Optimization of Liganded Polyethylenimine Polyethylene Glycol Vector for Nucleic Acid Delivery

Abstract

The delivery of nucleic acids into cells is an attractive approach for cancer therapy. Polyethylenimine (PEI) is among the most efficient nonviral carriers. Recent studies have demonstrated that PEI can be conjugated to targeting ligands, such as epidermal growth factor (EGF) and transferrin (Schaffert et al., 2011; Abourbeh et al., 2012; Ogris et al., 1999). Herein we present a simplified protocol for producing homogeneous preparations of PEGylated linear PEI: LPEI-PEG2k. We generated two well-characterized copolymers, with ratios of LPEI to PEG of 1:1 and 1:3. These copolymers were further conjugated through disulfide bonds to a Her-2 targeting moiety, Her-2 affibody. This reaction yielded two triconjugates that target Her-2 overexpressing tumors. Polyplexes were formed by complexing plasmid DNA with the triconjugates. We characterized the biophysical properties of the conjugates, and found that the triconjugate 1:3 polyplex had lower ζ potential, larger particle size, and more heterogeneous shape than the triconjugate 1:1 polyplex. Triconjugate 1:1 and triconjugate 1:3 polyplexes were highly selective toward cells that overexpress Her-2 receptors, but triconjugate 1:1 polyplex was more efficient at gene delivery. Our studies show that the biophysical and biological properties of the conjugates can be profoundly affected by the ratio of LPEI:PEG2k:ligand. The procedure described here can be adapted to generate a variety of triconjugates, simply by changing the targeting moiety.

HER2-Targeted Polyinosine/Polycytosine Therapy Inhibits Tumor Growth and Modulates the Tumor Immune Microenvironment

“…PPHAffibody and PPCys were generated as described (24). Throughout the text, the concentration of "pIC/PPHAffibody" or "pIC/PPCys" refers to the concentration of pIC in the complex.…”

“…The N:P ratio (amine:phosphate) was 6 for in vitro studies and 6.6 for in vivo studies. pIC/PPHAffibody complexes were 206 nm as measured using a Nano-ZS Zetasizer (24).…”

“…Because the HER2 receptor lacks a natural ligand that can be used for targeting, we conjugated the nonviral vector, PP (24), to a HER2 affibody (25) to generate PPHAffibody (24). Affibody molecules are small, soluble, robust affinity ligands that were developed from the B domain of staphylococcal protein A (26).…”

“…The HER2 affibody ZHER2:2891 binds to HER2 with high affinity and is internalized by HER2-expressing cells (25,28). The chemical and physicochemical properties of the HER2-targeting vector used in this study have been described (24).…”

Recent progress in gene therapy to deliver nucleic acids with multivalent cationic vectors

“…3 shows a tipical plot of size vs the charge ratio of the nano-aggregate formed by the (C 12 Am) 2 C 12 -siRNA lipoplex [114]. This procedure has been followed to get the size for: (a) lipoplexes formed by pDNA with GCLs [64,66,111,115,137] or MVCLs [138,139]; (b) polyplexes formed by pDNA with cationic polymers [95,98,[124][125][126]140] or polycations [134,141,142]; (c) lipoplexes formed by siRNA with GCLs [114] or MVCLs [139,143]; and (d) polyplexes formed by siRNA with cationic polymers [90,105,144] or polycations [145]. Additionally, SLS and DLS methods also provide an estimation of the electroneutrality ratio of the nanoaggregates because, at this point (see Fig.…”