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Dexamethasone-Conjugated Polyamidoamine Dendrimer for Delivery of the Heme Oxygenase-1 Gene into the Ischemic Brain Pureum Jeon, Manbok Choi, Jungju Oh, Minhyung Lee* Heme oxygenase-1 (HO-1) has anti-apoptotic and anti-inflammatory effects. In this study, the HO-1 gene was delivered into the brain using dexamethasone-conjugated polyamidoamine generation 2 (PAMAM G2-Dexa) for the treatment of ischemic stroke. PAMAM G2-Dexa formed stable complexes with plasmid DNA (pDNA). The pDNA delivery efficiency of PAMAM G2-Dexa was higher than that of polyethylenimine (PEI25k, 25 kDa), dexamethasone-conjugated PEI (PEI-Dexa), and PAMAM G2 in Neuro2A cells. Therapeutic effect of PAMAM G2-Dexa/pHO-1 complexes was evaluated in a stroke animal model. PAMAM G2-Dexa delivered pHO-1 more efficiently into the ischemic brain than PEI25k and PEI-Dexa with higher therapeutic effect. Therefore, PAMAM G2-Dexa/pHO-1 complexes may be useful for ischemic stroke gene therapy.

1. Introduction Gene therapy is a promising clinical method for the treatment of various diseases, including acquired and genetic diseases. There are two requirements for successful gene therapy: an efficient gene carrier and a target-specific therapeutic gene. Current gene carriers can be divided into non-viral and viral carriers, each of which have distinct advantages and disadvantages.[1] For example, viral carriers, such as adenoviral vectors, have high gene delivery efficiency. However, they induce immune responses, which limit the application of adenoviral vectors in a therapeutic setting. Non-viral carriers such as liposomes and polymers have low immunogenicity and toxicity.[1] However, their gene delivery efficiency is much lower than those of viral carriers. Therefore, much research has been dedicated toward increasing the transfection efficiency of non-viral carriers. M. Lee, P. Jeon, M. Choi, J. Oh BK21 Plus Future Biopharmaceutical Human Resources Training and Research Team, Department of Bioengineering, College of Engineering, Hanyang University, Seoul 133-791, Korea E-mail: [email protected] Macromol. Biosci. 2015, 15, 1021–1028 © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

Dexamethasone has been reported to be a useful molecule for non-viral gene delivery.[2–6] First, dexamethasone can bind to glucocorticoid receptors in the cytosol. On binding to these receptors, dexamethasone enters the nucleus. During this process, dexamethasone–glucocorticoid receptor complexes dilate the nuclear pores up to 20 nm.[7] This dilation of the nuclear pores has positive effects on gene delivery. Second, dexamethasone-conjugated polymeric gene carriers have a higher nuclear localization effect because dexamethasone–glucocorticoid receptor complexes enter the nucleus with dexamethasone-conjugated carriers and their bound DNA.[4,5] Dexamethasone has, therefore, been used in the development of non-viral gene carriers. Dexamethasone has been conjugated to DNA for efficient translocation of the DNA into the nucleus.[2] Similarly, dexamethasone has been conjugated to low molecular weight PEI (2 kDa), yielding dexamethasone-conjugated PEI (PEI-Dexa).[5,6,8,9] PEI-Dexa had a higher transfection efficiency than PEI2k, while the transfection efficiency of PEI-Dexa was similar to that of high molecular weight PEI (25 kDa).[5,8] Another important advantage of dexamethasone as a non-viral gene carrier is its anti-inflammatory effect.[6,8,9]

