We investigated a series of small-sized polyethylenimine (PEI, 0.8/1.2 k)-conjugated pluronic copolymers (PCMs) for their potential to enhance delivery of an antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in dystrophic mdx mice. PCM polymers containing pluronics of molecular weight (Mw) ranging 2-6 k, with hydrophilic-lipophilic balance (HLB) 7-23, significantly enhanced PMO-induced exon-skipping in a green fluorescent protein (GFP) reporter-based myoblast culture system. Application of optimized formulations of PCMs with PMO targeted to dystrophin exon 23 demonstrated a significant increase in exon-skipping efficiency in dystrophic mdx mice. Consistent with our observations in vitro, optimization of molecular size and the HLB of pluronics are important factors for PCMs to achieve enhanced PMO delivery in vivo. Observed cytotoxicity of the PCMs was lower than Endo-porter and PEI 25 k. Tissue toxicity of PCMs in muscle was not clearly detected with the concentrations used, indicating the potential of the PCMs as effective and safe PMO carriers for treating diseases such as muscular dystrophy.

Hyperbranched poly(ester amine)s (PEAs) based on tris[2-(acryloyloxy)ethyl]isocyanurate (TAEI) cross-linked low-molecular-weight polyethylenimine (Mw: 0.8k/1.2k/2.0k) have been evaluated for delivering antisense phosphorodiamidate morpholino oligomer (PMO) in vitro and in vivo in the dystrophic mdx mouse. The results show that the PEAs constructed with polyethylenimine (PEI) 2.0k (C series) improved PMO delivery more efficiently than those constructed with PEI 0.8k (A series) or 1.2k (B series) in a GFP reporter-based C2C12 mouse myoblast culture system. The highest efficiency of exon-skipping in vitro with the PMO oligonucleotide targeting human dystrophin exon 50 was obtained when the PEA C12 [TAEI-PEI 2.0k (1:2)] was used. Nearly all of the PEAs improved dystrophin expression in mdx mice by local injection with a 2-4-fold increase when compared with PMO alone. Improved transfection efficiency and lower toxicity indicate the potential of the biodegradable PEA polymers as safe and efficient PMO delivery vectors for in vivo applications.

A series of low molecular weight polyethyleneimine (LPEI) conjugated Pluronic copolymers (PCMs) were synthesized and evaluated in C2C12 myoblasts and CHO cells in vitro and in dystrophic mdx mice in vivo as gene delivery carriers. Pluronics with different molecular weights (M w ) and hydrophiliclipophilic-balance (HLB), and two LPEIs (M w : 0.8k, 1.2k Da) as composition of PCMs have been synthesized, and the dynamics between the structures and properties were investigated. The conjugation efficiency of PEI ranged from 77.5-95.4% with relatively higher levels of PEI conjugation to the Pluronic size in PCMs, with the Pluronics of smaller size achieving relatively higher levels of PEI conjugation. Almost all of the PCM polymers were able to bind and condense plasmid DNA effectively into particles of approximately 200 nm in solution at the polymer/DNA ratio of 2 and above. The PCMs composed of relatively moderate size (M w : 2000-5000 Da), intermediate HLB (12-23) of Pluronics, and LPEI produce much higher gene delivery efficacy and less cytotoxicity as compared with PEI 25k in C2C12 myoblasts and CHO cells in vitro. The PCMs were also able to enhance gene delivery in mdx mice in vivo, indicating their potential as biocompatible gene delivery carriers.