Nanosac, a Noncationic and Soft Polyphenol Nanocapsule, Enables Systemic Delivery of siRNA to Solid Tumors

Abstract

For systemic delivery of small interfering RNA (siRNA) to solid tumors, the carrier must circulate avoiding premature degradation, extravasate and penetrate tumors, enter target cells, traffic to the intracellular destination, and release siRNA for gene silencing. However, existing siRNA carriers, which typically exhibit positive charges, fall short of these requirements by a large margin; thus, systemic delivery of siRNA to tumors remains a significant challenge. To overcome the limitations of existing approaches, we have developed a carrier of siRNA, called "Nanosac", a noncationic soft polyphenol nanocapsule. A siRNA-loaded Nanosac is produced by sequential coating of mesoporous silica nanoparticles (MSNs) with siRNA and polydopamine, followed by removal of the sacrificial MSN core. The Nanosac recruits serum albumin, co-opts caveolae-mediated endocytosis to enter tumor cells, and efficiently silences target genes. The softness of Nanosac improves extravasation and penetration into tumors compared to its hard counterpart. As a carrier of siRNA targeting PD-L1, Nanosac induces a significant attenuation of CT26 tumor growth by immune checkpoint blockade. These results support the utility of Nanosac in the systemic delivery of siRNA for solid tumor therapy.

Keywords: immune checkpoint blockade; noncationic; polyphenol nanocapsules; siRNA; soft; solid tumors; systemic delivery.

Fig. 1.. Preparation and characterization of Nanosac.

(a) Schematic of O/siRNA/pD (Nanosac) preparation. MSN: mesoporous silica nanoparticles; MSN^a: MSN conjugated with (3-Aminopropyl)triethoxysilane (APTES); MSN^a/siRNA: MSN^a with siRNA loaded on the surface; MSN^a/siRNA/pD: MSN^a/siRNA covered with pD; O/siRNA/pD (Nanosac): siRNA-loaded nanocapsules after MSN core removal. (b) Transmission electron micrographs (TEM) of Nanosac and the precursors. Visualized by negative staining with 1% uranyl acetate. Scale bars: 50 nm. (c) Zeta potential and Z-average of Nanosac and the precursors (n = 3 independently and identically prepared batches, mean ± s.d) (d) Force-distance curves for MSN^a/pD and O/pD (Nanosac) measured by AFM, and Young’s moduli of MSN^a/PD and Nanosac (n = 15 tests of a representative batch, mean ± s.d.). ****: p < 0.0001 vs. MSN^a/pD by unpaired t-test.

Fig. 2.. Stability of Nanosac.

(a) Gel electrophoresis of siLuc, MSN^a/siLuc, MSN^a/siLuc/pD, and Nanosac with/without RNase or SDS challenge; (b) Gene silencing by MSN^a/siLuc, MSN^a/siLuc/pD, and Nanosac with/without RNase challenge (n = 3 test of a representative batch, mean ± s.d.). ***: p < 0.001; ns: not significant by Sidak’s multiple comparisons test following one-way ANOVA. Gene silencing by MSN^a/siRNA, MSN^a/siRNA/pD, and Nanosac, measured after 48 h treatment in complete medium. (c) siGAPDH in CT26 cells, (d) siLuc in 4T1-Luc cells, and (e) siPD-L1 in IFN-γ-activated CT26 cells. (n = 3 test of a representative batch, mean ± s.d.). ***: p < 0.001, ****: p < 0.0001 vs. No siRNA by Dunnett’s multiple comparisons test following two-way ANOVA. (f) Top: Representative western blot of PD-L1 expression in IFN-γ-activated CT26 cells by MSN^a/siPD-L1, MSN^a/siPD-L1/pD, and Nanosac. Bottom: Quantitative presentation of western blotting.

Fig. 3.. Endocytosis pathway of NPs.

(a) Effects of endocytosis inhibitors on NP uptake in serum-supplemented medium. CT26 cells were preincubated for 30 min with each inhibitor at a subtoxic concentration, followed by treatment of NPs in 10% FBS-supplemented medium for 2 h (n = 3 tests of a representative batch, mean ± s.d.). ****: p < 0.0001, ns: not significant vs. 37 °C by Dunnett’s multiple comparisons test following two-way ANOVA. CPZ: chloropromazine. Albuminylation of NPs. (b) Zeta potential of MSN^a, MSN^a/pD, and Nanosac before and after incubation in 50% FBS (n = 3 tests of a representative batch, mean ± s.d.). (c) SDS-PAGE of protein corona composition formed on MSN^a, MSN^a/pD, and Nanosac. NPs (4 mg/mL) were incubated in 50% FBS for 2 h and rinsed with PBS twice. (d) Spectral counts of proteins, analyzed by LC-MS/MS, bound on the MSN^a, MSN^a/pD, and Nanosac after 2 h exposure to 50% FBS. (e) Representative SDS-PAGE gel of albumin after pulse proteolysis. Native albumin (nAlb), denatured albumin (dAlb), MSN^a incubated with albumin (MSN^a+Alb) and MSN^a/pD with albumin (MSN^a/pD+Alb) were treated with thermolysin for 3 min. Lane 1: nAlb; Lane 2: dAlb; Lane 3: MSN^a+Alb; Lane 4: MSN^a/pD+Alb. % digestion albumin was defined as (1-albumin band intensity after proteolysis/albumin band intensity before proteolysis) × 100. n = 3 independently and identically performed experiments (mean ± s.d.). ***: p < 0.001 and ****: p < 0.0001 vs. nAlb by Dunnett’s multiple comparisons test following one-way ANOVA. Intracellular trafficking of NPs. (f) Confocal microscope images locating cy5-labeled MSN^a, MSN^a/pD, and Nanosac relative to lysosomes in CT26 cells. Green: Lysotracker (lysosome); Red: cy5-labeled NPs; Blue: Hoechst 33342 (nuclei). Scale bars: 10 μm. (g) Pearson’s correlation coefficients indicating the degree of NP/lysosome colocalization in confocal images: R=1 (perfect colocalization), R=0 (no colocalization). n = 5 tests of a representative batch (mean ± s.d). ***: p < 0.001 and ****: p < 0.0001 by Tukey's multiple comparisons test following one-way ANOVA. (h) Fluorescence intensity profiles along the white lines in (f). (i) Release kinetics of siRNA from Nanosac, performed in different pHs or H₂O₂ (100 μM) with constant agitation at 37 °C. n=3 tests with representative batches (mean ± s.d.).

