Fig 1.
The scheme of synthesis of pH-responsive liposome and the proposed intracellular drug release pathway.
Fig 2.
A: Fluorescent microscopy images of MiaPaCa-2 cells treated with Rhodamin-labeled liposomes for 4 hours. Green fluorescence shows lysosomes, stained with LysoTracker (Life Technologies). Red fluorescence shows Rhodamin-labeled liposomes. B: Dynamic light scattering of GGTI-loaded liposome is shown. GGTI-loaded liposomes were prepared as described in Materials and Methods. C: Low pH release of GGTI from GGTI-loaded liposomes.
GGTI-loaded liposomes were prepared and then exposed to solution adjusted to different pH values for 15 min and the release of GGTI was examined by HPLC. TritonX-100 was used as a full release control. The symbol * indicates statistically significant difference at P value < 0.05.
Fig 3.
Release of dye and drug from liposome is dependent on low pH of acidic organelles.
A: Fluorescent microscopy images of MCF7 cells incubated with Doxorubicin-loaded liposomes for 6 hours (red fluorescence). Control cells did not receive liposomes. B: Release of Pyranine dye in MiaPaCa-2 cells is examined by its fluorescence with or without the treatment with Bafilomycin A1. Phase contrast picture is shown. C: Effects of Bafilomycin A1 on pH of intracellular organelles are shown using Acridine Orange.
Fig 4.
A: Inhibition of protein geranylgeranylation by liposomal GGTI was examined by the use of an antibody that specifically detects unprenylated form of Rap1A. MiaPaCa-2 cells were treated for 3 hours with GGTI-loaded liposomes with or without Bafilomycin pretreatment and the amount of unprenylated Rap1A was examined by Western. Lipo: empty liposome. Lipo-GGTI: GGTI loaded liposome. Baf/Lipo-GGTI: GGTI loaded liposome was added to cells pretreated with Bafilomycin A. B: Cell proliferation inhibition by GGTI-loaded liposomes after 72 hour treatment. Empty liposomes and free GGTI are used as comparison. C: Cell cycle effect of liposomal-GGTI (Lipo-GGTI) was examined by FACS analysis after treating MiaPaCa-2 cells with liposomal-GGTI for 24 hours. Similar level of G1 accumulation was observed with that seen with free GGTI. D: Effect of liposomal-GGTI on p21CIP1/WAF1 expression was examined by Western analysis using MiaPaCa-2 cells treated with liposomal-GGTI for 24 hours.
Fig 5.
Cell proliferation inhibition of GGTI-loaded liposomes was examined with human non-small cell lung cancer cells H596, H358 and A549 as well as a normal lung cell.
Cell numbers after 72 hours’ treatments are shown as percentage of untreated cells. Empty liposomes do not affect proliferation. The symbol * indicates statistically significant difference at P value < 0.05.
Fig 6.
Effect of combination of liposomal GGTI and FTI on MiaPaCa-2 cells.
A: Effects of GGTI or FTI alone as well as the combination of the two on ERK phosphorylation were examined by Western blot after 12 hours of treatment. Total ERK is used as loading control. B: the same experiment was repeated with liposomal GGTI (Lipo-GGTI) and FTI. C: Effects of the combination of liposomal-GGTI and FTI on proliferation of MiaPaCa-2 cells after the treatment for 48 hours. FTI and liposomal GGTI concentrations are varied.