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Gemcitabine is a nucleoside analogue effective against a number of cancers. However, the full potential of this drug has not been realised, in part due to low oral bioavailability and frequent dosing requirements. This study reports the synthesis, in-vitro, ex-vivo and in-vivo evaluation of trimethyl chitosan (TMC) - CSKSSDYQC (CSK) peptide conjugates capable of enhancing the oral bioavailability of gemcitabine due to the ability to target intestinal goblet cells and promote intestinal cellular uptake. TMC was synthesized by a novel two-step methylation method to improve quanternization and yield. The CSK-TMC conjugates were prepared by ionic gelation to achieve particles sized at 173.6 ± 6.8 nm, zeta potential of +18.5 ± 0.2 mV and entrapment efficiency of 66.4 ± 0.1%, capable of sustained drug release. By encapsulating gemcitabine into CSK-TMC conjugates, an increased amount of drug permeated through porcine intestinal epithelial membranes compared with the unconjugated TMC nanoparticles (NPs). The rate of cellular uptake of drug loaded conjugates into HT29-MTX-E12 intestinal goblet cells, was time- and concentration-dependant. The conjugates underwent active transport associated with adsorptive mediated, clathrin and caveolae mediated endocytosis. In cellular transport studies, drug loaded conjugates had greater drug transport capability compared with drug solution and TMC NPs over the co-cultured Caco-2/HT29-MTX-E12 cell monolayer. The drug loaded conjugates exhibited electrostatic interaction with the intestinal epithelial cells. Both P-glycoprotein (P-gp) and multiple resistance protein-2 (MRP2) efflux affected the cellular transport of the conjugates. Importantly, during the pharmacokinetic studies, the orally administrated drug loaded into TMC NPs showed an improved oral bioavailability of 54.0%, compared with gemcitabine solution of 9.9%. Notable, the CSK-TMC conjugates further improved oral bioavailability to 60.1% and reduced the tumour growth rate in a BALB/c nude mouse model, with a 5.1-fold and 3.3-fold reduction compare with the non-treated group and gemcitabine solution group. Furthermore, no major evidence of toxicity was discernible on histologic studies of selected organs. In conclusion, the presented CSK-TMC conjugates and TMC nanoparticles both significantly improve the oral bioavailability of gemcitabine and have the potential for the treatment of breast cancer.