Neuroprotection by biodegradable PAMAM ester (e-PAM-R)-mediated HMGB1 siRNA delivery in primary cortical cultures and in the postischemic brain.
Although RNA interference (RNAi)-mediated gene silencing provides a powerful strategy for modulating specific gene functions, difficulties associated with siRNA delivery have impeded the development of efficient therapeutic applications. In particular, the efficacy of siRNA delivery into neurons has been limited by extremely low transfection efficiencies. e-PAM-R is a biodegradable arginine ester of PAMAM dendrimer, which is readily degradable under physiological conditions (pH 7.4, 37 degrees C). In the present study, we investigated the efficiency of siRNA delivery by e-PAM-R in primary cortical cultures and in rat brain. e-PAM-R/siRNA complexes showed high transfection efficiencies and low cytotoxicities in primary cortical cultures. Localization of fluorescence-tagged siRNA revealed that siRNA was delivered not only into the nucleus and cytoplasm, but also along the processes of the neuron. e-PAM-R/siRNA complex-mediated target gene reduction was observed in over 40% of cells and it was persistent for over 48 h. The potential use of e-PAM-R was demonstrated by gene knockdown after transfecting High mobility group box-1 (HMGB1, a novel cytokine-like molecule) siRNA into H(2)O(2)- or NMDA-treated primary cortical cultures. In these cells, HMGB1 siRNA delivery successfully reduced both basal and H(2)O(2)- or NMDA-induced HMGB1 levels, and as a result of that, neuronal cell death was significantly suppressed in both cases. Furthermore, we showed that e-PAM-R successfully delivered HMGB1 siRNA into the rat brain, wherein HMGB1 expression was depleted in over 40% of neurons and astrocytes of the normal brain. Moreover, e-PAM-R-mediated HMGB1 siRNA delivery notably reduced infarct volume in the postischemic rat brain, which is generated by occluding the middle cerebral artery for 60 min. These results indicate that e-PAM-R, a novel biodegradable nonviral gene carrier, offers an efficient means of transfecting siRNA into primary neuronal cells and in the brain and of performing siRNA-mediated gene knockdown.
Kim
Il-Doo
ID
Department of Anatomy and Center for Advanced Medical Education (BK21 project), Inha University School of Medicine, Inchon, Republic of Korea.
Lim
Chae-Moon
CM
Kim
Jung-Bin
JB
Nam
Hye Yeong
HY
Nam
Kihoon
K
Kim
Seung-Woo
SW
Park
Jong-Sang
JS
Lee
Ja-Kyeong
JK
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D001672:Biocompatible Materials / Q000737:chemistry*; D020520:Brain Infarction / Q000235:genetics / Q000473:pathology / Q000628:therapy; D002545:Brain Ischemia / Q000235:genetics / Q000473:pathology / Q000628:therapy*; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D050091:Dendrimers / Q000737:chemistry*; D004195:Disease Models, Animal; D004337:Drug Carriers / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D020868:Gene Silencing / Q000187:drug effects; D024243:HMGB1 Protein / Q000235:genetics*; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D018696:Neuroprotective Agents / Q000008:administration & dosage*; D009757:Nylons / Q000737:chemistry; D011091:Polyesters; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D001672:Biocompatible Materials; D050091:Dendrimers; D004337:Drug Carriers; D024243:HMGB1 Protein; C104200:Hbp1 protein, rat; D018696:Neuroprotective Agents; D009757:Nylons; C104700:PAMAM Starburst; C520550:PAMAM-G4; D011091:Polyesters; D034741:RNA, Small Interfering