Nanosized Multifunctional Polyplexes for Receptor-Mediated SiRNA Delivery

Although our understanding of RNAi and our knowledge on designing and synthesizing active and safe siRNAs significantly increased during the past decade, targeted delivery remains the major limitation in the development of siRNA therapeutics. On one hand, practical considerations dictate robust chemistry reproducibly providing precise carrier molecules. On the other hand, the multistep delivery process requires dynamic multifunctional carriers of substantial complexity. We present a monodisperse and multifunctional carrier system, synthesized by solid phase supported chemistry, for siRNA delivery <i>in vitro</i> and <i>in vivo</i>. The sequence-defined assembly includes a precise cationic (oligoethanamino)amide core, terminated at the ends by two cysteines for bioreversible polyplex stabilization, at a defined central position attached to a monodisperse polyethylene glycol chain coupled to a terminal folic acid as cell targeting ligand. Complexation with an endosomolytic influenza peptide-siRNA conjugate results in nanosized functional polyplexes of 6 nm hydrodynamic diameter. The necessity of each functional substructure of the carrier system for a specific and efficient gene silencing was confirmed. The nanosized polyplexes showed stability <i>in vivo</i>, receptor-specific cell targeting, and silencing of the EG5 gene in receptor-positive tumors. The nanosized appearance of these particles can be precisely controlled by the oligomer design (from 5.8 to 8.8 nm diameter). A complete surface charge shielding together with the high stability result in good tolerability <i>in vivo</i> and the absence of accumulation in nontargeted tissues such as liver, lung, or spleen. Due to their small size, siRNA polyplexes are efficiently cleared by the kidney

Chondramide does not influence EGF-R signalling.

<p>MDA-MB-231 cells were treated with Chondramide (1 or 24 h), stimulated with EGF (5 min) and analyzed via Western blot analysis on EGFR, Akt and Erk. Left panel: one representative Western blot is shown. Right: Densitometric analysis of Western blots. *, p<0.05 One-way ANOVA, Tukey post-test, n = 3.</p

Chondramide diminishes metastasis <i>in vivo</i>.

<p>1×10⁵ 4T1-Luc cells were injected intravenously into pretreated (0.5 mg/kg ChB) and untreated BALB/cByJRj mice. (A) 8 days after cell inoculation mice were sacrificed, lungs were harvested and used for recording bioluminescence signals. Each lung was imaged from the dorsal and ventral side. Color bar scales were equalized. (B) Quantitative evaluation of metastasis to the lungs. Region of interest were defined (ROI) and total luciferin signal in ROIs was calculated as photons/second/cm² (total flux/area). Ten lungs per group, *, p<0.05 (t-test, unpaired). (C) Mouse weight over treatment period. Weight of treated (ChB) and untreated (DMSO) mice from day of cell inoculation (day 0) to euthanasia (day8) is shown for each group.</p

Chondramide diminishes contractility.

<p>(A) MDA-MB-231 cells were pretreated for 24 h as indicated, embedded in matrigel containing fluorescent beads and pictures were taken over 4 h every 15 min. Cellular, contractile force on the surrounding matrix was visualized via bead movement towards the cell and analyzed using PIV analysis. Upper panel: Representative images of cells (red) and fluorescent beads (green) t = 0. Lower panel: PIV analysis of bead velocity in the 2D projection. Color codes show bead velocity indicating applied force. Direction of vectors indicates averaged direction of bead movement. (B) For quantitative analysis the radial velocity (bead velocity towards the cell center) per data point was calculated. Minimum 13 cells per condition were analyzed. *, p<0.05 One-way ANOVA, Tukey post-test. (C) Phosphorylation of the Rho-GEF Vav2 was tested via Western blot analysis upon EGF stimulation (n = 3); * p<0.05 vs. control, One-way ANOVA, Tukey post-test.</p

Chondramide affects activation of the RhoGTPase Rho.

<p>(A) A Rac1 pull down was performed for untreated and Chondramide treated cells (24 h) upon EGF-stimulation (5 min). (B) After same treatment a Rho pull down was conducted. (C) Myosin light chain 2 (MLC2) was analyzed on its activation state via Western blot analysis upon EGF stimulation (5 min). A,B,C: Left panel: one representative Western blot is shown. Right panel: Densitometric analysis of Western blots. *, p<0.05 One-way ANOVA, Tukey post-test, n = 3.</p

Treatment with Chondramide reduces breast cancer cell migration, invasion and adhesion.

<p>(A) Chondramide treated and untreated MDA-MB-231 cells were allowed to migrate in a Boyden chamber for 16 h. (B) Chondramide inhibits invasion of MDA-MB-231 cells through matrigel in Boyden chamber (48 h). A,B: For positive control (PC) lower compartment was filled with medium plus 10% FCS, for negative control (NC) only medium without FCS was added. *, p<0.05 One-way ANOVA, Tukey post-test, n = 3. (C) Pretreated MDA-MB-231 cells were seeded freshly on indicated surfaces and counted after fixation. *, p<0.05 One-way ANOVA, Tukey post-test, n = 3. (D) Cells from C were stained for F-actin, nuclei and vinculin. Bar represents 50 µm.</p