Fig. 4.. Comparison of MSN^a-cy5/pD and Nanosac in macrophage uptake, extravasation, and tumor spheroid penetration.

(a) Quantitative measurement and confocal microscope images of J774a.1 macrophage taking up MSN^a-cy5/pD and Nanosac. n = 3 tests of a representative batch (mean ± s.d). ***: p < 0.001 and ****: p < 0.0001 by Sidak's multiple comparisons test following two-way ANOVA. Green: Wheat Germ Agglutinin (cell membrane); Red: cy5-labeled NPs; Blue: Hoechst 33342 (nuclei). Scale bar: 50 μm. (b) Time-lapse intravital microscopic images of MSN^a-cy5/pD and Nanosac circulating in CT26 tumor-bearing BALB/c mice. Green: Dextran-FITC (locating blood vessel), Red: cy5-labeled NPs. (c) Z-section images of CT26 tumor spheroids incubated with MSN^a-cy5/pD or Nanosac. Scale bars: 500 μm. (d) Spheroid depth-wise fluorescence intensity profiles. n = 3 tests of a representative batch (mean ± s.d). ****: p < 0.0001 vs. MSN^a-cy5/pD by Sidak’s multiple comparisons test following two-way ANOVA. (e) Horizontal fluorescence intensity profile at 160 μm.

Fig. 5.. Anti-tumor activity of 5% dextrose (D5W), MSN^a/siPD-L1/pD, and Nanosac in Balb/c mice bearing CT26 tumors (siPD-L1: 0.75 mg/kg/time, q2d×10).

(a) Average tumor size (mm³). (b) Average body weight (g, grams). *: p < 0.05 and **: p < 0.01 between average tumor sizes on day 20 post-first injection by Tukey's multiple comparisons test following two-way repeated measures ANOVA. Arrowheads indicate times of treatment. n = 5 mice per treatment (mean ± s.d). (c) Expression of PD-L1 in CT26 tumors (Supporting Fig. 24). n = 4 mice per group (mean ± s.d). *: p < 0.05; ns: not significant vs. D5W by Dunnett's multiple comparisons test following one-way ANOVA. (d) %CD8⁺ cells, %CD4⁺ cells, and CD8⁺/CD4⁺ ratio in TDLNs of treated animals. n = 5 mice per treatment (mean ± s.d). *: p < 0.05, ***: p < 0.001, ****: p < 0.0001, and ns: not significant vs. D5W by Dunnett's multiple comparisons test following one-way ANOVA. (e) Fluorescence micrographs of tumor sections showing FITC-lectin-stained vessels (green) and MSN^a/siRNA-cy5/pD or Nanosac (red) at 24 h from IV injection. Scale bars: 50 μm. See Supporting Fig. 27 for additional micrographs. (f) quantitative analysis of micrographs in (e): % NPs departing from the lectin-positive endothelial cells was calculated as the area of free NPs (red) divided by the area of the total NP fluorescence (free NPs and NPs overlapping with endothelial cells: red + yellow). Three fields were randomly selected and analyzed by the Nikon A1R confocal microscope analysis software. (g) Photomicrographs of hematoxylin and eosin (H&E)-stained liver and spleen sections. No significant lesions were observed in either organ microscopically examined in all treatment groups. See Supporting Fig. 28 and 29 for high magnification photomicrographs.

Fig. 6.. Anti-tumor activity of D5W, anti-PD-L1 antibody, and Nanosac in Balb/c mice bearing CT26 tumors

(anti-PD-L1 antibody: 200 μg/mouse/time, intraperitoneal injection; siPD-L1: 1.5 mg/kg/time, IV injection; q2d×5). *: p < 0.05, **: p < 0.01, and ***: p < 0.001 between average tumor sizes on day 20 post-first injection by Tukey's multiple comparisons test following two-way repeated measures ANOVA. n = 5 mice per group (mean ± s.d).