Hyaluronate siRNA nanoparticles with positive charge display rapid attachment to tumor endothelium and penetration into tumors.

A cationizable sequence-defined lipo-oligoaminoamide (lipo-OAA) conferring stable assembly of siRNA into ~200 nm sized complexes contains an N-terminal azidolysine for covalent coating of formed nanoparticles with dibenzocyclooctyne-amine (DBCO)-modified hyaluronic acid (HA). Depending on the applied equivalents of DBCO-HA, stable nanoparticles with either cationic or anionic surface charge can be formed. The unmodified and two types of covalent HA-modified siRNA nanoparticles differ in their biological characteristics. Both types of HA coated siRNA complexes show an enhanced cellular uptake over uncoated complexes and facilitate efficient gene silencing, but differ in intracellular uptake pathways and distribution. Upon intravenous administration in mice, beyond our expectation and in contrast to the in vitro findings, only the cationic HA nanoparticles but neither the non-coated cationic nor the anionic HA complexes were able to target subcutaneous Huh 7 tumors and exert potent (78%) gene silencing. The favorable and very fast accumulation of cationic HA nanoparticles was confirmed in another subcutaneous tumor model. As evidenced by 3D nanoparticle distribution within Huh 7 tumors evaluated at early time points of 5 min and 45 min, only the cationic HA-based nanoparticles rapidly attach to the tumor endothelium and subsequently penetrate into tumor, in contrast to the analogous anionic HA coated or the cationic non-coated formulation.

Folate receptor-directed orthogonal click-functionalization of siRNA lipopolyplexes for tumor cell killing in vivo.

The delivery of small interfering RNA (siRNA) and its therapeutic usage as an anti-cancer agent requires a carrier system for selective internalization into the cytosol of tumor cells. We prepared folate-bearing formulations by first complexing siRNA with the novel azido-functionalized sequence-defined cationizable lipo-oligomer 1106 (containing two cholanic acids attached to an oligoaminoamide backbone in T-shape configuration) into spherical, ∼100-200 nm sized lipopolyplexes, followed by surface-functionalization with various folate-conjugated DBCO-PEG agents. Both the lipo-oligomer and the different defined shielding and targeting agents with mono- and bis-DBCO and varying PEG length were generated by solid phase supported synthesis. A bivalent DBCO surface agent with a PEG24 spacer was identified as the optimal formulation in terms of nanoparticle size, folate receptor (FR) targeting, cellular uptake and gene silencing in vitro. Notably, near-infrared fluorescence bioimaging studies showed that double-click incorporation of bivalent DBCO-PEG24 resulted in siRNA/1106/DBCO2-ss2-PEG24-FolA lipopolyplexes with extended biodistribution and intratumoral delivery in a subcutaneous FR-positive leukemia mouse model. Intravenous administration of analogous therapeutic siRNA lipopolyplexes (directed against the kinesin spindle motor protein EG5) mediated tumoral EG5 mRNA knockdown by ∼60% and, in combination with the novel antitubulin drug pretubulysin, significantly prolonged survival of aggressive leukemia bearing mice without noticeable side effects.