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

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, <i>N</i>-[3-(<i>N</i>,<i>N</i>-dimethylamino) propyl] methacrylamide (DMAPMA), or a primary amine-containing unit, <i>N</i>-(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

Poly(2-deoxy-2-methacrylamido glucopyranose)‑<i>b</i>‑Poly(methacrylate amine)s: Optimization of Diblock Glycopolycations for Nucleic Acid Delivery

A series of nine poly­(2-deoxy-2-methacrylamido glucopyranose)-<i>b</i>-poly­(methacrylate amine) diblock copolycations has been synthesized as new colloidally stable polynucleotide vehicles. The cationic block was varied in length and in the degree of methyl group substitution (secondary, tertiary, quaternary) on the pendant amine in an effort to optimize the structure and activity for plasmid DNA (pDNA) delivery. Upon a thorough kinetic study of polymerization for each polymer, the glycopolymers were prepared with well-controlled <i>M</i>_n and Đ. The binding and colloidal stability of the polymer–pDNA nanocomplexes at different N/P ratios and in biological media have been investigated using gel electrophoresis and light scattering techniques. The toxicity and transfection efficiency of the polyplexes have been evaluated with Hep G2 (human liver hepatocellular carcinoma) cells; several polymers displayed excellent delivery and toxicity profiles justifying their further development for in vivo gene therapy

Poly(trehalose): Sugar-Coated Nanocomplexes Promote Stabilization and Effective Polyplex-Mediated siRNA Delivery

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly­(methacrylamidotrehalose) or “poly­(trehalose)” and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (<i>n</i> = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P­(AEMA) and P­(AEMA)]. First, we demonstrate that the poly­(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly­(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly­(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly­(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC₅₀ of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly­(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications

Poly(trehalose): Sugar-Coated Nanocomplexes Promote Stabilization and Effective Polyplex-Mediated siRNA Delivery

When nanoparticles interact with their environment, the nature of that interaction is governed largely by the properties of its outermost surface layer. Here, we exploit the exceptional properties of a common disaccharide, trehalose, which is well-known for its unique biological stabilization effects. To this end, we have developed a synthetic procedure that readily affords a polymer of this disaccharide, poly­(methacrylamidotrehalose) or “poly­(trehalose)” and diblock copolycations containing this polymer with 51 repeat units chain extended with aminoethylmethacrylamide (AEMA) at three degrees of polymerization (<i>n</i> = 34, 65, and 84). Two series of experiments were conducted to study these diblock copolymers in detail and to compare their properties to two control polymers [PEG-P­(AEMA) and P­(AEMA)]. First, we demonstrate that the poly­(trehalose) coating ensures colloidal stability of polyplexes containing siRNA in the presence of high salt concentrations and serum proteins. Poly­(trehalose) retains the ability of trehalose to lower the phase transition energy associated with water freezing and can protect siRNA polyplexes during freeze-drying, allowing complete nanoparticle resuspension without loss of biological function. Second, we show that siRNA polyplexes coated with poly­(trehalose) have exceptional cellular internalization into glioblastoma cells that proceeds with zero-order kinetics. Moreover, the amount of siRNA delivered by poly­(trehalose) block copolycations can be controlled by the siRNA concentration in cell culture media. Using confocal microscopy we show that trehalose-coated polyplexes undergo active trafficking in cytoplasm upon internalization and significant siRNA-induced target gene down-regulation was achieved with an IC₅₀ of 19 nM. These findings coupled with a negligible cytotoxicity suggests that poly­(trehalose) has the potential to serve as an important component of therapeutic nanoparticle formulations of nucleic acids and has great promise to be extended as a new coating for other nanobased technologies and macromolecules, in particular, those related to nanomedicine applications

Trehalose-Based Block Copolycations Promote Polyplex Stabilization for Lyophilization and in Vivo pDNA Delivery

The development and thorough characterization of nonviral delivery agents for nucleic acid and genome editing therapies are of high interest to the field of nanomedicine. Indeed, this vehicle class offers the ability to tune chemical architecture/biological activity and readily package nucleic acids of various sizes and morphologies for a variety of applications. Herein, we present the synthesis and characterization of a class of trehalose-based block copolycations designed to stabilize polyplex formulations for lyophilization and in vivo administration. A 6-methacrylamido-6-deoxy trehalose (MAT) monomer was synthesized from trehalose and polymerized via reversible addition–fragmentation chain transfer (RAFT) polymerization to yield pMAT₄₃. The pMAT₄₃ macro-chain transfer agent was then chain-extended with aminoethylmethacrylamide (AEMA) to yield three different pMAT-<i>b</i>-AEMA cationic-block copolymers, pMAT-<i>b</i>-AEMA-1 (21 AEMA repeats), -2 (44 AEMA repeats), and -3 (57 AEMA repeats). These polymers along with a series of controls were used to form polyplexes with plasmids encoding firefly luciferase behind a strong ubiquitous promoter. The trehalose-coated polyplexes were characterized in detail and found to be resistant to colloidal aggregation in culture media containing salt and serum. The trehalose-polyplexes also retained colloidal stability and promoted high gene expression following lyophilization and reconstitution. Cytotoxicity, cellular uptake, and transfection ability were assessed in vitro using both human glioblastoma (U87) and human liver carcinoma (HepG2) cell lines wherein pMAT-<i>b</i>-AEMA-2 was found to have the optimal combination of high gene expression and low toxicity. pMAT-<i>b</i>-AEMA-2 polyplexes were evaluated in mice via slow tail vein infusion. The vehicle displayed minimal toxicity and discouraged nonspecific internalization in the liver, kidney, spleen, and lungs as determined by quantitative polymerase chain reaction (qPCR) and fluorescence imaging experiments. Hydrodynamic infusion of the polyplexes, however, led to very specific localization of the polyplexes to the mouse liver and promoted excellent gene expression in vivo

A Supramolecular Vaccine Platform Based on α‑Helical Peptide Nanofibers

A supramolecular peptide vaccine system was designed in which epitope-bearing peptides self-assemble into elongated nanofibers composed almost entirely of α-helical structure. The nanofibers were readily internalized by antigen presenting cells and produced robust antibody, CD4+ T-cell, and CD8+ T-cell responses without supplemental adjuvants in mice. Epitopes studied included a cancer B-cell epitope from the epidermal growth factor receptor class III variant (EGFRvIII), the universal CD4+ T-cell epitope PADRE, and the model CD8+ T-cell epitope SIINFEKL, each of which could be incorporated into supramolecular multiepitope nanofibers in a modular fashion