Filomicelles were prepared from hydration of di-block copolymers with no residual co-solvent13,23
. Block copolymers of PEG-polyethylethylene (EOm
, designated OE) or PEG-polycaprolactone (EOm
, designated OCL) were synthesized by standard polymerizations8
; provides details of the di-blocks used here. Note that OE7′ is a copolymer related to that used in previous studies of vesicles12
, but the present copolymer has a slightly higher volume fraction of PEG (fEO
) that is more consistent with cylinder micelle formation8
and also with cylinder micelles from the two OCL copolymers with similar fEO
. Cryo-transmission electron microscopy allows visualization of the hydrophobic core of the micelles, and the total diameter do
is estimated to be about twice the core diameter. Filomicelles were visualized by optical microscopy with a hydrophobic membrane marker, PKH26 (Sigma), which partitions into the cores of the filomicelles when added to a hydrated sample23
. Cell membrane probe, Fluorescein DHPE, nuclei Hoechst stain and Lysotracker Blue were from Molecular Probes.
Fluorescent and bright-field images were recorded using an Olympus IX71 inverted microscope with a CCD camera (Cascade 512, Roper Scientific). Repeated extrusion of filomicelle samples at 100−200 p.s.i. through a 400-nm membrane gently fragments the cylinders, leading under these conditions to a maximum contour length <10 μm, which can be controlled (, histograms) by the repetitions in extrusion.
For circulation studies, we followed our previous polymer vesicle protocols and assessed performance in two rodent species for comparison with previous studies12,15,16,18
. Male Sprague–Dawley rats were injected with 0.5 ml of 5 mg ml−1
copolymer in phosphate buffered saline; alternatively, equal numbers of male or female C57 mice (with similar results) were injected with 0.1 ml of the same. Orbital bleeds into heparin tubes were taken at various times during the study. The plasma (containing the filomicelles) was separated from the other blood components by centrifugation at 7,000 g
for 10 min to determine the number, N
, and contour lengths of the filomicelles in circulation. By comparison to non-centrifuged control samples, this level of centrifugation has no effect on measured length distributions and essentially separates into the supernatant plasma all of the micelles (or vesicles). N0
is the number of filomicelles from a one-hour bleed for the figures shown. Additional circulation studies in four mice show that (1) filomicelles in blood are statistically the same in number at timepoints of 1−2, 10, 30 and 60 min, and (2) the preinjected concentration is statistically the same as that at 1−2 min when corrected for dilution into the typical blood volume of mice. Organs were retrieved and sectioned (5-μm slices) using a microtome. Each data point in and represents results from at least 4 rats or mice; organ distribution data in is from 2−4 rats per condition. In any studies with blood, citrate or EDTA was used as anticoagulant.
In vitro phagocytosis assays were performed on blood-drawn human neutrophils and also a human macrophage cell line, THP1 (ATCC). Filomicelle suspensions of 0.1 mg copolymer (large excess for the number of cells) were incubated with the macrophage cell line for 24 h. In vitro assays of internalization of inert filomicelles by human lung-derived cells A549 (ATCC) were performed by incubation of cells prelabelled with fluorescein-phosphatidylethanolamine (FL-DHPE, Molecular Probes) with filomicelles (red dye, PKH26) for preset times. This was followed by removing the supernatant of the remaining filomicelles and washing the cells with PBS three times before imaging. Subsequent fluorescent intensity analysis was used to quantify uptake. Uptake by macrophages depends on activation and is not just a passive adsorption process, as omitting the PMA (phorbol-12-myristate-13-acetate) activation led to cells with control intensities.
Tumour studies were conducted in a similar way to those recently reported39
, with the use here of A549 cells. Briefly, cultured cells were injected subcutaneously onto the backs of nude mice and allowed to grow until they reached a mean size of 0.52 cm2
). Mice were then injected in the tail vein either with saline or filomicelle controls, paclitaxel in ethanol, or the same mass of worm-like filomicelles as above, except that the filomicelles were preloaded with paclitaxel45
. Each group consisted of four mice. No group of mice showed any significant differences in weight change, and the maximum tolerated dose (MTD) was determined in separate studies to be the dose that causes 10% weight loss within 24 h (average from three mice). For paclitaxel-loaded filomicelles, the MTD exceeds the 8 mg kg−1
used here by more than twofold. For unloaded OCL3 filomicelles (8 μm long), we have injected up to 300 mg kg−1
of copolymer without any significant weight loss in mice.