Discovery of siRNA lipid nanoparticles to transfect suspension leukemia cells and provide in vivo delivery capability

Abstract

As a powerful research tool, siRNA's therapeutic and target validation utility with leukemia cells and long-term gene knockdown is severely restricted by the lack of omnipotent, safe, stable, and convenient delivery. Here, we detail our discovery of siRNA-containing lipid nanoparticles (LNPs) able to effectively transfect several leukemia and difficult-to-transfect adherent cell lines also providing in vivo delivery to mouse spleen and bone marrow tissues through tail-vein administration. We disclose a series of novel structurally related lipids accounting for the superior transfection ability, and reveal a correlation between expression of Caveolins and successful transfection. These LNPs, bearing low toxicity and long stability of >6 months, are ideal for continuous long-term dosing. Our discovery represents the first effective siRNA-containing LNPs for leukemia cells, which not only enables high-throughput siRNA screening with leukemia cells and difficult-to-transfect adherent cells but also paves the way for the development of therapeutic siRNA for leukemia treatment.

Figure 1

Newly discovered SNALP-like lipid nanoparticles (SLPs) efficiently deliver siRNA into suspension leukemia cells. (a) Schematic representation of SLPs. (b) Composition of optimized SLPs with different N/P ratios (SLP100s), mechanical mixing rates (SLP200s), and percentages of PEG-c-DMG components (SLP300s). The differences among formulations were colored in blue or red. (c) Discovery of SLP300s able to efficiently knockdown KIF11 in suspension Molm13 leukemia cells. SLP100s-SLP300s were tested in easy-to-transfect adherent PC3 cells (upper panel) and hard-to-transfect suspension Molm13 cells (lower panel). KIF11 mRNA knockdown was measured by quantitative RT-PCR after 24 hours of siRNA transfection. Error bars represent mean ± SD. P value is calculated using a Student's t-test. SLP201 and SLP301, prepared from the same formula, are boxed in red. (d) Only SLP301R is able to efficiently knockdown KIF11 in Molm13 cells. SLP201R, repeat of SLP201 with Lot A of Dlin-KC2-DMA; SLP301R, repeat of SLP301 with Lot B of Dlin-KC2-DMA.

Figure 2

Alkylated Dlin-KC2-DMA lipids are the key components of novel SNALP-like lipid nanoparticles (SLPs) for efficient siRNA transfection into leukemia cells. (a) Chemical structures of Dlin-KC2-DMA (DMA) and Dlin-KC2-ClMDMA (ClMDMA). (b) Addition of ClMDMA into DMA-based SLPs leads to potent siRNA transfection of suspension Molm13 leukemia cells. ClMDMA was mixed with DMA at indicated ratios, and resulting SLPs were tested on Molm13 cells at indicated siRNA concentrations. KIF11 mRNA knockdown was measured by quantitative RT-PCR after 24 hours of siRNA transfection. (c) Chemical structures of Dlin-KC2-TMA (TMA) and Dlin-KC2-DEMA (DEMA). (d) Addition of TMA- or DEMA- to DMA-based SLPs leads to the similar siRNA transfection efficiency as addition of ClMDMA. ClMDMA, TMA, or DEMA was mixed with DMA at indicated ratio, and resulting SLPs were tested on Molm13 cells at indicated siRNA concentrations. (e) Comparison of biophysical properties of SLP201R and SLP301R. (f) Differential scanning calorimetry measurement of SLPs as indicated. In ClMDMA-containing SLP301R, the peak of the lamellar-to-inverted hexagonal phase transition shifted from lower temperature (~70 °C indicated by read line) to higher temperature (~90 °C indicated by blue line) compared with SLP201R.

Figure 3

Alkylated DMA-containing SNALP-like lipid nanoparticles (SLPs) displayed superior in vitro siRNA transfection efficiency, low cellular toxicity and high stability in both adherent cells and suspension leukemia cells. (a) ClMDMA-containing SLP301R showed superior transfection efficiency compared with leading commercial siRNA transfection reagents. Control siRNA or increasing concentration of KIF11 siRNA were transfected using Dharmafect (Thermo Fisher), RNAiMAX (Invitrogen), or SLP301R into easily transfected HCT116 (upper panel) and poorly transfected Colo205 adherent cells (lower panel). KIF11 mRNA knockdown was measured by quantitative RT-PCR after 24 hours of siRNA transfection. Error bars represent mean ± SD. P value is calculated using a Student's t-test. (b) SLP301R is able to potently transfect multiple leukemia cell lines. Increasing concentration of KIF11 siRNA were transfected using SLP301R in six leukemia cell lines as indicated. (c) SLP301R encapsulating siRNA is not toxic for cells. Increasing concentration of luciferase siRNA as indicated was transfected into PC3 cells using SLP301R, and cell viability was measured by CellTiter-Glo at 24 hours after transfection. (d) SLP301R maintained Molm13 transfection efficiency after storage in 4 °C for 6 months. The efficiency of knockdown is measured by quantitative RT-PCR. (e) Incorporating biocompatible polymers into alkylated DMA-containing SLPs (Supplementary Table S1) as indicated leads to more than fivefold enhancement of Molm13 transfection efficiency. HA, hyaluronic acid; INF7, a glutamic acid-enriched cell penetrating peptide derived from influenza virus; PPAA, poly(propylacrylic acid).

