Free Chitosan Research Articles

Supplemental Magnesium Gluconate Enhances Scaffold-Mediated New Bone Formation and Natural Bone Healing by Angiogenic- and Wnt Signal-Associated Osteogenic Activation.

Local implantation or supplementation of magnesium gluconate (MgG) is being investigated as an effective approach to bone repair. Although studies have highlighted the possible mechanisms in Mg ion-stimulated new bone formation, the role of MgG in healing bone defects and the signaling mechanisms are not yet completely understood. In this study, we explored how supplemental MgG has bone-specific beneficial effects by delivering MgG locally and orally in animal models. We fabricated MgG-incorporated (CMC-M) and -free chitosan (CMC) scaffolds with good microstructures and biocompatible properties. Implantation with CMC-M enhanced bone healing in rat model of mandible defects, compared with CMC, by activating Wnt signals and Wnt-related osteogenic and angiogenic molecules. Oral supplementation with MgG also stimulated bone healing in mouse model of femoral defects along with the increases in Wnt3a and angiogenic and osteogenic factors. Supplemental MgG did not alter nature bone accrual and bone marrow (BM) microenvironment in adult mouse model, but enhanced the functioning of BM stromal cells (BMSCs). Furthermore, MgG directly stimulated the induction of Wnt signaling-, angiogenesis-, and osteogenesis-related molecules in cultures of BMSCs, as well as triggered the migration of endothelial cells. These results suggest that supplemental MgG improves bone repair in a way that is synergistically enhanced by Wnt signal-associated angiogenic and osteogenic molecules. Overall, this study indicates that supplemental MgG might ameliorate oxidative damage in the BM, improve the functionality of BM stem cells, and maintain BM-microenvironmental homeostasis.

Chitosan-enclosed SLN improved SV-induced hepatocellular cell carcinoma death by modulation of IQGAP gene expression, JNK, and HDAC activities.

Hepatocellular carcinoma (HCC) is a global life-threatening problem and therapeutic interventions are still encountered. IQGAP genes are involved in HCC oncogenesis. The modulatory effect of statins on the expression of IQGAP genes is still unclear. This study aims to study the effect of free SV and chitosan (CS) decorated simvastatin (SV) loaded solid lipid nanoparticles (C-SV-SLNs) on HCC mortality. Plain, SV-SLN, and C-SV- SLN were prepared and characterized in terms of particle size (PS), zeta potential (ZP), and polydispersity index (PDI). The biosafety of different SLN was investigated using fresh erythrocytes, moreover, cytotoxicity was investigated using HepG2 cell lines. The effect of SLNs on IQGAPs gene expression as well as JNK, HDAC6, and HDAC8 activity was investigated using PCR and MOE-docking. The current results displayed that SV-SLNs have nanosized, negative ZP and are homogenous, CS decoration shifts the ZP of SLN into cationic ZP. Furthermore, all SLNs exhibited desirable biosafety in terms of no deleterious effect on erythrocyte integrity. SV solution and SV-SLN significantly increase the mortality of HepG2 compared to undertreated cells, however, the effect of SV-SLN is more pronounced compared to free SV. Remarkably, C-SV-SLN elicits high HepG2 cell mortality compared to free SV and SV-SLN. The treatment of HepG2 cells with SV solution, SV-SLN, or C-SV-SLN significantly upregulates the IQGAP2 gene with repression of IQGAP1 and IQGAP3 genes. MOE-docking studies revealed both SV and tenivastatin exhibit interactions with the active sites of JNK, HDAC6, and HDAC8. Moreover, tenivastatin exhibited greater interactions with magnesium and zinc compared to SV. This research provides novel insights into the therapeutic potential of SV, SV-SLN and C-SV-SLNs in HCC treatment, modulating critical signaling cascades involving IQGAPs, JNK, and HDAC. The development of C-SV-SLNs presents a promising strategy for effective HCC therapy.

