Dexamethasone-loaded peptide micelles for delivery of the heme oxygenase-1 gene to ischemic brain

The R3V6 peptides, which are composed of a 3-arginine block and a 6-valine block, formed self-assembled micelles in aqueous solution. Dye quenching assays showed that a hydrophobic fluorescent dye, 5-dodecanoylaminofluorescein (DAF), interacted with and was loaded into the hydrophobic core of the micelles. In this study, dexamethasone-loaded R3V6 peptide micelles (R3V6-Dexa) were evaluated as a gene carrier. R3V6-Dexa had higher gene delivery efficiency in human embryonic kidney 293 cells compared to those of the R3V6 peptides and poly-L-lysine (PLL). Dexamethasone might stabilize the micelle structure of the R3V6 peptides by forming strong hydrophobic cores and enhanced the transfection efficiency. Furthermore, R3V6-Dexa reduced the expression of an inflammatory cytokine, interleukin-6 (IL-6), more efficiently in lipopolysaccharide (LPS)-induced Raw264.7 cells than did dexamethasone, suggesting that R3V6-Dexa is also a useful carrier for dexamethasone delivery. A focal brain ischemia-reperfusion model was produced by middle cerebral artery occlusion (MCAO). A heme oxygenase-1 (HO-1) expression plasmid DNA, pSV-HO-1, was delivered into the brain using R3V6-Dexa as a carrier. The pSV-HO-1/R3V6-Dexa complex was injected into the brain 1hr prior to MCAO. Twenty-four hours later, the HO-1 expression of the pSV-HO-1/R3V6-Dexa injection group was higher than those of the MCAO control, pβ-Luc/R3V6-Dexa, and pSV-HO-1/PEI25k injection groups. In addition, the infarct size was reduced due to the delivery of pSV-HO-1/R3V6-Dexa complex. Therefore, R3V6-Dexa may be a useful carrier for HO-1 gene delivery and stroke gene therapy.     

Molecular imaging of a cancer-targeting theragnostics probe using a nucleolin aptamer- and microRNA-221 molecular beacon-conjugated nanoparticle

MicroRNAs (miRNA, miR) have been reported as cancer biomarkers that regulate tumor suppressor genes. Hence, simultaneous detecting and inhibiting of miRNA function will be useful as a cancer theragnostics probe to minimize side effects and invasiveness. In this study, we developed a cancer-targeting therangostics probe in a single system using an AS1411 aptamer - and miRNA-221 molecular beacon (miR-221 MB)-conjugated magnetic fluorescence (MF) nanoparticle (MFAS miR-221 MB) to simultaneously target to cancer tissue, image intracellularly expressed miRNA-221 and treat miRNA-221-involved carcinogenesis. AS1411 aptamer-conjugated MF (MFAS) nanoparticles displayed a great selectivity and delivery into various cancer cell lines. The miR-221 MB detached from the MFAS miR-221 MB in the cytoplasm of C6 cells clearly imaged miRNA-221 biogenesis and simultaneously resulted in antitumor therapeutic effects by inhibiting miRNA function, indicating a successful astrocytoma-targeting theragnostics. MFAS miRNA MB can be easily applied to other cancers by simply changing a targeted miRNA highly expressed in cancers.     

DNA delivery to the mitochondria sites using mitochondrial leader peptide conjugated polyethylenimine

Some genetic diseases are associated with the defects of the mitochondrial genome. Direct DNA delivery to the mitochondrial matrix has been suggested as an approach for mitochondrial gene therapy for these diseases. We hypothesized that a mitochondrial leader peptide (LP) conjugated polyethylenimine (PEI) could deliver DNA to the mitochondrial sites. PEI-LP was synthesized by the conjugation of LP to PEI using disulfide bond. The complex formation of PEI-LP with DNA was confirmed by a gel retardation assay. In this study, DNA was completely retarded at a 0.4/1 PEI-LP/DNA weight ratio. In vitro delivery tests into isolated mitochondria or living cells were performed with rhodamin-labeled DNA and PEI-LP. In vitro cell-free delivery assay with isolated mitochondria showed that PEI-LP/DNA complexes were localized at mitochondria sites. Furthermore, the PEL-LP/DNA complexes were localized at the mitochondrial sites in living cells. However, a control carrier, PEI, did not show this effect. In addition, MTT assay showed that PEI-LP showed lower cytotoxicity than PEI. These results suggest that PEI-LP can deliver DNA to the mitochondrial sites and may be useful for the development of mitochondrial gene therapy.     

