Adam E. Smith

Membrane and Nuclear Permeabilization by Polymeric pDNA Vehicles: Efficient Method for Gene Delivery or Mechanism of Cytotoxicity?

The aim of this study is to compare the cytotoxicity mechanisms of linear PEI to two analogous polymers synthesized by our group: a hydroxyl-containing poly(l-tartaramidoamine) (T4) and a version containing an alkyl chain spacer poly(adipamidopentaethylenetetramine) (A4) by studying the cellular responses to polymer transfection. We have also synthesized analogues of T4 with different molecular weights (degrees of polymerization of 6, 12, and 43) to examine the role of molecular weight on the cytotoxicity mechanisms. Several mechanisms of polymer-induced cytotoxicity are investigated, including plasma membrane permeabilization, the formation of potentially harmful polymer degradation products during transfection including reactive oxygen species, and nuclear membrane permeabilization. We hypothesized that since cationic polymers are capable of disrupting the plasma membrane, they may also be capable of disrupting the nuclear envelope, which could be a potential mechanism of how the pDNA is delivered into the nucleus (other than nuclear envelope breakdown during mitosis). Using flow cytometry and confocal microscopy, we show that the polycations with the highest amount of protein expression and toxicity, PEI and T4(43), are capable of inducing nuclear membrane permeability. This finding is important for the field of nucleic acid delivery in that direct nucleus permeabilization could be not only a mechanism for pDNA nuclear import but also a potential mechanism of cytotoxicity and cell death. We also show that the production of reactive oxygen species is not a main mechanism of cytotoxicity, and that the presence or absence of hydroxyl groups and polymer length play a role in polyplex size and charge in addition to protein expression efficiency and toxicity.

Diblock glycopolymers promote colloidal stability of polyplexes and effective pDNA and siRNA delivery under physiological salt and serum conditions

A series of glycopolymers composed of 2-deoxy-2-methacrylamido glucopyranose (MAG) and the primary amine-containing N-(2-aminoethyl) methacrylamide (AEMA) were synthesized via aqueous reversible addition-fragmentation chain transfer (RAFT) polymerization. The colloidal stability of the polyplexes formed with three diblock glycopolymers and pDNA was assessed using dynamic light scattering, and the polyplexes were found to be stable against aggregation in the presence of salt and serum over the 4 h time period studied. Delivery experiments were performed in vitro to examine the cellular uptake, transfection efficiency, and cytotoxicity of the glycopolymer/pDNA polyplexes in cultured HeLa cells and the diblock copolymer with the shortest AEMA block was found to be the most effective. Additionally, the ability of the diblock glycopolymers to deliver siRNA to U-87 (glioblastoma) cells was screened, and the diblock copolymer with the longest AEMA block was found to have gene knockdown efficacy similar to Lipofectamine 2000.

Phosphonium-containing diblock copolymers for enhanced colloidal stability and efficient nucleic acid delivery.

RAFT polymerization successfully controlled the synthesis of phosphonium-based AB diblock copolymers for nonviral gene delivery. A stabilizing block of either oligo(ethylene glycol(9)) methyl ether methacrylate or 2-(methacryloxy)ethyl phosphorylcholine provided colloidal stability, and the phosphonium-containing cationic block of 4-vinylbenzyltributylphosphonium chloride induced electrostatic nucleic acid complexation. RAFT polymerization generated well-defined stabilizing blocks (M(n) = 25000 g/mol) and subsequent chain extension synthesized diblock copolymers with DPs of 25, 50, and 75 for the phosphonium-containing block. All diblock copolymers bound DNA efficiently at ± ratios of 1.0 in H(2)O, and polyplexes generated at ± ratios of 2.0 displayed hydrodynamic diameters between 100 and 200 nm. The resulting polyplexes exhibited excellent colloidal stability under physiological salt or serum conditions, and they maintained constant hydrodynamic diameters over 24 h. Cellular uptake studies using Cy5-labeled DNA confirmed reduced cellular uptake in COS-7 and HeLa cells and, consequently, resulted in low transfection in these cell lines. Serum transfection in HepaRG cells, which are a predictive cell line for in vivo transfection studies, showed successful transfection using all diblock copolymers with luciferase expression on the same order of magnitude as Jet-PEI. All diblock copolymers exhibited low cytotoxicity (>80% cell viability). Promising in vitro transfection and cytotoxicity results suggest future studies involving the in vivo applicability of these phosphonium-based diblock copolymer delivery vehicles.