Evaluation of fungicidal efficacy of free and nano-encapsulated chitosan against <i>Aspergillus flavus</i> isolated from rice (<i>Oryza sativa</i>) seed

Grains are exposed to contamination by mycotoxins, both in the field and during storage. Aflatoxins produced by Aspergillus species are the most harmful and carcinogenic mycotoxins in grains. Synthetic fungicides are widely used for the control of mycotoxigenic fungi in grains. However, rising public awareness about the toxicological effects of fungicides on human health necessitates the development of non-toxic bio-fungicides. In this connection, reports have shown that chitosan synthesized from shell waste has the potential to serve as an alternative fungicide. Therefore, this study investigated the fungicidal efficacy of free and nano-encapsulated chitosan against aflatoxigenic fungus (Aspergillus flavus). High molecular weight chitosan was purchased, and the in-vitro antifungal efficacy of chitosan against A. flavus was tested using the food poisoning method. Nano-encapsulated chitosan was synthesized using the ionic gelation method, the particle size was determined, and invitro inhibition against A. flavus was investigated. The results revealed that nano-encapsulated chitosan with particle sizes of 525.4 nm, 468.3 nm, and 711.7 nm were obtained. In-vitro mycelial A. flavus growth inhibition of 100% was recorded at 1.5% and 2.0% of free chitosan, while at 0.5% and 1.0% of free chitosan, 45% and 75% inhibition were observed, respectively. Nano-encapsulated chitosan with particle size (nm) of 525.4, 468.3, and 711.7 at 0.50% recorded 61%, 84% and 87% inhibition, respectively. However, at 0.25%, the rate of inhibition was 64%, 78% and 67%, respectively. This study showed that free chitosan and nano-encapsulated chitosan are potential antifungal agents for the control of A. flavus.

Comparison of chitosan nanoparticles containing Lippia citriodora essential oil and citral on the induction of apoptosis in A375 melanoma cells

BackgroundUsing nanoparticles containing L. citriodora EO and citral has shown potential in treating skin disorders such as melanoma.MethodsIn this study, GC‒MS was used to analyze the chemical composition of L. citriodora essential oil (EO). The ion gelation method prepared free chitosan nanoparticles and chitosan nanoparticles containing L. citriodora EO and citral. The successful loading of the EO and citral was evaluated using ATR-FTIR. The DPPH assay measured the antioxidant effect of citral, L. citriodora EO, Citral-ChiNPs, L. citriodora-ChiNPs, and Free-ChiNPs. A375 melanoma cell viability was assessed using the MTT assay. The qPCR technique was employed to evaluate the expression of apoptotic genes, and flow cytometry was used to detect apoptosis.ResultsThis study showed that in equal concentrations, the antioxidant properties of chitosan nanoparticles containing citral were greater than those of chitosan nanoparticles containing L. citriodora. The IC50 values of chitosan nanoparticles containing citral, L. citriodora EO, and their nonformulated states were 105.6, 199.9, 136.9, and 240 µg/ml, respectively. The gene expression results showed that the ratio of the expression of the apoptosis gene to the inhibitory gene was higher than 1 in all the samples, indicating that the conditions for apoptosis were present. Flow cytometry confirmed cell apoptosis, with 93.5 ± 0.3% in chitosan nanoparticles containing citral, 80 ± 0.2% in chitosan nanoparticles containing L. citriodora EO, 63 ± 0.3 in citral, and 42.03% in L. citriodora EO-treated cells.ConclusionThe results showed that using the Nano form of L. citriodora and citral increased their efficiency in apoptosis pathways and their toxicity against 375 melanoma cancer cells.

Removal of Cr (III) by using Chitosan-Grafting-Poly(Acryl Acid-Crotonic Acid) Characterization and Kinetic Study

Hydrogel nanocomposites were prepared using free radical polymerization chitosan and acrylic acid (AA) in combination with crotonic acid. KPS was employed as the initiating agent, while MBA functioned as the cross-linking agent. The nanocomposite of Ch-g-P(AA-co-CA) is a powerful pollutant absorber. Cr (III) was removed from the water using the combination. Field emission scanning electron microscopy (FESEM) and infrared spectroscopy (FTIR) were used to examine the nanocomposites’ structure and morphology. The kinetics of Cr (III) adsorption was studied using these rates. Pseudo-second order kinetics characterize the adsorption process. Hydrogel nanocomposite efficiently removes Cr by adsorption (III).

