Objectives: The objective of this study was to utilize a chitosan-based nanoparticle system as the delivery carrier for glutamic acid, a model for encapsulated biomolecules to visualize the in vitro release and accumulation of the encapsulated glutamic acid from chitosan nanoparticle (CNP) systems.
Methods: CNP was synthesized via ionic gelation routes utilizing tripolyphosphate (TPP) as a cross-linker. In order to track glutamic acid release, the glutamic acid was fluorescently-labeled with fluorescein isothiocyanate prior encapsulation into CNP.
Results: Light Scattering data concluded the successful formation of small-sized and mono-dispersed CNP at a specific volume ratio of chitosan to TPP. Encapsulation of glutamic acid as a model cargo into CNP led to an increase in particle size to >100 nm. The synthesized CNP exhibited spherical shape under Electron Microscopy. The formation of CNP was reflected by the reduction in free amine groups of chitosan following ionic crosslinking reactions. The encapsulation of glutamic acid was further confirmed by Fourier Transform Infrared (FTIR) analysis. Cell viability assay showed 70% cell viability at the maximum concentration of 0.5 mg/mL CS and 0.7 mg/mL TPP used, indicating the low inherent toxicity property of this system. In vitro release study using fluorescently-tagged glutamic acids demonstrated the release and accumulation of the encapsulated glutamic acids at 6 hours post treatment. A significant accumulation was observed at 24 hours and 48 hours later. Flow cytometry data demonstrated a gradual increase in intracellular fluorescence signal from 30 minutes to 48 hours post treatment with fluorescently-labeled glutamic acids encapsulated CNP.
Conclusion: These results therefore suggested the potential of CNP system towards enhancing the intracellular delivery and release of the encapsulated glutamic acids. This CNP system thus may serves as a potential candidate vector capable to improve the therapeutic efficacy for drugs and biomolecules in medical as well as pharmaceutical applications through the enhanced intracellular release and accumulation of the encapsulated cargo.
MATERIALS AND METHODS: In this study, we focus on two important drugs used for TB treatment - rifampicin (RIF) and isoniazid (INH) - and report a detailed study of RIF-loaded poly lactic-co-glycolic acid (PLGA) NPs and INH modified as INH benz-hydrazone (IH2) which gives the same therapeutic effect as INH but is more stable and enhances the drug loading in PLGA NPs by 15-fold compared to INH. The optimized formulation was characterized using particle size analyzer, scanning electron microscopy and transmission electron microscopy. The drug release from NPs and stability of drug were tested in different pH conditions.
RESULTS: It was found that RIF and IH2 loaded in NPs release in a slow and sustained manner over a period of 1 month and they are more stable in NPs formulation compared to the free form. RIF- and IH2-loaded NPs were tested for antimicrobial susceptibility against Mycobacterium tuberculosis H37Rv strain. RIF loaded in PLGA NPs consistently inhibited the growth at 70% of the minimum inhibitory concentration (MIC) of pure RIF (MIC level 1 µg/mL), and pure IH2 and IH2-loaded NPs showed inhibition at MIC equivalent to the MIC of INH (0.1 µg/mL).
CONCLUSION: These results show that NP formulations will improve the efficacy of drug delivery for TB treatment.