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DOI: 10.1002/mabi.201500058

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Dexamethasone is a steroid anti-inflammatory drug and widely used to treat various inflammatory diseases. Especially, it has been used to alleviate brain edema in ischemic stroke.[10] In our study with PEI-Dexa, we showed that PEI-Dexa had a strong anti-inflammatory effect and reduced inflammatory reactions in the ischemic brain.[8] The effect of PEI-Dexa on inflammation has also been studied in the context of acute lung injury, which is accompanied by a severe inflammatory reaction.[9] PEIDexa reduced inflammation and protected cells in both stroke and acute lung injury. In another study, dexamethasone-conjugated polyamidoamine generation 2 (PAMAM G2-Dexa) was synthesized as a gene carrier.[11] PAMAM G2-Dexa had higher transfection efficiency than PEI-Dexa in the mouse neuroblastoma Neuro2A (N2A) cells. This suggests that PAMAM G2-Dexa may be more efficient than PEI-Dexa for therapeutic gene delivery to the ischemic brain. Furthermore, PAMAM, unlike PEI, has been reported to have an antiinflammatory effect.[12] Therefore, PAMAM G2-Dexa may have a greater anti-inflammatory effect than PEI-Dexa. Heme oxygenease-1 (HO-1) is an anti-oxidant enzyme that catalyzes the degradation of heme into downstream products such as biliverdin, CO, and Fe heme.[13] These products mediate anti-inflammatory and immunomodulatory effects.[14] Therefore, the combination delivery of the HO-1 gene and dexamethasone may have an additive effect on reduction of infarction in the ischemic brain. In our previous report, dexamethasone and the HO-1 gene were co-delivered into the brain using PEI-Dexa, resulting in reduction of infarction volume.[8] In the current study, we evaluated PAMAM G2-Dexa as an HO-1 gene carrier for the treatment of ischemic stroke. The antiinflammatory effect and gene delivery efficiency of PAMAM G2-Dexa were compared with those of PEI-Dexa. The HO-1 gene was delivered to the brain in a middle cerebral artery occlusion (MCAO) animal model to evaluate the therapeutic effects of PAMAM G2-Dexa/ pHO-1 complexes. Our results indicate that PAMAM G2-Dexa is an efficient carrier of the HO-1 gene and dexamethasone to the ischemic brain.

2. Experimental Section 2.1. Materials PAMAM G2, PEI2k, PEI25k, and [4,5-dimetylthiazol-2-yl]-2,5diphenyltetrazoliumbromide (MTT) were obtained from Sigma– Aldrich (Saint Louis, MO). Dexamethasone-21-mesylate was purchased from Steraloids Inc. (Newport, RI). Traut’s reagent and BCA assay kit were purchased from Pierce (Iselin, NJ). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were obtained from Welgene (Seoul, Korea). Luciferase assay kit and reporter lysis buffer were purchased from Promega (Madison,

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WI). Tumor necrosis factor-a (TNF-a) and interleukin-6 (IL-6) ELISA kits were purchased from eBioscience (San Diego, CA). HO-1 ELISA kit was obtained from Enzo (Farmingdale, NY).

2.2. Synthesis of Dexamethasone-Conjugated PAMAM G2 The synthesis of PAMAM G2-Dexa and PEI-Dexa has been described previously.[5,11] Briefly, PAMAM G2 or PEI2k was dissolved in anhydrous dimethyl sulfoxide (DMSO) with a twofold molar surplus of Traut’s reagent and dexamethasone-21-mesylate. The cross-linking reaction was allowed to proceed at room temperature for 14 h. Then, the mixture was dialyzed using dialysis membrane (MWCO 1 000 Da). After 2 d, PAMAM G2-Dexa was lyophilized using a freeze dryer.

2.3. Preparation of Plasmid DNA pb-Luc and pHO-1 were constructed as reported previously.[6,15] In pb-Luc and pHO-1, the luciferase and HO-1 cDNAs are located downstream of the b-actin enhancer and promoter. Plasmid DNAs (pDNAs) were transformed into the JM109 strain of Escherichia coli and the bacteria were cultured in Luria broth. pDNA was extracted from the bacteria using the Qiagen Maxi kit (Valencia, CA, USA). The concentration and purity of the pDNA were determined by measuring absorbance at 260 and 280 nm.

