S4). To evaluate the targeting efficiency of t-L, three v6 integrin positive cell lines were used. tumour mouse models. A 839977 Immuno-compromised mice bearing A375P6 experimental metastatic lung tumours were treated with L-ALD or t-L-ALD as monotherapies or in combination with but t-L-ALD offered no added advantage compared to L-ALD. studies [20], [21], [22], [23], [24], [25], [26], [27]. The encapsulation of ALD in liposomes (L-ALD), has been shown to increase A 839977 its therapeutic efficacy [24]. Long-circulating liposomes passively target the tumour due to the enhanced permeation and retention (EPR) effect [28], leading to a greater amount of the encapsulated drug reaching the tumour cells. The aim of this study is to formulate v6 integrin targeted ALD liposomes (t-L-ALD), using the peptide A20FMDV2. It A 839977 is Rabbit polyclonal to ANKRD33 hypothesised that A20FMDV2 conjugation to liposomal alendronate will promote v6-receptor mediated endocytosis and improved therapeutic efficacy in combination with T cell immunotherapy and possibly overnight dialysis against PBS using a dialysis bag with a MWCO of 10,000?kD at room temperature. For cellular uptake studies, fluorescent liposomes were formed as above but with the inclusion of 1% mol CF-DOPE to give a final liposome composition of DSPC:CF-DOPE:cholesterol:DSPE-PEG2000:DSPE-PEG2000-maleimide (54:1:40:4:1?molar ratio). Liposomes containing alendronate (L-ALD and t-L-ALD) were prepared as above, but the lipid film was hydrated with 1?ml of 100?mM solution of ALD in HEPES Buffered Saline (HBS, 20?mM HEPES, 150?mM NaCl). Un-encapsulated ALD was removed by overnight dialysis against HBS using a dialysis bag with a MWCO of 10,000?kD. 2.3. Peptide quantification The amount of peptide conjugated to the liposomes was determined by LavaPep? Protein and Peptide quantification kit. A calibration curve was obtained in the range 0.122C500?g/ml using free A20FMDV2. Liposomes were diluted 100 times in deionised water and the amount of peptide quantified according to the manufacturer’s instructions. Briefly, 50?l of the diluted sample was A 839977 incubated with 50?l of LavaPep working solution for 60?min in the dark at RT. The fluorescence intensity was then measured using 540??10?nm and 630??10?nm excitation and emission filters, respectively (FLUOStar Omega, BMG Lab Tech). The per cent peptide conjugated to the liposomes was calculated by quantifying the amount of peptide in the liposome sample before and after purification. 2.4. Cell culture conditions The cell lines PANC-1 (CRL-1469?, pancreatic), PANC0403 (CRL-2555?, pancreatic) and 4T1 (CRL-2539?, breast) were obtained from ATCC?. A375Ppuro and A375P6puro cell lines were created using the human A 839977 melanoma cell line A375P (CRL-3224?, melanoma), which was infected with pBabe retroviruses encoding puromycin resistance alone or in combination with cDNA for human 6, as previously reported [12]. The A375Ppuro and A375P6 cell lines were a kind gift from Prof. John Marshall (QMUL). The A375P6 cell line was subsequently transfected with firefly luciferase (luc) using an SFG retroviral vector whereby luc was co-expressed with dsTomato red fluorescent protein. Transduced cells were then flow sorted for red fluorescence to obtain a pure A375P6-luc cell line [24]. All cell lines were maintained at 37?C, 5% CO2 and 5% relative humidity. Advanced RPMI (PANC-1, PANC0403, 4T1) or DMEM media (A375Ppuro, A375P6puro) were used, both of these were supplemented with 10% FBS, 1% GlutaMAX? and 1% Penicillin/Streptomycin. 2.5. Characterisation of cell lines for v6 integrin expression v6 integrin receptor expression was confirmed by 10D5 antibody staining and flow cytometry. Cells (1??105/100?l) were incubated with 5?l of 10D5 or the isotype control (IgG FITC) for 30?min at 4?C, washed twice with 1?ml PBS before 30?min incubation with 2.5?l of the FITC labelled IgG secondary antibody at 4?C then washed with PBS. Using the FL1 detector, 10,000 cells were gated and the fluorescence was analysed under live gating. The cells were read on a BD FACS Calibur? flow cytometer obtained from BD Bioscience (US) and analysed using FlowJo software. 2.6. Cellular uptake of liposomes using flow cytometry Cells were plated in a 24 well plate at a density of 50,000 cells/well and left overnight to allow the cells to attach. Cells were treated with 32.5C130?M CF-DOPE containing liposomes (L or t-L) in complete media (10% FBS) for 1 or 4?h. In order to determine if the increased uptake of t-L was v6 receptor specific, peptide inhibition studies were carried out. Cells were incubated at 4?C for 10?min and were then treated with 0.2?ml of 50?g/ml free peptide in complete media for a further 10?min on ice. The fluorescently labelled L and t-L (32.5C130?M) were then additionally incubated with the cells for 1 or 4?h. Additional peptide (50?g in 25?l PBS) was added after 1?h to ensure that the v6 receptors.