Overcoming endosomal barrier by amphotericin B-loaded dual pH-responsive PDMA-b-PDPA micelleplexes for siRNA delivery

Abstract

The endosomal barrier is a major bottleneck for the effective intracellular delivery of siRNA by nonviral nanocarriers. Here, we report a novel amphotericin B (AmB)-loaded, dual pH-responsive micelleplex platform for siRNA delivery. Micelles were self-assembled from poly(2-(dimethylamino)ethyl methacrylate)-block-poly(2-(diisopropylamino)ethyl methacrylate) (PDMA-b-PDPA) diblock copolymers. At pH 7.4, AmB was loaded into the hydrophobic PDPA core, and siRNA was complexed with a positively charged PDMA shell to form the micelleplexes. After cellular uptake, the PDMA-b-PDPA/siRNA micelleplexes dissociated in early endosomes to release AmB. Live cell imaging studies demonstrated that released AmB significantly increased the ability of siRNA to overcome the endosomal barrier. Transfection studies showed that AmB-loaded micelleplexes resulted in significant increase in luciferase (Luc) knockdown efficiency over the AmB-free control. The enhanced Luc knockdown efficiency was abolished by bafilomycin A1, a vacuolar ATPase inhibitor that inhibits the acidification of the endocytic organelles. These data support the central hypothesis that membrane poration by AmB and increased endosomal swelling and membrane tension by a "proton sponge" polymer provided a synergistic strategy to disrupt endosomes for improved intracellular delivery of siRNA.

Figure 1

Schematic diagram of AmB-loaded dual pH-responsive micelleplexes for siRNA delivery with enhanced siRNA endosomal escape ability. (A) Production of AmB-loaded PDMA-b-PDPA micelleplexes. AmB was loaded in the hydrophobic PDPA core, and siRNA was complexed with the PDMA corona shell. (B) AmB-facilitated endosome disruption and siRNA cytoplasmic release (a: AmB-loaded micelleplexes dissociated in early endosomes after cell uptake, and AmB molecules are inserted into endosomal membranes; b: protonated PDMA-b-PDPA unimers complexed with siRNA and trafficked from early endosomes into late endosome/lysosomes, causing vesicle swelling; c: AmB-enhanced siRNA release from endosomes into cytoplasm via membrane destabilization). (C) In the case of AmB-free micelleplexes, polymer/siRNA complexes were entrapped in late endosomes or lysosomes without efficient cytoplasmic siRNA release.

Figure 2

Physical characterization of the dual pH-responsive PDMA-b-PDPA micelles and micelleplexes. (A) Chemical structures of the diblock copolymers used in this study. (B) Titration curves of PDMA-b-PDPA, PEG-b-PDPA, and PEG-b-PDMA copolymers. Change of hydrodynamic diameter (C) and zeta-potential (D) of the PDMA-b-PDPA micelles as a function of buffer pH (*no particles were detected by DLS for PEG-b-PDPA and PDMA-b-PDPA micelles at pH 6.2, indicating their dissociation at acidic pH). The surface charge of PSt-b-PDMA micelles reached a plateau at pH 6.2 owing to the complete protonation of the PDMA segment. (E) Gel shift assay shows the siRNA binding stability by the PDMA₅₀-b-PDPA₆₅ micelleplexes. (F) TEM images of AmB-free or 1.0 wt % AmB-loaded PDMA-b-PDPA micelleplexes at pH 7.4 and 6.0, respectively (scale bars = 200 nm).

Figure 3

CLSM examination of intracellular dissociation of TMR-labeled PDMA-b-PDPA micelleplexes. (A) Intracellular dissociation of PDMA-b-PDPA micelleplexes in A549-Luc cells containing Rab5a-GFP-labeled early endosomes (top panel) or Lamp1-GFP-labeled late endosomes/lysosomes (middle panel). Dissociation of micelleplexes resulted in the activation of TMR signal (red), which showed colocalization with late endosomes/lysosomes. (B) Baf-A1 inhibited the intracellular dissociation of the PDMA-b-PDPA micelleplexes in A549-Luc cells, as indicated by the lack of TMR fluorescence from the pH-activatable micelles (bottom panel) (scale bar = 10 µm). siRNA molecules were labeled with Alexa dye and are shown as the blue color.

Figure 4

CLSM examination of siRNA endosomal escape in the cells treated with AmB-loaded micelleplexes. The images were taken 12 h after micelleplex incubation. Compared to the AmB-free micelleplexes, AmB-loaded micelleplexes significantly increased the siRNA endosomal escape, as indicated by the diffusive distribution of Alexa-labeled siRNA molecules (control: untreated cells; AmB-0: AmB-free micelleplex-treated cells; AmB-0.5: 0.5 wt % AmB-loaded micelleplexes; AmB-1.0: 1.0 wt % AmB-loaded micelleplexes, scale bar = 20 µm). TMR-dextran polymers were used as an endosomal marker.

Figure 5

siRNA transfection study of AmB-loaded PDMA-b-PDPA micelleplexes in A549-Luc cells. (A) Influence of AmB loading on Luc knockdown efficiency by the PDMA-b-PDPA micelleplexes (siRNA concentration = 75 nM, or 100 ng per well). The Rel Luc knockdown was determined by normalizing the decreased Luc activity in siRNA-Luc-treated cells over that of the siRNA-Scr-treated control. (B) Relative cell viability of A549-Luc cells transfected by AmB-loaded micelleplexes as determined by the MTT assay. (C) Comparison of AmB-free vs AmB-loaded siRNA/micelleplexes as a function of micelleplex dose and PDMA-b-PDPA/siRNA ratio. Significant increase in Luc knockdown efficiency was observed with AmB loading. (D) Erythrocytic activity of AmB-loaded micelleplexes. At lower pH (6.2), significantly increased hemolytic activities were observed with the combination of AmB and PDMA-b-PDPA copolymer, demonstrating the synergistic effect in membrane destabilization.

Figure 6

Influence of Baf-A1 treatment on Luc knockdown efficiency by the AmB-loaded PDMA-b-PDPA micelleplexes. (A) Relative Luc knockdown efficiency of AmB-loaded micelleplexes in Baf-A1-pretreated A549-Luc cells. (B) Relative Luc knockdown efficiency vs Baf-A1 addition times. The cells were first treated with micelleplex, and then Baf-A1 was added at different time points (0, 3, 6, or 12 h) after micelleplex addition (1.0 wt % AmB loading, w/w 10, and siRNA concentration of 75 nM were applied) (*p < 0.05, **p < 0.01). Rel Luc knockdown was determined by normalizing the decreased Luc activity in siRNA-Luc-treated cells over that of the siRNA-Scr-treated control.