Lipid-like Nanoparticles for Small Interfering RNA Delivery to Endothelial Cells

Abstract

Here we develop nanoparticles composed of lipid-like materials (lipidoids) to facilitate non-viral delivery of small interfering RNA (siRNA) to endothelial cells (ECs). Nanoparticles composed of siRNA and lipidoids with small size (~200 nm) and positive charge (~34 mV) were formed by self assembly of lipidoids and siRNA. Ten lipidoids were synthesized and screened for their ability to facilitate the delivery of siRNA into ECs. Particles composed of leading lipidoids show significantly better delivery to ECs than a leading commercially-available transfection reagent, Lipofectamine 2000. As a model of potential therapeutic application, nanoparticles composed of the top performing lipidoid, NA114, were studied for their ability to deliver siRNA targeting anti-angiogenic factor (SHP-1) to human ECs. Silencing of SHP-1 expression significantly enhanced EC proliferation and decreased EC apoptosis under a simulated ischemic condition.

Figure 1

Synthesis of lipidoids for siRNA delivery. (A) Alkyl-acrylamide and amine molecules were used to synthesize a combinatorial library of lipidoids. (B) Lipidoid synthesis (114N₉-5; NA114) was performed through the conjugate addition of amine (114) to acrylamide (N₉; NA). Depending on the number of addition sites in the amino monomer, lipidoids can be formed with anywhere from 1 to 7 tails. Lipidoids are named as follows; (amine number)(acrylamide name)-(the number of tails). For example, 100N₁₂-3 (ND100) indicates “the reaction of 100 with N₁₂ where 3 of 4 tails are present”. (C) The molecular structures of 110N₉-5 (NA110) and 98N₁₂-5 (ND98).

Figure 2

Characterization of lipidoid nanoparticles. TEM images of (A) lipidoid (NA114) nanoparticles in the absence of siRNA and (B) lipidoid (NA114) nanoparticles complexed with siRNA (5:1 weight ratio of lipidoid to siRNA). Scale bars indicate 200 nm. (C) Zeta potential and particle size of lipidoid (NA114) and siRNA-lipidoid (NA114) nanoparticles (5:1 weight ratio of lipidoid to siRNA).

Figure 3

Screening of lipidoids for siRNA delivery to HUVECs. (A) GAPDH activity of HUVECs (n=4) at 2 days after siGAPDH transfection. The % reduction in GAPDH activity following transfection with siRNA-lipidoid complexes at various lipidoid/siRNA weight ratios is shown. Only lipidoids showing no significant cytotoxicity are included in this graph. (B) The viability of siRNA-transfected HUVECs (n=4) cultured for 2 days under hypoxic (1% oxygen) and serum-deprived condition (*; p<0.01, **; p<0.05, compared to siGFP group with respective lipidoids or Lipofectamine 2000).

Figure 4

Silencing of SHP-1 by siSHP-1 delivery in HUVECs. RT-PCR and quantitative real-time PCR (n=3) to examine SHP-1 expression in HUVECs at 2 days after siRNA transfection using NA114 and Lipofectamine 2000 under culture condition with (A) 2% FBS and (B) 10% FBS (*; p<0.01, **; p<0.05, compared to all siGFP-transfected groups and #; p<0.01, compared to siSHP-1-transfected group with Lipofectamine 2000). The relative expression of SHP-1 in each group was normalized to that of siGFP group using Lipofectamine 2000.

Figure 5

Gene expression profiles in siRNA-transfected HUVECs under hypoxic (1% oxygen) and serum-deprived (endothelial basal medium-2 (EBM-2) with no serum and growth factors) condition. (A) RT-PCR for SHP-1, KDR/Flk-1, and eNOS of siRNA-transfected HUVECs at day 1 and 2 after culture. Quantitative real-time PCR for (B) SHP-1, (C) KDR/Flk-1, and (D) eNOS. The gene expression in siRNA-transfected HUVECs (n=3) at day 1 and 2 was normalized to that in siGFP-transfected HUVECs using Lipofectamine 2000 at day 1 (*; p<0.01, **; p<0.05, versus all siGFP groups at comparable time point).

Figure 6

Inhibition of apoptotic activity in siSHP-1-transfected HUVECs cultured under hypoxic (1% oxygen) and serum-deprived (EBM-2 with no serum and growth factors) condition. (A) TUNEL staining of siRNA-transfected HUVECs and capillary formation (arrows) by siRNA-transfected HUVECs at 2 days after culture (×100). Scale bars indicate 200 µm. (B) The percentage ratio of TUNEL-positive cells (red; apoptotic cells) in DAPI-positive cells (blue; total cells) at day 1 and 2 after culture (n=4, *; p<0.01 versus all siGFP groups at comparable time point). (C) The viability of siRNA-transfected HUVECs at day 1 and 2 after culture (n=4, *; p<0.01 versus all siGFP groups at comparable time point).