Oral administration of encapsulated catechin in chitosan-alginate nanoparticles improves cognitive function and neurodegeneration in an aluminum chloride-induced rat model of Alzheimer's disease.

The present study aimed to investigate the effect of catechin-loaded Chitosan-Alginate nanoparticles (NPs) on cognitive function in an aluminum chloride (AlCl3)-induced rat model of Alzheimer's disease (AD). The Catechin-loaded Chitosan-Alginate nanocarriers were synthesized through ionotropic gelation (IG) method. Physio-chemical characterization was conducted with the Zetasizer Nano system, the scanning electron microscope, and the Fourier transform infrared spectroscopy. The experiments were performed over 21 days on six groups of male Wistar rats. The control group, AlCl3 treated group, Catechin group, nanocarrier group, treatment group 1 (AlCl3 + Catechin), and treatment group 2 (AlCl3 + nanocarrier). A behavioral study was done by the Morris water maze (MWM) test. In addition, the level of oxidative indices and acetylcholine esterase (AChE) activity was determined by standard procedures at the end of the study. AlCl3 induced a significant increase in AChE activity, along with a significant decrease in the level of Catalase (CAT) and total antioxidant capacity (TAC) in the hippocampus. Moreover, the significant effect of AlCl3 was observed on the behavioral parameters of the MWM test. Both forms of Catechin markedly improved AChE activity, oxidative biomarkers, spatial memory, and learning. The present study indicated that the administration of Catechin-loaded Chitosan-Alginate NPs is a beneficial therapeutic option against behavioral and chemical alteration of AD in male Wistar rats.

Activation of the BMP2/SMAD4 signaling pathway for enhancing articular cartilage regeneration of mesenchymal stem cells utilizing chitosan/alginate nanoparticles on 3D extracellular matrix scaffold.

This study investigated the efficacy of using chitosan/alginate nanoparticles loaded with recombinant human bone morphogenetic-2 (rhBMP-2) and SMAD4 encoding plasmid to enhance the chondrogenesis of human bone marrow mesenchymal stem cells (hBM-MSCs) seeded on an extracellular matrix (ECM). The research treatments included the stem cells treated with the biological cocktail (BC), negative control (NC), hBM-MSCs with chondrogenic medium (MCM), hBM-MSCs with naked rhBMP-2 and chondrogenic medium (NB/C), and hBM-MSCs with naked rhBMP-2 and chondrogenic medium plus SMAD4 encoding plasmid transfected with polyethyleneimine (PEI) (NB/C/S/P). The cartilage differentiation was performed with real-time quantitative PCR analysis and alizarin blue staining. The data indicated that the biological cocktail (BC) exhibited significantly higher expression of cartilage-related genes compared to significant differences with MCM and negative control (NC) on chondrogenesis. In the (NB/C/S/P), the expression levels of SOX9 and COLX were lower than those in the BC group. The expression pattern of the ACAN gene was similar to COL2A1 changes suggesting that it holds promising potential for cartilage regeneration.

Development of Biocompatible Nanoparticles of Tizanidine Hydrochloride in Orodispersible Films: In vitro Characterization, Ex vivo Permeation, and Cytotoxic Study on Carcinoma Cells.