Combinational therapy of ischemic brain stroke by delivery of heme oxygenase-1 gene and dexamethasone

Combinational therapies using genes and drugs are promising therapeutic strategies for various diseases. In this research, a co-delivery carrier of dexamethasone and plasmid DNA (pDNA) was developed by conjugation of dexamethasone to polyethylenimine (2 kDa, PEI2k) for combinational therapy of ischemic brain. Dynamic light scattering, atomic force microscopy and flow cytometry studies showed that the pDNA/dexamethasone-conjugated PEI2k (PEI2k-Dexa) complex was 150 nm in size and was taken up by cells more easily than PEI2k-Dexa only. The tumor necrosis factor-α (TNF-α) level was decreased more efficiently by pDNA/PEI2k-Dexa complex than dexamethasone only in hypoxia activated Raw 264.7 macrophage cells, suggesting that pDNA/PEI2k-Dexa complex increased the delivery efficiency and therapeutic effect of dexamethasone. In in vitro transfection assay, PEI2k-Dexa had higher transfection efficiency than PEI2k and lipofectamine. However, the simple mixture of PEI2k and dexamethasone did not show this effect, suggesting that the conjugation of dexamethasone to polyethylenimine increased DNA delivery efficiency of PEI2k. To evaluate the effects of combinational therapy in vivo, pDNA/PEI2k-Dexa complex was applied to a transient focal ischemia animal model. At 24 h after the injection, mean infarction volume and the TNF-α level were reduced more efficiently in the pDNA/PEI2k-Dexa injection group, compared with the control, pDNA/PEI2k, or dexamethasone injection group. The infarction volume and inflammatory cytokines were further decreased by delivery of pSV-HO-1 using PEI2k-Dexa. Magnetic resonance imaging and microPET studies confirmed the therapeutic effect of pSV-HO-1/PEI2k-Dexa complex at 10 days after the injection. Therefore, pSV-HO-1/PEI2k-Dexa complexes may be useful in combinational therapy for ischemic diseases such as stroke.     

Apatite-coated collagen scaffold for bone morphogenetic protein-2 delivery

Bone morphogenetic proteins (BMPs) are the most potent osteoinductive growth factors. BMP-2 is clinically used for spine fusion and bone fracture healing. Commercially available BMP-2 uses a type I collagen scaffold as a carrier, but it only releases BMP-2 for a short period of time, which may release the bone formation efficacy. In the present study, we hypothesize that apatite coating of a collagen scaffold increases the release period as well as the osteogenic efficacy of BMP-2. Apatite coating was achieved by incubating collagen scaffolds in simulated body fluids (SBFs). Apatite coating on collagen scaffolds was confirmed by X-ray diffraction, electron spectroscopy for chemical analysis, attenuated total reflectance-Fourier transform infrared spectroscopy, and scanning electron microscopy. The rate and period of BMP-2 release from apatite-coated collagen scaffolds varied depending on the concentration of SBFs used. The 5× and 10× SBF apatite-coated collagen scaffolds released 91.8%±11.5% and 82.2%±13.1% of their loaded BMP-2 over 13 days in vitro, respectively, whereas noncoated collagen scaffold released 98.3%±2.2% over the initial one day. BMP-2 released from apatite-coated collagen scaffold significantly increased the alkaline phosphatase activity of cultured osteoblasts, compared with BMP-2 released from noncoated collagen scaffold. Computed tomography and histomorphometry showed that BMP-2 delivery using apatite-coated collagen scaffolds resulted in 2.5-fold higher bone formation volume and 4.0-fold higher bone formation area than BMP-2 delivery using noncoated collagen scaffolds. This study shows that simple apatite coating of a collagen scaffold results in a BMP-2 carrier that renders long-term release of BMP-2 and dramatically enhances osteogenic efficacy.     

Dexamethasone conjugation to polyamidoamine dendrimers G1 and G2 for enhanced transfection efficiency with an anti-inflammatory effect

Polyamidoamine (PAM) dendrimers with low generation such as PAM generation 1 (PAMG1) and PAM generation 2 (PAMG2) have been widely used as a gene carrier due to low toxicity, albeit their low transfection efficiency. In this study, dexamethasone was conjugated to PAMG1 and PAMG2 in order to increase the transfection efficiency. In a gel retardation assay, the dexamethasone conjugated PAMG1 and PAMG2 (PAMG1-Dexa and PAMG2-Dexa) retarded plasmid DNA (pDNA) completely at 5:1 and 3:1 weight ratios (polymer:pDNA), respectively. In transfection assays, PAMG1-Dexa and PAMG2-Dexa had the highest transfection efficiency at 20:1 and 10:1 weight ratios, respectively. In addition, PAMG1-Dexa and PAMG2-Dexa had higher transfection efficiencies than PAMG1, PAMG2, PEI25k, and lipofectamine. In a MTT assay, PAMG1-Dexa and PAMG2-Dexa were less cytotoxic than lipofectamine. In addition, PAMG1-Dexa and PAMG2-Dexa decreased the TNF-α level more efficiently than dexamethasone only in the lipopolysaccharide (LPS)-induced Raw264.7 cells. Therefore, PAMG1-Dexa and PAMG2-Dexa may prove to be useful as gene delivery carriers with an anti-inflammatory effect.