However, after the third treatment, CTX-CPT-NPs were shown to confer a distinct therapeutic advantage over the non-targeted formulation. of rhodamine 6G. Alternatively, as a control formulation, blank NPs contained neither drug nor fluorophore. 50 g/mL of CTX per mg polymer was added to the NP formulation in MES buffer. NPs were stirred at 80 SC 560 rpm at room temperature for 2 hours to allow protein conjugation, and unbound antibody was removed by centrifugation at 15000 rcf for 15 minutes, followed by resuspension in PBS. CTX conjugation to NPs was quantified using the bicinchoninic acid (BCA) protein assay (Thermo Scientific, UK), using CTX as a protein standard in solution containing blank-NPs to account for polymer interference. CPT encapsulation was assessed in the NP pellet by measurement of CPT fluorescence at 330Ex/460Em. A CPT standard curve was used which contained blank-NPs to account for polymer interference. NP physical characteristics were analyzed in deionised water by dynamic light scattering using the NanoBrook Omni Particle Sizer and Zeta Potential Analyser, and by Scanning Electron Micrography using a FEI quanta FEG environmental scanning electron microscope. CPT release was assessed in Slide-A-Lyzer dialysis cassettes having a 7 kDa pore size (Thermo Scientific, UK). 15 mg CPT-loaded polymer in 1 mL PBS were dialyzed against 29 mL of PBS/ 2 % Tween-80 (v/v) while shaking (180 rpm) at 37 C. At the indicated time points, 1 mL of release medium was collected and analysed for CPT content, and concentrations were determined by comparison to a CPT standard by measuring fluorescence at 330Ex/460Em. SDS-PAGE gel electrophoresis. 1 mg non-conjugated or CTX-conjugated NPs were boiled in 10x Laemmli buffer for 5 minutes before electrophoretic separation on a reducing pre-cast Novex WedgeWell 4C20 % Tris-glycine gel (Invitrogen, UK). Cell culture. HCT116 (donated by the Volgelstein laboratory), A549 (ATCC), HKH-2 (donated by the Sasazuki laboratory where the cell line was generated19), HCC827 (ATCC) and PANC-1 (ATCC) cell lines were cultured in high glucose Dulbeccos Modified Eagle Medium (DMEM) containing 10 %10 % fetal calf serum (FCS) (Life Technologies, UK), and BxPC-3 cells (ATCC) were cultured in RPMI 1640 containing 10 %10 % FCS. Cell surface EGFR expression. To measure EGFR cell surface expression, cells were stained with either 1 g FITC-conjugated anti-EGFR antibody (Santa Cruz sc: 120) or 1 g SC 560 FITC-conjugated IgG2a Isotype control (Santa Cruz sc: 2856) for 45 minutes at 4 C in PBS/ 5 % FCS. Cells were washed twice in 5 mL of PBS / 5% FCS and then once in 5 mL of PBS. Samples were resuspended in 300 l of PBS and analysed by FACS using either BD LSRII or BD Accuri C6 Plus flow cytometers (BD, UK). Measurement of NP binding to the cell surface. Cells were seeded at 4 104 cells per well in a black 96-well plate SC 560 or 2 105 cells per well in a 6-well plate. Prior to NP treatment, Rabbit polyclonal to ERO1L cells were incubated in serum-free media, chilled to 4 C, and treated with 25 g/mL fluorescent rhodamine-6G -loaded NPs for different time periods. Cells were then washed three times with ice-cold PBS. For measuring total fluorescence, cells were lysed in 50 l of 0.2 M NaOH / 0.05 % Triton X-100, and fluorescence was measured at 525Ex/555Em using a plate reader. Fluorescence was also measured by FACS using a BD Accuri C6 plus instrument. Assessing cell viability. For free drug treatments, viability was measured by MTT assay. For NP treatments, viability was measured using the Cell Titre-Glo assay (Promega) or by staining with 0.4 % crystal violet and measuring absorbance at 590 nm. Viability was calculated as a percentage cell growth relative to the untreated control. Assessing colony formation by clonogenic assay. Cells were seeded at 2.5 105 cells per well in 6-well plates. After 1 hour of treatment, the cells were re-seeded at 250 cells per well in triplicate in 6-well plates and were incubated for 11C13 days (cell line-dependent) to allow colonies to form. The cells were then stained with 1 mL of 0.4 % crystal violet for.