Cosilencing Intestinal Transglutaminase-2 and Interleukin-15 Using Gelatin-Based Nanoparticles in an in Vitro Model of Celiac Disease.

In this study, we have developed a type B gelatin nanoparticle based siRNA delivery system for silencing of intestinal transglutaminase-2 (TG2) and interleukin-15 (IL-15) genes in cultured human intestinal epithelial cells (Caco-2) and murine alveolar macrophage cells (J774A.1). Small interfering RNA (siRNA) targeting the TG2 or IL-15 gene was encapsulated within gelatin nanoparticles using ethanol-water solvent displacement method. Size, charge, and morphology of gelatin nanoparticles were evaluated using a Zetasizer instrument and transmission electron microscopy. siRNA encapsulation efficiency was determined using an siRNA specific stem-loop quantitative polymerase chain reaction (qPCR) assay. Cellular uptake of siRNA-containing gelatin nanoparticles was determined using fluorescent microscopy and stem-loop qPCR assay. siRNA loading in the RISC (RNA-induced silencing complex) was determined by immunoprecipitation of argonaute 2 (AGO2) protein followed by stem-loop qPCR for siRNA quantification. Gene expression analysis of TG2, IL-15, and the proinflammatory cytokines, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), was performed using qPCR assays. Efficacy of silencing TG2 and IL-15 knockdown was evaluated in an in vitro model of celiac disease by utilizing immunogenic α-gliadin peptide p31-43 in cultured J774A.1 cells. siRNA-containing gelatin nanoparticles were spherical in shape with mean particle size and charge of 217 ± 8.39 nm and -6.2 ± 0.95 mV, respectively. siRNA loading efficiency within gelatin nanoparticles was found to be 89.3 ± 3.05%. Evaluations of cellular uptake using fluorescent microscopy showed rapid internalization of gelatin nanoparticles within 2 h of dosing, with cytosolic localization of delivered siRNA in Caco-2 cells. Gelatin nanoparticles showed greater intracellular siRNA exposure with a longer half-life, when compared to Lipofectamine-mediated siRNA delivery. Approximately 0.1% of total intracellular siRNA was associated in the RISC complex. A maximum knockdown of 60% was observed at 72 h post siRNA treatment for both TG2 and IL-15 genes, which corresponded to ∼200 copies of RISC associated siRNA. Further, efficacy of gelatin nanoparticle mediated knockdown of TG2 and IL-15 mRNA was tested in an in vitro model of celiac disease. Significant suppression in the levels of proinflammatory cytokines (TNF-α and IFN-γ) was observed in p31-43 stimulated J774A.1 cells upon either IL-15 or IL-15 + TG2 siRNA treatment. The results from this study indicate that gelatin nanoparticle mediated TG2 and IL-15 siRNA gene silencing is a very promising approach for the treatment of celiac disease.

Highly Efficient Synthesis of Type B Gelatin and Low Molecular Weight Chitosan Nanoparticles: Potential Applications as Bioactive Molecule Carriers and Cell-Penetrating Agents.

Gelatin and chitosan nanoparticles have been widely used in pharmaceutical, biomedical, and nanofood applications due to their high biocompatibility and biodegradability. This study proposed a highly efficient synthesis method for type B gelatin and low-molecular-weight (LMW) chitosan nanoparticles. Gelatin nanoparticles (GNPs) were synthesized by the double desolvation method and the chitosan nanoparticles (CNPs) by the ionic gelation method. The sizes of the obtained CNPs and GNPs (373 ± 71 nm and 244 ± 67 nm, respectively) and zeta potential (+36.60 ± 3.25 mV and -13.42 ± 1.16 mV, respectively) were determined via dynamic light scattering. Morphology and size were verified utilizing SEM and TEM images. Finally, their biocompatibility was tested to assure their potential applicability as bioactive molecule carriers and cell-penetrating agents.

Tailorable hydrogel of gelatin with silk fibroin and its activation/crosslinking for enhanced proliferation of fibroblast cells.

Gelatin based hydrogel (Gel) possess remarkable cytocompatibility profile rendering it appropriate for tissue engineering applications. Herein, the questionable mechanical property of Gel was tuned by tailoring with different loading concentrations of silk fibroin (SF). The as tailored matrix was reconnoitred for its physico-mechanical, chemical and biological properties in order to investigate the effect of SF loading. Ethanol treatment lead to enhance ß-sheet formation of silk and subsequently, carbodiimide coupling was deployed to covalently crosslink the matrix. Substantial increase in cohesive energy with amplifying concentration of SF in the Gel matrix. As evidenced by Raman spectroscopy, right shift of the Amide I peak and stretching of COO- confirms activation of fibroin moiety along with crosslinking of gelatin, respectively. Moreover, with addition of SF, surface properties were tuned to attain maximum cell adhesion and proliferation. Further, MTT assay corroborated the same with definite increase in mitochondrial activities of L929 fibroblast cells for SF containing matrix as compared to its bare counterfeit while enhanced proliferation was confirmed by Rhodamine-DAPI staining. The alteration in mechanical and textural tribology of SF tailored Gel matrix certainly portrayed improved stability along with excellent cytocompatibility thus making it a plausible alternative in regenerative medicine.

Influence of process parameters on microcapsule formation from chitosan-Type B gelatin complex coacervates.

A series of chitosan/gelatin based microcapsules containing n-hexadecane was synthesized through complex phase coacervation from chitosan (CH) and type-B gelatin (GB), and crosslinked by glutaraldehyde (GTA). This research was conducted to clarify the influence of different parameters on the encapsulation process, i.e., the emulsion formation and the shell formation, using zeta potential and surface tension measurements, attenuated total reflectance (ATR), and thermal analysis such as differential scanning calorimetry (DSC). The optimal values of biopolymer ratios (TBP), crosslinker amount, emulsion time and feeding weight ratio of core/shell polymer (RCS) were identified. The stability of the emulsion was depended on the surface activity and TBP ratio, which also affected the droplet size distribution and the thickness of the shell. Furthermore, core content, encapsulation efficiency and thermal properties of the microcapsules were related to TBP and RCS; with the lowest RCS giving the best microcapsules features.

Surface behavior and bulk properties of aqueous chitosan and type-B gelatin solutions for effective emulsion formulation.

The behavior of aqueous chitosan (CH), type-B gelatin (GB) and CH-GB coacervate was studied on oil-in-water emulsion formulation at various pH and concentration ratio. The coacervate was formed by phase separation at ratios CH:GB, 1:10 to 1:1 with total biopolymer concentrations of 0.55%-1.0% (w/v) at pH 4.0-5.5. Soluble complexes were formed below pH 5.0 and coacervate formation was confirmed at pH 5.0 and above by zeta potential and UV-spectroscopy measurements. The coacervate formation was found maximum at the CH-GB ratios of 1:10 and 1:5 at pH 5.5. Formulated emulsions (>10µm droplets) using 1% (w/v) chitosan and GB were found stable (+52.5mv and creaming index 86%) and unstable respectively. Emulsion stabilized by mixed CH:GB 1:5 (3%w/v) had no creaming effect. The instability was attributed to the lower surface activity (K=5.0Lg-1) of pure GB compared to CH (K=14.3Lg-1). The formulation and methods can successfully tune the stability of the emulsions.