GalNAc- or Mannose-PEG-Functionalized Polyplexes Enable Effective Lectin-Mediated DNA Delivery.

A cationic, dendrimer-like oligo(aminoamide) carrier with four-arm topology based on succinoyl tetraethylene pentamine and histidines, cysteines, and N-terminal azido-lysines was screened for plasmid DNA delivery on various cell lines. The incorporated azides allow modification with various shielding agents of different polyethylene glycol (PEG) lengths and/or different ligands by copper-free click reaction, either before or after polyplex formation. Prefunctionalization was found to be advantageous over postfunctionalization in terms of nanoparticle formation, stability, and efficacy. A length of 24 ethylene oxide repetition units and prefunctionalization of ≥50% of azides per carrier promoted optimal polyplex shielding. PEG shielding resulted in drastically reduced DNA transfer, which could be successfully restored by active lectin targeting via novel GalNAc or mannose ligands, enabling enhanced receptor-mediated endocytosis of the carrier system. The involvement of the asialoglycoprotein receptor (ASGPR) in the uptake of GalNAc-functionalized polyplexes was confirmed in the ASGPR-positive hepatocarcinoma cell lines HepG2 and Huh7. Mannose-modified polyplexes showed superior cellular uptake and transfection efficacy compared to unmodified and shielded polyplexes in mannose-receptor-expressing dendritic cell-like DC2.4 cells.

Systemic Delivery of Folate-PEG siRNA Lipopolyplexes with Enhanced Intracellular Stability for In Vivo Gene Silencing in Leukemia.

Protection of small interfering RNA (siRNA) against degradation and targeted delivery across the plasma and endosomal membranes to the final site of RNA interference (RNAi) are major aims for the development of siRNA therapeutics. Targeting for folate receptor (FR)-expressing tumors, we optimized siRNA polyplexes by coformulating a folate-PEG-oligoaminoamide (for surface shielding and targeting) with one of three lipo-oligoaminoamides (optionally tyrosine-modified, for optimizing stability and size) to generate ∼100 nm targeted lipopolyplexes (TLPs), which self-stabilize by cysteine disulfide cross-links. To better understand parameters for improved tumor-directed gene silencing, we analyzed intracellular distribution and siRNA release kinetics. FR-mediated endocytosis and endosomal escape of TLPs was confirmed by immuno-TEM. We monitored colocalization of TLPs with endosomes and lysosomes, and onset of siRNA release by time-lapse confocal microscopy; analyzed intracellular stability by FRET using double-labeled siRNA; and correlated results with knockdown of eGFPLuc protein and EG5 mRNA expression. The most potent formulation, TLP1, containing lipopolyplex-stabilizing tyrosine trimers, was found to unpack siRNA in sustained manner with up to 5-fold higher intracellular siRNA stability after 4 h compared to other TLPs. Unexpectedly, data indicated that intracellular siRNA stability instead of an early endosomal exit dominate as a deciding factor for silencing efficiency of TLPs. After i.v. administration in a subcutaneous leukemia mouse model, TLP1 exhibited ligand-dependent tumoral siRNA retention, resulting in 65% EG5 gene silencing at mRNA level without detectable adverse effects. In sum, tyrosine-modified TLP1 conveys superior protection of siRNA for an effective tumor-targeted delivery and RNAi in vivo.

Minicircle Versus Plasmid DNA Delivery by Receptor-Targeted Polyplexes.

Due to its minimal size and lack of bacterial backbone sequences, minicircle (MC) DNA presents a promising alternative to plasmid DNA (pDNA) for non-viral gene delivery in terms of biosafety and improved gene transfer. Here, luciferase pDNA (pCMV-luc) and analogous MC DNA (MC07.CMV-luc) were formulated into polyplexes with c-Met targeted, PEG-shielded sequence-defined oligoaminoamides, or linear PEI (linPEI) as standard transfection agent. Distinct physicochemical and biological characteristics were observed for polyplexes formed with either pDNA or MC DNA as vectors. The carriers were found to dominate the shape of polyplexes, whereas the DNA type was decisive for the nanoparticle size. c-Met-targeted, tyrosine trimer-containing polyplexes were optimized into compacted rod structures with a size of 65-100 nm for pDNA and 35-40 nm for MC. Notably, these MC polyplexes display a lack of cell cycle dependence of transfection and a ∼200-fold enhanced gene transfer efficiency in c-Met-positive DU145 prostate carcinoma cultures over their tyrosine-free pDNA analogues.

Combined atomic force microscopy and optical microscopy measurements as a method to investigate particle uptake by cells.

We propose a combination of atomic force microscopy (AFM) and optical microscopy for the investigation of particle uptake by cells. Positively and negatively charged polymer microcapsules were chosen as model particles, because their interaction with cells had already been investigated in detail. AFM measurements allowed the recording of adhesion forces on a single-molecule level. Due to the micrometer size of the capsules, the number of ingested capsules could be counted by optical microscopy. The combination of both methods allowed combined measurement of the adhesion forces and the uptake rate for the same model particle. As a demonstration of this system, the correlation between the adhesion of positively or negatively charged polymer microcapsules onto cell surfaces and the uptake of these microcapsules by cells has been investigated for several cell lines. As is to be expected, we find a correlation between both processes, which is in agreement with adsorption-dependent uptake of the polymer microcapsules by cells.

Tumoral gene silencing by receptor-targeted combinatorial siRNA polyplexes.

Small interfering RNA (siRNA) promises high efficacy and excellent specificity to silence the target gene expression, which shows potential for cancer treatment. However, systemic delivery of siRNA with selectivity to the tumor site and into the cytosol of tumor cells remains a major limitation. To achieve this, we generated oligoaminoamide-based sequence-defined polycationic oligomers by solid-phase assisted synthesis, which can form polyplexes with anionic siRNA by electrostatic interaction to serve as siRNA carrier. Targeting for folate receptor (FR)-overexpressing tumors, we optimized the physicochemical properties of polyplexes by combinatorial optimization of PEGylated folate-conjugated oligomer (for FR targeting and shielding of surface charges) and 3-arm oligomer (for size modification and particle stability). For uni-directional fast coupling between the two groups of oligomers, we activated the cysteine thiol groups of one of the oligomers with 5,5'-dithio-bis(2-nitrobenzoic acid) to achieve a fast chemical linkage through disulfide formation with the free thiol groups of the other oligomer. These targeted combinatorial polyplexes (TCPs) are homogeneous spherical particles with favorable size and surface charge, which showed strong siRNA binding activity. TCPs were internalized into cells by FR-mediated endocytosis, triggered significant eGFP-luciferase marker gene silencing, and transfection with antitumoral EG5 siRNA suppressed cell proliferation in FR-expressing tumor cells. Moreover, the most promising formulation TCP1 after i.v. administration in tumor-bearing mice exhibited siRNA delivery into the tumor, resulting in EG5 gene silencing at mRNA level. Therefore, by covalent combination of two sequence-defined functional oligomers, we developed a siRNA carrier system with optimized size and surface charge for efficient tumor cell-directed gene silencing and cytotoxicity in vitro and in vivo.