title: Hyperthermia-triggered intracellular delivery of anticancer agent to HER2(+) cells by HER2-specific affibody (ZHER2-GS-Cys)-conjugated thermosensitive liposomes (HER2(+) affisomes).
abstract: We previously reported the formulation and physical properties of HER2 (human epidermal growth factor receptor 2)-specific affibody (ZHER2:342-Cys) conjugated thermosensitive liposomes (HER2(+)affisomes). Here we examined localized delivery potential of these affisomes by monitoring cellular interactions, intracellular uptake, and hyperthermia-induced effects on drug delivery. We modified ZHER2:342-Cys by introducing a glycine-serine spacer before the C-terminus cysteine (called ZHER2-GS-Cys) to achieve accessibility to cell surface expressed HER2. This modification did not affect HER2-specific binding and ZHER2-GS-Cys retained its ability to conjugate to the liposomes containing dipalmitoyl phosphatidyl choline: DSPE-PEG2000-Malemide, 96:04 mole ratios (HER2(+)affisomes). HER2(+)affisomes were either (i) fluorescently labeled with rhodamine-PE and calcein or (ii) loaded with an anticancer drug doxorubicin (DOX). Fluorescently labeled HER2(+) affisomes showed at least 10-fold increase in binding to HER2(+) cells (SK-BR-3) when compared to HER2(-) cells (MDA-MB-468) at 37°C. A competition experiment using free ZHER2-GS-Cys blocked HER2(+) affisome-SK-BR-3 cell associations. Imaging with confocal microscopy showed that HER2(+) affisomes accumulated in the cytosol of SK-BR-3 cells at 37°C. Hyperthermia-induced intracellular release experiments showed that the treatment of HER2(+) affisome/SK-BR-3 cell complexes with a 45°C (±1°C) pre-equilibrated buffer resulted in cytosolic delivery of calcein. Substantial calcein release was observed within 20min at 45°C, with no effect on cell viability under these conditions. Similarly, DOX-loaded HER2(+)affisomes showed at least 2- to 3-fold higher accumulation of DOX in SK-BR-3 cells as compared to control liposomes. DOX-mediated cytotoxicity was more pronounced in SK-BR-3 cells especially at lower doses of HER2(+)affisomes. Brief exposure of liposome-cell complexes at 45°C prior to the onset of incubations for cell killing assays resulted in enhanced cytotoxicity for affisomes and control liposomes. However, Doxil (a commercially available liposome formulation) showed significantly lower toxicity under identical conditions. Therefore, our data demonstrate that HER2(+)affisomes encompass both targeting and triggering potential and hence may prove to be viable nanodrug delivery carriers for breast cancer treatment.
authors.affiliation: CCR Nanobiology Program, NCI, Frederick, MD, USA.
authors.forename: Brandon
authors.initials: B
authors.lastname: Smith
authors.forename: Ilya
authors.initials: I
authors.lastname: Lyakhov
authors.forename: Kristin
authors.initials: K
authors.lastname: Loomis
authors.forename: Danielle
authors.initials: D
authors.lastname: Needle
authors.forename: Ulrich
authors.initials: U
authors.lastname: Baxa
authors.forename: Amichai
authors.initials: A
authors.lastname: Yavlovich
authors.forename: Jacek
authors.initials: J
authors.lastname: Capala
authors.forename: Robert
authors.initials: R
authors.lastname: Blumenthal
authors.forename: Anu
authors.initials: A
authors.lastname: Puri
mesh_terms: D000903:Antibiotics, Antineoplastic / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy* / Q000378:metabolism; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability; D002470:Cell Survival / Q000187:drug effects; D004317:Doxorubicin / Q000008:administration & dosage* / Q000493:pharmacokinetics / Q000494:pharmacology; D016503:Drug Delivery Systems; D005260:Female; D006801:Humans; D006979:Hyperthermia, Induced; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*
chemical_list: D000903:Antibiotics, Antineoplastic; D004317:Doxorubicin; D008081:Liposomes; D018719:Erb-b2 Receptor Tyrosine Kinases; C508053:ERBB2 protein, human