Clove Oil-Nanostructured Lipid Carriers: A Platform of Herbal Anesthetics in Whiteleg Shrimp (Penaeus vannamei).

Whiteleg shrimp (Penaeus vannamei) have been vulnerable to the stress induced by different aquaculture operations such as capture, handling, and transportation. In this study, we developed a novel clove oil-nanostructured lipid carrier (CO-NLC) to enhance the water-soluble capability and improve its anesthetic potential in whiteleg shrimp. The physicochemical characteristics, stability, and drug release capacity were assessed in vitro. The anesthetic effect and biodistribution were fully investigated in the shrimp body as well as the acute multiple-dose toxicity study. The average particle size, polydispersity index, and zeta potential value of the CO-NLCs were 175 nm, 0.12, and -48.37 mV, respectively, with a spherical shape that was stable for up to 3 months of storage. The average encapsulation efficiency of the CO-NLCs was 88.55%. In addition, the CO-NLCs were able to release 20% of eugenol after 2 h, which was lower than the standard (STD)-CO. The CO-NLC at 50 ppm observed the lowest anesthesia (2.2 min), the fastest recovery time (3.3 min), and the most rapid clearance (30 min) in shrimp body biodistribution. The results suggest that the CO-NLC could be a potent alternative nanodelivery platform for increasing the anesthetic activity of clove oil in whiteleg shrimp (P. vannamei).

Improvement of the multi-performance biocharacteristics of cordycepin using BiloNiosome-core/chitosan-shell hybrid nanocarriers.

Cordycepin, a derivative of the nucleotide adenosine, has displayed several pharmacological activities including enhanced apoptosis and cancer cells inhibition. However, oral administration of cordycepin has limited practical use due to its poor bioavailability in the intestine. Herein, we developed and demonstrated a hybrid nanocarrier system in the form of biloniosome-core/chitosan-shell hybrid nanocarriers (HNCs) in order to improve the bio-characteristics of cordycepin. In this study, HNCs were prepared by using a solvent (ethanol) injection method involving cordycepin as the biloniosome core and mucoadhesive chitosan biopolymer as a coating shell. Our results showed that the cordycepin-loaded HNCs were positively charged with enhanced mucoadhesive characteristics and highly stable in gastric fluid. The increased permeability of cordycepin-loaded HNCs compared with standard cordycepin was confirmed by in vitro intestinal permeation study across the human intestinal barrier. In addition, we demonstrated that the cordycepin-loaded HNCs are able to release their components in an active form resulting in enhanced anti-cancer activity in two-dimensional (2D) cell cultures as well as in three-dimensional (3D) multi-cellular spheroids of colon cancer cells. Further, quantitative real time PCR analysis of apoptotic gene expression revealed that cordycepin HNCs can induce apoptosis in cancer cells by negatively regulating the expression of B-cell lymphoma-extra large (BCL-XL). I Overall our results showed that the hybrid nanocarrier systems represent a promising strategy for improving the bio-characteristics of cordycepin which can be considered as a potential anti-cancer agent for colorectal cancer chemotherapy.

Enhanced efficacy of immersion vaccination in tilapia against columnaris disease by chitosan-coated "pathogen-like" mucoadhesive nanovaccines.

Red tilapia (Oreochromis sp.) has become one of the most important fish in aquaculture. Bacterial infection caused by Flavobacterium columnare, the causative agent of columnaris disease, has been now identified as one of the most serious infectious diseases in farmed red tilapia and cause major financial damage to the producers. Among the effective prevention and control strategies, vaccination is one of the most effective approach. As the surface of living fish is covered by mucus and directly associated with the mucosal immunity, we therefore hypothesized that better adsorption on mucosal surfaces and more efficient vaccine efficacy could be enhanced biomimetic nanoparticles mimicking the mucoadhesive characteristic of live F. columnare. In this work, we describe an effective approach to targeted antigen delivery by coating the surface of nanoparticles with mucoadhesive chitosan biopolymer to provide "pathogen-like" properties that ensure nanoparticles binding on fish mucosal membrane. The physiochemical properties of nanovaccines were analyzed, and their mucoadhesive characteristics and immune response against pathogens were also evaluated. The prepared vaccines were nano-sized and spherical as confirmed by scanning electron microscope (SEM). The analysis of hydrodynamic diameter and zeta-potential also suggested the successful modification of nanovaccines by chitosan as indicated by positively charged and the overall increased diameter of chitosan-modified nanovaccines. In vivo mucoadhesive study demonstrated the excellent affinity of the chitosan-modified nanovaccines toward fish gills as confirmed by bioluminescence imaging, fluorescent microscopy, and spectrophotometric quantitative measurement. Following vaccination with the prepared nanovaccines by immersion 30 min, the challenge test was then carried out 30 and 60 days post-vaccination and resulted in high mortalities in the control. The relative percent survival (RPS) of vaccinated fish was greater than 60% for mucoadhesive nanovaccine. Our results also suggested that whole-cell vaccines failed to protect fish from columnaris infection, which is consistent with the mucoadhesive assays showing that whole-cell bacteria were unable to bind to mucosal surfaces. In conclusion, we could use this system to deliver antigen preparation to the mucosal membrane of tilapia and obtained a significant increase in survival compared to controls, suggesting that targeting mucoadhesive nanovaccines to the mucosal surface could be exploited as an effective method for immersion vaccination.

The potential of mucoadhesive polymer in enhancing efficacy of direct immersion vaccination against Flavobacterium columnare infection in tilapia.

Vaccination is the most effective approach for prevention of infectious diseases in aquaculture. Although immersion vaccination is more applicable compared to in-feed/oral administration and injection, this method suffers from low potency as the efficiency of uptake of antigens through mucosal membranes is limited. In this study, we have successfully developed a mucoadhesive vaccine delivery system to enhance the efficacy of direct immersion vaccination against Flavobacterium columnare, the causative agent of columnaris disease in red tilapia. A formalin-killed negatively charged, bacterial cell suspension was used to prepare a mucoadhesive vaccine by electrostatic coating with positively charged chitosan. Our results demonstrate that the chitosan-complexed vaccine greatly increases its mucoadhesiveness, thus increasing the chances of vaccine uptake by the gill mucosa and improving the protection obtained against columnaris infection. The surface charge of the chitosan-complexed vaccine was altered from anionic to cationic after chitosan modification. Tilapia were vaccinated with the prepared chitosan-complexed vaccine by immersion. The challenge test was then carried out 30 and 60 days post vaccination, which resulted in a high level of mortalities in the non-vaccinated and uncomplexed vaccine groups. A high relative percentage survival (RPS) of vaccinated fish was noted with the mucoadhesive vaccine. Our results indicated that the naked vaccine failed to protect the fish from columnaris infection, which is consistent with the mucoadhesive assays performed during the study showing that the naked vaccine was unable to bind to mucosal surfaces. This system is therefore an effective method for immersion vaccination in order to deliver the antigen preparation to the mucosal surface membrane of the fish.