BACKGROUND: The main limitations of the therapeutic effectiveness of tizanidine hydrochloride (TNZ) are its low bioavailability due to its tendency to undergo first-pass metabolism and short biological half-life. These factors make it an ideal candidate for formulating orally disintegrating films. OBJECTIVE: The present study was aimed to prepare nanoparticles of tizanidine hydrochloride using biodegradable polymers and loading them on orodispersible films to obtain a sustained release dissolution profile with improved permeability and further study the cytotoxicity on A549 lung carcinoma cells, MCF7 breast cancer cells, and HOP 92 non-small lung adenocarcinoma cells. METHODS: The fast-dissolving film of TNZ HCl was prepared by the solvent-casting method and characterized using scanning electron microscopy, FTIR, and XRD, and evaluated for critical quality attributes for this type of dosage form such as disintegration time, tensile strength, drug content, dissolution, and ex vivo permeability. In vitro cytotoxicity studies were also conducted on cancer cell lines to confirm the cytotoxic effect. RESULTS: The polymeric matrix containing the drug provided a rapid disintegration time varying between 7±2 and 30±2 seconds, adequate tensile strength between 1.4 and 11.25 N/mm2, and improved permeability through porcine buccal mucosa when compared to the reference product. CONCLUSION: A study of the cytotoxic effect on the MCF-7 breast cancer cells and A549 lung carcinoma cells revealed that tizanidine hydrochloride nanoparticles at 2.3 mg/film exhibited an IC50 value of 65.1 % cytotoxicity on MCF-7, approximately 100% on HOP92, and 83.5 % on A549 lung carcinoma cells, thus paving the way for a new paradigm of research for a cytotoxic study on MCF-7, HOP92, and A549 cell lines using the subject drug model prepared as oral films or biodegradable nanoparticles in oral films for site-specific targeting.

Chitosan/Alginate Nanoparticles for the Enhanced Oral Antithrombotic Activity of Clam Heparinoid from the Clam Coelomactra antiquata.

Chitosan/alginate nanoparticles (DG1-NPs and DG1/Cur-NPs) aiming to enhance the oral antithrombotic activity of clam heparinoid DG1 were prepared by ionotropic pre-gelation. The influence of parameters, such as the concentration of sodium alginate (SA), chitosan (CTS), CaCl2, clam heparinoid DG1, and curcumin (Cur), on the characteristics of the nanoparticles, were investigated. Results indicate that chitosan and alginate can be used as polymer matrices to encapsulate DG1, and nanoparticle characteristics depend on the preparation parameters. Nano-particles should be prepared using 0.6 mg/mL SA, 0.33 mg/mL CaCl2, 0.6 mg/mL CTS, 7.2 mg/mL DG1, and 0.24 mg/mL Cur under vigorous stirring to produce DG1-NPS and DG1/Cur-NPS with small size, high encapsulation efficiency, high loading capacity, and negative zeta potential from approximately -20 to 30 mV. Data from scanning electron microscopy, Fourier-transform infrared spectrometry, and differential scanning calorimetry analyses showed no chemical reaction between DG1, Cur, and the polymers; only physical mixing. Moreover, the drug was loaded in the amorphous phase within the nanoparticle matrix. In the acute pulmonary embolism murine model, DG1-NPs enhanced the oral antithrombotic activity of DG1, but DG1/Cur-NPs did not exhibit higher antithrombotic activity than DG1-NPs. Therefore, the chitosan/alginate nanoparticles enhanced the oral antithrombotic activity of DG1, but curcumin did not further enhance this effect.

Applications of Chitosan-Alginate-Based Nanoparticles-An Up-to-Date Review.

Chitosan and alginate are two of the most studied natural polymers that have attracted interest for multiple uses in their nano form. The biomedical field is one of the domains benefiting the most from the development of nanotechnology, as increasing research interest has been oriented to developing chitosan-alginate biocompatible delivery vehicles, antimicrobial agents, and vaccine adjuvants. Moreover, these nanomaterials of natural origin have also become appealing for environmental protection (e.g., water treatment, environmental-friendly fertilizers, herbicides, and pesticides) and the food industry. In this respect, the present paper aims to discuss some of the newest applications of chitosan-alginate-based nanomaterials and serve as an inception point for further research in the field.

Improvement of the Antimicrobial Activity of Oregano Oil by Encapsulation in Chitosan-Alginate Nanoparticles.

