Mastering Dendrimer Self-Assembly for Efficient siRNA Delivery: From Conceptual Design to In Vivo Efficient Gene Silencing

Abstract

Self-assembly is a fundamental concept and a powerful approach in molecular science. However, creating functional materials with the desired properties through self-assembly remains challenging. In this work, through a combination of experimental and computational approaches, the self-assembly of small amphiphilic dendrons into nanosized supramolecular dendrimer micelles with a degree of structural definition similar to traditional covalent high-generation dendrimers is reported. It is demonstrated that, with the optimal balance of hydrophobicity and hydrophilicity, one of the self-assembled nanomicellar systems, totally devoid of toxic side effects, is able to deliver small interfering RNA and achieve effective gene silencing both in cells - including the highly refractory human hematopoietic CD34(+) stem cells - and in vivo, thus paving the way for future biomedical implementation. This work presents a case study of the concept of generating functional supramolecular dendrimers via self-assembly. The ability of carefully designed and gauged building blocks to assemble into supramolecular structures opens new perspectives on the design of self-assembling nanosystems for complex and functional applications.

Keywords: amphiphilic dendrons; nanomicelles; self-assembly; siRNA delivery; supramolecular dendrimers.

Figure 1

Schematic presentation of self-assembly of amphiphilic dendrons into supramolecular dendrimers to resemble covalent high-generation dendrimers for functional siRNA delivery and gene silencing.

Figure 2

(A) Dendrons 1–8 investigated in this study; (B) The experimental/predicted values of self-assembled micellar supramolecular dendrimer diameter (D_exp, D_cal) and critical micelle concentration (CMC_exp, CMC_cal), the calculated packing parameter (P) and free energy of micellization (ΔG_mic) for dendrons 1–6. (C) TEM images of the self-assembled supramolecular micellar structures formed by 3, 4, 5 and 6 in buffer at pH 7.4.

Figure 3

siRNA/dendrimer binding complexes revealed by (A) ethidium bromide assay; (B) gel retardation, (C) dynamic light scattering and (D) mesoscale modeling. Color legend in D: siRNA, red sticks; purple and lilac, hydrophilic dendron beads; light grey, hydrophobic tail beads; green, linker beads. Water and counterions are portrayed as a light blue field for clarity.

Figure 4

Dendrimer-mediated siRNA delivery and gene silencing of heat shock protein 27 (Hsp27) and translationally controlled tumor protein (TCTP) in human prostate cancer PC-3 (A, B) and C4-2 (C, D) cells. Hsp27 and TCTP protein levels were quantified by western blotting 72 h after treatment with the corresponding siRNA/dendrimer complexes at an N/P ratio of 10. (E) Hsp27 expression in human prostate cancer PC-3 cells treated with 50 nM Hsp27 siRNA and 4 at an N/P ratio of 10 compared with Hsp27 expression in cells treated with the high-generation dendrimer G₇. (F) CD4 expression in human hematopoietic CD34⁺ stem cells treated with 50 nM CD4 dsiRNA and 4 at an N/P ratio of 5. The commercial transfection reagents Oligofectamine (oligo) and TransIT-TKO (TKO) was used as controls.

Figure 5

(A) siRNA release from complexes with 4–6 was assessed using heparin-coupled ethidium bromide fluorescent assays. (B) siRNA/dendrimer binding data as derived from molecular dynamics simulations for 4–6: free energy of effective binding (ΔG_bind,eff), number of effective charges (N_eff) and effective-charge-normalized free energy of binding (ΔG_{bind,eff/Neff}) for siRNA/dendrimer complexes. (C) Atomistic MD simulations of amphiphilic dendron self-assembly in the presence of siRNA. In all panels, the dendrons 4–6 are shown as forest green, with the terminal charged amine groups highlighted as chartreuse green sticks-and-balls. The siRNA is represented as a red ribbon and some representative Cl⁻ and Na⁺ ions and counterions are portrayed as light and dark gray spheres, respectively. Water is omitted in all panels for clarity. The binding interface region between each micelle and siRNA is also shown in each complex.

Figure 6

Effective 4-mediated systemic siRNA delivery and gene silencing in mice. Nude mice bearing prostate cancer PC-3 tumors of 30–50 mm³ were randomly selected for treatment with Hsp27 siRNA/4 and with PBS, 4 alone, and scrambled siRNA/4 as controls (3 mg/kg siRNA and 4 at an N/P ratio of 5). Treatments were administered by intraperitoneal injection during a period of 4 weeks with an injection frequency of twice per week (A–D). Effective gene silencing of Hsp27 was measured by western blotting and quantified by Image J software (A). (B) Inhibition of tumor growth and (C) the antiproliferation activity in tumors assessed by measuring tumor size and by Ki-67 immunohistochemical staining, respectively. (D) The body weight of the mice was recorded during the whole treatment period. (E) In vivo toxicity assessment of 4/siRNA by sectioning and HES staining of the major mouse organs including heart, kidney, liver, lung, and spleen.