Nanoparticle based siRNA formulations often suffer from aggregation and loss of function during storage. We in this study report a frozen targeted RGD-polyethylene glycol (PEG)-ECO/siβ3 nanoparticle formulation with a prolonged shelf life and preserved nanoparticle functionality. The targeted RGD-PEG-ECO/siβ3 nanoparticles are formed by step-wised self-assembly of RGD-PEG-maleimide, ECO, and siRNA. The nanoparticles have a diameter of 224.5 ± 9.41 nm and a zeta potential to 45.96 ± 3.67 mV in water and a size of 234.34 ± 3.01 nm and a near neutral zeta potential in saline solution. The addition of sucrose does not affect their size and zeta potential and substantially preserves the integrity and biological activities of frozen and lyophilized formulations of the targeted nanoparticles. The frozen formulation with as low as 5% sucrose retains nanoparticle integrity (90% siRNA encapsulation), size distribution (polydispersity index [PDI] ≤20%), and functionality (at least 75% silencing efficiency) at −80°C for at least 1 year. The frozen RGD-PEG-ECO/siβ3 nanoparticle formulation exhibits excellent biocompatibility, with no adverse effects on hemocompatibility and minimal immunogenicity. As RNAi holds the promise in treating the previously untreatable diseases, the frozen nanoparticle formulation with the low sucrose concentration has the potential to be a delivery platform for clinical translation of RNAi therapeutics.
Get full access to this article
View all access options for this article.
References
1.
KulkarniJA, CullisPR and van der MeelR. (2018). Lipid nanoparticles enabling gene therapies: from concepts to clinical utility. Nucleic Acid Ther, 28:146–157.
2.
GaudetD, Drouin-ChartierJP and CoutureP. (2017). Lipid metabolism and emerging targets for lipid-lowering therapy. Can J Cardiol, 33:872–882.
3.
Barrett ADT. (2018). Current status of Zika vaccine development: Zika vaccines advance into clinical evaluation. NPJ Vaccines, 3:24.
4.
AdamsD, Gonzalez-DuarteA, O'RiordanWD, YangCC, UedaM, KristenAV, TournevI, SchmidtHH, CoelhoT, et al. (2018). Patisiran, an RNAi therapeutic, for hereditary transthyretin amyloidosis. N Engl J Med, 379:11–21.
5.
LorenzerC, DirinM, WinklerAM, BaumannV and WinklerJ. (2015). Going beyond the liver: progress and challenges of targeted delivery of siRNA therapeutics. J Control Release, 203:1–15.
6.
TatipartiK, SauS, KashawSK and IyerAK. (2017). siRNA delivery strategies: a comprehensive review of recent developments. Nanomaterials (Basel), 7:pii:E77.
7.
GujratiM, VaidyaA and LuZR. (2016). Multifunctional pH-sensitive amino lipids for siRNA delivery. Bioconjug Chem, 27:19–35.
8.
ChakrabortyC, SharmaAR, SharmaG, DossCGP and LeeSS. (2017). Therapeutic miRNA and siRNA: moving from bench to clinic as next generation medicine. Mol Ther Nucleic Acids, 8:132–143.
9.
GarbaAO and MousaSA. (2010). Bevasiranib for the treatment of wet, age-related macular degeneration. Ophthalmol Eye Dis, 2:75–83.
10.
LusthausJA and GoldbergI. (2016). Investigational and experimental drugs for intraocular pressure reduction in ocular hypertension and glaucoma. Expert Opin Investig Drugs, 25:1201–1208.
11.
WittrupA and LiebermanJ. (2015). Knocking down disease: a progress report on siRNA therapeutics. Nat Rev Genet, 16:543–552.
12.
LeungRK and WhittakerPA. (2005). RNA interference: from gene silencing to gene-specific therapeutics. Pharmacol Ther, 107:222–239.
13.
OgrisM, SteinleinP, KursaM, MechtlerK, KircheisR and WagnerE. (1998). The size of DNA/transferrin-PEI complexes is an important factor for gene expression in cultured cells. Gene Ther, 5:1425–1433.
14.
BallRL, BajajP and WhiteheadKA. (2017). Achieving long-term stability of lipid nanoparticles: examining the effect of pH, temperature, and lyophilization. Int J Nanomed, 12:305–315.
15.
AbdelwahedW, DegobertG, StainmesseS and FessiH. (2006). Freeze-drying of nanoparticles: formulation, process and storage considerations. Adv Drug Deliv Rev, 58:1688–1713.
16.
AndersenMO, HowardKA, PaludanSR, BesenbacherF and KjemsJ. (2008). Delivery of siRNA from lyophilized polymeric surfaces. Biomaterials, 29:506–512.
17.
MalamasAS, GujratiM, KummithaCM, XuR and LuZR. (2013). Design and evaluation of new pH-sensitive amphiphilic cationic lipids for siRNA delivery. J Control Release, 171:296–307.
ParvaniJG, GujratiMD, MackMA, SchiemannWP and LuZR. (2015). Silencing beta3 integrin by targeted ECO/siRNA nanoparticles inhibits EMT and metastasis of triple-negative breast cancer. Cancer Res, 75:2316–2325.
20.
GujratiM, VaidyaAM, MackM, SnyderD, MalamasA and LuZR. (2016). Targeted dual pH-sensitive lipid ECO/sirna self-assembly nanoparticles facilitate in vivo cytosolic sieIF4E delivery and overcome paclitaxel resistance in breast cancer therapy. Adv Healthc Mater, 5:2882–2895.
21.
