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

Abstract

Background Promising plasmid-based treatments have limited value without an effective delivery system. Recently, the linear H2K with a repeating –KHHK pattern was determined to be an effective plasmid carrier to tumor xenografts in vivo. Although unpacking of the H2K polyplex within the tumor may have a role, the mechanism for the enhanced efficacy remains unclear. Methods After solid-phase synthesis of linear and branched histidine-lysine (HK) peptide carriers of plasmids, the peptides were compared for their ability to lyse endosomes with a red blood cell model and to transfect MDA-MB-435 xenografts in the presence or absence of neuropilin-1 receptor (NRP-1) antibodies. To examine stability, polyplexes were incubated with trypsin or NaCl and then analyzed by electrophoresis. Results After screening peptides with a model for endosomal lysis at 2 pHs, the 33-mer H3K peptide lysed red blood cells effectively at the lower pH. Combining H3K and H2K peptides as carriers of plasmids expressing luciferase were more effective than H2K alone. Based on the repeating –KHHK- sequences of H2K, we studied whether the widespread gene expression in the tumor may be mediated by NRP-1. By blocking NRP-1 in tumor-bearing mice, luciferase activity in tumors delivered by HK polyplexes was reduced by 96% whereas activity in normal tissues was minimally reduced. Conclusions Combining an endosomolytic peptide, H3K with H2K polyplexes as a carrier further enhanced transfection in vivo. Moreover, the widespread distribution of H2K polyplexes is mediated by NRP-1, suggesting that transcytosis of these polyplexes through the tumor endothelium may lead to efficient transfection.

NRP1 transport of cancer therapeutics mediated by tumor-penetrating peptides

“…Under these conditions, however, it is also possible that excess NPs may enter by a less preferred endocytic pathway. Although recent studies with TPP-labeled silver particles or proteins showed that these enter only by the NRP1-mediated endocytosis (39), polyplexes, which are dependent on NRP1 for widespread distribution in the tumor, enter cells (at least in vitro ) through numerous endocytic pathways (16). How much NRP1 receptor mediated therapy is limited by saturation of receptors on tumor cells may be dependent on multiple factors including the specific TPP-drug (or NP) conjugate, the pegylation pattern, and the level of non-NRP1 mediated endocytosis in the cell.…”

“…For instance, Sugahara et al determined that levels of a doxorubicin conjugate or an NP such as Abraxane accumulated in tumors significantly more with TPP-targeting (7). 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).…”

“…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.…”