A new fluorescence-based optical imaging method to non-invasively monitor hepatic myofibroblasts in vivo

Graphical abstract

previously reported by Moase [2]. The mixture is then applied to a Sephadex G-50 column eluted with Hepes buffer (25 mM Hepes, 140 mM NaCl), pH7.4, to remove the unloaded DOX (DOX-liposomes) or to exchange the buffer (empty liposomes). The loading efficiency of DOX is around 95% and liposomes routinely contained DOX at a concentration of 150-180 μg DOX/μmol PL [3]. Finally, C1-3 or CSDB9 scFv fragments are coupled to the maleimide terminus of DSPE-PEG2000-MAL using the previously described methods of coupling for whole antibodies and for Fab' fragments with slightly modifications [4,5].
Briefly, to activate the C1-3 and CSDB9 fragments for reactivity toward the maleimide, we utilized 2-iminothiolane (Traut's reagent) to convert exposed amino groups on the antibody into free sulfhydryl groups. A 20:1 mole ratio of 2-iminothiolane to ScFv fragments and 1 hour of incubation at room temperature with occasional mixing gave optimal ScFv activation. After separation of thiolated ScFv from iminothiolane, with the use of Sephadex G-25 column chromatography, the ScFv was slowly added to the liposomes in the presence of a small magnetic stirring bar. Oxygen was displaced by running a slow stream of nitrogen over the reaction mixture. The tube was capped and sealed with Teflon tape, and the reaction mixture was incubated overnight at room temperature with continuous slow stirring. The resulting immunoliposomes were separated from unreacted ScFv by chromatography with the use of Sepharose CL-4B, sterilized by filtration through 0.2-μm pore cellulose membranes (Millipore Corp., Bedford, MA), and stored at 4 °C until use. The antibody density was evaluated by BioRad (Richmond, CA) protein assay.
Zeta potential values were recorded following dilution in distilled water or in PBS. These results are presented as the average values ± standard deviation. Unilamellar Liposomes possess a hydrodynamic diameter of about 155 nm (C1-3-lipo (DOX): 157.2 ± 3.164 nm; CSDB9: 158.2 ± 3.72 nm, with a mean PdI value 0.054 ± 0.025 for C1-3 and 0.072 ± 0.027 for CSDB9, indicating a very good monodisperse liposomal preparation.
Moreover, the Z-potential values in water as well as in PBS were similar for the two formulations: C1-3 -25.8 ± 1.48 mV and 2.1 ± 0.665 mV and CSBD9 -29.2 ±2 .4 mV and -3 ± 0.53, respectively; indicating good stability properties in a medium resembling physiological conditions. These two parameters, taken together, warrant loss of aggregation during liposomes storage, and possibly, increased half-life in systemically conditions as expected due to the PEG shield [11].

Cellular binding studies in vitro: 3 H[CHE]
-labeled liposomes were used to measure cellular binding, as described previously [3,10,12]. Briefly, cells (1×10 6 /mL) were incubated with 600 nmol PL/mL of [3H]CHE-empty liposomal formulations, with or without coupled C1-3 and maintained at 4°C in a total volume of 200 µL. After a 1-hour incubation, the cells were extensively washed, and lysed with 1 N NaOH for evaluation of protein and for β-counting. In vivo activated HSC isolation: Mice were given CCl4 biweekly at (1:3 v/v CCl4:Olive Oil) for two weeks, 24h after the final injection mice were humanely killed and then the livers removed.

Analysis of in vivo
In vivo activated HM were then isolated as previously described by [13] SDS-PAGE: Total protein was fractionated by 9% SDS-PAGE, transferred onto nitrocellulose and then blocked blots with Tris-buffered saline and Tween 20 (0.1%) containing 5% BSA before incubation overnight with primary antibodies to synaptophysin (Ab32127 Abcam,

Mouse Hepatic stellate cell isolation: Mouse hepatic stellate cells were isolated from
C57Bl6 mice as previously described [14] Sirius Red: Formalin-fixed and paraffin-embedded sections were stained with H&E, 0.1% Sirius Red Picric solution following standard procedures.
Immunohistochemistry: Staining was performed on formalin-fixed paraffin-embedded 5µm cut sections. Sections were dewaxed, dehydrated then endogenous peroxidase was blocked with 2% H2O2 in methanol. Antigen retrieval was achieved using proteinase K (20ug/ml) for Immunocytochemistry: Cells were isolated from C75Bl6 murine livers and culture activated.
Hepatic myofibroblasts were used for ICC staining between passages 1-4. Briefly, 22mmx22mm coverslips were sterilised with 70% ethanol and placed into a well of a 6 well plate. Cell numbers were determined using a haemocytometer and an equal number of cells seeded per well. Once ~70% confluency was reached, cells were incubated with 60μg/ml C1-3-AF for 2Hrs in HEPES/HBSS for 2Hrs at 37°C. Following this cells were fixed with 2% formaldehyde/0.2% glutaraldehyde in PBS and permeabilised with ice cold methanol. Cells were co-stained with aSMA-FITC. Finally, cell nuclei were stained with DAPI, mounted on slides and visualised using a Zeiss fluorescent microscope. Images taken at x200 magnification.