2.4. Gel Retardation Assays Carrier/pDNA complexes were prepared by mixing the carrier and pDNA at various weight ratios. Mixtures were incubated for 30 min at room temperature to allow complex formation. Complexes were analyzed by electrophoresis through a 1% agarose gel. After electrophoresis, pDNA was visualized using an UV transilluminator.

2.5. Heparin Competition Assays Carrier/pDNA complexes were prepared with PAMAM G2, PAMAM G2-dexa, PEI25k, and PEI-Dexa at their optimal weight ratios for the highest transfection. The complex mixtures were incubated at room temperature for 30 min. Then, various amounts of heparin were added to the complex mixtures and the mixtures were incubated at room temperature for an additional 30 min. Complexes were analyzed by electrophoresis through 1% agarose gels.

2.6. Size and Zeta Potential PAMAM G2, PAMAM G2-dexa, PEI25k, and PEI-Dexa were mixed with 5 mg of pb-Luc in distilled water. PAMAM G2-dexa/pDNA complexes were prepared at various weight ratios. PAMAM G2/ pDNA, PEI25k/pDNA, and PEI-Dexa/pDNA complexes were prepared at their optimal weight ratios for the highest transfection

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efficiency based on previous reports.[5,11,16] Complexes were incubated for 30 min at room temperature to allow for complex formation. Then, particle size and zeta potential were analyzed using the Zetasizer Nano ZS system (MALVERN Instrument, Worcestershire, UK).

2.7. Cell Culture and Transfection N2A cells were cultured in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum and antibiotics. N2A cells were seeded in a 12-well plate at a density of 1  105 cells  well 1 and incubated for 24 h at 37 8C in a humidified 5% CO2 incubator. To determine the optimal weight ratio of the PAMAM G2-dexa/pDNA complexes, complexes were prepared at various weight ratios. The amount of pDNA was fixed at 1 mg  well 1. Culture medium was replaced with serum-free medium just before transfection. Complexes were added to the wells and the cells were incubated for 4 h at 37 8C. After the 4-h incubation, the serum-free medium containing the complexes was replaced with DMEM with 10% FBS. Then, the cells were incubated for an additional 20 h at 37 8C in a humidified 5% CO2 incubator. To compare the transfection efficiencies of the carriers, carrier/ pDNA complexes were prepared at their optimum weight ratios. The optimum weight ratios were determined previously.[11] Complexes of PAMAM G2, PAMAM G2-dexa, PEI25k, and PEI-Dexa with pDNA were prepared at 20:1, 4:1, 1:1, and 8:1 weight ratios, respectively. Transfection assays were performed with N2A cells as described above.

2.8. Luciferase Assay

N2A cells were seeded in 24-well plates at a density of 5  104 cells  well 1. After 24 h, transfection was performed as described above. Then, 40 ml of 5 mg  ml 1 MTT solution was added to the wells and the cells were incubated for an additional 4 h at 37 8C. After the incubation, the medium containing MTT was removed and 700 ml of dimethyl sulfoxide (DMSO) was added to the cells. The absorbance at 570 nm was measured using a microplate reader (BioRad, Hercules, CA, USA).

2.11. Preparation Mouse Peritoneal Monocytes and Enzyme-Linked Immunosorbent Assays (ELISA) for Pro-Inflammatory Cytokines Mouse abdominal cavity was separated from abdominal wall. Ten milliliters of PBS was injected into the peritoneal cavity. After the injection, peritoneal immune response was induced by tapping around the peritoneum. Then, the fluid was removed from the peritoneal cavity. This procedure was repeated three times. The monocytes were isolated from the fluid by the centrifugation. Mouse peritoneal monocytes were seeded onto 12-well plates at a density of 1.5  105 cells. After 24 h, the cells were treated with 20 ng of lipopolysaccharide (LPS) for 2 h. Then, the medium with LPS was removed. Complexes between carriers and empty plasmid (pEmtpy) were prepared at ratios to obtain the highest transfection efficiency, and carrier/pEmpty complexes were then added to the cells. After 1 d, the levels of the pro-inflammatory cytokines such as TNF-a and IL-6 were detected using ELISA kits.

