Development of novel nanoparticulate delivery system for oral delivery of gemcitabine to treat breast cancer

Abstract

Background and Aim: In New Zealand women, breast cancer has a highest rate of incidence of any cancer. In 2015, there were approximate 60,300 new cases of breast cancer diagnosed globally, which is second most common cancer overall. To address breast cancer, chemotherapy is typically administered parenterally. However, this is an unpleasant and inconvenient administration route, and often leads to high peak levels of drug in the systemic circulation above the maximum tolerated concentration (MTC) resulting in a multitude of side effects. Oral chemotherapy is attractive with better patient acceptability, good therapeutic efficacy, and low cost. However, there are many obstacles to achieve oral drug delivery including physical and biochemical barriers, such as the epithelial barrier of the small intestine, degradation through the acidic environment of the stomach and digestive enzymes throughout the gastrointestinal tract, as well as efflux pumps which limit oral drug absorption. Gemcitabine is a promising drug candidate with proven activity against breast cancer, however, it has an oral bioavailability of less than 10%, due to its high hydrophilicity and low permeability through intestinal epithelium. Therefore, the aim of this project was to develop a novel nanoparticulate drug delivery system for oral delivery of gemcitabine, to improve its oral bioavailability. Methods: Two different polymeric nanoparticulate delivery systems were designed suitable for the oral delivery of gemcitabine. The first was gemcitabine-loaded TMC modified PLGATPGS nanoparticles (NPs) prepared through a modified solvent evaporation technique. The PLGA-TPGS random copolymer was synthesized prior to the fabrication of NPs. A central composite design (CCD) was applied to optimize the formulation parameters. The second delivery system of gemcitabine loaded TMC-CSK NP was fabricated via an ionic gelation method. The TMC polymer was synthesized by using a new two-step methylation method prior to preparing the TMC based NPs. The physical and chemical properties of both nanoparticulate delivery systems were determined including particles size, zeta potential, entrapment efficiency, in-vitro drug release and ex-vivo drug permeation over the porcine epithelial membrane, and the optimal formulations were selected. A co-cultured Caco-2 and HT29-MTX-E12 cell model was set up to determine cytotoxicity, cellular uptake and transport studies of the drug solution and optimal drug loaded NPs. Finally, the pharmacokinetic parameters associated with different formulation were determined using a Sprague-Dawley (SD) rat model. The tumour growth rate associated with the drug solution and the drug loaded NPs were investigated using a BALB/c nude mouse model. Results and discussion: The optimal formulations of drug loaded TMC modified PLGATPGS NPs and drug loaded TMC-CSK NPs showed particle size of 243.21 ± 21.72 nm, and 173.60 ± 6.82 nm, zeta potential of +14.70 ± 1.31 mV, and +18.50 ± 0.22 mV, entrapment efficiency of 76.43 ± 0.21%, and 66.43 ± 0.13%, respectively. Particles of less than 500 nm show significantly higher absorption than larger particles across intestinal epithelium, thus the particle sizes of both NPs are suitable for oral absorption. NPs with zeta potentials more positive than +15 mV or more negative than -15 mV are considered stable, thus the two NPs are considered having good steric stability. In addition, the positive charged NPs promote mucoadhesion with the negatively charged intestinal mucosa, through electrostatic interaction. The high entrapment efficiency results were promising and are higher than most polymeric NPs delivery systems reported. Scanning electron microscopy (SEM) showed the TMC modified PLGA-TPGS NPs were spherical with a smooth particle surface, while the TMC-CSK NPs had more irregular shape with a craggy particle surface. They both showed sustain drug release profiles during in vitro drug release studies, and greater drug permeation compared to drug solution over porcine epithelial membrane in the ex-vivo drug permeation studies. Moreover, both optimal drug loaded NPs exhibited good stability in terms of particle size and drug entrapment over 3 months stored at 4°C. The cytotoxicity of gemcitabine solution, gemcitabine loaded TMC modified PLGA-TPGS NPs, and gemcitabine loaded TMC-CSK NPs on Caco-2/HT29-MTX-E12 cells showed dose dependence and with IC50s of 529.4 ± 67.2 μg.mL-1, 1881.4 ± 51.5 μg.mL-1 and 1682.4 ± 27.9 μg.mL-1 respectively, indicating the drug loaded NPs were less toxic to the intestinal epithelial cells compared to the drug solution. The rate of cellular uptake of both optimal drug loaded NPs was time-, temperature-, and concentration- dependant. Cellular uptake for the gemcitabine loaded TMC modified PLGA-TPGS NPs undergo active transport involving adsorptive mediated endocytosis and caveloae mediated endocytosis, while the gemcitabine loaded TMC-CSK NPs was through active transport associate with adsorptive mediated, clathrin and caveolae mediated endocytosis. In cellular transport studies, both drug loaded NPs had greater drug transport capability compared to drug solution over the Caco-2/HT29- MTX-E12 cell membrane. For the transport mechanism studies, both NP formulations showed electrostatic interaction with the intestinal epithelial cells. P glycoprotein (P-gp) efflux affected the cellular transport for both NPs. By blocking the P-gp efflux pump, more drug loaded NPs were transported through the cell membrane. The multiple resistance protein-2 (MRP2) only affected TMC-CSK NPs to some extent. Interestingly, for the TMC modified PLGA-TPGS NPs, the addition of the MRP2 inhibitor resulted in a reduction in the efflux of gemcitabine suggesting that the role of MRP2 in the efflux of gemcitabine loaded TMC modified PLGA-TPGS NPs can be neglected. Moreover, EDTA is able to activate the cellular protein kinase C (PKC) by depletion of extracellular calcium via chelation, resulting in tight junction opening. The addition of EDTA significantly enhanced the cellular transport for both drug loaded NPs, facilitating the transport of the NPs via the paracellular route. In the in vivo pharmacokinetic studies, the half-life (t1/2) and oral bioavailability of gemcitabine were significantly improved in drug loaded NPs compared to drug solution group. The t1/2 of gemcitabine loaded TMC-CSK NPs and gemcitabine loaded TMC modified PLGA-TPGS NPs were of 77.16 ± 24.20 hr and 69.98 ± 20.50 hr, respectively, compared with 9.40 ± 2.13 hr for the gemcitabine solution. The absolute oral bioavailability of gemcitabine loaded TMC-CSK NPs (55.20%), was 1.1-fold and 6.1-fold higher than that of gemcitabine loaded TMC modified PLGA-TPGS NPs (49.92%) and gemcitabine solution (9.86%), respectively. In pharmacodynamics studies, the drug loaded NPs had greater inhibition of tumour growth rate compared with the drug solution (p < 0.01). The gemcitabine loaded TMC-CSK NPs group had the greatest inhibition of tumour growth, with 3.12-fold and 1.78-fold reduction compared to saline control group and gemcitabine solution group, respectively. This result corresponds to the pharmacokinetic studies with greater oral bioavailability and longer plasma half-life of gemcitabine loaded TMC-CSK NPs group compared to all other groups. Conclusion: This project has demonstrated that TMC modified PLGA-TPGS NPs and TMCCSK NPs can be utilised as controlled release drug delivery systems for the oral delivery of gemcitabine. Encapsulated gemcitabine is able to overcome the physical and biochemical barriers in GIT, to enhance the drug absorption over the intestinal epithelial membrane, therefore improving the oral bioavailability of gemcitabine, and promoting the anticancer therapeutic efficacy. The promising results confirmed the two developed NPs are promising platforms for developing future oral chemotherapy products loaded with gemcitabine.