Prevention of bacterial biofilm formation on orthodontic brackets by non-crosslinked chitosan coating

During orthodontic treatment, the patients are susceptible to dental caries as a result of the bacterial adhesion and biofilm formation around the orthodontic brackets. Prevention of the caries-related biofilm formation is of significance for maintaining both aesthetics and health of the teeth. Herein, the brackets were functionalized with antibacterial activity via coating a layer of non-crosslinked chitosan (CS). We firstly demonstrated the ability of free CS scaffolds (not coated on brackets) to inhibit the formation of Streptococcus mutans biofilms (inhibition rate 94.3 % for CS-0.3 mg) and to eradicate the mature biofilms (biofilm loss rate 99.8 % for CS-1.2 mg). Further, the inhibition of S. mutans biofilm formation on brackets by CS coating was investigated for the first time. As a result, the CS-coated brackets (Br-CS) kept the great biofilm inhibition capacity of free CS scaffolds. In detail, the Br-CS, prepared by immersing brackets in CS solutions (containing 1.0, 2.5, 5.0 and 10 mg/mL CS) and freeze-drying, showed the biofilm inhibition rate of 48.5 %, 88.6 %, 96.4 % and 99.6 %, respectively. In conclusion, coating orthodontic brackets with the non-crosslinked CS is a potential approach for inhibiting biofilm formation and protecting patients from dental caries.

Construction of three-dimensional mesh porous Mo2C/carbon composites by chitosan salting-out for efficient microwave absorption

Constructing conductive network and adjusting impedance matching are effective ways to enhance the microwave absorption capacity of absorbers. However, the experimental procedures for preparing three-dimensional porous network are complex and often involve the use of hazardous agents. We proposed a simple and safe strategy to prepare porous three-dimensional network Mo2C/C composites. The salting-out method can change the aggregation state of chitosan macromolecules and make the free chitosan molecules form a porous three-dimensional network structure autonomously. In addition, the plus-charged amino on chitosan is able to attract minus-charged molybdate ions, which makes the surface of the 3D structured chitosan precursor covered with molybdenum-containing species. The result shows that the porous mesh structure and the heterogeneous interfaces combine to achieve excellent absorption performance of Mo2C/C with effective absorption bandwidth of 5.04 GHz at 1.8 mm. This work demonstrates that salting-out method broadens the idea of porous three-dimensional network electromagnetic wave absorbing materials.

Towards understanding the interaction of quercetin with chitosan-phytate complex: An experimental and computational investigation

Spectroscopic and structural properties of quercetin (QE) encapsulated in chitosan-phytate anion (CT-PA) nanoparticles were investigated, and an all-atom model was employed for molecular dynamics simulations to broaden the perspective on the conformational and energetics aspects regarding the quercetin-chitosan complexation. Quercetin binding to the free chitosan chains and its encapsulation in the nanoparticle obtained in the presence of the phytate anion was confirmed using dynamic light scattering and Fourier-transform infrared spectroscopy, and a considerably hindered rotational motion of the probe bound to the chitosan strand was demonstrated by the steady-state and anisotropy fluorescence measurements. While morphologies ranging from nearly spherical nanoparticles to nanofibrils were observed through atomic force microscopy upon increasing the chitosan content, a high encapsulation efficiency of QE, weakly dependent on the size and shape of CT-PA nanoparticles, was found. Simulation-based structural and energetics predictions indicated a similar H bond pattern for the QE binding to the chitosan strands, either in a free state or reticulated with the aid of the phytic anion. Additional crosslinker-assisted retention of quercetin in the chitosan network, as well as an enhanced electrostatic contribution to the binding free energy obtained at encapsulation, were derived from molecular dynamics simulations.