2.12. Stereotaxic Injection

After transfection, cells were washed with phosphate-buffered saline (PBS) and 150 ml of reporter lysis buffer was added to each well. After 10-min incubation at room temperature, the cell extracts were harvested and transferred to micro-centrifuge tube. Then, the cells were centrifuged at 13 000 rpm for 10 min. Cell supernatants were transferred to new micro-centrifuge tubes and luciferase activity was measured using a 96-well plate luminometer (Berthold Detection System GmbH, Pforzheim, Germany). The protein concentration of the lysate was measured using a BCA assay kit. Luciferase activity was expressed as relative light units (RLU)/mg total protein.

2.9. Flow Cytometry Analysis N2A cells were seeded onto six-well plates at a density of 3  105 cells  well 1 24 h prior to transfection. pcDNA-EGFP was complexed with various carriers. Carrier/pDNA complexes were prepared as described above. Carrier/pDNA complexes were added to the cells and the cells were incubated for 24 h in a humidified CO2 incubator. Then, the cells were washed, suspended in DPBS, and centrifuged at 1 300 rpm for 5 min. The washing steps were repeated. Finally, the cells were resuspended in FACS buffer. These samples were analyzed by a flow cytometry (BD FACS Calibur TM, BD Bioscience Immunocytometry Systems, Franklin Lakes, NJ, USA).

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2.10. Cytotoxicity Assay

All experimental procedures were performed in accordance with the institutional guidelines of the IACUC of Hanyang University. Male Sprague Dawley (SD) rats (280–300 g) were anesthetized using 5% isoflurane in 70% N2O and 30% O2. The monitoring point was placed 4 mm lateral from the bregma. A hole was drilled in the skull carefully with gentle saline (0.89% NaCl) to prevent damage to the dura mater. A 10-ml aliquot of the carrier/ pHO-1 complexes (15 mg pHO-1) was stereotaxically injected into the striatum 4 mm interior to the surface of a hole for 10 min using a 26-gauge Hamilton syringe (80330; Hamilton, Reno, NV, USA) and a microinjector.

2.13. Middle Cerebral Artery Occlusion (MCAO) Model and 2,3,5-Triphenyl Tetrazolium Chloride (TTC) Staining One hour after injection, rats were anesthetized as described above. The neck skin was incised about 2 cm. The right external carotid artery (ECA) and internal carotid artery (ICA) were isolated, divided in a carotid bifurcation, and ligatured with a silk suture, respectively. After the ECA was incised, 4-0 nylon suture was inserted into the ICA through the ECA and a suture was placed up to the MCA. The blood stream was occluded using a clip. One hour after occlusion, the clip and nylon suture were removed to induce reperfusion. After a day,

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the rats were decapitated and their brains harvested. Brains were sliced into 2 mm sections and stained in 2% 2,3,5-triphenyl tetrazolium chloride (TTC) solution for 10 min. Afterward, the brain slices were kept in 4% paraformaldehyde (PFA) at 4 8C.

2.14. Evaluation of Infarct Volume Infarct total size was measured using Image J 1.42 software (NIH, National Institutes of Health). Infarct volume was calculated by multiplying the area by a thickness of 2 mm.

2.15. HO-1 ELISA

Figure 1. (A) Gel retardation assay. Carrier/pDNA complexes were prepared with increasing amount of carrier. Complexes were analyzed by electrophoresis through a 1% agarose gel. (B) Heparin competition assay. Carrier/pDNA complexes were prepared at ratios optimal for highest transfection. An increasing amount of heparin was added to the complexes. Complexes were analyzed by electrophoresis through a 1% agarose gel.

