Increased tumor distribution and expression of histidine‐rich plasmid polyplexes

Abstract

Background Selecting nonviral carriers for in vivo gene delivery is often dependent on determining the optimal carriers from transfection assays in vitro. The rationale behind this in vitro strategy is to cast a net sufficiently wide to identify the few effective carriers of plasmids for in vivo studies. Nevertheless, many effective in vivo carriers may be overlooked by this strategy because of the marked differences between in vitro and in vivo assays. Methods After solid-phase synthesis of linear and branched histidine/lysine (HK) peptides, the two peptide carriers were compared for their ability to transfect MDA-MB-435 tumor cells in vitro and then in vivo. Results By contrast to their transfection activity in vitro, the linear H2K carrier of plasmids was far more effective in vivo compared to the branch H2K4b. Surprisingly, negatively-charged polyplexes formed by the linear H2K peptide gave higher transfection in vivo than did those with a positive surface charge. To examine the distribution of plasmid expression within the tumor from H2K polyplexes, we found widespread expression by immunohistochemical staining. With a fluorescent tdTomato expressing-plasmid, we confirmed a pervasive distribution and gene expression within the tumor mediated by the H2K polyplex. Conclusions Although mechanisms underlying the efficiency of gene expression are probably multifactorial, unpacking of the H2K polyplex within the tumor appears to have a significant role. Further development of these H2K polyplexes represents an attractive approach for plasmid-based therapies of cancer.

NRP1 transport of cancer therapeutics mediated by tumor-penetrating peptides

“…Still, EPR and/or tumor interstitial pressure may have a role in tumor targeting and biodistribution of molecules mediated by TPP targeting of NRP1 (16, 44). For instance, in addition to targeting NRP1, EPR may provide a necessary component for the increased accumulation of polyplexes that did not contain a targeting ligand (15, 16). Further study of these physical factors will be of interest to determine if they have any effect on the distribution of TPP-mediated cargo within the tumor.…”

“…Interestingly, in contrast to the cyclic LyP-1, the truncated linear form does not bind p32, yet it still targets the lymph vessels of tumors (12). In addition to these TPP, there are other peptides with the CendR sequence which appear to have similar tumor penetrating properties (15, 16). …”

“…Although linear HK peptides are 20-mers and are not TPP, these two classes of peptides likely share transport systems that are mediated by NRP1. By injecting a NRP1 antibody prior to administering HK polyplexes (with luciferase-expressing plasmid), tumor transport of HK polyplexes was shown to be mediated by NRP1 with the marked reduction of luciferase expression (96%) in the tumor (15, 16). Although the linear HK (H2K) has multiple –KHHK-repeats, its C-terminal end has a –KHK-sequence with an amide cap, which consequently would prevent H2K from binding to NRP1.…”

“…In contrast to H2K, the branched peptide H2K4b, which also has a -KHHK-motif, does not transport plasmids effectively to tumors. In addition to differences in biodistribution, reduced enzymatic processing of H2K4b, which has a high affinity for DNA, may explain the inability of branched peptides to be effective carriers in vivo (15, 16). Moreover, since H2K polyplexes achieve tumor specificity without targeting ligands, multiple factors, including increased NRP1-expression and/or EPR, may be necessary for the specificity of polyplexes.…”

The neuropilin‐1 receptor mediates enhanced tumor delivery of H2K polyplexes

“…Because specificity toward tumors of non‐ligand, nonpegylated polyplexes was highly unusual , we considered whether unique mechanisms other than enhanced permeability and retention (EPR) may be involved in the tumor delivery of plasmids by HK peptides. Because peptides contain the ‐KXXK‐ sequence, where X is any amino acid are known to activate NRP‐mediated transport, we considered whether H2K (or H3K) with multiple ‐KHHK‐ repeats may similarly augment the NRP‐mediated pathway.…”