Research ArticleGENETICS

A supramolecular platform for controlling and optimizing molecular architectures of siRNA targeted delivery vehicles

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Science Advances  29 Jul 2020:
Vol. 6, no. 31, eabc2148
DOI: 10.1126/sciadv.abc2148
  • Fig. 1 Schematic illustration of the engineering platform for controlling, screening, and optimizing molecular architectures of siRNA targeted delivery vehicles.

    Supramolecular siRNA delivery vehicles are formed by host-guest complexation between CD-SS-pDMAEMA host polymer and Ad-PEG or Ad-PEG-FA guest polymers, which subsequently condense siRNA to form core-shell PNPs. The host polymer is responsible to condense and load siRNA, to assist endosomal escape induced by the “proton sponge” effect, and to unload siRNA in the cytoplasm in the target tumor cells, while the guest polymer is responsible to shield the vehicles from nonspecific cellular uptake, to prolong the circulation time, and to actively target the tumor cells. By switching the host and guest in different combinations, the molecular architectures of the PNPs are precisely controlled, which are subsequently evaluated in vitro in cell cultures to screen out the optimized PNPs, followed by further in vivo validation for targeted delivery of therapeutic siRNA-Bcl2 in a mouse tumor model.

  • Fig. 2 Characterization of CD-SS-P host polymer and its properties for siRNA condensation and cytoplasmic delivery.

    (A) 1H NMR spectrum of CD-SS-P in D2O. (B) GPC curve of CD-SS-P host polymer. Phosphate-buffered saline (PBS) (0.5×) was used as eluent. (C) siRNA condensation by CD-SS-P evaluated by gel retardation assay. (D) Three-dimensional (z-stack) confocal microscopic images showing intracellular distribution of rhodamin-B-isothiocyanate (RITC)–labeled polymer vehicles (red) and fluorescein isothiocyanate (FITC)–labeled siRNA (green) in KB cells at 1, 2, and 24 hours after transfection. RITC dye was labeled to hydroxyl groups of β-CD of CD-SS-P and CD-P. Cell nucleus was stained by Hoechst 33342 (blue). Scale bars, 10 μm.

  • Fig. 3 Screening and optimizing of PEG architectures and ligand densities for efficient siRNA targeted delivery.

    (A) Linear and comb-shaped Ad-PEG host polymers are used to screen the architectures (length, size, and shape) of PEG shell for minimized nonspecific binding. (B) Ad-PEG with distal FA ligands are used to screen and optimize the architectures and overall effects of nonspecific binding and ligand-induced specific binding. (C) Particle sizes and zeta potentials of siRNA-loaded PNPs formed by H/G carriers with various linear and comb-shaped Ad-PEG guests. Host polymer CD-SS-P without a guest polymer (H) was used as a control. PNPs were prepared at N/P ratio of 20 in PBS. (D) Mean fluorescence intensity (MFI) of KB and A549 cells at 4 hours after transfection with FITC-siRNA–loaded PNPs formed by various H/G and H carriers, measured by flow cytometry. **P < 0.01, when compared with H. (E) Fluorescence intensity of KB cells at 1 hour after incubation on ice with FITC-siRNA–loaded H/G targeted carriers, plotted against siRNA concentration, after subtracting that of corresponding H/G without FA. (F) Plots of fluorescence intensity of KB cells versus time during the dissociation of the bound PNPs from the surfaces of the cells. The KB cells were preincubated to equilibrium with PNPs formed by FITC-siRNA–loaded H/G targeted carriers, followed by reincubation in PBS. For (C) to (F), all data were presented in means ± SD (n = 3). a.u., arbitrary units.

  • Fig. 4 Cellular uptake of PNPs formed by FITC-siRNA and H or H/G(PC8kF6) in FR+ KB and FR− A549 cells.

