Intracellular trafficking and drug release from fluorescently-labeled chitosan nanoparticle systems for development of innovative drug delivery systems

The increased bioavailability of essential biomolecules such as drugs, DNA and peptides is pre-requisite for efficient intracellular efficacy on drug delivery systems. Nanotechnological-based approaches for drug delivery applications potentially promotes a better distribution of energy in vivo, increasing the intracellular uptake of biomolecules for enhanced therapeutic uptake. Realising the ubiquitous utilization of nanoparticles in an increasing myriad of research fields, investigations into nanoparticle uptake, cargo release, as well as nanoparticle carrier persistence are pertinent towards their consequent optimization and development. We describe in this work, the elucidation of nanoparticle uptake and sustained release of its encapsulated cargo in colon cancer cells to model a nanoparticle-mediated drug delivery system. Chitosan nanoparticles were synthesized through ionic gelation routes and characterized by means of light scattering, electron microscopy, and infrared spectroscopic analysis. The nanoparticles were encapsulated with a fluorescently-modified amino acid for in vitro tracking, and its intracellular release was quantitated in a time-dependent study using flow cytometry and fluorescent microscopy. Cytotoxic analysis was subsequently performed to evaluate any inherent efficacy of the nanoparticle for use as a candidate delivery system. Findings arising from our analyses showed that intracellular uptake of nanoparticles occurred within 30 mins of cell treatment; and continually took place up to 48 hours post treatment. Interestingly, release of cargo only occurred 6 hours post treatment and a controlled release system was exhibited up to 48 hours without extracellular leakage. MTT assay showed very low toxicity of the 60-180nm size particles; demonstrating a potential of the chitosan nanoparticle system for use as a systemic, slow release system for drug delivery. Conclusions derived from this study is hoped to provide sufficient data towards more critical developments of nanoparticle delivery systems for targeted and enhanced drug delivery parameters, most clinically relevant in the pharmaceutical and medical fields

Synthesis of a nanoparticle system for the enhanced accumulation of fluorescently-labelled amino acids encapsulated in monodispersed chitosan nanoparticle system

Poor in vivo bioavailability of nutrient is a major challenge in efficient delivery of nutraceutics. The increased bioavailability of nutraceuticals is prerequisite for efficient absorption by the gastrointestinal system. Nanotechnology-based approaches for nutraceutical applications could potentially increase absorption of nutrients and enhance its cellular accumulation due to its nanosize and promote better in vivo energy biodistribution. However, the dynamics of intracellular cell trafficking of nanoparticles and nutraceutical release has remain scarcely studied. This study describes a non-efficacious nanoparticle-mediated system for the encapsulation and delivery of fluorescently-labelled amino acids using tripolyphosphate as a crosslinker. Light scattering data showed successful formation of particle size as small as 65.69 nm with low polydispersity index (PDI) value of 0.178 at specific volume ratios of chitosan to tripolyphosphate. Following encapsulation, nanoparticle size and PDI value increased to 182.73 nm and 0.257 respectively discern successful accommodation of the fluorescently-labelled amino acid within its core. In vitro visualization of amino acids release and accumulation via fluorescence microscopy suggested that encapsulated amino acids were efficiently accumulated into Vero3 cell cytoplasm at 24 hours post treatment with localization in proximity to the cell nucleus. These results therefore suggested that the chitosan nanoparticle system developed was able to enhance the intracellular accumulation of glutamic acids and may serve as a suitable carrier for nutraceuticals delivery

Development of a nanoparticle-mediated delivery system to study release of fluorescently-labeled glutamic acid encapsulated in chitosan nanoparticles

Inefficient cellular delivery and intracellular accumulation are major drawbacks towards achieving favorable therapeutic responses for many therapeutic drugs and biomolecules. To tackle this issue, nanoparticle-mediated delivery vectors have been aptly explored as a promising delivery strategies capable to enhance the bioavailability and cellular localization of biomolecules and therefore, improve their therapeutic efficacies. However, the dynamics of intracellular biomolecule release and accumulation from such nanoparticle systems has remain scarcely studied. Therefore, in this study, chitosan nanoparticle (CNP) was synthesized to serve as the delivery carrier for glutamic acid, a model for encapsulated biomolecules. Various chemical and morphological analyses were conducted to verify the nanoparticle formation and glutamic acid loading. In order to track the glutamic acid release and accumulation, the glutamic acid was then fluorescently-labeled with fluorescein isothiocyanate (FITC) prior encapsulation into CNP. This study therefore describes the encapsulation, release and accumulation of fluorescently labeled-glutamic acid from a robust, non-efficacious chitosan-based nanoparticle delivery system using tripolyphosphate (TPP) as a cross-linker. Light Scattering data concluded the formation of small-sized and monodispersed CNP at a specific volume ratio of chitosan to TPP. Following encapsulation, nanoparticle size increased exponentially to >100 nm to accommodate the glutamic acids within its core. Electron microscopy images revealed a discrete and spherical shape of CNP populations. The particle size increased over 100 nm following glutamic acids loading as reflected by Light Scattering data. Formation of CNP was further reflected by reduction in free amine groups in chitosan, and Fourier Transform Infrared detected peaks of functional groups belonging to both chitosan and TPP. Approximately 60% glutamic acid were efficiently encapsulated into CNP, which further suggested the potential of CNP as a drug delivery vehicle. Cell viability assay demonstrated a low toxicity property of CNP; conferring about 70% cell viability of 786-O cancer cells at the highest concentration used. In vitro tracking of glutamic acids release via fluorescence microscopy revealed a time-dependent release and controlled accumulation of fluorescently modified-glutamic acids from CNP into 786-O cells from 6 hours to 48 hours treatment points. The fluorescently-labeled glutamic acids was found to be release into cells as early as 6 hours post treatment. The fluorescence was gradually increased at 24 hours later and persisted inside the treated cells up to 48 hours. 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. These results therefore suggested the potential of CNP system towards enhancing the intracellular delivery and release of the encapsulated glutamic acids as well as controlling their accumulation and retention over prolong period of time. 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