Figure 4

Alkylated DMA-containing SNALP-like lipid nanoparticles (SLPs) are efficient and convenient in vitro siRNA screening tools for suspension leukemia cells. (a) Screening of FLT3 siRNAs in Molm13 cells. Twenty FLT3 siRNAs were transfected into Molm13 cells at two concentrations as indicated by SLP401. FLT3 knockdown was measured by quantitative RT-PCR at 24 hours after transfection. (b) Compared with control siRNA, a potent FLT3 siRNA, FLT3_14, encapsulated in SLP401 showed dose-dependent FLT3 knockdown. (c) FLT3_14 siRNAs encapsulated in SLP401 showed dose-dependent growth inhibition in a 5-day growth assay. Cell growth was measured by CellTiter-Glo.

Figure 5

Efficient siRNA delivery to mouse hematopoietic tissues in vivo with alkylated DMA-containing SNALP-like lipid nanoparticles (SLPs). (a) Examination of syntelically oriented chromosomes, characteristic of KIF11 knockdown, from representative spleen and bone marrow sections of mice at 48 hours after receiving one tail-vein injection of KIF11 siRNA encapsulated in SLP301R. Arrows point to irregular mitotic figures resulted from KIF11 knockdown. Some part of sections from spleen and bone marrow were enlarged to provide a better view. (b) Quantitative RT-PCR analysis of mouse KIF11 mRNA expression in spleens and bone marrows (n = 5) from mice 48 hours after receiving one tail-vein injection of control luciferase siRNA and KIF11 siRNA encapsulated with SLP401. KIF11 mRNA knockdown was measured by quantitative RT-PCR. P value is calculated using a Student's t-test.

Figure 6

Expression of Caveolin 1 and 2 correlates with efficient siRNA transfection with alkylated DMA-containing SNALP-like lipid nanoparticles (SLPs). (a) Transfection efficiency of three tested leukemia cell lines, K562 (easily transfected), Molm13 (modestly transfected) and KG1 (poorly transfected), correlates with the amount of siRNAs entering into cells. Cy3-labeled control luciferase siRNAs were transfected into K562, Molm13 and KG1 cells using SLP301R. The cellular entry of siRNA was measured by quantitative fluorescent imaging (ImageStream^X, Amnis) at 2 hours after transfection. Mean Cy3 florescent intensity with SD was shown on the left. Two representative images from each cell line were shown on the right. (b) Four endocytosis-related genes were identified to be significantly underexpressed in poorly transfected KG1 and Mv4-11 cells compared with modestly transfected Molm13 and THP1 cells by comparative microarray analysis (Supplementary Table S2). (c) The expression levels of candidate genes identified in microarray were confirmed by quantitative RT-PCR in cell lines as indicated. Cav1, Cav2, and Rab13 were confirmed as underexpressed genes in poorly transfected KG1 and Mv4-11 cells compared with modestly and easily transfectd cell lines Molm13, THP1, HEL, and K562. (d) Upper panel, three groups of cell lines including easily transfected and poorly transfected adherent cell lines as well as hardest-to-transfect suspension leukemia cells, were subjected to comparative microarray analysis (Supplementary Tables S3 and S4). Lower panels, Cav1 and Cav2 were confirmed by quantitative RT-PCR as overexpressed genes in easily transfected adherent cell line HCT116, as compared with poorly transfected adherent cell lines HT29 and Colo205, and suspension leukemia cell line K562. (e) Colocalization of siRNA and Caveoloae. Cy3-labeled control luciferase siRNAs encapsulated in SLP301R were coadministered into K562 cells with Alexa647-labeled Albumins, which have been known to enter cells through Caveolae-mediated endocytosis. The cellular entry of siRNAs and Albumins was measured by quantitative fluorescent imaging (ImageStream^X, Amnis) at 30 minutes after administration. Two representative colocalization images were shown.