Oregano oil (OrO) possesses well-pronounced antimicrobial properties but its application is limited due to low water solubility and possible instability. The aim of this study was to evaluate the possibility to incorporate OrO in an aqueous dispersion of chitosan-alginate nanoparticles and how this will affect its antimicrobial activity. The encapsulation of OrO was performed by emulsification and consequent electrostatic gelation of both polysaccharides. OrO-loaded nanoparticles (OrO-NP) have small size (320 nm) and negative charge (-25 mV). The data from FTIR spectroscopy and XRD analyses reveal successful encapsulation of the oil into the nanoparticles. The results of thermogravimetry suggest improved thermal stability of the encapsulated oil. The minimal inhibitory concentrations of OrO-NP determined on a panel of Gram-positive and Gram-negative pathogens (ISO 20776-1:2006) are 4-32-fold lower than those of OrO. OrO-NP inhibit the respiratory activity of the bacteria (MTT assay) to a lower extent than OrO; however, the minimal bactericidal concentrations still remain significantly lower. OrO-NP exhibit significantly lower in vitro cytotoxicity than pure OrO on the HaCaT cell line as determined by ISO 10993-5:2009. The irritation test (ISO 10993-10) shows no signs of irritation or edema on the application site. In conclusion, the nanodelivery system of oregano oil possesses strong antimicrobial activity and is promising for development of food additives.

Potential of chitosan/alginate nanoparticles as a non-viral vector for gene delivery: Formulation and optimization using D-optimal design.

Chitosan/alginate (Chi/Alg) nanoparticles as a non-viral vector for the Smad4 encoding plasmid were optimized utilizing D-optimal design based on the nanoparticles/plasmid ratio, Chi/Alg MW, and preparation method type. Following the optimization and validation of the best formula, morphology studies and FTIR measurements were performed to evaluate the optimized Chi/Alg/S NPs. Toxicity (MTT assay) and transfection studies were performed for the best formula in comparison with Lipofectamine 2000, and Polyethyleneimine (PEI) and evaluated using Green Fluorescence Protein (GFP) assay, Flow cytometry, and RT-PCR. The model predicted a particle size of 111 nm, loading efficacy (LE) of 43%, cumulative release (CMR) of 39%, the ζ-potential of +50 mV, and PDI of 0.13. The predicted point condition was as follows: NP ratio = 13, Chi/Alg MW ratio = 2.35, and preparation method type = 1. Microscopic findings revealed that the shape of nanoparticles was spherical. The Chi/Alg/S nanoparticles showed no toxicity and transfection efficacy of 29.9% was observed in comparison with Lipofectamine (35.5%) and PEI (30.9%).

Chitosan-alginate nanoparticles as effective oral carriers to improve the stability, bioavailability, and cytotoxicity of curcumin diethyl disuccinate.

Curcumin diethyl disuccinate (CDD) is an ester prodrug of curcumin that has better chemical stability in phosphate buffer (pH 7.4) and anticancer activities against MDA-MB-231 human breast cancer cells and Caco-2 cells than curcumin. However, a major drawback of CDD is its poor water solubility and low bioavailability in the gastrointestinal tract. To overcome these problems, a nanoformulation was developed using chitosan/alginate nanoparticles (CANPs) under the optimal condition as previously derived by statistical optimization. The CDD-loaded CANPs (CDD-CANPs) were found to exhibit good stability after exposure to simulated digestive fluids and ultraviolet light, and a sustained-release profile of CDD in the simulated digestive and body fluids. The in vitro release pattern fitted well to the Peppas-Sahlin model, indicating that the release of CDD was mainly governed by diffusion. Compared to free CDD, the CDD-CANPs showed better stability, bioaccessibility, bioavailability, cellular uptake, and cytotoxicity against HepG2 cells.

Response Surface Methodology for Statistical Optimization of Chitosan/Alginate Nanoparticles as a Vehicle for Recombinant Human Bone Morphogenetic Protein-2 Delivery.

