor growth by systemic delivery of PEI-g-PEG-RGD/pCMV-sFlt-1 complexes in tumor-bearing mice.

Vascular endothelial growth factor (VEGF) is an endogenous mediator of tumor angiogenesis. Blocking associations of the VEGF with its corresponding receptors (Flt-1, KDR/flk-1) have become critical for anti-tumor angiogenesis therapy. Previously, we synthesized PEI-g-PEG-RGD conjugate and evaluated as an angiogenic endothelial polymeric gene carrier. In this study, PEI-g-PEG-RGD/pCMV-sFlt-1 complexes are evaluated in terms of tumor growth inhibition in vivo. Complexes were repeatedly injected systemically via tail vein into subcutaneous tumor-bearing mice. As a result, tumor growth was inhibited in the PEI-g-PEG-RGD/pCMV-sFlt-1 injected group. However, this effect was not identified in PEI-g-PEG/pCMV-sFlt-1 or PEI-g-PEG-RGD/pCMV-GFP control groups. Moreover, the survival rate increased in the PEI-g-PEG-RGD/pCMV-sFlt-1 group compared with the controls group. These results suggest that delivery of pCMV-sFlt-1 using PEG-g-PEG-RGD may be effective for anti-angiogenic gene therapy.