Tailored Design of Au Nanoparticle-siRNA Carriers Utilizing Reversible Addition-Fragmentation Chain Transfer Polymers

The facile synthesis of polymer-stabilized Au nanoparticles (AuNPs) capable of forming neutral, sterically stable complexes with small interfering RNA (siRNA) is reported. The amine-containing cationic block of poly(N-2-hydroxypropyl methacrylamide(70)-block-N-[3-(dimethylamino)propyl] methacrylamide(24)) [P(HPMA(70)-b-DMAPMA(24))] was utilized to promote the in situ reduction of Au(3+) to AuNPs and subsequently bind small interfering RNA, while the nonimmunogenic, hydrophilic block provided steric stabilization. The ratio of [DMAPMA](0)/[Au(3+)](0) utilized in the reduction reaction was found to be critical to the production of polymer-stabilized AuNPs capable of complexing siRNA. Significant protection ( approximately 100 times) against nucleases was demonstrated by enzymatic tests, while gene down-regulation experiments indicated successful delivery of siRNA to cancerous cells.

Facile 'One-Pot' Preparation of Reversible, Disulfide-Containing Shell Cross-Linked Micelles from a RAFT-Synthesized, pH-Responsive Triblock Copolymer in Water at Room Temperature

A pH-responsive triblock copolymer, α-methoxy poly(ethylene oxide)-b-poly(N-(3-aminopropyl) methacrylamide)-β-poly(2-(diisopropylamino)ethyl methacrylate) (mPEO-PAPMA-PDPAEMA), was synthesized via aqueous RAFT polymerization. This triblock copolymer dissolves in aqueous solution at low pH (<5.0) due to protonation of primary amine residues on the PAPMA block and tertiary amine residues on the PDPAEMA block. Above pH 6.0, the copolymer unimers self-assemble into micelles consisting of PDPAEMA cores, PAPMA shells, and mPEO coronas. Dynamic light scattering studies indicated a hydrodynamic diameter of 92 nm at pH 9.0. A bifunctional, reversible cross-linker, dimethyl 3,3′-dithiobispropionimidate (DTBP), was used to cross-link the micelles. The ‘one-pot’ formation of shell cross-linked (SCL) micelles was accomplished at room temperature in water by mixing the triblock copolymers and DTBP at pH 3.0, and slowly increasing the solution pH to 9.0 leading to the simultaneous formation of micelles and cross-linking. These SCL micelles are readily cleaved by the addition of the reducing agent, dithiothreitol, and can be re-cross-linked simply by exposure to air. Such SCL micelles have potential as nanocarriers for controlled release of therapeutic and diagnostic agents because the in situ cleavage of the disulfide linkages would not only allow release of bioactive agents, but also permit renal clearance of the resulting unimeric components.

Glucose-Containing Diblock Polycations Exhibit Molecular Weight, Charge, and Cell-Type Dependence for pDNA Delivery

A series of diblock glycopolycations were created by polymerizing 2-deoxy-2-methacrylamido glucopyranose (MAG) with either a tertiary amine-containing monomer, N-[3-(N,N-dimethylamino) propyl] methacrylamide (DMAPMA), or a primary amine-containing unit, N-(2-aminoethyl) methacrylamide (AEMA). Seven structures were synthesized via aqueous reversible addition–fragmentation chain transfer (RAFT) polymerization that varied in the block lengths of MAG, DMAPMA, and AEMA along with two homopolymer controls of DMAPMA and AEMA that lacked a MAG block. The polymers were all able to complex plasmid DNA into polyplex structures and to prevent colloidal aggregation of polyplexes in physiological salt conditions. In vitro transfection experiments were performed in both HeLa (human cervix adenocarcinoma) cells and HepG2 (human liver hepatocellular carcinoma) cells to examine the role of charge type, block length, and cell type on transfection efficiency and toxicity. The glycopolycation vehicles with primary amine blocks and PAEMA homopolymers revealed much higher transfection efficiency and lower toxicity when compared to analogs created with DMAPMA. Block length was also shown to influence cellular delivery and toxicity; as the block length of DMAPMA increased in the glycopolycation-based polyplexes, toxicity increased while transfection decreased. While the charge block played a major role in delivery, the MAG block length did not affect these cellular parameters. Lastly, cell type played a major role in efficiency. These glycopolymers revealed higher cellular uptake and transfection efficiency in HepG2 cells than in HeLa cells, while homopolycations (PAEMA and PDMAPMA) lacking the MAG blocks exhibited the opposite trend, signifying that the MAG block could aid in hepatocyte transfection.

Reversible gold “locked” synthetic vesicles derived from stimuli-responsive diblock copolymers

Polymersomes derived from a RAFT-generated, thermally responsive diblock copolymer, P(DMAEMA165-b-NIPAM435), were shell cross-linked by in situ gold nanoparticle formation. The cross-linking was subsequently reversed by the addition of the thiols capable of inducing a ligand exchange on the surface of the gold nanoparticle.