PURPOSE: In this study, chitosan/alginate nanoparticles are prospected as a carrier for controlled release of recombinant human bone morphogenetic protein-2 (rhBMP-2). MATERIALS AND METHODS: The rhBMP-2-loaded chitosan/alginate nanoparticles (Cs/Alg/B NPs) were prepared using the ionic gelation (IG) method. The current research was conducted to optimize the effective factors for entrapping rhBMP-2 in Cs/Alg NPs using response surface methodology (RSM) and the Box-Behnken design (BBD). The variables were the Cs/Alg molecular weight (Mw) ratios (1-3), pH (4.8-5.5), stirring rates (900-1300 rpm) and the responses included size, ζ-potential, polydispersity index (PDI), loading efficacy (LE), cumulative release (CR), and morphological degradation time (MDE). Then, the morphological properties of optimum formulation were studied for post-characterization. In the next step, the MTT assay for the optimized run was done for 24 and 48 hours. RESULTS: The results revealed that the optimum conditions for the mentioned variables were stirring rate=1100 rpm, pH=5.15, and Cs/Alg Mw ratio=1.75 based on numerical optimization. It was shown that the average particle size and loading efficacy at optimum conditions were 253 nm and 67%, respectively. Other responses were as follows: CR=66%, ζ-potential=+35mV, PDI=0.5, and MDT=7 days. CONCLUSION: The results have suggested that the statistical optimization of rhBMP-2 offers the possibility of preparing Cs/Alg/B NPs with a favorable size, controlled release characteristics, and high loading efficiency. It is expected that the acquired optimum conditions will be useful for efficient rhBMP-2 delivery.

Synergistic Effects of Photo-Irradiation and Curcumin-Chitosan/Alginate Nanoparticles on Tumor Necrosis Factor-Alpha-Induced Psoriasis-Like Proliferation of Keratinocytes.

Psoriasis is a chronic inflammatory skin disease characterized by hyperproliferation of the epidermal cells and is clinically presented as thick, bright red to pink plaques with a silvery scale. Photodynamic therapy (PDT) using visible light has become of increasing interest in the treatment of inflammatory skin diseases. In this study, we demonstrate that a combination of curcumin-loaded chitosan/alginate nanoparticles (Cur-CS/Alg NPs) and blue light emitting diodes (LED) light irradiation effectively suppressed the hyperproliferation of tumor necrosis factor-alpha (TNF-α)-induced cultured human kerlatinocyte (HaCaT) cells. The Cur-CS/Alg NPs were fabricated by emulsification of curcumin in aqueous sodium alginate solution and ionotropic gelation with calcium chloride and chitosan using an optimized formulation derived from a Box-Behnken design. The fabricated Cur-CS/Alg NPs were characterized for their particle size, zeta potential, encapsulation efficiency, and loading capacity. The surrogate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, to measure the relative number of viable cells, showed that the CS/Alg NPs were nontoxic to normal HaCaT cells, while 0.05 µg/mL and 0.1 µg/mL of free curcumin and Cur-CS/Alg NPs inhibited the hyperproliferation of HaCaT cells induced by TNF-α. However, the Cur-CS/Alg NPs demonstrated a stronger effect than the free curcumin, especially when combined with blue light irradiation (10 J/cm²) from an LED-based illumination device. Therefore, the Cur-CS/Alg NPs with blue LED light could be potentially developed into an effective PDT system for the treatment of psoriasis.

Chitosan/alginate nanoparticles as a promising carrier of novel curcumin diethyl diglutarate.

Chitosan/alginate nanoparticles (CANPs) were formulated to encapsulate curcumin diethyl diglutarate (CDG) for oral delivery. CDG-loaded CANPs (CDG-CANPs) were prepared by o/w emulsification and ionotropic gelation. The optimization of CDG-CANPs was successfully performed by response surface methodology. The characteristics including photostability, storage stability, digestive stability, in vitro digestibility, bioaccessibility and in vitro uptake in Caco-2 cells of free CDG and CDG-CANPs were investigated. The optimal CDG-CANPs could be prepared by chitosan/alginate mass ratio of 0.065:1, 1% (w/v) Pluronic®F127 and 4.5 mg/mL of CDG. The optimized nanoparticles had the particle size, zeta potential, encapsulation efficiency and loading capacity of 215 nm, -24.1 mV, 85% and 27%, respectively. The CDG-CANPs showed better stability under UV irradiation and thermal exposure compared with free CDG. The CDG-CANPs had stability up to 3 months at 4 °C. The in vitro release profile showed sustained-release manner and best fit with the Korsmeyer-Peppas kinetic model, indicating the Fickian diffusion mechanism. Nanoparticle encapsulation significantly enhanced in vitro digestibility and bioaccessibility under simulated gastrointestinal conditions and cellular uptake of CDG. The overall results suggest that CANPs are promising candidates for encapsulation, protection and controlled release of CDG, a hydrophobic compound, with an improvement of physicochemical stabilities, digestibility, bioaccessibility and cellular uptake.