The HO-1 plasmid (pHO-1) was transfected into N2A cells using various carriers. For brain tissue extract preparation, 5-mm-thick coronal brain slices located between 5 and 10 mm from the front were made using a brain matrix, and the slices included the section affected by the carrier/pDNA complex. Tissue was ground in reporter lysis buffer. HO-1 gene expression was measured using the human HO-1 ELISA following the manufacturer’s manual.

2.16. Statistical Analysis ANOVA followed by the Newman–Keuls test was used to assess the significance of differences among groups. All data are presented as averages  standard errors, and P values less than 0.05 were considered statistically significant.

3. Results and Discussion 3.1. Physical Characterization of DNA/PAMAM G2Dexa Complexes PAMAM G2-Dexa was synthesized as described previously.[11] Synthesis of PAMAM G2-Dexa was confirmed by 1 H NMR. The degree of dexamethasone conjugation and the critical micelle concentration (CMC) was reported in the previous paper.[11] The molar ratios between the conjugated dexamethasone and PAMAM G2 were 2.4:1 and the CMC of PAMAM G2-Dexa was 1.14 mg  ml 1.[11] Complex formation of PAMAM G2-Dexa with pDNA was confirmed by gel retardation assays (Figure 1). PAMAM G2, PEI25k, and PEI-Dexa were used as controls. All carriers formed complexes with pDNA. In particular, PAMAM G2Dexa retarded pDNA completely at a 1:2 weight ratio (carrier:pDNA), while PAMAM G2 did this at a 1:7 weight ratio. PAMAM G2-Dexa and pDNA formed complexes more efficiently than PAMAM G2, suggesting that conjugation of dexamethasone may facilitate complex formation

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between PAMAM G2 and pDNA. This may be because PAMAM G2-Dexa forms micelle structures in aqueous solution and behaves like higher molecular weight PAMAM. Stability of the carrier/pDNA complexes was evaluated by heparin competition assays. The PAMAM G2/pDNA and PEI25k/pDNA complexes began to release pDNA in response to the addition of a 10-fold and 15-fold excess heparin. However, the PAMAM G2-Dexa/pDNA and PEIDexa/pDNA complexes did not release pDNA, even in the presence of 30-fold excess heparin. These results suggest that PAMAM G2-Dexa and PEI-Dexa form more stable complexes with pDNA than PAMAM G2 and PEI25k. The size of the PAMAM G2-Dexa/pDNA complexes tended to decrease with an increasing ratio of PAMAM G2-Dexa (Table 1). The size of the PAMAM G2-Dexa/pDNA

Table 1. Particle size and zeta potential measurement of complexes.

Group

Avg. size  SE Avg. zeta [d.nm] potential [mV]

PAMAM G2-Dexa/pDNA (1:1)

208.4  12.1

–3.7  1.3

PAMAM G2-Dexa/pDNA (2:1)

113.6  9.4

45.1  1.6

PAMAM G2-Dexa/pDNA (4:1)

104.1  8.7

50.5  1.5

87.4  6.3

52.7  1.6

PAMAM G2/pDNA

126.6  16.4

35.1  3.0

PEI25k/pDNA

212.6  6.6

59.5  0.4

PEI-Dexa/pDNA

185.4  20.4

33.9  4.1

PAMAM G2-Dexa/pDNA (6:1)

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complexes was 104 nm at a 4:1 weight ratio, similar to that of PAMAM G2/pDNA (126 nm) and smaller than that of the PEI25k/pDNA and PEI-Dexa/pDNA complexes. The zeta potential of the PAMAM G2-Dexa/pDNA complexes also increased with increasing amount of PAMAM G2Dexa (Table 1). At a 4:1 weight ratio, the zeta potential of the complexes was 50 mV, which was slightly lower than that of PEI25k/pDNA complex.

