title: Stability of lyophilized siRNA nanosome formulations.
abstract: The goal of this study is to evaluate the stability of lyophilized siRNA formulations. The gene silencing efficiency of a stored lyophilized siRNA formulation (i.e. siRNA nanosomes) was evaluated in interferon-α (IFN-α) resistant hepatitis C virus (HCV) at different time points up to three months in an in vitro cell culture model and compared with freshly prepared siRNA formulations. Novel siRNA sequences were encapsulated within nanosize liposomes following condensation with protamine sulfate. The siRNA encapsulated nanosomes were lyophilized and stored at 4 °C for 3 months, along with liquid liposomes (L) and lyophilized liposome powder (P) which were subsequently used to prepare siRNA nanosomes (L) and siRNA nanosomes (P), respectively at different time points. Physiochemical and biological properties of all three formulations were compared at different time points up to 3 months. The particle size of the stored siRNA nanosomes (642 ± 25 nm) was considerably larger initially in comparison with the liquid liposomes (134 ± 5 nm) and lyophilized liposomes (118 ± 3). However, the particle size gradually became smaller over time (413 ± 128 nm by the third month). The zeta potential of all three formulations was initially very high (> +40 mV), followed by a gradual decrease over time. The amount of siRNA in the stored siRNA nanosomes decreased ∼18 % during the 3 month storage period (1.16 ± 0.03 nmol initially on day 1 vs. 0.95 ± 0.04 nmol after 3 months). With respect to biological potency, all three formulations were significantly effective to knock-down HCV throughout the storage time. The cell viability was well-maintained throughout this period. Thus, this study indicates that the stored lyophilized siRNA formulation is as effective as the fresh preparation and that long-term storage could be a viable option to treat deadly diseases such as cancer and viral infection.
authors.affiliation: Center for Nanomedicine and Drug Delivery, Xavier University College of Pharmacy, New Orleans, LA 70125, United States.
authors.forename: Anup K
authors.initials: AK
authors.lastname: Kundu
authors.forename: Partha K
authors.initials: PK
authors.lastname: Chandra
authors.forename: Sidhartha
authors.initials: S
authors.lastname: Hazari
authors.forename: Grace
authors.initials: G
authors.lastname: Ledet
authors.forename: Yashoda V
authors.initials: YV
authors.lastname: Pramar
authors.forename: Srikanta
authors.initials: S
authors.lastname: Dash
authors.forename: Tarun K
authors.initials: TK
authors.lastname: Mandal
mesh_terms: D000998:Antiviral Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D024882:Drug Resistance, Viral; D005612:Freeze Drying; D015316:Genetic Therapy / Q000379:methods*; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics / Q000254:growth & development; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D008055:Lipids / Q000737:chemistry; D008081:Liposomes; D053758:Nanoparticles; D036103:Nanotechnology; D009690:Nucleic Acid Conformation; D010316:Particle Size; D011479:Protamines / Q000737:chemistry; D034622:RNA Interference; D020871:RNA Stability; D034741:RNA, Small Interfering / Q000737:chemistry*; D013997:Time Factors; D014162:Transfection; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D016898:Interferon-alpha; D008055:Lipids; D008081:Liposomes; D011479:Protamines; D034741:RNA, Small Interfering