Influence of free and immobilized chitosan on a defined human gut microbial ecosystem

In this work, the influence of different forms of presentation of chitosan in the human gut microbiota with a defined bacterial community was evaluated. First, the susceptibility of individual gut bacterial isolates against chitosan was studied within a concentration range between 0.125 and 1 mg/mL. Then, the impact of chitosan (0.25 and 1 mg/mL) on a defined human gut microbial ecosystem was studied by metagenomic and metabonomic analyses. The results showed that chitosan in its free form had a high impact on individual isolates with a minimum inhibitory concentration below 1 mg/mL for most of the strains studied. In comparison, chitosan immobilized in the different carriers displayed a diverse effect on gut microbiota. The most susceptible strains were Agathobacter rectalis strain 16-6-I 1 FAA, Clostridium spiroforme strain 16-6-I 21 FAA and Mediterraneibacter faecis strain 16-6-I 30 FAA. The impact of the different modes of presentation of chitosan was strain-specific and species-specific when compared to results obtained from analysis of other strains within the genera Agathobacter, Clostridium and Mediterraneibacter, and therefore a study using a defined ecosystem was needed to extrapolate the results. Significant decreases in defined community richness and diversity and changes in metabolic profile were observed after exposure to free chitosan. Free chitosan produced significant reductions in the abundance of the genera Lachnoclostridium, Anaerotignum, Blautia, Enterococcus, Eubacterium and Ruthenibacterium together with a slight decrease of the production of SCFAs, among other fermentation by-products. The immobilized chitosan significantly alleviated the impact caused by the antimicrobial polymer and significantly increased the relative abundance of the Bacteroidetes phylum compared to free chitosan. These results suggest the significance of assessing the impact of new ingredients and materials included in food on the human gut microbiota with models that simulate the gastrointestinal environment, such as in vitro bioreactor systems.

3D Chitosan-Gallic Acid Complexes: Assessment of the Chemical and Biological Properties.

Three-dimensional chitosan-gallic acid complexes were proposed and prepared for the first time by a simple adsorption process of gallic acid (GA) on three-dimensional chitosan structures (3D chitosan). Highly porous 3D devices facilitate a high GA load, up to 2015 mmol/kg at pH 4.0. The preservation of the redox state of GA released from 3D chitosan was confirmed by spectroscopic analyses. The antioxidant activity of 3D chitosan-GA complexes was assessed using the DPPH radical scavenging assay and was found to be dramatically higher than that of free chitosan. The mechanical property of 3D chitosan–GA complexes was also evaluated using a compression test. Finally, 3D chitosan–GA complexes showed a significant antimicrobial capacity against E. coli and S. aureus, selected, respectively, as a model strain for Gram-negative and Gram-positive bacteria. Our study demonstrated a new, simple, and eco-friendly approach to prepare functional chitosan-based complexes for nutraceutical, cosmeceutical, and pharmaceutical applications.

Fabrication and Characterization of Thymol-Loaded Chitosan Nanogels: Improved Antibacterial and Anti-Biofilm Activities with Negligible Cytotoxicity.

Thymol is a monoterpene phenolic derivative extracted from the Thymus vulgaris which has antimicrobial effects. In the present study, thymol-loaded chitosan nanogels were prepared and their physicochemical properties were characterized. The encapsulation efficiency of thymol into chitosan and its stability were determined. The in vitro antimicrobial and anti-biofilm activities of thymol-loaded chitosan nanogel (Ty-CsNG), free thymol (Ty), and free chitosan nanogel (CsNG) were evaluated against both Gram-negative and Gram-positive multidrug-resistant (MDR) bacteria including Staphylococcus aureus, Acinetobacter baumanii, and Pseudomonas aeruginosa strains using the broth microdilution and crystal violet assay, respectively. After treatment of MDR strains with sub-minimum inhibitory concentration (Sub-MIC) of Ty-CsNG, free Ty and CsNG, biofilm gene expression analysis was studied. Moreover, cytotoxicity of Ty-CsNG, free Ty, and CsNG against HEK-293 normal cell line was determined using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method. The average size of Ty-CsNG was 82.71±9.6 nm, encapsulation efficiency was 76.54±0.62 % with stability up to 60 days at 4 °C. Antibacterial activity test revealed that Ty-CsNG reduced the MIC by 4-6 times in comparison to free thymol. In addition, the expression of biofilm-related genes including ompA, and pgaB were significantly down-regulated after treatment of strains with Ty-CsNG (P<0.05). In addition, free CsNG displayed negligible cytotoxicity against HEK-293 normal cell lines and presented a biocompatible nanoscale delivery system. Based on the results, it can be concluded that Ty-CsNG can be considered a promising candidate for enhancing antimicrobial and anti-biofilm activities.