Figure 2. Transfection efficiency of PAMAM G2-Dexa in N2A cells. (A) Optimization of the ratio between PAMAM G2-Dexa and pDNA. PAMAM G2-Dexa/pDNA complexes were prepared at various weight ratios. Complexes were transfected into N2A cells. Transfection efficiency was measured by luciferase assay. Luciferase activity is presented as mean  standard error of quadruplicated experiments. *P < 0.05 as compared with the others except for the complex at a 5:1 weight ratio. (B) Comparison of the transfection efficiency of PAMAM G2-Dexa with those of other carriers. Carrier/pDNA complexes were prepared under optimal conditions. Complexes were then transfected into N2A cells. Transfection efficiency was measured by luciferase assay. Luciferase activity is presented as mean  standard error of quadruplicated experiments. **P < 0.05 as compared with all others.

3.2. In vitro Transfection of PAMAM G2-Dexa Transfection efficiency of PAMAM G2Dexa in N2A cells was evaluated by luciferase assays. First, the ratio of PAMAM G2-Dexa/pDNA was optimized by transfection assays with complexes at various weight ratios. The highest transfection efficiency was obtained at a 4:1

Figure 3. Flow cytometry. Carrier/pcDNA-EGFP complexes were prepared under optimal conditions. Complexes were transfected into N2A cells. After 24 h, the expression of EGFP was measured by flow cytometry.

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Figure 4. Cytotoxicity of PAMAM G2-dexa in N2A cells. Carrier/ pDNA complexes were prepared under optimal conditions. Naked pDNA or carrier/pDNA complexes were added to the cells. After 24 h, the cytotoxicities of the carriers were measured by MTT assay. Cell viability is presented as mean  standard error of quadruplicated experiments. *P < 0.05 as compared with control, naked DNA, and PEI-Dexa, but not significant as compared with PAMAM G2 and PEI25k.

weight ratio (Figure 2A). Therefore, PAMAM G2-Dexa/ pDNA complexes were prepared at a 4:1 weight ratio in subsequent experiments. The transfection efficiency of PAMAM G2-Dexa was compared with that of other carriers. PAMAM G2, PEI25k, and PEI-Dexa were used as controls.

Figure 6. HO-1 ELISA in vitro. Carrier/pHO-1 complexes were prepared at their optimal ratios. Complexes were transfected into N2A cells. After 24 h, HO-1 expression was measured by ELISA. The HO-1 level is presented as mean  standard error of quadruplicated experiments.

PAMAM G2-Dexa had a higher transfection efficiency than the other carriers (Figure 2B). In particular, the transfection efficiency of PAMAM G2-Dexa was higher than that of PEIDexa, despite the similarity of their structures. PAMAM G2 has a higher molecular weight than PEI2k. Therefore, PAMAM G2-Dexa has a higher charge density and forms smaller complexes with pDNA than PEI-Dexa (Table 1), which may account for the higher transfection efficiency of PAMAM G2-Dexa than PEIDexa. The transfection efficiency of PAMAM G2-Dexa was confirmed by EGFP gene delivery. Treatment of cells with PAMAM G2-Dexa resulted in transfection of more cells with pcDNA-EGFP than treatment of cells with the other carriers (Figure 3). The cytotoxicity of PAMAM G2-Dexa in N2A cells was measured by MTT assay. Cells treated with PAMAM G2Dexa had a higher viability compared to cells treated with PEI25k, suggesting that PAMAM G2-Dexa was less cytotoxic than PEI25k (Figure 4).

Figure 5. Pro-inflammatory cytokine expression. Mouse peritoneal monocytes were activated by LPS treatment. After activation, carrier/pDNA complexes were transfected into activated cells. After 24 h, (A) TNF-a and (B) IL-6 levels were measured by ELISA. Cytokine concentrations are presented as means  standard errors of quadruplicated experiments. *P < 0.05 as compared with the others except for dexamethasone alone. **P < 0.05 as compared with all others.

