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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. © 2014 The Authors.  The Journal of Gene Medicine  published by John Wiley & Sons, Ltd.

Increased tumor distribution and expression of histidine‐rich plasmid polyplexes