ENHANCING THE ANTIBACTERIAL EFFECT OF BACTERIOCIN FROM LACTOCOCCUS LACTIS SUBSP. LACTIS USING CHITOSAN NANOPARTICLES

Chitosan, a cationic polymer derived from the hot alkali deacetylation of chitin, has numerous biological applications with non-toxicity, biocompatibility and biodegradability. Chitosan nanoparticles were prepared and encapsulated with bacteriocin extracted from Lactococcus lactis subsp lactis to produce chitosan nanoparticles conjugate bacteriocin using ionic gelation method. This formulation was examined for its antibacterial activity representing food bio-preservative against Salmonella typhimurium, Escherichiae coli, Bacillus cereus and Staphylococcus aureus, compared with chitosan nanoparticles, crud chitosan and free bacteriocin. Agar diffusion method was applied to evaluate the in-vitro drug release, effect of pH and temperature on its stability. The results revealed that the In-vitro release within 24 hours of chitosan nanoparticles conjugate bacteriocin was controlled by about (79%) with cumulative and sustained effect when compared with free bacteriocin (94%). Chitosan nanoparticles conjugate bacteriocin exhibit the highest antibacterial activity (with significant difference p &lt; 0.05) followed by free bacteriocin and chitosan nanoparticles, while crud chitosan was the lowest representing thermal stability (≤ 70ºC) when subjected to low pH. Gram-positive bacteria were more susceptible than Gram-negative bacteria to all of the components. Chitosan nanoparticles contribute successful safe food preservative enhancement when incorporated with bacteriocin against the common food-borne pathogenic bacteria.

Comparative efficacy of amphotericin B-loaded chitosan nanoparticles and free amphotericin B drug against Leishmania tropica

BackgroundThe preparation of an effective drug delivery formulation is an urgent need to treat cutaneous leishmaniasis (CL). Pentavalent antimonials and Amphotericin B (AmB) are considered to treat leishmaniasis; however, their clinical usage is hampered by poor solubility, high cost, toxicity, and the emergence of drug-resistant Leishmania spp. The drug delivery systems (DDS) could be used as an alternative treatment option for the treatment of CL to circumvent these problems. We tested the antileishmanial efficacies of free AmB and amphotericin B-loaded chitosan nanoparticles (AmB-CNPs) under in vitro conditions.ResultsChitosan nanoparticles (CNPs) were synthesized using the ionic gelation method with negatively charged tripolyphosphate (TPP). During the synthesis of CNPs, AmB was incorporated into the nanoparticles (NPs). The NPs were characterized for their size, surface morphology, encapsulation efficacy (EE), drug loading content (DLC), and surface charge using different techniques. Their efficacy was evaluated against promastigotes and axenic amastigotes forms of Leishmania tropica using MTT assay. The synthesized AmB-CNPs displayed a spherical shape with a mean particle size of 118 nm, a positive zeta potential of (+ 6.21 ± 2.02 mV), and an encapsulation efficacy of 88%. Dynamic light scattering technique (DLS) shows that the average size of prepared AmB-CNPs was 95.5 nm. Free AmB presented very low efficacy (only 65% and 67% inhibition of the promastigotes and axenic amastigotes parasite load), whereas AmB-CNPs exhibited 90% and 84% parasite inhibition after 72 h incubation. The AmB-CNPs exhibited significantly higher efficacy than free AmB in terms of reduction in parasite viability. Half-maximal inhibitory concentration (IC50) measured values of the AmB-CNPs were significant lowers than free AmB.ConclusionsThe present data indicated that AmB-CNPs exhibited vigorous anti-leishmanial activity than free AmB by dose and time-dependent manner. This formulation can be used for local therapy of CL after in vivo efficacy conformational studies.

