Nanoparticles assembled from fucoidan and trimethylchitosan as anthrax vaccine adjuvant: In vitro and in vivo efficacy in comparison to CpG.

Fucoidan/trimethylchitosan nanoparticles (FUC-TMC-NPs) have the potential to improve the immunostimulating efficiency of anthrax vaccine adsorbed (AVA). FUC-TMC-NPs with positive (+) or negative (-) surface charges were prepared via polyelectrolyte complexation, both charged NP types permitted high viability and presented no cytotoxicity on L929, A549 and JAWS II dendritic cells. Flow cytometry measurements indicated lower (+)-FUC-TMC-NPs internalization levels than (-)-FUC-TMC-NPs, yet produced high levels of pro-inflammatory cytokines IFN-γ, IL12p40, and IL-4. Moreover, fluorescence microscope images proved that both charged NP could deliver drugs into the nucleus. In vivo studies on A/J mice showed that (+)-FUC-TMC-NPs carrying AVA triggered an efficient response with a higher IgG anti-PA antibody titer than AVA with CpG oligodeoxynucleotides, and yielded 100 % protection when challenged with the anthracis spores. Furthermore, PA-specific IgG1 and IgG2a analysis confirmed that (+)-FUC-TMC-NPs strongly stimulated humoral immunity. In conclusion, (+)-FUC-TMC-NP is promising anthrax vaccine adjuvant as an alternative to CpG.

A fucoidan-quaternary chitosan nanoparticle adjuvant for anthrax vaccine as an alternative to CpG oligodeoxynucleotides.

We examined the efficacy of fucoidan-N-(2-hydroxy-3-trimethylammonium)propylchitosan nanoparticles (FUC-HTCC NPs) as adjuvants for anthrax vaccine adsorbed (AVA). Positively and negatively surface-charged FUC-HTCC NPs were prepared via polyelectrolyte complexation by varying the mass ratio of FUC and HTCC. When cultured with L929 cells or JAWS II dendritic cells, both charged NPs showed high cell viability and low cytotoxicity, observed via MTT assay and lactate dehydrogenase release assay, respectively. In addition, we have monitored excellent NPs uptake efficacy by dendritic cells and observed that combining FUC-HTCC NPs with AVA significantly increases the magnitude of IgG-anti-protective antigen titers in A/J mice compared to that by CpG oligodeoxynucleotides plus AVA or AVA alone, and PA-specific IgG1 and IgG2a analysis confirmed that FUC-HTCC NPs strongly stimulated humoral immunity. Furthermore, FUC-HTCC NPs plus AVA provided a superior survival rate (100%) of A/J mice compared to CpG oligodeoxynucleotides plus AVA (75%) or AVA alone (50%) following anthrax lethal toxin challenge. The findings support FUC-HTCC NPs as a potential adjuvant of AVA for rapid induction of protective immunity.

Chondroitin sulfate-stabilized silver nanoparticles: Improved synthesis and their catalytic, antimicrobial, and biocompatible activities.

Herein, we describe an improved procedure for the green synthesis of chondroitin sulfate stabilized silver nanoparticles (ChS-AgNPs). Glucose was used as a reducing agent under alkaline conditions to obtain a small particle size (<10 nm), and the reduction was complete within one hour at room temperature. The concentration of NaOH affected the reaction rate, formation yield, and particle size of ChS-AgNPs. The formation of AgNPs was confirmed using UV-vis, TEM, XRD, and XPS. ChS-AgNPs showed excellent catalytic activities in the reduction of 4-nitrophenol by NaBH4, and the reaction rate increased linearly with increasing catalyst amounts. The antimicrobial activities of ChS-AgNPs against A. baumannii (including multidrug-resistant strains), E. coli, P. aeruginosa, and S. aureus were evaluated using the broth microdilution method. Finally, from the morphological observations and cell cycle analysis of L929 cells, we found that ChS-AgNPs exhibited antimicrobial and biocompatible activities.

Positively charged gold nanoparticles capped with folate quaternary chitosan: Synthesis, cytotoxicity, and uptake by cancer cells.

