HER2-Specific Affibody-Conjugated Thermosensitive Liposomes (Affisomes) for Improved Delivery of Anticancer Agents

Abstract

Thermosensitive liposomes are attractive vehicles for delivery and release of drugs to tumors. To improve targeting efficacy for breast cancer treatment, an 8.3 kDa HER2-specific Affibody molecule (Z HER2:342 -Cys) was conjugated to the surface of liposomes. The effects of this modification on physical characteristics and stability of the resulting nano particles denoted "Affisomes" were investigated. Thermo-sensitive small unilamellar vesicles (SUV) liposomes of (80-100 nm) diameter consisting of dipalmitoyl phosphatidylcholine (DPPC, Tm 41 °C) as the matrix lipid and a maleimide conjugated pegylated phospholipid (DSPE-MaL-PEG2000) were prepared by probe sonication. Fluorescent probes were incorporated into liposomes for biophysical and/or biochemical analysis and/or triggered release assays. Affibody was conjugated to these liposomes via its C-terminal cysteine by incubation in the presence of a reducing agent (tributylphosphine) for 16-20 hours under argon atmosphere. Lipid conjugated affibody molecule was visible as an 11.3-kDa band on a 4-12% Bis/Tris gel under reducing conditions. Affibody conjugation yields were ~70% at a protein/lipid ratio of 20 μg/mg, with an average number of 200 affibody molecules per Affisome. Affibody conjugation to thermosensitive liposomes did not have any significant effect on the hydrodynamic size distribution of the liposomes. Thermo-sensitivity of Affisomes was determined by monitoring release of entrapped calcein (a water-soluble fluorescent probe, λex/em 490/515 nm) as a function of temperature. Calcein was released from Affisomes (thermosensitive liposomes with affibody-Targeted SUV) as well as non-targeted SUV (thermosensitive liposomes without affibody) in a temperature-dependent manner, with optimal leakage (90-100%) at 41°C. In contrast, liposomes prepared from Egg-phosphatidyl choline (Tm 0 °C) under similar conditions released only 5-10% calcein at 41°C. Affisomes, when stored at room temperature, retained >90% entrapped calcein up to 7 days. Moreover, incubation of liposomes in PBS supplemented with 10% heat-inactivated serum (FBS) did not result in destabilization of liposomes. Therefore, Affisomes present promising and novel drug delivery candidates for breast cancer targeting.

Affibody molecules: Engineered proteins for therapeutic, diagnostic and biotechnological applications

“…Enzyme fusion RNase [64][65][66][67] Blocking Nuclide Nanoparticle [60,61] Liposome [62,63] Enzyme fusion -gal, HRP [85,86] Separation Detection Blocking SPIO [46] Enzyme DNA pol [Finnzymes] [ 86,90,91] [ [77][78][79][80][81][82][83] IP [86,87] Affinity chromatography Microarray, FRET Fig. 1.…”

“…The authors could show temperature-induced opening of the liposomes at 41°C, which can be achieved in vivo using focused ultrasound. Here, the liposomes were conjugated to a HER2-specific Affibody molecule that mediated binding and internalization into HER2-expressing cells but not HER2-negative ones [63]. The notion that further specificity by a secondary controlled opening step may be needed in addition to tumor cell targeting is important.…”

Exploding vesicles

“…Fatty acid vesicles have been studied as models of primitive cell membranes at the origin of life [1][2][3][4][5][6][7], and phospholipid vesicles (liposomes) have been widely studied as models of modern cell and organelle membranes [8,9] and as drug delivery vehicles [10][11][12]. We first observed the phenomenon of "exploding vesicles" during microscopic observations of large (approximately 4 μm in diameter) oleate vesicles containing 10 mM HPTS (8-hydroxypyrene-1,3,6-trisulfonic acid trisodium salt, a water-soluble, membrane-impermeable fluorescent dye), in 0.2 M Nabicine buffer, pH 8.5.…”

PET-CT Imaging in Breast Cancer Patients: New Tracers, Future Directions

“…The preliminary results of this study are expected to be published in the autumn of 2013, the end of the study will be in 2015 [46]. [47][48][49][50]. However other randomized studies with larger number of enrolled patients will also be required to further analyze the significance of HER2-imaging in treatment planning and daily oncological practice.…”