Cryo-electron microscopy of ready liposomes. Images of final dehydration-rehydration vesicle (DRV) formulations were obtained by cryo-EM (A-E). Zoom on DRV + CPZ (B) shows undulating bilayer, while zoom on DRV + HP-γ-CD/CPZ shows example of lipid discs. Scale bars represent 200 nm (A, D, E) or 400 nm (B, C).

HL-60 cells are unaffected by CDs and empty liposomes but sustain a dose-dependent reduction in viability when treated with CPZ and CD/CPZ liposome formulations. HL-60 cells were measured for viability relative to control after incubation for 48 h with either: A) cyclodextrins (CD), B) empty dehydration-rehydration vesicles (DRV), or C) either free chlorpromazine (CPZ), CPZ-loaded DRV, or DRV loaded with CD/CPZ complexes. The results are shown as the mean ± SD of four wells measured by metabolic conversion of the MTS reagent.

DRV formulations demonstrate dose-dependent cytotoxicity towards AML and normal cell lines after various incubation times. The AML cell lines MOLM-13, OCI-AML3, and MV4–11, and the NRK cell line, were measured for viability after incubation with various concentrations of either empty dehydration-rehydration vesicles (DRV), free chlorpromazine (CPZ), CPZ-loaded DRV, or DRV loaded with different cyclodextrin (CD)/CPZ-complexes. Cells were either incubated for 24 or 72 h. The results are shown as viability relative to untreated cells as measured by the metabolic conversion of the WST-1 reagent and represent the mean ± SD from 2 to 5 wells obtained from two separate experiments.

DRV formulations demonstrate CD-dependent cell death kinetics. MOLM-13 cells were incubated for up to 12 h with 100 μM chlorpromazine (CPZ) in either free or encapsulated form, or empty (dehydration-rehydration vesicles) DRV of equivalent lipid concentration, before being fixed and stained with 2 % formaldehyde (in pH 7.4 PBS) containing 0.005 mg/mL Hoechst33342. Nuclei-stained cells were imaged with an Axiovert 200M fluorescence microscopy and counted for apoptosis. The results are shown as the mean ± SD of a triplicate experiment.

DRV formulations are quickly internalised into MOLM-13 AML cells. MOLM-13 cells were incubated with rhodamine-labelled fluorescent dehydration-rehydration vesicles (F-DRV), that were either empty or loaded with cyclodextrins (CD), at 2 % v/v concentration for up to 8 h. After incubation, the cells were washed twice in PBS and their red fluorescent intensity analysed using an Accuri C6 flow cytometer (A, B). At least 10,000 events were collected, and the median fluorescence from each sample was determined. See Supplementary Fig. S2 for examples of the gating strategy. The results represent the mean ± SD of either two experiments (F-DRV, F-DRV + SGM) or a single experiment (F-DRV + HP-γ-CD, F-DRV + SBE-β-CD), all performed in triplicates. Results are provided as direct measurements from the flow cytometer (A) or adjusted for the relative fluorescence intensity of each formulation (B). Cells incubated for 2 h were also washed twice in PBS, fixed in 2 % buffered formaldehyde (pH 7.4 PBS), and imaged with an Andor Dragonfly 505 confocal microscope fitted with a CFI SR HP Apo TIRF 100× objective (C-G). Scale bars in the confocal micrographs represent 15 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Distribution of DRV formulations after intravenous injection into zebrafish larvae. Zebrafish larvae were injected into the posterior cardinal vein at two days post-fertilisation (dpf) with approximately 4 nL of different rhodamine-labelled, fluorescent dehydration-rehydration vesicles (F-DRV). Zebrafish larvae were kept in fresh E3 medium at 28.5 °C and anesthetised in a 0.7 mM tricaine solution before injection and during imaging. Images were acquired at the day of injection, or zero days post-injection (dpi), and at one and two dpi using an Andor Dragonfly 505 confocal microscope fitted with a CFI Plan Apochromat Lambda 10× objective, 561 nm excitation laser, and 600/25 nm band pass filter. Fluorescence images of whole larvae (A-F) are a Z-projection obtained by summation of overlying pixels in the confocal Z-stack. Images also show large sections of the head of one dpi zebrafish larvae, with 3D representations of the blood vessels in the brain (G-J) obtained by pre-processing using a 3D-median filter and rendered using the ImageJ 3D Viewer plugin. All image processing was performed in ImageJ ver. 1.52p. The scale bars represent 500 μm (A-F) and 250 μm (G-J).