In this study, we synthesized various quaternary chitosan derivatives and used them to stabilize gold nanoparticles (AuNPs). These chitosan derivatives comprised N-(2-hydroxy)propyl-3-trimethylammonium chitosan chloride (HTCC), folate-HTCC, galactosyl-HTCC, and their fluorescein isothiocyanate-conjugated derivatives. Various positively surface-charged AuNPs were prepared under alkaline conditions using glucose as a reducing agent in the presence of the HTCC derivatives (HTCCs). The effects of the concentration of NaOH, glucose, and HTCCs on the particles size, zeta potential, and stability were studied in detail. Cell cycle assays verify that none of the HTCCs or HTCCs-AuNPs was cytotoxic to human umbilical vein endothelial cells. Flow cytometry analysis showed that the folate HTCC-AuNPs were internalized in Caco-2, HepG2, and HeLa cancer cells to a significantly greater extent than AuNPs without folate. But, galactosyl HTCC-AuNPs only showed high cell uptake by HepG2 cells.

Positively and negatively surface-charged chondroitin sulfate-trimethylchitosan nanoparticles as protein carriers.

Positively and negatively surface-charged nanoparticles (NPs) were prepared with chondroitin sulfate (ChS) and trimethylchitosan (TMC). NP size, surface charge, formation yield, and water content were investigated as a function of weight ratio and concentration. Size and zeta potential were controlled by varying the ChS/TMC mass ratio. FTIR spectra revealed interactions among composite NP constituents. TEM images showed that the NPs were nearly spherical, with an average size of ∼ 300 nm. Encapsulation efficiency increased in positively charged NPs with increases in fluorescein isothiocyanate-bovine serum albumin concentration. Negatively charged NPs had only 10-20% encapsulation efficiency. The release profile, release kinetics and mechanism of positively charged ChS-TMC NPs were studied in vitro. NP cytocompatibility and uptake were verified ex vivo. Both types of NPs were taken up and retained in cells. A549 cells took up more positively charged (49.4%) than negatively charged (35.5%) NPs.

Novel protein-loaded chondroitin sulfate-N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan nanoparticles with reverse zeta potential: preparation, characterization, and ex vivo assessment.

A facile polyelectrolyte complexation method for the preparation of both positively and negatively surface charged nanoparticles composed of chondroitin sulfate (ChS) and N-[(2-hydroxy-3-trimethylammonium)propyl]chitosan (HTCC) is reported. Production of ChS-HTCC nanoparticles with reverse zeta potential was easily controlled by varying the ChS/HTCC mass ratio. The encapsulation efficiency increased with the increase in initial FITC-BSA concentration in positively charged NPs and reached 75%. However, a maximum of 20% encapsulation efficiency was achieved in the case of negatively charged NPs. In vitro release studies of positively charged ChS-HTCC NPs showed a small burst effect followed by a continued and controlled release. Both charges of ChS-HTCC NPs showed no cytotoxicity in HUVECs. The confocal images showed that ChS-HTCC NPs of both charges can be incorporated and retained by the A549 cells. Flow cytometric analysis data demonstrated that ChS-HTCC NPs of both charges were detected in more than 80% of the A549 cells.

Green synthesis of chondroitin sulfate-capped silver nanoparticles: characterization and surface modification.

A one-step route for the green synthesis of highly stable and nanosized silver metal particles with narrow distribution is reported. In this environmentally friendly synthetic method, silver nitrate was used as silver precursor and biocompatible chondroitin sulfate (ChS) was used as both reducing agent and stabilizing agent. The reaction was carried out in a stirring aqueous medium at the room temperature without any assisted by microwave, autoclave, laser irradiation, γ-ray irradiation or UV irradiation. The transparent colorless solution was converted to the characteristics light red then deep red-brown color as the reaction proceeds, indicating the formation of silver nanoparticles (Ag NPs). The Ag NPs were characterized by UV-visible spectroscopy (UV-vis), photon correlation spectroscopy, laser Doppler anemometry, transmission electron microscopy (TEM), and Fourier-transform infrared spectroscopy (FT-IR). The results demonstrated that the obtained metallic nanoparticles were Ag NPs capped with ChS. In this report, dynamic light scattering (DLS) was used as a routinely analytical tool for measuring size and distribution in a liquid environment. The effects of the reaction time, reaction temperature, concentration and the weight ratio of ChS/Ag+ on the particle size and zeta potential were investigated. The TEM image clearly shows the morphology of the well-dispersed ChS-capped Ag NPs are spherical in shape, and the average size (<20 nm) is much smaller than the Z-average value (76.7 nm) measured by DLS. Meanwhile, the ChS-capped Ag NPs coated with N-[(2-hydroxy-3-trimethylammonium) propyl] chitosan chloride (HTCC) were prepared by an ionic gelation method and the surface charge of Ag NPs was switched from negative to positive.

Studies on a novel gelatin sponge: preparation and characterization of cross-linked gelatin scaffolds using 2-chloro-1-methylpyridinium iodide as a zero-length cross-linker.

