Polycations are one of the most frequently used classes of materials for non-viral gene transfer in vivo. Several studies have demonstrated a sensitive relationship between polymer structure and delivery activity. In this work, we used reverse addition-fragmentation chain transfer (RAFT) polymerization to build a panel of N-(2-hydroxypropyl(methacrylamide) (HPMA)-oligolysine copolymers with varying peptide length and polymer molecular weight. The panel was screened for optimal DNA-binding, colloidal stability in salt, high transfection efficiency, and low cytotoxicity. Increasing polyplex stability in PBS correlated with increasing polymer molecular weight and decreasing peptide length. Copolymers containing K5 and K10 oligocations transfected cultured cells with significantly higher efficiencies than copolymers of K15. Four HPMA-oligolysine copolymers were identified that met desired criteria. Polyplexes formed with these copolymers demonstrated both salt stability and transfection efficiencies on-par with poly(ethylenimine) PEI in cultured cells.

CONSPECTUS Nucleic acid-based therapeutics can potentially address otherwise untreatable genetic disorders and have significant potential for a wide range of diseases. Therapeutic gene delivery can restore protein function by replacing defunct genes to restore cellular health while RNA interference (RNAi) can mask mutated and harmful genes. Cationic polymers have been extensively studied for nucleic acid delivery applications due to their self-assembly with nucleic acids into virus-sized nanoparticles and high transfection efficiency in vitro, but toxicity and particle stability have limited their clinical applications. The advent of controlled radical polymerization has improved the quality, control and reproducibility of synthesized materials. Controlled radical polymerization yields well-defined, narrowly disperse materials of designable architectures and molecular weight, allowing study of the effects of polymer architecture and molecular weight on transfection efficiency and cytotoxicity for improved design of next-generation vectors. Robust methods such as atom transfer radical polymerization (ATRP), reverse addition-fragmentation chain transfer polymerization (RAFT), and ring-opening metastasis polymerization (ROMP) have been used to engineer materials that specifically enhance extracellular stability, cellular specificity, and decrease toxicity. This Account reviews findings from structure-function studies that have elucidated key design motifs necessary for the development of effective nucleic acid vectors. In addition, polymers that are biodegradable, form supramolecular structures, target specific cells, or facilitate endosomal release are also discussed. Finally, promising materials with in vivo applications ranging from pulmonary gene delivery to DNA vaccines are described.

Non-viral gene delivery systems capable of transfecting cells in the brain are critical in realizing the potential impact of nucleic acid therapeutics for diseases of the central nervous system. In this study, the membrane-lytic peptide melittin was incorporated into block copolymers synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. The first block, designed for melittin conjugation, was composed of N-(2-hydroxypropyl)methacrylamide (HPMA) and pyridyl disulfide methacrylamide (PDSMA) and the second block, designed for DNA binding, was composed of oligo-L-lysine (K10) and HPMA. Melittin modified with cysteine at the C-terminus was conjugated to the polymers through the pyridyl disulfide pendant groups via disulfide exchange. The resulting pHgMelbHK10 copolymers are more membrane-lytic than melittin-free control polymers, and efficiently condensed plasmid DNA into salt-stable particles (~ 100–200 nm). The melittin-modified polymers transfected both HeLa and neuron-like PC-12 cells more efficiently than melittin-free polymers although toxicity associated with the melittin peptide was observed. Optimized formulations containing the luciferase reporter gene were delivered to mouse brain by intraventricular brain injections. Melittin-containing polyplexes produced about 35-fold higher luciferase activity in the brain compared to polyplexes without melittin. Thus, the melittin-containing block copolymers described in this work are promising materials for gene delivery to the brain.

One of the major intracellular barriers to non-viral gene delivery is efficient endosomal escape. The incorporation of histidine residues into polymeric constructs has been found to increase endosomal escape via the proton sponge effect. Statistical and diblock copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA), oligolysine, and oligohistidine were synthesized via reversible-addition fragmentation chain transfer (RAFT) polymerization, and tested for in vitro transfection efficiency, buffering ability, and polyplex uptake mechanism via the use of chemical endocytic inhibitors. Interestingly, histidine-containing statistical and diblock polymers exhibited increased buffer capacity in different endosomal pH ranges. Statistical copolymers transfected better than block copolymers that contained similar amounts of histidine. In addition, only the polymer containing the highest incorporation of oligohistidine residues led to increases in transfection efficiency over the HPMA-oligolysine base polymer. Thus, for these polymer architectures, high histidine incorporation may be required for efficient endosomal escape. Furthermore, uptake studies indicate that non-acidified caveolae-mediated endocytosis may be the primary route of transfection for these copolymers, suggesting that alternative approaches for increasing endosomal escape may be beneficial for enhancing transfection efficiency with these HPMA-oligolysine copolymers.

Successful adaptation of in vitro optimized polymeric gene delivery systems for in vivo use remains a significant challenge. Most in vivo applications require particles that are sterically stabilized but doing so significantly compromises transfection efficiency of materials shown to be effective in vitro. In this communication, we present a multi-functional well-defined block copolymer that forms particles with the following properties: cell targeting, reversible shielding, endosomal release, and DNA condensation. We show that targeted and stabilized particles retain transfection efficiencies comparable to the non-stabilized formulations. The block copolymers are synthesized using a novel, double-head agent (CPADB-SS-iBuBr) that combines a RAFT CTA and an ATRP initiator through a disulfide linkage. Using this double-head agent, a well-defined cationic block copolymer P(OEGMA)15-SS-P(GMA-TEPA)50 containing a hydrophilic oligoethyleneglycol (OEG) block and a tetraethylenepentamine (TEPA)-grafted polycation block was synthesized. This material effectively condenses plasmid DNA into salt-stable particles that deshield under intracellular reducing conditions. In vitro transfection studies showed that the reversibly shielded polyplexes afforded up 10-fold higher transfection efficiencies compared to the analogous stably-shielded polymer in four different mammalian cell lines. To compensate for reduced cell uptake caused by the hydrophilic particle shell, a neuron-targeting peptide was further conjugated to the terminus of theP(OEGMA) block. Transfection of neuron-like, differentiated PC-12 cells demonstrated that combining both targeting and deshielding in stabilized particles yields formulations that are suitable for in vivo delivery without compromising in vitro transfection efficiency. These materials are therefore promising carriers for in vivo gene delivery applications.