Brij-functionalized chitosan nanocarrier system enhances the intestinal permeability of P-glycoprotein substrate-like drugs

The highly expressed P-glycoprotein (Pgp) in the intestine plays a key role in preventing drugs across the intestinal epithelium, which linked by tight junctions (TJs). Thus increasing the oral bioavailability of Pgp substrate-like drugs (PSLDs) remains a great challenge. Herein, we construct a nanocarrier system derived from Brij-grafted-chitosan (BC) to enhance the oral bioavailability and therapeutic effect of berberine (BBR, a typical PLSD) against diabetic kidney disease. The developed BC nanoparticles (BC-NPs) are demonstrated to improve the intestinal permeability of BBR via transiently and reversibly modulating the intercellular TJs (paracellular pathway) and Pgp-mediated drug efflux (transcellular pathway). As compared to free BBR and chitosan nanoparticles, the BC-NPs enhanced the relative oral bioavailability of BBR in rats (4.4- and 2.7-fold, respectively), and the therapeutic potency of BBR in renal function and histopathology. In summary, such strategy may provide an effective nanocarrier system for oral delivery of BBR and PSLDs.

Application of Lactobacillus helveticus CNRZ 32 to Control the Microbial Contaminants in Cheese by Addition of Bacterial Extracts or by In Situ Production of Bioactive Metabolites

Microbial spoilage of cheese represents a major concern from both health and economic views. The addition of food preservatives is considered the most applied strategy to ensure food quality and to control microbial contamination. It well established that natural preservatives such as Nisin and Natamycin are of great effectiveness against a wide range of microbial concerns, but the lack of wide spectrum effect induces looking for more efficient alternatives. This research suggests alternative treatments to be evaluated side by side with both Nisin and Natamycin within real cheese models to control microbial contamination during the storage period. To evaluate this, two varieties of cheese were manufactured and inoculated with a set of pathogen and cheese spoiling microorganisms. Talaga cheese batches were separately treated with extract of MRS that previously fermented by Lactobacillus helveticus CNRZ 32, Nisin and Natamycin at Free State and Chitosan Nanoparticles-loaded state to become 6 treatments (T1 – T6) other than the control (C). The same treatments were applied to Karish cheese batches, in addition to inoculation of Lactobacillus helveticus CNRZ 32 1% (v/v) in the seventh batch. Upon microbiological analyses, results show that T2; the extract loaded on Chitosan Nanoparticles completely reduced the count of all pathogens and spoiling populations after two weeks of cold storage (2 – 6oC) in Talaga cheese. In the case of Karish cheese, the 7th batch treated with Lactobacillus helveticus CNRZ 32 inoculation had no pathogenic nor spoiler growth after one week of cold storage (2 – 6oC). These results suggest that Lactic acid bacteria especially Lactobacillus helveticus CNRZ 32 can efficiently ensure the safety and quality of cheese if applied in appropriate form.

A potential antibiofilm, antimicrobial and anticancer activities of chitosan capped gold nanoparticles prepared by γ–irradiation

ABSTRACT Recently, nanotherapy has been considered a promising and rapidly evolving field to combat cancer. Both gold nanoparticles and chitosan are interesting materials with biological and antitumor properties. The present study aimed to synthesise chitosan/gold nanocomposites (Cs/Au) using gamma irradiation technique and to study their anticancer, antibiofilm and antimicrobial activities. The synthesized Cs/Au nanocomposites were characterized using various analytical tools such as UV-vis, XRD, and TEM. UV-vis absorption spectra exhibited a surface plasmon resonance (SPR) peak at 525 nm that confirming the successful synthesis of colloidal AuNPs. The TEM image demonstrates well-dispersed spherical shaped Au nanoparticles. The cytotoxic activity of Cs/Au nanocomposites was examined against HepG-2 and CACO-2 cell lines in comparison to free chitosan using neutral red assay test. The Cs/Au nanocomposites inhibited the proliferation of cancer cells more than chitosan. Moreover, the prepared Cs/Au nanocomposites showed antibacterial and antibiofilm activities encouraging its potential candidature for biomedical and pharmaceutical purposes.