Hepatoprotective and antioxidant activity of quercetin loaded chitosan/alginate particles in vitro and in vivo in a model of paracetamol-induced toxicity.

The toxic liver impairment caused by free radical injury or excessive reactive oxigen species (ROS) formation could be effectivelly attenuated by natural antioxidants. The present study aimed to explore and compare the hepatoprotective and antioxidant effects of free and encapsulated quercetin in in vitro and in vivo models of hepatotoxicity. Thus, quercetin was encapsulated in chitosan/alginate nanoparticles by gelation method. Both empty and quercetin-loaded nanoparticles revealed good safety profile in vitro, determined by the lack of cytotoxicity in human hepatoma HepG2 cells. The pretreatment of HepG2 cells with encapsulated quercetin (10µg/ml) significantly attenuated the decrease in cell viability in H2О2-induced oxidative stress (0.1mM H2О2), thus showing an effective in vitro protection. In vivo evaluation of the antioxidant and hepatoprotective potential of free and encapsulated quercetin was performed in a model of paracetamol - induced liver injury in male Wistar rats. The oral pretreatment with encapsulated quercetin (0.18mg/kg b.w., 7days) significantly diminished the increased levels of serum transaminases ALT and AST, attenuated the lipid peroxidation and restored the levels of gluthation (a marker of cell antioxidant defence system). The protective effects of quercetin encapsulated in chitosan-based nanoformulation were superior to those of free quercetin. The results of the study suggest that the encapsulation of quercetin in chitosan/alginate nanoformulations might represent an effective therapeutic approach against oxidative stress induced liver injury.

Еvaluation of biocompatibility and antioxidant efficiency of chitosan-alginate nanoparticles loaded with quercetin.

The present study deals with development and evaluation of the safety profile of chitosan/alginate nanoparticles as a platform for delivery of a natural antioxidant quercetin. The nanoparticles were prepared by varying the ratios between both biopolymers giving different size and charge of the formulations. The biocompatibility was explored in vitro in cells from different origin: cultivated HepG2 cells, isolated primary rat hepatocytes, isolated murine spleen lymphocytes and macrophages. In vivo toxicological evaluation was performed after repeated 14-day oral administration to rats. The study revealed that chitosan/alginate nanoparticles did not change body weight, the relative weight of rat livers, liver histology, hematology and biochemical parameters. The protective effects of quercetin-loaded nanoparticles were investigated in the models of iron/ascorbic acid (Fe2+/AA) induced lipid peroxidation in microsomes and tert-butyl hydroperoxide oxidative stress in isolated rat hepatocytes. Interesting finding was that the empty chitosan/alginate nanoparticles possessed protective activity themselves. The antioxidant effects of quercetin loaded into the nanoparticles formulated with higher concentration of chitosan were superior compared to quercetin encapsulated in nanoparticles with higher amount of sodium alginate. In conclusion, chitosan/alginate nanoparticles can be considered appropriate carrier for quercetin, combining safety profile and improved protective activity of the encapsulated antioxidant.

Release profile and stability evaluation of optimized chitosan/alginate nanoparticles as EGFR antisense vector.