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3.3. Anti-Inflammatory Effect of PAMAM G2-Dexa One of the advantages of PAMAM over PEI is its anti-inflammatory effect.[12] Therefore, we expected PAMAM G2-Dexa/

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G2/pEmpty complexes were used as controls. The results showed that PEI-Dexa/pEmpty complexes reduced cytokine levels (Figure 5). The cytokine levels were also reduced by treatment with PAMAM G2/pEmpty complexes, suggesting that PAMAM G2 has an anti-inflammatory effect. Treatment of cells with PAMAM G2-Dexa/pEmpty complexes revealed that these complexes had the highest anti-inflammatory effect among the carriers evaluated (Figure 5). These results suggest that conjugation of dexamethasone to PAMAM increases the anti-inflammatory effect of PAMAM. 3.4. HO-1 Gene Delivery to the Ischemic Brain

Figure 7. HO-1 expression in vivo after gene delivery. Carrier/pHO1 complexes were prepared at their optimal ratios. Complexes were injected into the brain 1 h prior to MCAO. After 24 h, brains were harvested and protein extracts were prepared from the brain tissues. HO-1 levels were measured by ELISA. *P < 0.05 as compared with all others.

pDNA complexes to have a greater anti-inflammatory effect than PEI-Dexa/pDNA complexes. Anti-inflammatory effect of PAMAM G2-Dexa was evaluated in LPS-activated monocytes. LPS-activated monocytes produced high levels of the pro-inflammatory cytokines TNF-a and IL-6 (Figure 5). The LPS-activated monocytes were treated with PAMAM G2Dexa/pEmpty complexes. PEI-Dexa/pEmpty and PAMAM

We hypothesized that the anti-inflammatory effect of PAMAM G2-Dexa would increase the therapeutic effectiveness of HO-1 gene delivery. To evaluate the effect of HO-1 gene delivery using PAMAM G2-Dexa, we performed both in vitro and in vivo assays. First, the in vitro HO-1 gene delivery efficiency of PAMAM G2-Dexa to N2A cells was measured by ELISA. PAMAM G2-Dexa had higher HO-1 gene delivery efficiency than PEI25k and PEI-Dexa (Figure 6). This result is in coincident with the luciferase assay results. Second, in vivo evaluation of PAMAM G2-Dexa was performed in an MCAO animal model. Carrier/pHO-1 complexes were injected into the brain using stereotaxic equipment 1 h prior to MCAO. Brain tissues were harvested 24 h after injection, and HO-1 expression in the tissues was measured by ELISA. PEI25k/ pHO-1 and PEI-Dexa/pHO-1 complexes were used as controls. PAMAM G2-Dexa induced higher HO-1 gene expression in the ischemic brain than the other complexes (Figure 7). TTC staining showed that the brains of rats treated with PAMAM G2-Dexa/pHO-1 group had the smallest infarct volume (Figure 8). This suggests that PAMAM G2-Dexa delivered the HO-1 plasmid into the ischemic brain effectively, and that elevated HO-1 expression protected brain cells against ischemia–reperfusion damage. As proof of concept, the effect of the HO-1 gene delivery with dexamethasone-conjugated carrier was evaluated by pre-injection of the complex. However, for the therapeutic application, the effect of the HO-1 gene delivery should be verified by a post-injection of the complex.

Figure 8. Effect of the PAMAM G2-Dexa/pHO-1 complexes in the MCAO model. Carrier/ pHO-1 complexes were prepared at their optimal ratios and injected in to the brain 1 h prior to MCAO. After 24 h, brains were harvested and subjected to local staining. (A) The infarction was visualized by TTC staining of brain slices. (B) Quantitation of the stained area. Data are presented as means  standard errors. *P < 0.05 as compared with all others.

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4. Conclusion The PAMAM G2-Dexa/pDNA complex had higher anti-inflammatory effect than dexamethasone alone and the PEIDexa/pDNA complex. PAMAM G2-Dexa

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forms micelles with positive surface charge, which facilitates the interaction with negatively charged cellular membrane. This effect increases the cellular uptake and anti-inflammatory effect of PAMAM G2-Dexa, compared with dexamethasone alone. In addition, PAMAM G2-Dexa had greater transfection efficiency than PEI25k, PAMAM, and PEI-Dexa in the N2A cells in vitro. These suggest that PAMAM G2-Dexa may be useful for drug and gene combination therapy of inflammatory diseases. This hypothesis was proved in an ischemic stroke model. In a stroke brain, PAMAM G2-Dexa/pHO-1 complexes reduced infarct volume more effectively than PEI/pHO-1 and PEI-Dexa/pHO-1 complexes. These results suggest that PAMAM G2-Dexa/pHO-1 complexes may be useful for ischemic stroke gene therapy.

