Expression, Microencapsulation of Recombinant Human Epidermal Growth Factor, and Release Study in Gastric Ulcer Representing Condition

Recombinant human epidermal growth factor (rhEGF) has been studied and expressed in various expression systems. It has been also commercialized and clinically used, yet limited to topical diseases. However, being naturally expressed in different tissues, the rhEGF is potential to be applied not only for external wound and skin disorders, but also to regenerates internal damaged epidermal cells such found in gastric ulcer. In the recent study, chitosan microparticles were developed to facilitate delivery of the rhEGF and to overcome gastric degradation that majorly interfere protein, particularly rhEGF oral administration. The rhEGF was expressed in E. coli BL21(DE3) and purified using Ni-NTA chromatography. The refolded rhEGF showed proliferation activity on MC7 cells. rhEGF loaded chitosan microparticles were stable in the gastric and specifically released the loaded rhEGF in the high oxidative environment in acidic pH representing gastric ulcer condition.

Nano-in-Micro-Particles Consisting of PLGA Nanoparticles Embedded in Chitosan Microparticles via Spray-Drying Enhances Their Uptake in the Olfactory Mucosa

Intranasal delivery has gained prominence since 1990, when the olfactory mucosa was recognized as the window to the brain and the central nervous system (CNS); this has enabled the direct site specific targeting of neurological diseases for the first time. Intranasal delivery is a promising route because general limitations, such as the blood-brain barrier (BBB) are circumvented. In the treatment of multiple sclerosis (MS) or Alzheimer’s disease, for example, future treatment prospects include specialized particles as delivery vehicles. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles are well known as promising delivery systems, especially in the area of nose-to-brain (N2B) delivery. Chitosan is also broadly known as a functional additive due to its ability to open tight junctions. In this study, we produced PLGA nanoparticles of different sizes and revealed for the first time their size-time-dependent uptake mechanism into the lamina propria of porcine olfactory mucosa. The intracellular uptake was observed for 80 and 175 nm within only 5 min after application to the epithelium. After 15 min, even 520 nm particles were detected, associated with nuclei. Especially the presence of only 520 nm particles in neuronal fibers is remarkable, implying transcellular and intracellular transport via the olfactory or the trigeminal nerve to the brain and the CNS. Additionally, we developed successfully specialized Nano-in-Micro particles (NiMPs) for the first time via spray drying, consisting of PLGA nanoparticles embedded into chitosan microparticles, characterized by high encapsulation efficiencies up to 51%, reproducible and uniform size distribution, as well as smooth surface. Application of NiMPs accelerated the uptake compared to purely applied PLGA nanoparticles. NiMPs were spread over the whole transverse section of the olfactory mucosa within 15 min. Faster uptake is attributed to additional paracellular transport, which was examined via tight-junction-opening. Furthermore, a separate chitosan penetration gradient of ∼150 µm caused by dissociation from PLGA nanoparticles was observed within 15 min in the lamina propria, which was demonstrated to be proportional to an immunoreactivity gradient of CD14. Due to the beneficial properties of the utilized chitosan-derivative, regarding molecular weight (150–300 kDa), degree of deacetylation (80%), and particle size (0.1–10 µm) we concluded that M2-macrophages herein initiated an anti-inflammatory reaction, which seems to already take place within 15 min following chitosan particle application. In conclusion, we demonstrated the possibility for PLGA nanoparticles, as well as for chitosan NiMPs, to take all three prominent intranasal delivery pathways to the brain and the CNS; namely transcellular, intracellular via neuronal cells, and paracellular transport.

Application of chitosan microparticles against human norovirus

Human norovirus (HuNoV) is the leading causative agent of foodborne outbreaks and is associated with the second most prevalent cause of waterborne infections in the United States. The goal of this research was to investigate the antiviral activity of chitosan microparticles (CM) against HuNoV GII.4 Sydney and its cultivable surrogate, Tulane virus (TuV), in suspensions mimicking fecally-contaminated water. CM was prepared by crosslinking chitosan molecules with sodium sulfate, and then its anti-noroviral activity was assessed using infectivity assay on TuV and RT-qPCR on TuV and HuNoV. A 3% CM suspension in PBS (pH 7.2) showed binding to TuV particles but with a negligible impact on virus infectivity (p>0.05). TuV and HuNoV suspended in fecal suspensions showed a 1.5-log10 reduction in genomic copies per ml following a 10-min contact time (p<0.05). Despite the negligible impact on viral infectivity, CM moderately binds to virus particles and helps purify environmental water by removing infectious virus particles. In this study, TuV served as a suitable surrogate for HuNoV by showing a similar log10 reduction in fecal suspension. Overall, the outcomes of thisresearch highlight the potential application of CM as a novel, natural treatment to minimize the spread of water-transmitted viral pathogens.