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With its nearly unrestricted possibilities, gene therapy attracts more and more significance in modern-day research. The only issue still seeming to hold back its clinical success is the actual effective delivery of genetic material. Nucleic acids are in general challenging to administer to their intracellular targets due to their unfavorable pharmaceutical characteristics. Polymeric nanogels present a promising delivery platform for oligonucleotide-based therapies, as the growing number of reports deliberated in this review represents. Within the scope of this article, recent progress in the employment of nanogels as gene delivery vectors is summarized and different examples of modified, stimuli-responsive, targeted and co-delivering nanogels are discussed in detail. Furthermore, major aspects of successful gene delivery are addressed and critically debated in regards to nanogels, giving insights into what progress has been made and which key issues still need to be further approached.

The block copolymer VIPER (virus-inspired polymer for endosomal release) has been reported to be a promising novel delivery system of DNA plasmids both in vitro and in vivo . VIPER is comprised of a polycation segment for condensation of nucleic acids as well as a pH-sensitive segment that exposes the membrane lytic peptide melittin in acidic environments to facilitate endosomal escape. The objective of this study was to investigate VIPER/siRNA polyplex characteristics, and compare their in vitro and in vivo performance with commercially available transfection reagents and a control version of VIPER lacking melittin. VIPER/siRNA polyplexes were formulated and characterized at various charge ratios and shown to be efficiently internalized in cultured cells. Target mRNA knockdown was confirmed by both flow cytometry and qRT-PCR and the kinetics of knockdown was monitored by live cell spinning disk microscopy, revealing knockdown starting by 4 h post-delivery. Intratracheal instillation of VIPER particles formulated with sequence specific siRNA to the lung of mice resulted in a significantly more efficient knockdown of GAPDH compared to treatment with VIPER particles formulated with scrambled sequence siRNA. We also demonstrated using pH-sensitive labels that VIPER particles experience less acidic environments compared to control polyplexes. In summary, VIPER/siRNA polyplexes efficiently deliver siRNA in vivo resulting in robust gene silencing (>75% knockdown) within the lung.

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New folic acid (FA) coupled three layered micelles (3LM) were designed to encapsulate DNA, and their application as delivery system that specifically targets activated macrophages was investigated for new treatment options in rheumatoid arthritis (RA). FA coupled poly(l-lactide)-b-poly(ethylene glycol) (FA-PEG-PLLA) was synthesized via the NHS-ester activated/amine coupling method. Fluorescein labeled folic acid was used for flow cytometric detection of the expression of functional folic receptor β in LPS-activated and resting macrophages. FA coupled 3LM were formulated in a two-step procedure and characterized regarding hydrodynamic diameters and zeta potentials. The presence of the targeting ligand was shown not to increase the size of the 3LM compared to their non-targeted counterparts. Targeted and non-targeted 3LM were used in vitro to optimize uptake conditions in the RAW 264.7 macrophage cell line. The amount of FA coupled polymer in the final formulation was found to be optimal at 75% FA-PEG-PLLA and 25% PLLA-PEG-PLLA. Subsequently, transgene expression in vitro in RAW 264.7 cells and ex vivo in primary activated and resting mouse macrophages was determined as a function of FR-mediated internalization of 3LM encapsulating GFP expressing plasmid. FR-overexpressing activated cells, as successfully identified by internalization of FA-fluorescein, showed significantly higher GFP expression in vitro and ex vivo than resting macrophages with only a basal level of FR expression. Lastly, injectable hydrogels as depot formulation were formed by stereocomplexation, and their degradation, DNA release profiles, and dissociation into intact 3LM were found to be beneficial for potential in vivo application. Our findings confirm that FA-3LM are taken up by activated macrophages via folate receptor mediated endocytosis and that their hydrogels release intact 3LM for efficient transfection of primary macrophages. Therefore, FA-3LM could become a promising delivery system for receptor-mediated drug or gene delivery and novel therapy for rheumatoid arthritis in an in situ forming gel formulation.

