Purpose: KRAS mutation is considered to be a common growth driver of malignant tumors, such as pancreatic cancer, colon cancer and lung cancer. It is difficult to develop therapeutics targeting KRAS mutation directly. Nanomedicine with functionalized properties could serve as a promising delivery system to target specific cell population or tissue. However, challenges of the functionalization and the complexity of tumor microenvironment impedes the transport and uptake of nanomedicine. It is important to develop approaches to improve this situation. It is found that oncogenic KRAS cells can actively scavenging extracellular albumin by micropinocytosis as a source of amino acid for survival. We synthesized nanoparticles to take advantage of this mechanism for albumin uptake to achieve natural targeted uptake of nanoparticles in oncogenic KRAS cancer cells.
Methods: Desolvation method was used to synthesize albumin nanoparticles. Synthesized nanoparticles were then purified by centrifugal filtration unit (molecular weight cut-off, 100 k). For labeling, nanoparticles were reacted with cyanine 7 (Cy7) NHS ester or fluorescein isothiocyanate (FITC) in sodium bicarbonate buffer. Nanoparticles were characterized by Zetasizer Nano ZS (Malvern) and transmission electron microscopy (TEM). Intracellular uptake of nanoparticles was studied in MDA-MB-231 (oncogenic KRAS) and MDA-MB-468 (wild-type KRAS) breast cancer cell lines. Cells were starved in serum free medium and then incubated with FITC-NPs. The uptake of FITC-NPs was measured by flow cytometry. To evaluate the effect of KRAS expression levels on the uptake of FITC-NPs, MDA-MB-231 cells were treated with Accell siRNA targeting KRAS to knock down the expression of KRAS protein. KRAS protein levels were confirmed by western blotting. Uptake of FITC-NPs in siRNA treated cells were also measured by flow cytometry. Tetramethylrhodamine-dextran (TMR-dextran, selectively labelling macropinosomes) and 5-(N-ethyl-N-isopropyl) amiloride (EIPA, an inhibitor of micropinocytosis) was used to assess the level of uptake via macropinocytosis pathway. The uptake of Cy7-NPs was qualitatively measure by fluorescent microscopy.
Results: The size of the nanoparticles was 58.47 ± 0.73 nm in diameter measured by dynamic light scattering with the polydispersity index of 0.166 ± 0.004. The zeta potential of the nanoparticles was -43.8±2.34 mV. The nanoparticles were uniform distributed in spherical shape. Both MDA-MB-231 and MDA-MB-468 cells took up more FITC-NPs than FITC-BSA at different doses. The uptake of FITC-NPs in MDA-MB-231 (oncogenic KRAS) cells were higher than that in MDA-MB-468 cells (wild-type KRAS). The expression of KRAS was decreased by 49.6% after treatment with 1.5 µM siRNA targeting KRAS. The uptake of FITC-NPs was significantly decreased by ~50% (p < 0.05). The Cy7-NPs taken up by cells can be colocalized with TMR-dextran labelled macropinosomes and the colocalization was decreased after treatment with EIPA, indicating macropinocytosis was involved in the uptake process.
Conclusion: We evaluated the uptake of albumin nanoparticles in both oncogenic KRAS cells and wild-type KRAS cells. Increased uptake of nanoparticles was observed in oncogenic KRAS cells. Cells with higher oncogenic KRAS protein expression level tend to take up more nanoparticles. The albumin nanoparticles can be served as a promising drug delivery vehicle for treatment targeting oncogenic KRAS cells without further functionalization. The nanoparticles taken up by cells can be colocalized with macropinosomes and be inhibited by micropinocytosis inhibitor. However, other alternative endocytosis pathways may also be involved. Further studies are needed to confirm if multiple endocytosis pathways were contributed to the increased uptake of albumin nanoparticles.