Bio-nanotechnological advancement of orally administered insulin nanoparticles: Comprehensive review of experimental design for physicochemical characterization.

Abstract

Therapeutic proteins are labile macromolecules that are prone to degradation during production, freeze-drying and storage. Recent studies showed that nanoparticles can enhance the stability and oral bioavailability of encapsulated proteins. Several conventional approaches (enzyme inhibitors, mucoadhesive polymers) and novel strategies (surface modification, ligand conjugation, flash nano-complexation, stimuli-responsive drug delivery systems) have been employed to improve the physiochemical properties of nanoparticles such as size, zeta potential, morphology, polydispersity index, drug release kinetics and cell-targeting capacity. However, clinical translation of protein-based nanoparticle is limited due to poor experimental design, protocol non-compliance and instrumentation set-up that do not reflect the physiological conditions, resulting in difficulties in mass production of nanoparticles and waste in research funding. In order to address the above concerns, we conducted a comprehensive review to examine the experimental designs and conditions for physical characterization of protein-based nanoparticles. Reliable and robust characterization is essential to verify the cellular interactions and therapeutic potential of protein-based nanoparticles. Importantly, there are a number of crucial factors, which include sample treatment, analytical method, dispersants, sampling grid, staining, quantification parameters, temperature, drug concentration and research materials, should be taken into careful consideration. Variations in research protocol and unreasonable conditions that are used in optimization of pharmaceutical formulations can have great impact in result interpretation. Last but not least, we reviewed all novel instrumentations and assays that are available to examine mucus diffusion capacity, stability and bioactivity of protein-based nanoparticles. These include circular dichroism, fourier transform infrared spectroscopy, X-ray diffractogram, UV spectroscopy, differential scanning calorimetry, fluorescence spectrum, Förster resonance energy transfer, NMR spectroscopy, Raman spectroscopy, cellular assays and animal models.