Formulation, Optimization and Efficacy of Gemcitabine Loaded Thermosensitive Liposomal Nanoparticles in the Treatment of Pancreatic Cance

Purpose: Pancreatic cancer has the lowest survival rate of all cancers. The current standard care for pancreatic cancer is gemcitabine. However, the success of this treatment is limited due to rapid systemic elimination following administration in vivo. Furthermore, early intervention has been impeded by difficulty in detection of early or small obscure tumors. To mitigate these challenges, increasing drug delivery efficiency and increasing plasma circulation time of Gem will remarkably improve the treatment of pancreatic cancer and quality of life of pancreatic cancer. We hypothesize that PEGylated thermosensitive liposomal nanoparticles loaded with either gemcitabine or gadolinium can penetrate deeper, increase the concentration of Gem in tumor site by applying an external mild hyperthermia and also aid in detection of small and obscure tumors.;Methods: PANC-1, BxPC-3, AsPC and MIA PaCa-2 cells lines were cultured in a required growth medium and at optimum growth conditions. Different heat sensitive and non-heat sensitive liposomes were investigated to determine the ideal lipid composition which best entraps Gem. The method of thin film hydration and extrusion was employed, and physicochemical properties determined. Gem-TSLnps, a thermosensitive liposome entrapped the highest amount of Gem. Also, release studies was performed at 28°C through to 45 °C. Gd-TSLnps phantom was also developed based on the lipid composition of Gem-TSLnps with little modifications to track early or obscure tumors noninvasively both in vitro and ex-vivo by MRI. Cell viability was conducted using Gem and Gem-TSLnps plus mild hyperthermia whereas, clonogenic assay was used to evaluate cell's reproductive death after treating cells with different concentrations of Gem and Gem-TSLnps plus mild hyperthermia at 42°C. To assess the effectiveness of our formulation in vivo, MIA PaCa-2 cells were chosen for in vivo studies as they have been reported to be one of most invasive cells lines with aggressive metastatic potential and most importantly highly tumorigenic with a latency period of 3 weeks for tumors to form. Injections were given every other day per week for two weeks. Measurement of tumor growth was determined every other day for 24 days. Systemic adverse effect of our formulation was determined by measuring serum levels of Liver enzymes; alanine amino transferase, aspartate amino transferase and alkaline phosphatase. The pharmacokinetic properties and biodistribution of Gd-TSLnps and Gem-TSLnps were investigated via inductively coupled plasma mass spectrometry and HPLC-UV/VIS respectively. Results: The TSLnps and Gem-TSLnps size values were 132.9 +/- 0.3 nm and 216.1 +/- 0.5 nm respectively. Entrapment efficiency of Gem into TSLnp was 41.10 +/- 2.02 %. The hydrodynamic diameter and surface charge for Gd-TSLnps were found to be 216.10 +/- 0.565 nm. MR imaging of Gd-TSLnps phantoms showed that strongest contrast for Gd-TSLnps was at Gd concentration of 14.3 mM while 0.3 mM Gd of Gd-TSLnps exhibited the weakest contrast comparable with the control agarose sample. Interestingly, Ex-vivo MR imaging revealed a bright contrast of the T1 weighted image which was observed after 30 min when injected with Gd-TSLnps only, nonetheless, images acquired after 60 and 90 min showed no visible contrast. Also, mice injected with equivalent amount of Gd only showed no visible contrast at 30, 60 and 90 min. In vitro release pattern of Gem-TSLnps showed a significant Gem release at 42°C compared to that released at 37°C. In cytotoxicity study, we observed that Gem-TSLnps showed significant cell growth inhibition in vitro in BxPC-3, AsPC and PANC-1 compared with Gem but to a lesser extent in MIA PaCa-2 cells. In clonogenic assay, we observed that cells treated with Gem showed a significant increase in cell survival compared with the groups that were exposed to Gem-TSLnps. Flow cytometric and confocal studies revealed a higher cellular uptake of Rho-TSLnps. Tumor volume of mice treated with Gem alone was 1.8-fold higher compared to the group treated with Gem-TSLnps + mHT. In general, tumor suppression by Gem-TSLnps + mHT was significantly higher compared to Gem or Gem-TSLnps. For liver functioning test, no significant difference in AST and ALT serum levels was observed among the control, Gem and Gem-TSLnps + mHT groups, however, ALP level was significantly elevated in Gem and Gem-TSLnps + mHT treated groups compared to the control group. But the ALP level in Gem and Gem-TSLnps + mHT was similar.;Conclusion: Overall, our nano-delivery system: i) enhanced the anti-tumor effect of gemcitabine ii) improved pharmacokinetic profile and tumor distribution of gemcitabine and, iii) increased MR sensitivity of gadolinium in tumor imaging. (Abstract shortened by ProQuest.).