Accumulation, internalization and therapeutic efficacy of neuropilin-1-targeted liposomes

Eric E. Paoli, Elizabeth S. Ingham, Hua Zhang, Lisa M. Mahakian, Brett Z. Fite, M. Karen Gagnon, Sarah Tam, Azadeh Kheirolomoom, Robert Cardiff, Katherine W. Ferrara

Research output: Contribution to journalArticlepeer-review

25 Scopus citations


Advancements in liposomal drug delivery have produced long circulating and very stable drug formulations. These formulations minimize systemic exposure; however, unfortunately, therapeutic efficacy has remained limited due to the slow diffusion of liposomal particles within the tumor and limited release or uptake of the encapsulated drug. Here, the carboxyl-terminated CRPPR peptide, with affinity for the receptor neuropilin-1 (NRP), which is expressed on both endothelial and cancer cells, was conjugated to liposomes to enhance the tumor accumulation. Using a pH sensitive probe, liposomes were optimized for specific NRP binding and subsequent cellular internalization using in vitro cellular assays. Liposomes conjugated with the carboxyl-terminated CRPPR peptide (termed C-LPP liposomes) bound to the NRP-positive primary prostatic carcinoma cell line (PPC-1) but did not bind to the NRP-negative PC-3 cell line, and binding was observed with liposomal peptide concentrations as low as 0.16 mol%. Binding of the C-LPP liposomes was receptor-limited, with saturation observed at high liposome concentrations. The identical peptide sequence bearing an amide terminus did not bind specifically, accumulating only with a high (2.5 mol%) peptide concentration and adhering equally to NRP positive and negative cell lines. The binding of C-LPP liposomes conjugated with 0.63 mol% of the peptide was 83-fold greater than liposomes conjugated with the amide version of the peptide. Cellular internalization was also enhanced with C-LPP liposomes, with 80% internalized following 3 h incubation. Additionally, fluorescence in the blood pool (~ 40% of the injected dose) was similar for liposomes conjugated with 0.63 mol% of carboxyl-terminated peptide and non-targeted liposomes at 24 h after injection, indicating stable circulation. Prior to doxorubicin treatment, in vivo tumor accumulation and vascular targeting were increased for peptide-conjugated liposomes compared to non-targeted liposomes based on confocal imaging of a fluorescent cargo, and the availability of the vascular receptor was confirmed with ultrasound molecular imaging. Finally, over a 4-week course of therapy, tumor knockdown resulting from doxorubicin-loaded, C-LPP liposomes was similar to non-targeted liposomes in syngeneic tumor-bearing FVB mice and C-LPP liposomes reduced doxorubicin accumulation in the skin and heart and eliminated skin toxicity. Taken together, our results demonstrate that a carboxyl-terminated RXXR peptide sequence, conjugated to liposomes at a concentration of 0.63 mol%, retains long circulation but enhances binding and internalization, and reduces toxicity.

Original languageEnglish (US)
Pages (from-to)108-117
Number of pages10
JournalJournal of Controlled Release
Issue number1
StatePublished - Mar 28 2014


  • CendR
  • Doxorubicin
  • Liposome
  • Neuropilin-1
  • Optical imaging
  • Toxicity

ASJC Scopus subject areas

  • Pharmaceutical Science


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