SUN-159 ORAL NANOCAPSULE FORMULATION FOR POLYCYSTIC KIDNEY DISEASE

Nanomaterials are promising drug delivery tools, but few have been successful for oral administration. Oral delivery has the highest patient compliance and is optimal for chronic diseases like polycystic kidney disease (PKD). PKD affects 12.5 million individuals, where diseased tubule cells produce cysts that destroy kidney function1. Drug candidates to slow cyst growth require high dosages, leading to side effects. For example, the only FDA-approved PKD drug, Tolvaptan, showed a 23% dropout during clinical trials due to nausea and abdominal pain2. To limit toxicity, we previously developed self-assembled kidney targeting micelles, (KM) and found they accumulated in the kidneys3. To augment this for oral delivery, herein, we load KMs and metformin (met) into chitosan nanocapsules, which have been reported to overcome the barriers of the gastrointestinal (GI) tract. Metformin is an FDA-approved drug for diabetes which can also slow cyst growth. We hypothesize that chitosan nanocapsules deliver met across the GI tract to show efficacy in PKD mice. Furthermore, we characterize KMs loaded into chitosan nanocapsules to serve as a platform for oral delivery of nanotherapeutics. Capsules were synthesized as previously described in literature4. Briefly, 2 mg/ml of chitosan was added dropwise to a 1 mg/ml solution of poly-glutamic acid crosslinker. Met was loaded into our chitosan nanocapsules (CS-NP met) by mixing with the crosslinker. To assess oral delivery performance, we administered 300 mg/kg met loaded in CS-NP or free met to Pkd1fl/fl;Pax8 rtTA;Tet-O cre mice. Pups were IP injected with doxycycline on P10-11 to induce a cystic phenotype. Mice were orally gavaged every three days starting on P12 until P22. Kidneys were excised to assess kidney to body weight ratio and stained with haemotoxylin and eosin (H&E) to compare cystic index. To confirm the feasibility of loading KMs into chitosan nanocapsules, diameter analysis was performed by dynamic light scattering (DLS, Wyatt) with the following: KMs loaded into chitosan nanocapsules (CS-KM), KMs mixed with unloaded chitosan nanocapsules, free chitosan nanocapsules (CS-NP), and free KM. DLS of unloaded CS-NP showed diameters of 148.5±0.3 nm, while KMs are 14.9±1.5 nm (Fig. 1Ai, ii). Nanoparticles of this size have been reported to pass through intestinal Peyer's patches, then into systemic circulation5. DLS results of the mixed condition shows both particle populations (Fig. 1Aiii), whereas loading KMs within CS-NPs removes free KMs (Fig. 1Aiv), demonstrating encapsulation within CS-NPs. TEM micrographs of KMs and CS-NP (Fig. 1B,1C) show spherical morphology. Kidney sections from PKD mice show smaller kidney size in the CS-NP group compared to free met (Fig. 1D). A lower reduction of kidney to body weight and cystic index was seen in CS-NP met groups compared to free met (Fig. 1E). This may be due to the reported intestinal permeation effects of chitosan5. These initial studies show promise that KMs can be loaded within CS-NPs to be an orally delivered nanotherapeutic. Oral gavage of CS-NP met showed higher efficacy in a PKD mouse model compared to free met. Currently, studies confirming KM morphology preservation within CS-NPs are being conducted and future studies will test CS-KM in diseased PKD mouse models. To our knowledge, our studies represent the first nanomedicine strategy for PKD.

