Intratracheal gene transfer of adrenomedullin using polyplex nanomicelles attenuates monocrotaline-induced pulmonary hypertension in rats

Abstract

Pulmonary arterial hypertension (PAH) is a life-threatening disease characterized by progressive PAH and right ventricular failure. Despite recent advances in therapeutic approaches using prostanoids, endothelin antagonists, and so on, PAH remains a challenging condition. To develop a novel therapeutic approach, we have established a nonviral gene delivery system of poly(ethylene glycol) (PEG)-based block catiomers, which form a polyplex nanomicelle with a nanoscaled core-shell structure in the presence of DNA. The polyplex nanomicelle from PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (PEG-b-P[Asp(DET)]), having ethylenediamine units at the side chain, showed ~100-fold increase in luciferase transgene expression activity in mouse lung via intratracheal administration with a minimal toxicity compared with the polyplex from linear poly(ethylenimine) (LPEI). The transfection activity was highest on day 3 after administration and remained detectable until day 14. PEG-b-P[Asp(DET)] polyplex nanomicelles were formulated with a therapeutic plasmid bearing the human adrenomedullin (AM) gene and intratracheally administered to rats with monocrotaline-induced pulmonary hypertension. The right ventricular pressure significantly decreased 3 days after administration as confirmed by a notable increase of pulmonary human AM mRNA levels. Intratracheal administration of PEG-b-P[Asp-(DET)] polyplex nanomicelles showed remarkable therapeutic efficacy with PAH animal models without compromising biocompatibility.

Figure 1

Luciferase gene expression by intratracheal administration of LPEI polyplex (N/P = 6) or PEG-b-P[Asp(DET)] polyplex nanomicelle (N/P = 80). Samples of the polyplex and the polyplex nanomicelle were prepared before the administration and left for 1 day. The mice (five mice per group) were anesthetized and the polyplex or the polyplex nanomicelle was administered intratracheally. At 24 hour postadministration, the lung tissues were harvested, homogenized, and measured for luciferase activity (mean ± SEM, N = 5). LPEI, linear poly(ethylenimine); PEG-b-P[Asp(DET)], PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}.

Figure 2

Time-dependent changes in gene expression after intratracheal administration of PEG-b-P[Asp(DET)] polyplex nanomicelle loaded with luciferase gene. The mice (five mice per group) were anesthetized and the polyplex nanomicelle was administered intratracheally. After the indicated time, the lung tissues were harvested, homogenized, and measured for luciferase activity (mean ± SEM, N = 5). PEG-b-P[Asp(DET)], PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}.

Figure 3

Charge-ratio-dependent changes in gene expression after intratracheal administration of the PEG-b-P[Asp(DET)] polyplex nanomicelle loaded with luciferase gene. The mice (five mice per group) were anesthetized and the polyplex nanomicelle was administered intratracheally. At 3 days postadministration, the lung tissues were harvested, homogenized, and measured for luciferase activity (mean ± SEM, N = 5). PEG-b-P[Asp(DET)], PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}.

Figure 4

Fluorescence photographs of lungs transfected by intratracheal administration of YFP gene using PEG-b-P[Asp(DET)] polyplex nanomicelle or LPEI polyplex. (a) PEG-b-P[Asp(DET)] polyplex nanomicelle (N/P = 80), (b) LPEI polyplex (N/P = 6). The lung specimens were observed under a fluorescence microscope (SZX12; Olympus). LPEI, linear poly(ethylenimine); PEG-b-P[Asp(DET)], PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}.

Figure 5

Representative photomicrographs of lung tissues 7 days postintratracheal administration of LPEI/pLuc (N/P = 6) (a–c) or PEG-b-P[Asp(DET)]/pLuc (N/P = 80) (d–f). The terminal bronchiole and alveoli of the lungs administered with LPEI polyplex (a,b) and PEG-b-P[Asp(DET)] polyplex nanomicelle (d,e) are shown. The neutrophil infiltration is indicated with arrows in the photomicrographs with higher magnification (b,e). Each inset is the picture with higher magnification. The bronchus of the lungs administered with LPEI polyplex (c) and PEG-b-P[Asp(DET)] polyplex nanomicelle (f) are also shown. LPEI, linear poly(ethylenimine); PEG-b-P[Asp(DET)], PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}.

Figure 6

mRNA expression of inflammatory cytokines in lung tissues 7 days postintratracheal administration of luciferase gene in saline; LPEI polyplex with luciferase gene (N/P = 6); or PEG-b-P[Asp(DET)] polyplex nanomicelle with luciferase gene (N/P = 80) (mean ± SEM, N = 4). (a) TNF-α, (b) IL-6, (c) IL-10, (d) Cox-2. IL, interleukin; LPEI, linear poly(ethylenimine); TNF, tumor necrosis factor; PEG-b-P[Asp(DET)], PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}.

Figure 7

Effect of gene transfer on the right ventricle pressure in the PAH rat model. At 4 weeks after subcutaneous monocrotaline injection, a hemodynamic study was performed to measure the RV pressure indicated as “Before”. The PEG-b-P[Asp(DET)] polyplex nanomicelle loaded with AM expression vector; AM expression vector in saline; PEG-b-P[Asp(DET)] polyplex micelle loaded with luciferase gene; or the LPEI polyplex loaded with AM expression vector were sprayed intratracheally. Three days later, a hemodynamic study was performed again to measure the RV pressure indicated as “After”. AM, adrenomedullin; LPEI, linear poly(ethylenimine); PAH, pulmonary arterial hypertension; PEG-b-P[Asp(DET)], PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}; RV, right ventricular.

Figure 8

mRNA expression of human AM in the PAH rat model postintratracheal administration of PEG-b-P[Asp(DET)] polyplex nanomicelles loaded with the AM expression vector; AM expression vector in saline; PEG-b-P[Asp(DET)] polyplex nanomicelles loaded with the luciferase gene; or LPEI polyplexes loaded with the AM expression vector. Rats were transfected intratracheally at 4 weeks after subcutaneous monocrotaline injection. Three days later, lungs were harvested, homogenized, and measured for AM mRNA using real-time RT-PCR (mean ± SEM, N = 4). AM, adrenomedullin; LPEI, linear poly(ethylenimine); PAH, pulmonary arterial hypertension; PEG-b-P[Asp(DET)], PEG-b-poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide}.