VaidyaAM, SunZ, AyatN, SchilbA, LiuX, JiangH, SunD, ScheidtJ, QianV, et al. (2019). Systemic delivery of tumor-targeting siRNA nanoparticles against an oncogenic lncRNA facilitates effective triple-negative breast cancer therapy. Bioconjug Chem, 30:907–919.
22.
AlekuM, SchulzP, KeilO, SantelA, SchaeperU, DieckhoffB, JankeO, EndruschatJ, DurieuxB, et al. (2008). Atu027, a liposomal small interfering RNA formulation targeting protein kinase N3, inhibits cancer progression. Cancer Res, 68:9788–9798.
23.
SchultheisB, StrumbergD, SantelA, VankC, GebhardtF, KeilO, LangeC, GieseK, KaufmannJ, KhanM and DrevsJ. (2014). First-in-human phase I study of the liposomal RNA interference therapeutic Atu027 in patients with advanced solid tumors. J Clin Oncol, 32:4141–4148.
24.
SunD, SahuB, GaoS, SchurRM, VaidyaAM, MaedaA, PalczewskiK and LuZR. (2017). Targeted multifunctional lipid ECO plasmid DNA nanoparticles as efficient non-viral gene therapy for Leber's congenital amaurosis. Mol Ther Nucleic Acids, 7:42–52.
25.
JonesLJ, YueST, CheungCY and SingerVL. (1998). RNA quantitation by fluorescence-based solution assay: RiboGreen reagent characterization. Anal Biochem, 265:368–374.
26.
SuzukiY, HyodoK, TanakaY and IshiharaH. (2015). siRNA-lipid nanoparticles with long-term storage stability facilitate potent gene-silencing in vivo. J Control Release, 220:44–50.
27.
GuptaK, AfoninKA, ViardM, HerreroV, KasprzakW, KagiampakisI, KimT, KoyfmanAY, PuriA, et al. (2015). Bolaamphiphiles as carriers for siRNA delivery: from chemical syntheses to practical applications. J Control Release, 213:142–151.
28.
GuptaK, MattinglySJ, KnippRJ, AfoninKA, ViardM, BergmanJT, SteplerM, NantzMH, PuriA and ShapiroBA. (2015). Oxime ether lipids containing hydroxylated head groups are more superior siRNA delivery agents than their nonhydroxylated counterparts. Nanomedicine (Lond), 10:2805–2818.
29.
KimT, AfoninKA, ViardM, KoyfmanAY, SparksS, HeldmanE, GrinbergS, LinderC, BlumenthalRP and ShapiroBA. (2013). In silico, in vitro, and in vivo studies indicate the potential use of bolaamphiphiles for therapeutic siRNAs delivery. Mol Ther Nucleic Acids, 2:e80.
30.
KasperJC, TroiberC, KuchlerS, WagnerE and FriessW. (2013). Formulation development of lyophilized, long-term stable siRNA/oligoaminoamide polyplexes. Eur J Pharm Biopharm, 85:294–305.
31.
KunduAK, ChandraPK, HazariS, LedetG, PramarYV, DashS and MandalTK. (2012). Stability of lyophilized siRNA nanosome formulations. Int J Pharm, 423:525–534.
32.
ChaconM, MolpeceresJ, BergesL, GuzmanM and AberturasMR. (1999). Stability and freeze-drying of cyclosporine loaded poly(d,l lactide-glycolide) carriers. Eur J Pharm Sci, 8:99–107.
33.
HansenSH, SandvigK and van DeursB. (1993). Clathrin and HA2 adaptors: effects of potassium depletion, hypertonic medium, and cytosol acidification. J Cell Biol, 121:61–72.
34.
ChenCL, HouWH, LiuIH, HsiaoG, HuangSS and HuangJS. (2009). Inhibitors of clathrin-dependent endocytosis enhance TGFbeta signaling and responses. J Cell Sci, 122:1863–1871.
35.
DobrovolskaiaMA and McNeilSE. (2013). Understanding the correlation between in vitro and in vivo immunotoxicity tests for nanomedicines. J Control Release, 172:456–466.
36.
DobrovolskaiaMA, PatriAK, SimakJ, HallJB, SemberovaJ, De Paoli LacerdaSH and McNeilSE. (2012). Nanoparticle size and surface charge determine effects of PAMAM dendrimers on human platelets in vitro. Mol Pharm, 9:382–393.
37.
IlinskayaAN and DobrovolskaiaMA. (2013). Nanoparticles and the blood coagulation system. Part II: safety concerns. Nanomedicine (Lond), 8:969–981.
38.
DobrovolskaiaMA, PatriAK, PotterTM, RodriguezJC, HallJB and McNeilSE. (2012). Dendrimer-induced leukocyte procoagulant activity depends on particle size and surface charge. Nanomedicine (Lond), 7:245–256.
39.
JulianoRL, HsuMJ, PetersonD, RegenSL and SinghA. (1983). Interactions of conventional or photopolymerized liposomes with platelets in vitro. Exp Cell Res, 146:422–427.
40.
DurrantTN, van den BoschMT and HersI. (2017). Integrin alphaIIbbeta3 outside-in signaling. Blood, 130:1607–1619.
41.
NieswandtB, Varga-SzaboD and ElversM. (2009). Integrins in platelet activation. J Thromb Haemost, 7 (Suppl. 1):206–209.
42.
NetoEH, CoelhoAL, SampaioAL, HenriquesM, MarcinkiewiczC, De FreitasMS and Barja-FidalgoC. (2007). Activation of human T lymphocytes via integrin signaling induced by RGD-disintegrins. Biochim Biophys Acta, 1773:176–184.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