"Three-layered micelles" (3LM) composed of two triblock copolymers, poly(L-lactide)-b-polyethyleneimine-b-poly(L-lactide) (PLLA-PEI-PLLA) and poly(L-lactide)-b-poly(ethylene glycol)-b-poly(L-lactide) (PLLA-PEG-PLLA) are designed to combine electrostatic interaction and solvent-induced condensation of DNA. The low molecular weight PLLA-PEI-PLLA is synthesized by a facile amine-protection/deprotection approach and employed as a gene vector, compacting DNA as a polyplex core in the organo-micelles. The individual organo-micelle is further encapsulated within a PLLA-PEG-PLLA amphiphilic micelle leading to an aqueous stable colloidal dispersion. The resulting spherical 3LM possess a hydrodynamic diameter of ca. 200 nm and zeta potential close to neutral and display excellent stability to competing polyanions such as dextran sulfate in neutral pH (7.4). Such high stability is attributed to the complete shielding of the PEI/DNA polyplex core with an impermeable hydrophobic intermediate layer. However, greater than 90% of the encapsulated DNA are released within 30 min when exposed to slightly acidic pH (4.5). Based on our findings, a new class of non-viral delivery system for nucleic acids with superb stability and stealth properties is identified.

Polymeric micelles have found a growing interest as gene vectors due to the serious safety concerns associated with viral vectors. In particular, the cationic polymer polyethylene imine (PEI) has shown relatively high condensation and transfection efficiencies. Additionally, polyethylene glycol (PEG) modification of polymeric gene vectors has dramatically improved their biological properties, including enhanced biocompatibility, prolonged circulation time, and increased bio-distribution. However, PEG grafting of PEI for subsequent condensation of nucleic acids (NAs) does not necessarily result in the formation of a PEI/NAs core with a PEG corona. But often times, the presence of PEG interferes with PEI's electrostatic interaction with NAs. We describe here a facile method to prepare multilayered biodegradable micelles which address some of the critical drawbacks associated with current PEI-based systems. The polyplex micelles have superb stability and stealth properties. Moreover, we describe a method to prepare fully biodegradable and biocompatible injectable hydrogels for use in localized gene therapy.

Polymeric micelles have found a growing interest as gene vectors due to the serious safety concerns associated with viral vectors. In particular, the cationic polymer polyethylene imine (PEI) has shown relatively high condensation and transfection efficiencies. Additionally, polyethylene glycol (PEG) modification of polymeric gene vectors has dramatically improved their biological properties, including enhanced biocompatibility, prolonged circulation time, and increased bio-distribution. However, PEG grafting of PEI for subsequent condensation of nucleic acids (NAs) does not necessarily result in the formation of a PEI/NAs core with a PEG corona. But often times, the presence of PEG interferes with PEI's electrostatic interaction with NAs. We describe here a facile method to prepare multilayered biodegradable micelles which address some of the critical drawbacks associated with current PEI-based systems. The polyplex micelles have superb stability and stealth properties. Moreover, we describe a method to prepare fully biodegradable and biocompatible injectable hydrogels for use in localized gene therapy.

"Three-layered micelles" (3LM) composed of two triblock copolymers, poly(L-lactide)-b-polyethyleneimine-b-poly(L-lactide) (PLLA-PEI-PLLA) and poly(L-lactide)-b-poly(ethylene glycol)-b-poly(L-lactide) (PLLA-PEG-PLLA) are designed to combine electrostatic interaction and solvent-induced condensation of DNA. The low molecular weight PLLA-PEI-PLLA is synthesized by a facile amine-protection/deprotection approach and employed as a gene vector, compacting DNA as a polyplex core in the organo-micelles. The individual organo-micelle is further encapsulated within a PLLA-PEG-PLLA amphiphilic micelle leading to an aqueous stable colloidal dispersion. The resulting spherical 3LM possess a hydrodynamic diameter of ca. 200 nm and zeta potential close to neutral and display excellent stability to competing polyanions such as dextran sulfate in neutral pH (7.4). Such high stability is attributed to the complete shielding of the PEI/DNA polyplex core with an impermeable hydrophobic intermediate layer. However, greater than 90% of the encapsulated DNA are released within 30 min when exposed to slightly acidic pH (4.5). Based on our findings, a new class of non-viral delivery system for nucleic acids with superb stability and stealth properties is identified.