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.

吉西他滨是对多种癌症有效的核苷类似物。但是，该药物的全部潜力尚未实现，部分原因是口服生物利用度低和需要频繁用药。这项研究报告了体外，离体和体内合成壳聚糖三甲基壳聚糖（TMC）-CSKSSDYQC（CSK）肽缀合物的评估，由于其靶向肠杯状细胞和促进肠道细胞摄取的能力，因此能够增强吉西他滨的口服生物利用度。通过新颖的两步甲基化方法合成TMC，以提高量化和产率。通过离子凝胶化制备CSK-TMC共轭物，以实现173.6±6.8 nm的粒径，+ 18.5±0.2 mV的ζ电位和66.4±0.1％的包封效率，能够持续释放药物。与未结合的TMC纳米颗粒（NPs）相比，通过将吉西他滨封装到CSK-TMC结合物中，药物通过猪肠上皮膜的渗透量增加。HT29-MTX-E12肠杯状细胞的药物结合缀合物的细胞摄取率，是时间和浓度依赖的。结合物经历了与吸附介导的网格蛋白和小窝介导的胞吞作用有关的主动转运。在细胞运输研究中，在共培养的Caco-2 / HT29-MTX-E12细胞单层上，与药物溶液和TMC NP相比，载有药物的结合物具有更高的药物运输能力。载有药物的结合物表现出与肠上皮细胞的静电相互作用。P-糖蛋白（P-gp）和多重耐药蛋白2（MRP2）外排均影响缀合物的细胞转运。重要的是，在药代动力学研究期间，与吉西他滨溶液的9.9％相比，口服加载到TMC NP中的药物的口服生物利用度提高了54.0％。值得注意的是，CSK-TMC偶联物进一步提高了口服生物利用度至60。与未治疗组和吉西他滨溶液组相比，在BALB / c裸鼠模型中肿瘤生长率降低了1％，降低了5.1倍和3.3倍。此外，在选定器官的组织学研究中，没有发现毒性的主要证据。总之，提出的CSK-TMC缀合物和TMC纳米颗粒均可以显着提高吉西他滨的口服生物利用度，并具有治疗乳腺癌的潜力。