Synthesis and characterisation of magnetic κ-carrageenan nanocomposites for chitinase33 enzyme immobilisation

Chitinases are chitin-degrading enzymes that have wide biotechnological applications in the fields of medicine, agriculture and the industry. Production of chitinase on an industrial scale requires high protein expression. Pichia pastoris yeast is an important host for the rapid production and high levels of recombinant proteins. In this project, the fungal chit33 enzyme was expressed and purified in Pichia pastoris X-33, and the purified enzyme was immobilised to enhance the sustainability, activity, and reuse on the magnetite κ-carrageenan/chitosan nanocomposite as a biocatalyst. The synthesised nanoparticle was characterised using FTIR, SEM and EDS analysis. According to the results of enzyme activity measurement under different pH conditions, the temperature and time of the stabilised enzyme showed better activity and stability than the free enzyme. Compared to free enzyme and chitosan beads, in the magnetic κ-carrageenan/chit36 substrate the specific activity, pH tolerance, optimum temperature of the enzyme was improved. The enzyme stabilised in the magnetic cariogenic/chit33 substrate at 70°C maintains about 80% of its enzyme activity relative to the magnetic chitosan/chit36 bead and maintains about 40% of its activity after eight cycles of re-use of the enzyme.

Synthesis and characterisation of magnetic κ-carrageenan nanocomposites for chitinase33 enzyme immobilisation

Chitinases are chitin-degrading enzymes that have wide biotechnological applications in the fields of medicine, agriculture and the industry. Production of chitinase on an industrial scale requires high protein expression. Pichia pastoris yeast is an important host for the rapid production and high levels of recombinant proteins. In this project, the fungal chit33 enzyme was expressed and purified in Pichia pastoris X-33, and the purified enzyme was immobilised to enhance the sustainability, activity, and reuse on the magnetite κ-carrageenan/chitosan nanocomposite as a biocatalyst. The synthesised nanoparticle was characterised using FTIR, SEM and EDS analysis. According to the results of enzyme activity measurement under different pH conditions, the temperature and time of the stabilised enzyme showed better activity and stability than the free enzyme. Compared to free enzyme and chitosan beads, in the magnetic κ-carrageenan/chit36 substrate the specific activity, pH tolerance, optimum temperature of the enzyme was improved. The enzyme stabilised in the magnetic cariogenic/chit33 substrate at 70°C maintains about 80% of its enzyme activity relative to the magnetic chitosan/chit36 bead and maintains about 40% of its activity after eight cycles of re-use of the enzyme.

Assembling of the interfacial layer affects the physical and oxidative stability of faba bean protein-stabilized oil-in-water emulsions with chitosan

This study investigated how different ways of assembling of faba bean protein isolate (FBPI) and chitosan (CH) affected the oil-in-water emulsion stability. Emulsions were prepared either by a soluble FBPI-CH complex to make EMC, or by FBPI first and then added non-interactive CH to make EMN, or by FBPI first and then added a secondary layer of CH via electrostatic attraction to make EML. A control emulsion EMFB was prepared by only FBPI. All emulsions contained 5% (w/v) oil and 0.5% (w/v) FBPI. EMC/EML/EMN contained 0.05% (w/v) CH. The emulsion stability was monitored at 37 °C for 7 days. EML not only showed better physical stability as indicated by the changes in droplet size, Turbiscan stability index, and microstructure (by confocal microscopy), but also exhibited superior oxidative stability as indicated by lipid oxidation products (less conjugated dienes and secondary oxidation products) and protein oxidation biomarkers (less tryptophan loss and carbonyls formation). This might be attributed to that CH enhanced formation of a layer-by-layer (LBL) interfacial structure, which increased the interfacial layer thickness/compactness and maintained the interfacial protein adsorption. In contrast, EMC demonstrated worse stability, albeit with thicker interfacial layer due to CH. This could be explained by the lower surface hydrophobicity and surface charge of soluble FBPI-CH complex. The free aqueous CH also impaired physical stability and promoted oxidation, as shown in EMN. In conclusion, CH improves the oxidative and physical stability of FBPI-containing emulsion when added as a LBL model.