Acknowledgements: This work was financially supported by grants from the National Research Foundation of Korea, funded by the Ministry of Science, ICT, and Future Planning (grant number, NRF-2013R1A1A2059236).

Received: March 4, 2015; Revised: April 28, 2015; Published online: May 29, 2015; DOI: 10.1002/mabi.201500058 Keywords: dexamethasone; gene therapy; heme oxygenase-1; polyamidoamine; stroke

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[1] H. C. Kang, M. Lee, Y. H. Bae, Crit. Rev. Eukaryot. Gene Expr. 2005, 15, 317. [2] A. Rebuffat, A. Bernasconi, M. Ceppi, H. Wehrli, S. B. Verca, M. Ibrahim, B. M. Frey, F. J. Frey, S. Rusconi, Nat. Biotechnol. 2001, 19, 1155. [3] J. A. Gruneich, A. Price, J. Zhu, S. L. Diamond, Gene Ther. 2004, 11, 668. [4] J. S. Choi, K. S. Ko, J. S. Park, Y. H. Kim, S. W. Kim, M. Lee, Int. J. Pharm. 2006, 320, 171. [5] Y. M. Bae, H. Choi, S. Lee, S. H. Kang, Y. T. Kim, K. Nam, J. S. Park, M. Lee, J. S. Choi, Bioconjug. Chem. 2007, 18, 2029. [6] H. Kim, H. A. Kim, Y. M. Bae, J. S. Choi, M. Lee, J. Gene Med. 2009, 11, 515. [7] V. Shahin, L. Albermann, H. Schillers, L. Kastrup, C. Schafer, Y. Ludwig, C. Stock, H. Oberleithner, J. Cell. Physiol. 2005, 202, 591. [8] H. Hyun, J. Lee, W. Hwang do, S. Kim, D. K. Hyun, J. S. Choi, J. K. Lee, M. Lee, Biomaterials 2011, 32, 306. [9] H. A. Kim, J. H. Park, S. Lee, J. S. Choi, T. Rhim, M. Lee, J. Control. Release 2011, 156, 60. [10] U. I. Tuor, C. S. Simone, J. D. Barks, M. Post, Stroke 1993, 24, 452. [11] J. Y. Kim, J. H. Ryu, H. Hyun, H. A. Kim, J. S. Choi, D. Yun Lee, T. Rhim, J. H. Park, M. Lee, J. Drug Target. 2012, 20, 667. [12] A. S. Chauhan, P. V. Diwan, N. K. Jain, D. A. Tomalia, Biomacromolecules 2009, 10, 1195. [13] F. H. Bach, Wien Klin. Wochenschr. 2002, 114, 1. [14] H. Hyun, Y. W. Won, K. M. Kim, J. Lee, M. Lee, Y. H. Kim, Biomaterials 2010, 31, 9128. [15] M. Lee, S. Oh, C. H. Ahn, S. W. Kim, B. D. Rhee, K. S. Ko, J. Control. Release 2006, 115, 316. [16] G. F. Lemkine, D. Goula, N. Becker, L. Paleari, G. Levi, B. A. Demeneix, J. Drug Target. 1999, 7, 305.

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Dexamethasone-Conjugated Polyamidoamine Dendrimer for Delivery of the Heme Oxygenase-1 Gene into the Ischemic Brain.

Heme oxygenase-1 (HO-1) has anti-apoptotic and anti-inflammatory effects. In this study, the HO-1 gene was delivered into the brain using dexamethason...
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