The development of an in vitro assay to screen lipid based nanoparticles for siRNA delivery.
In order to rapidly screen and select lead candidates for in vivo evaluation of lipid nanoparticles (LNPs) for systemic small interfering RNA (siRNA) delivery, an in vitro assay amenable to high-throughput screening (HTS) is developed. The strategy is to mimic the in vivo experience of LNPs after systemic administration, such as interactions with serum components, exposure to endosomal pH environments, and interactions with endosomal membrane lipids. It is postulated that the amount of siRNA released from LNPs after going through these treatments can be used as a screening tool to rank order the effectiveness of siRNA delivery by lipid nanoparticles in vivo. LNPs were incubated with 50% serum from different species (i.e. mouse, rat, or rhesus) at 37°C. The resulting samples were then reacted with anionic, endosomal-mimicking lipids at different pHs. The amount of siRNA released from LNPs was determined using spectrophotometry employing the fluorescent indicator SYBR Gold. Our results indicated that the amount of siRNA liberated was highly dependent upon the species of serum used and the pH to which it was exposed. LNPs treated with mouse serum showed higher levels of siRNA release, as did those subjected to endosomal pH (6.0), compared to physiological pH. Most interestingly, a good correlation between the amount of siRNA released and the in vivo efficacy was observed. In conclusion, an in vitro siRNA release assay was developed to screen and rank order LNPs for in vivo evaluation.
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA. Electronic address: zhangye101@gmail.com.
Ye
Y
Zhang
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Leticia
L
Arrington
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
David
D
Boardman
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Jared
J
Davis
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Yan
Y
Xu
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Katie
K
DiFelice
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Steve
S
Stirdivant
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Weimin
W
Wang
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Brian
B
Budzik
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Jack
J
Bawiec
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
James
J
Deng
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Greg
G
Beutner
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Darla
D
Seifried
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Matthew
M
Stanton
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Marian
M
Gindy
Department of RNAi Therapeutics, Merck Research Laboratories, 770 Sumneytown Pike, West Point, 19486, USA.
Anthony
A
Leone
D000818:Animals; D001055:Apolipoproteins B / Q000235:genetics; D001681:Biological Assay; D011992:Endosomes / Q000737:chemistry; D005260:Female; D006863:Hydrogen-Ion Concentration; D008055:Lipids / Q000737:chemistry*; D008081:Liposomes; D008099:Liver / Q000378:metabolism; D008253:Macaca mulatta; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000008:administration & dosage* / Q000737:chemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D044967:Serum / Q000737:chemistry
D001055:Apolipoproteins B; D008055:Lipids; D008081:Liposomes; D012333:RNA, Messenger; D034741:RNA, Small Interfering
Endosome escape; Lipid nanoparticles; Serum stability; siRNA delivery; siRNA release