Purpose: Pancreatic cancer is the fourth leading cause of cancer related death in the United States with a five-year survival rate of 6%. Although surgery may be an effective way to treat pancreatic cancer, according to the American Cancer Society, 80% of pancreatic cancer is unresectable and local recurrence can develop after tumor resection in many cases. Delivery of a chemotherapeutic drug specifically to pancreatic cancer cells has a potential to enhance the treatment efficacy and limit adverse side effects upon healthy organs. Somatostatin receptor 2 (SSTR2) abundantly expresses in pancreatic cancer cells, and therefore SSTR2 ligand somatostatin or its analogs can be used as a targeting moiety for a pancreatic cancer targeted drug delivery system. In the present study, a modified synthetic somatostatin (SST) analog, 5-pentynecarbonyl-octreotide (OCT), was employed as a SSTR2 targeting agent for a liposomal paclitaxel delivery system (OCT-PTX-Liposome). Human pancreatic cancer cells with different expression of SSTR2 receptors have been used for testing of the proposed pancreatic cancer targeted drug delivery system in vitro.
Methods: Human pancreatic cell lines PANC-1 and CFPAC-1 with substantially different expression of SSTR2 were used. Both cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA). To measure the gene expression of SSTR2, both cell lines were incubated in cell medium, and cells were harvested until 70% confluence. Total RNA was extracted from both cell lines using RNeasy® Mini Kit (Qiagen, Valencia, CA) according to manufacturer’s instructions. The harvested mRNAs were reverse-transcribed into cDNAs using the High Capacity cDNA Reverse Transcription Kits (Applied biosystems, Carlsbaad, CA) with Veriti 96 well thermal cycler (Applied Biosystems, Carlsbaad,CA). Then cDNA levels for SSTR-2 in PANC-1 cells and CFPAC-1 cells were measured by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using Step One Plus System (Applied Biosystems, Carlsbad, CA).
Liposome was prepared by thin film method. Lipids ratio for the liposomes was 15:5:2:1 mole% of EPC : DPPC : Cholesterol :1,2,-distearoyl-sn-glycero-3-phosphoethanolamine-N-aminopolyethelenglycol- 2000 ammonium salt (DSPE-PEG), respectively. Paclitaxel was incorporated in to the liposomal membrane. Customized 5-pentynecarbonyl-octreotide (with alkyne group) was conjugated to DSPE-PEG-Azide at 1.5:1 mole ratio to produce targeted liposomal drug formulation. In an aliquot of delivery system, Oregon Green R 488 Taxol (green fluorescence) and Egg Liss Rhod PE (Red fluorescence) were used to formulate paclitaxel containing liposomes. Free paclitaxel and empty liposomes were used for comparison. Cellular uptake of liposomal paclitaxel by both cell lines were observed by Confocal Microscope (Leica TCS SP8, CarlZeiss, Germany) and images were analyzed by LAS AF Lite Leica software. Particle size and zeta potential of formulated samples were measure by Malvern Zetasizer NanoSeries (Marvern Instruments, UK) at 25 °C. MTT assay was used to measure cytotoxicity of tested compounds in human PANC-1 and CFPAC-1 pancreatic cancer cells.
Results: RT-qPCR data showed that the expression of SSTR2 mRNA in PANC-1 cells was almost seven fold higher when compared with CFPAC-1 cells. The results of confocal microscope study clearly indicated that fluorescence liposomes (red fluorescence) were internalized into both of cells and localized in the cytoplasm; paclitaxel (green fluorescence) was successfully released from liposomes and localized in the cytoplasm and nuclei (labeled with a blue fluorescence dye). Liposomes and paclitaxel were distributed homogenously from the top to the bottom of the cells. The IC50 values of liposomal PTX was significantly lower when compared with free drug while this difference was more pronounced in PANC-1 with higher SSTR2 expression.
Conclusion: A chemically modified somatostatin analog (5-pentynecarbonyl-octreotide) conjugated to paclitaxel-loaded liposomes were formulated for targeted chemotherapy of pancreatic cancer. It was found that delivery of paclitaxel by liposomes improved intracellular internalization of this hydrophobic chemotherapeutic drug. Moreover, liposomal dosage form of Paclitaxel induced more pronounced anticancer effect on human pancreatic cancer cells when compared with free drug. Therefore, the proposed 5-pentynecarbonyl-octreotide conjugated liposomal paclitaxel represents a promising delivery system specific to pancreatic cancer cells and tissues with high expression of SSTR2.
YIngjia Chen– Rutgers University Ernest Mario School of Pharmacy
Andriy Kuzmov– Graduate Student, Rutgers University, Piscataway
Olga Garbuzenko– Rutgers University Ernest Mario School of Pharmacy
Olena Taratula– Oregon State University
Tamara Minko– Rutgers University Ernest Mario School of Pharmacy