Synthesis and characterization of 4-vinylimidazole ABA triblock copolymers utilizing a difunctional RAFT chain transfer agent

Reversible addition–fragmentation chain transfer (RAFT) polymerization strategies enabled the unprecedented synthesis of 4-vinylimidazole (4VIM)-containing ABA triblock copolymers in glacial acetic acid. The synthesis of a novel, difunctional trithiocarbonate RAFT chain transfer agent (CTA) controlled the divergent RAFT polymerization of methacrylic and 4VIM monomers with controlled molecular weights and narrow polydispersity indices (PDIs). The triblock copolymers consisted of a low-Tg di(ethylene glycol) methyl ether methacrylate (DEGMEMA) center block (Mn = 26 000 g mol−1) and an amphoteric 4VIM external, mechanically reinforcing block (Mn = 6500–16 500 g mol−1). Varying the 4VIM content probed the influence of the triblock copolymer composition on the macromolecular thermomechanical and morphological properties. Dynamic mechanical analysis (DMA) of the triblock copolymers exhibited a rubbery plateau region over a wide temperature range (∼200 °C), which confirmed the establishment of microphase-separated morphologies with flow temperatures above 200 °C. Transmission electron microscopy (TEM), atomic force microscopy (AFM), and small-angle X-ray scattering (SAXS) collectively probed the solid state morphologies of the triblock copolymers; all techniques revealed phase separation at nanoscale dimensions. The triblock copolymers with 40 wt% 4VIM formed lamellar morphologies. Well-defined, amphoteric, 4VIM ABA triblock copolymers (PDIs < 1.10) with microphase-separated morphologies now permit imidazole-containing macromolecules of controlled architectures for emerging applications.

The antiproliferative cytostatic effects of a self-activating viridin prodrug

Although viridins like wortmannin (Wm) have long been examined as anticancer agents, their ability to self-activate has only recently been recognized. Here, we describe the cytostatic effects of a self-activating viridin (SAV), which is an inactive, polymeric prodrug. SAV self-activates to generate a bioactive, fluorescent viridin NBD-Wm with a half-time of 9.2 hours. With cultured A549 cells, 10 μmol/L SAV caused growth arrest without inducing apoptosis or cell death, a cytostatic action markedly different from other chemotherapeutic agents (vinblastine, camptothecin, and paclitaxel). In vivo, a SAV dosing of 1 mg/kg once in 48 hours (i.p.) resulted in growth arrest of an A549 tumor xenograft, with growth resuming when dosing ceased. With a peak serum concentration of SAV of 2.36 μmol/L (at 2 hours post i.p. injection), the concentration of bioactive NBD-Wm was 41 nmol/L based on the partial inhibition of neutrophil respiratory burst. Therefore, SAV was present as an inactive prodrug in serum (peak = 2.36 μmol/L), which generated low concentrations of active viridin (41 nmol/L). SAV is a prodrug, the slow release and cytostatic activities of which suggest that it might be useful as a component of metronomic-based chemotherapeutic strategies. [Mol Cancer Ther 2009;8(6):1666–75]

Lipophilic Polycation Vehicles Display High Plasmid DNA Delivery to Multiple Cell Types

A class of cationic poly(alkylamidoamine)s (PAAAs) containing lipophilic methylene linkers were designed and examined as in vitro plasmid DNA (pDNA) delivery agents. The PAAAs were synthesized via step-growth polymerization between a diamine monomer and each of four different diacid chloride monomers with varying methylene linker lengths, including glutaryl chloride, adipoyl chloride, pimeloyl chloride, and suberoyl chloride, which served to systematically increase the lipophilicity of the polymers. The synthesized polymers successfully complexed with pDNA in reduced serum medium at N/P ratios of 5 and greater, resulting in polyplexes with hydrodynamic diameters of approximately 1 μm. These polyplexes were tested for in vitro transgene expression and cytotoxicity using HDFa (human dermal fibroblast), HeLa (human cervical carcinoma), HMEC (human mammary epithelial), and HUVEC (human umbilical vein endothelial) cells. Interestingly, select PAAA polyplex formulations were found to be more effective than Lipofectamine 2000 at promoting transgene expression (GFP) while maintaining comparable or higher cell viability. Transgene expression was highest in HeLa cells (∼90% for most formulations) and lowest in HDFa cells (up to ∼20%) as measured by GFP fluorescence. In addition, the cytotoxicity of PAAA polyplex formulations was significantly increased as the molecular weight, N/P ratio, and methylene linker length were increased. The PAAA vehicles developed herein provide a new delivery vehicle design strategy of displaying attributes of both polycations and lipids, which show promise as a tunable scaffold for refining the structure-activity-toxicity profiles for future genome editing studies.