title: Accumulation, internalization and therapeutic efficacy of neuropilin-1-targeted liposomes.
abstract: 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 0.16mol%. 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 (2.5mol%) peptide concentration and adhering equally to NRP positive and negative cell lines. The binding of C-LPP liposomes conjugated with 0.63mol% 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 3h incubation. Additionally, fluorescence in the blood pool (~40% of the injected dose) was similar for liposomes conjugated with 0.63mol% of carboxyl-terminated peptide and non-targeted liposomes at 24h 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 0.63mol%, retains long circulation but enhances binding and internalization, and reduces toxicity.
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA. Electronic address: eepaoli@ucdavis.edu.
authors.forename: Eric E
authors.initials: EE
authors.lastname: Paoli
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA. Electronic address: esingham@ucdavis.edu.
authors.forename: Elizabeth S
authors.initials: ES
authors.lastname: Ingham
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA. Electronic address: hchzhang@ucdavis.edu.
authors.forename: Hua
authors.initials: H
authors.lastname: Zhang
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA. Electronic address: lmmahakian@ucdavis.edu.
authors.forename: Lisa M
authors.initials: LM
authors.lastname: Mahakian
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA. Electronic address: bzfite@ucdavis.edu.
authors.forename: Brett Z
authors.initials: BZ
authors.lastname: Fite
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA. Electronic address: mkgagnon@ucdavis.edu.
authors.forename: M Karen
authors.initials: MK
authors.lastname: Gagnon
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA. Electronic address: smjohns@ucdavis.edu.
authors.forename: Sarah
authors.initials: S
authors.lastname: Tam
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA. Electronic address: akheirolomoom@ucdavis.edu.
authors.forename: Azadeh
authors.initials: A
authors.lastname: Kheirolomoom
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA. Electronic address: rdcardiff@ucdavis.edu.
authors.forename: Robert D
authors.initials: RD
authors.lastname: Cardiff
authors.affiliation: University of California, Davis, Department of Biomedical Engineering, 451 Health Sciences Drive, Davis, CA 95616, USA. Electronic address: kwferrar@ucdavis.edu.
authors.forename: Katherine W
authors.initials: KW
authors.lastname: Ferrara
mesh_terms: D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage* / Q000737:chemistry; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D003287:Contrast Media / Q000008:administration & dosage / Q000737:chemistry; D004317:Doxorubicin / Q000008:administration & dosage* / Q000737:chemistry; D005260:Female; D005682:Gadolinium / Q000008:administration & dosage / Q000737:chemistry; D006571:Heterocyclic Compounds / Q000008:administration & dosage / Q000737:chemistry; D006801:Humans; D008081:Liposomes; D051379:Mice; D009369:Neoplasms / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D039942:Neuropilin-1 / Q000737:chemistry / Q000378:metabolism*; D009842:Oligopeptides / Q000008:administration & dosage* / Q000737:chemistry; D009942:Organometallic Compounds / Q000008:administration & dosage / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D047368:Tumor Burden / Q000187:drug effects
chemical_list: D000903:Antibiotics, Antineoplastic; D003287:Contrast Media; D006571:Heterocyclic Compounds; D008081:Liposomes; D009842:Oligopeptides; D009942:Organometallic Compounds; C507807:cysteinyl-arginyl-prolyl-prolyl-arginine; C062402:gadoteridol; D039942:Neuropilin-1; D011092:Polyethylene Glycols; D004317:Doxorubicin; D005682:Gadolinium
keywords: CendR; Doxorubicin; Liposome; Neuropilin-1; Optical imaging; Toxicity