5-Amino-1-pentanol

One of the most significant challenges in the development of polymer materials for gene delivery is to understand how topological structure influences their transfection properties. Poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (C32) has proven to be the top-performing gene delivery vector developed to date. Here, we report the development of branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (HC32) as a novel gene vector and elucidate how the topological structure affects gene delivery properties. We found that the branched structure has a big impact on gene transfection efficiency resulting in a superior transfection efficiency of HC32 in comparison to C32 with a linear structure. Mechanistic investigations illustrated that the branched structure enhanced DNA binding, leading to the formation of toroidal polyplexes with smaller size and higher cationic charge. Importantly, the branched structure offers HC32 a larger chemical space for terminal functionalization (e.g., guanidinylation) to further enhance the transfection. Moreover, the optimized HC32 is capable of transfecting a diverse range of cell types including cells that are known to be difficult to transfect such as stem cells and astrocytes with high efficiency. Our study provides a new insight into the rational design of poly(β-amino ester) (PAE) based polymers for gene delivery.

Gene vectors for oral delivery encounter harsh conditions throughout the gastrointestinal tract, and the continuous peristaltic activity can quickly remove the vectors, leading to inefficient intestinal permeation. Therefore, vectors have demanding property requirements, such as stability under various pH and, more importantly, efficient uptake in different intestinal segments. In this study, a functional polymer, cholesterol-grafted poly(β-amino ester) (poly[hexamethylene diacrylate-β-(5-amino-1-pentanol)] (CH-PHP)), is synthesized and electrostatically interacted with plasmid DNA to form a CH-PHP/DNA complex (CPNC). This complex is designed to target the Niemann-Pick C1-like receptor, a cholesterol receptor, to improve oral gene delivery efficacy. With the presence of cholesterol, CH-PHP shows mitigated cytotoxicity, enhanced enzyme resistance, and improved gene condensing ability. CPNC further contributes to ≈43.1- and 2.3-fold increases in luciferase expression in Caco-2 cells compared with PNC and Lipo 2000/DNA complexes, respectively. In addition, the in vivo transfection efficacy of CPNC is ≈4.1-, 2.1-, and 1.6-fold higher than that of Lipo 2000/DNA complexes in rat duodenum, jejunum, and ileum, respectively. Therefore, CPNC may be a promising delivery vector for gene delivery, and using a cholesterol-specific endocytic pathway can be a novel approach to achieve efficient oral gene transfection.

The top-performing linear poly(β-amino ester) (LPAE), poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (C32), has demonstrated gene transfection efficiency comparable to viral-mediated gene delivery. Herein, we report the synthesis of a series of highly branched poly(5-amino-1-pentanol-co-1,4-butanediol diacrylate) (HC32) and explore how the branching structure influences the performance of C32 in gene transfection. HC32 were synthesized by an "A2 + B3 + C2" Michal addition strategy. Gaussia luciferase (Gluciferase) and green fluorescent protein (GFP) coding plasmid DNA were used as reporter genes and the gene transfection efficiency was evaluated in human cervical cancer cell line (HeLa) and human recessive dystrophic epidermolysis bullosa keratinocyte (RDEBK) cells. We found that the optimal branching structure led to a much higher gene transfection efficiency in comparison to its linear counterpart and commercial reagents, while preserving high cell viability in both cell types. The branching strategy affected DNA binding, proton buffering capacity and degradation of polymers as well as size, zeta potential, stability, and DNA release rate of polyplexes significantly. Polymer degradation and DNA release rate played pivotal parts in achieving the high gene transfection efficiency of HC32-103 polymers, providing new insights for the development of poly(β-amino ester)s-based gene delivery vectors.