Nanoparticle morphology has been shown to affect cellular uptake, but there are few studies investigating the impact of particle shape on biologic drug delivery. Recently, our group synthesized a series of N-(2-hydroxypropyl) methacrylamide (HPMA)-oligolysine brush polymers for nucleic acid delivery that varied in oligolysine peptide length and polymer molecular weight. Interestingly, a 50% longer peptide (K15) transfected very poorly compared to the optimized polymer comprised of K10 peptide despite similar chemical composition and molecular weight. We hypothesized that differences in particle morphology contributed to the differences in plasmid DNA delivery. We found that particles formed with plasmid DNA and a polymer with the longer oligolysine peptide (pHK15) had larger aspect ratios than particles formed with optimized polymer (pHK10). Even though both formulations showed similar percentages of cellular association, particles of a higher aspect ratio were internalized to a lesser extent. Furthermore, the rod-like particles accumulated more in endosomal/lysosomal compartments, leading to delayed nuclear delivery. Other parameters, such as particle surface charge, unpackaging ability, uptake mechanism, intracellular trafficking, nor the presence of heparan sulfate proteoglycans did not significantly differ between the two polymer formulations. These results indicate that, for this system, polyplex morphology primarily impacts nucleic acid delivery efficiency through differences in cellular internalization rates.

The tumor microenvironment in the majority of cancers is known to favor polarization of tumor-associated macrophages (TAMs) to alternatively activated M2 phenotype, promoting disease progression and reducing patient survival. Effective therapy targeting this M2 macrophage population is thus a promising adjuvant to approved cancer therapies. One of the challenges in targeting M2-like TAMs is a lack of high affinity targeting ligand with good selectivity over anti-tumor M1-like TAMs. We have previously identified an M2 macrophage-targeting peptide (M2pep) that binds preferentially to murine M2 macrophages and M2-like TAMs. A fusion peptide of M2pep with pro-apoptotic peptide KLA (M2pepKLA) was further used to reduce TAM population in vivo but high concentrations and frequent dosing were required due to low binding affinity of M2pep for M2 macrophage. The goal of this study was to develop more potent TAM depletion constructs by increasing the valency of both the M2pep targeting and KLA drug domains. Divalent and tetravalent displays of M2pep ([M2pep]2-Biotin and [M2pep]4-Biotin) were synthesized and evaluated for improvement in binding avidity to the murine macrophages. High avidity and selective binding of [M2pep]2-Biotin to M2 macrophages was achieved with at least 10-fold lower concentration than required for monovalent M2pep activity. Increasing M2pep valency to four, however, resulted in a reduction in both binding activity and selectivity. Surprisingly, both divalent and tetravalent M2pep, without conjugation of any cytotoxic drug cargo, exhibited M2 macrophage-selective toxicity not observed in monovalent M2pep treatment. We next synthesized divalent M2pep with monovalent and divalent KLA ([M2pep]2-[KLA] and [M2pep]2-[KLA]2) to evaluate its enhanced potency compared to M2pepKLA. While both constructs were significantly more toxic than M2pepKLA to primary, bone marrow-derived M2 macrophage, desired selectivity was retained only with [M2pep]2-[KLA]. Finally, we evaluated all multivalent M2pep and M2pepKLA analogs using a syngeneic CT-26 tumor cell suspension. In this setting, [M2pep]4-Biotin and [M2pep]2-[KLA]2 exhibited selective toxicity to both M2-like TAMs and malignant cells but not to M1-like TAMs. Therefore, these constructs are promising anti-cancer constructs with dual-modality mechanisms: malignant cell killing and TAM-based immunomodulation.

Biodegradability can be incorporated into cationic polymers via use of disulfide linkages that are degraded in the reducing environment of the cell cytosol. In this work, N-(2-hydroxypropyl)methacrylamide (HPMA) and methacrylamido-functionalized oligo-L-lysine peptide monomers with either a non-reducible 6-aminohexanoic acid (AHX) linker or a reducible 3-[(2-aminoethyl)dithiol]propionic acid (AEDP) linker were copolymerized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Both of the copolymers and a 1:1 (w/w) mixture of copolymers with reducible and non-reducible peptides were complexed with DNA to form polyplexes. The polyplexes were tested for salt stability, transfection efficiency, and cytotoxicity. The HPMA-oligolysine copolymer containing the reducible AEDP linkers was less efficient at transfection than the non-reducible polymer and was prone to flocculation in saline and serum-containing conditions, but was also not cytotoxic at charge ratios tested. Optimal transfection efficiency and toxicity was attained with mixed formulation of copolymers. Flow cytometry uptake studies indicated that blocking extracellular thiols did not restore transfection efficiency and that the decreased transfection of the reducible polyplex is therefore not primarily caused by extracellular polymer reduction by free thiols. The decrease in transfection efficiency of the reducible polymers could be partially mitigated by the addition of low concentrations of EDTA to prevent metal-catalyzed oxidation of reduced polymers.