    (A) Western blot analysis of FR (α-FR) expression in KB and A549 cells. I, Cells were not pretreated. II, FR receptors of the cells were silenced by pretreating cells with Lipofectamine 2k/siRNA-α-FR. The β-actin expression was used as a control. (B) Flow cytometry analysis showing the relationships between cellular uptake of FITC-siRNA–loaded PNPs and the expressions of FR on cells of KB and A549 cells. Cells were first treated with FITC-loaded PNPs for 4 hours and then fixed and stained using anti-rabbit α-FR as primary antibody and Alexa Fluor 546–labeled goat anti-rabbit antibody as secondary antibody. (C) Flow cytometric histograms showing the effect of FR receptor silencing and blocking of FR on cellular uptake of PNPs. I, Blank cells without pretreatment. II, The FR receptors of the cells were knocked down by pretreating cells with Lipofectamine 2k/siRNA-α-FR and then transfected with PNPs for 4 hours. III, Cells were first blocked with 600 μM free FA for 4 hours and then transfected with PNPs for 4 hours. IV, Cells directly transfected with PNPs for 4 hours without pretreatment. MFIs of KB and A549 under various conditions and treatment were quantitatively analyzed. Data were presented as means ± SD (n = 3). (D) Confocal laser scanning microscopic analysis of the cellular uptake of PNPs in FR+ KB cells and FR− A549 cells. Green, FITC-labeled siRNA. Blue, nucleus stained with DAPI. Scale bars, 20 μm.

  • Fig. 5 Delivery of Bcl-2 siRNA.

    (A) Down-regulation of Bcl-2 mRNA levels as quantified by RT-qPCR (n = 3). The relative level of mRNA expression was calculated over the untreated control (blank). (B) Suppression of Bcl-2 protein expression levels as evaluated by Western blot. β-Actin was used as negative controls. (C) Representative scatter dot-plot of apoptotic cells obtained by FITC–annexin V/PI costaining assay in flow cytometry experiments. (D) Quantitative analysis of the percentage of apoptotic cells using annexin V–FITC/PI costaining assay (n = 3) by flow cytometry.

  • Fig. 6 Antitumor efficacy of siRNA-Bcl2–loaded H/G(PC8kF6) PNPs against KB tumors and whole-body and ex vivo fluorescence imaging of KB tumor–bearing mice injected with PNPs loaded with Cy5-siRNA.

    (A) Timeline of treatment of BALB/c nude mice. Ten days after subcutaneous inoculation of KB cells, male BALB/c nude mice were injected with PNPs loaded with siRNA-Bcl2 via tail vein injection every 2 days. All survival mice were euthanized at day 21. (B) The KB tumor growth curves of mice treated with PNPs loaded with siRNA-Bcl2 (dosage, 1000 pmol/kg) based on H/G(PC8kF6) and other control groups [PBS and siRNA-Bcl2–loaded PNPs based on PEI 25k, H, and H/G(PC8k)]. One injection every 2 days until day 20 (6 mice per group). Data are presented as means ± SD; six mice per group. Data of the H/G(PC8kF6) and H/G(PC8k) groups were compared using one-tailed t test. *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Body weight change of KB tumor–bearing mice during 21-day anticancer treatment. Body weight percentage (%) = Body weight (Dayn)/Body weight (Day0) × 100. Data of the H/G(PC8kF6) and H/G(PC8k) groups were compared using one-tailed t test. *P < 0.05, **P < 0.01. (D) Images of the tumors harvested from the mice at day 21 after the initial treatment (or at the end point of the experiment). (E) Survival curves of KB tumor–bearing mice in response to treatment via tail vein injection with PNPs loaded with siRNA-Bcl2. (F) In vivo fluorescence imaging of KB tumor–bearing mice assessing the biodistribution, tumorous targeting, and retention of PNPs. Mice were imaged at different time points (2, 8, 24, and 36 hours) using IVIS live imaging system. Red circles indicate tumor areas. (G) Fluorescence intensity of Cy5-siRNA in tumor area at various time points. The tumor accumulation was calculated based on the quantification of Cy5 intensity in red circles. Photo credit: H.B., Zhejiang University.

Supplementary Materials

  • Supplementary Materials

    A supramolecular platform for controlling and optimizing molecular architectures of siRNA targeted delivery vehicles

    Yuting Wen, Hongzhen Bai, Jingling Zhu, Xia Song, Guping Tang, Jun Li

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