We prepared a novel porous gelatin (GEL) sponge which was cross-linked (CL) with a zero-length crosslinker of 2-chloro-1-methylpyridinium iodide (CMPI), and compared CPMI with 1-ethyl-3,3-dimethylaminoproplycarbodiimide (EDC). The ninhydrin assay indicated that the CMPI-CL-GEL sponge had a higher degree of cross-linking than the EDC-CL-GEL sponge at cross-linking saturation. In contrast, the EDC-CL-GEL sponge demonstrated poor water uptake and a much slower enzymatic degradation rate than the CMPI-CL-GEL sponge. Scanning electron microscopy (SEM) images of the gelatin sponge fabricated using a gradient frozen-lyophilization method showed uniformly distributed and interconnected pores. Human 3T3 fibroblasts were successfully seeded onto the scaffolds, and cell proliferation was sustained on all CL-GEL sponges. CMPI-CL-GEL sponges demonstrated significantly increased cell numbers after day 1, and cell numbers steadily rose from day 1 to 12. Meanwhile, the CMPI-CL-GEL sponge had a higher cell number than the EDC-CL-GEL sponge (P < 0.05) by day 4. In vitro studies with 3T3 fibroblasts demonstrated an increased cell viability for those cells grown on sponges cross-linked with CMPI compared to those cross-linked with EDC. SEM images revealed attachment and spreading of cells, the CMPI-CL-GEL sponges had more cells that had elongated, migrated, and formed interconnected networks with neighboring cells.

Novel protein-loaded chondroitin sulfate-chitosan nanoparticles: preparation and characterization.

In this study, the potential of chondroitin sulfate (ChS)-chitosan (CS) nanoparticles (NPs) for the delivery of proteins was investigated. ChS-CS NPs were prepared by ionic cross-linking of CS solution with ChS. The aggregation line, particle size and zeta potential were investigated as a function of the pH, weight ratio and concentration. The water content and formation yield of the NPs were measured by gravimetry. Results indicated that ChS-CS NPs showed a higher degree of ionic cross-linking and formation yield than sodium tripolyphosphate-CS NPs. Fluorescein isothiocyanate conjugate bovine serum albumin (FITC-BSA), a model protein drug, was incorporated into the ChS-CS NPs. The encapsulation efficiency was obviously increased with the increase in initial FITC-BSA concentration and was as high as 90%. In vitro release studies of ChS-CS NPs showed a small burst effect following a continued and controlled release. Cytotoxicity tests with Caco-2 cells showed no toxic effects of ChS-CS NPs. The ex vivo cellular uptake studies using Caco-2 and HEK-293 cells indicated that NPs were found to be endocytosed into the cells. In conclusion, ChS-CS NPs are a potential new delivery system for the transport of hydrophilic compounds such as proteins.

Preparation of cross-linked hyaluronic acid film using 2-chloro-1-methylpyridinium iodide or water-soluble 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide.

In order to obtain much slower biodegradable films, which are often required for biomedical applications, we have developed a series of studies on heterogeneous cross-linking of hyaluronic acid (HA) films by using 2-chloro-1-methylpyridinium iodide (CMPI) or 1-ethyl-(3,3-dimethylaminopropyl)carbodiimide (EDC) as cross-linking reagents. From the in vitro degradation rate, we found that EDC cross-linked HA films completely dissolved in PBS at 37 degrees C during the period of 4-6 days. However, CMPI cross-linked HA films showed only a low percentage of weight loss over 30 days. This phenomenon could be explained from the mechanism of reaction between carboxyl group of HA and EDC. The latter reacted with carboxyl group to form an unstable intermediate O-acylurea, which showed a relatively low reactivity and quickly rearranged to form a stable N-acylurea. Thus, most of the EDC-activated carboxyl groups in HA were chemically transferred into N-acylurea or left as unreactive O-acylurea, and only a few of cross-linking bonds were formed between HA. On the other hand, the intermediate obtained from the reaction between carboxyl group and CMPI showed a relatively high reactivity and reacted with the hydroxyl group of the same and/or different molecules of HA to form an inter- and intramolecular esterification. Apparently, CMPI cross-linked HA films have a much higher cross-linking density and constructed a more rigid three-dimensional network. Therefore, it produced HA films, which dramatically increased its enzymatic stability in aqueous solution of hyaluronidase. The obtained results from elemental analyses, FT-IR spectra and NMR spectra also indicate that acylurea groups were introduced into EDC-cross-linked HA films.