Chitosan/alginate nanoparticles which had been optimized in our previous study using two different N/P ratios were chosen and their ability to release epidermal growth factor receptor (EGFR) antisense was investigated. In addition, the stability of these nanoparticles in aqueous medium and after freeze-drying was investigated. In the case of both N/P ratios (5, 25), nanoparticles started releasing EGFR antisense as soon as they were exposed to the medium and the release lasted for approximately 50 hours. Nanoparticle size, shape, zeta potential, and release profile did not show any significant change after the freeze-drying process (followed by reswelling). The nanoparticles were reswellable again after freeze-drying in phosphate buffer with a pH of 7.4 over a period of six hours. Agarose gel electrophoresis of the nanoparticles with the two different N/P ratios showed that these nanoparticles could protect EGFR antisense molecules for six hours.

A Comparative Study Between the Antibacterial Effect of Nisin and Nisin-Loaded Chitosan/Alginate Nanoparticles on the Growth of Staphylococcus aureus in Raw and Pasteurized Milk Samples.

The aim of this study was to evaluate the antibacterial effect of nisin-loaded chitosan/alginate nanoparticles as a novel antibacterial delivery vehicle. The nisin-loaded nanoparticles were prepared using colloidal dispersion of the chitosan/alginate polymers in the presence of nisin. After the preparation of the nisin-loaded nanoparticles, their physicochemical properties such as size, shape, and zeta potential of the formulations were studied using scanning electron microscope and nanosizer instruments, consecutively. FTIR and differential scanning calorimetery studies were performed to investigate polymer-polymer or polymer-protein interactions. Next, the release kinetics and entrapment efficiency of the nisin-loaded nanoparticles were examined to assess the application potential of these formulations as a candidate vector. For measuring the antibacterial activity of the nisin-loaded nanoparticles, agar diffusion and MIC methods were employed. The samples under investigation for total microbial counts were pasteurized and raw milks each of which contained the nisin-loaded nanoparticles and inoculated Staphylococcus aureus (ATCC 19117 at 10(6) CFU/mL), pasteurized and raw milks each included free nisin and S. aureus (10(6) CFU/mL), and pasteurized and raw milks each had S. aureus (10(6) CFU/mL) in as control. Total counts of S. aureus were measured after 24 and 48 h for the pasteurized milk samples and after the time intervals of 0, 6, 10, 14, 18, and 24 h for the raw milk samples, respectively. According to the results, entrapment efficiency of nisin inside of the nanoparticles was about 90-95%. The average size of the nanoparticles was 205 nm, and the average zeta potential of them was -47 mV. In agar diffusion assay, an antibacterial activity (inhibition zone diameter, at 450 IU/mL) about 2 times higher than that of free nisin was observed for the nisin-loaded nanoparticles. MIC of the nisin-loaded nanoparticles (0.5 mg/mL) was about four times less than that of free nisin (2 mg/mL). Evaluation of the kinetic of the growth of S. aureus based on the total counts in the raw and pasteurized milks revealed that the nisin-loaded nanoparticles were able to inhibit more effectively the growth of S. aureus than free nisin during longer incubation periods. In other words, the decrease in the population of S. aureus for free nisin and the nisin-loaded nanoparticles in pasteurized milk was the same after 24 h of incubation while lessening in the growth of S. aureus was more marked for the nisin-loaded nanoparticles than the samples containing only free nisin after 48 h of incubation. Although the same growth reduction profile in S. aureus was noticed for free nisin and the nisin-loaded nanoparticles in the raw milk up to 14 h of incubation, after this time the nisin-loaded nanoparticles showed higher growth inhibition than free nisin. Since, generally, naked nisin has greater interactions with the ingredients present in milk samples in comparison with the protected nisin. Therefore, it is concluded that the antibacterial activity of nisin naturally decreases more during longer times of incubation than the protected nisin with the chitosan/alginate nanoparticles. Consequently, this protection increases and keeps antibacterial efficiency of nisin in comparison with free nisin during longer times of storage. These results can pave the way for further research and use of these nanoparticles as new antimicrobial agents in various realms of dairy products.