Estudio de biocompatibilidad de matrices poliméricas combinadas, responsivas al pH, con aplicación en Ingeniería de Tejido Óseo / Biocompatibility study of combined pH-responsive polymeric matrices with application in Bone Tissue Engineering

El presente trabajo muestra la obtención de un material a partir de un polímero sintético (TerP) y otro natural, mediante entrecruzamiento físico y su caracterización fisicoquímica y biológica, con el fin de emplearlos para regeneración de tejido óseo. Las membranas fueron obtenidas por la técnica de evaporación del solvente y caracterizadas por espectroscopia FTIR, ensayos de hinchamiento, medidas de ángulo de contacto y microscopia electrónica de barrido (SEM). Se encontró que la compatibilidad entre los polímeros que la constituyen es estable a pH fisiológico y que, al incorporar mayor cantidad del TerP a la matriz, esta se vuelve más hidrofóbica y porosa. Además, teniendo en cuenta la aplicación prevista para dichos materiales, se realizaron estudios de biocompatibilidad y citotoxicidad con células progenitoras de médula ósea (CPMO) y células RAW264.7, respectivamente. Se evaluó la proliferación celular, la producción y liberación de óxido nítrico (NO) al medio de cultivo durante 24 y 48 horas y la expresión de citoquinas proinflamatorias IL-1ß y TNF-α de las células crecidas sobre los biomateriales variando la cantidad del polímero sintético. Se encontró mayor proliferación celular y menor producción de NO sobre las matrices que contienen menos proporción del TerP, además de poseer una mejor biocompatibilidad. Los resultados de este estudio muestran que el terpolímero obtenido y su combinación con un polímero natural es una estrategia muy interesante para obtener un biomaterial con posibles aplicaciones en medicina regenerativa y que podría extenderse a otros sistemas estructuralmente relacionados. (AU)

In the present work, the preparation of a biomaterial from a synthetic terpolymer (TerP) and a natural polymer, physically crosslinked, is shown. In order to evaluate the new material for bone tissue regeneration, physicochemical and biological characterizations were performed. The membranes were obtained by solvent casting and characterized using FTIR spectroscopy, swelling tests, contact angle measurements, and scanning electron microscopy (SEM). It was found that the compatibility between the polymers is stable at physiological pH and the incorporation of a higher amount of TerP into the matrix increases hydrophobicity and porosity.Furthermore, considering the intended application of these materials, studies of biocompatibility and cytotoxicity were conducted with Bone Marrow Progenitor Cells (BMPCs) and RAW264.7 cells, respectively. Cell proliferation, NO production and release into the culture medium for 24 and 48 hours, and proinflammatory cytokine expression of IL-1ß and TNF-α from cells grown on the biomaterials while varying the amount of the synthetic polymer were evaluated. Greater cell proliferation and lower NO production were found on matrices containing a lower proportion of TerP, in addition to better biocompatibility. The results of this study demonstrate that the obtained terpolymer and its combination with a natural polymer is a highly interesting strategy for biomaterial preparation with potential applications in regenerative medicine. This approach could be extended to other structurally related systems. (AU)

Bringing pathology to nanomedicine: a comparison of in vivo toxicity of polymeric nanoparticle carriers with and without chitosan coating.

Over the last years, there has been an increasing number of proposals for the use of nanomaterials in medicine. The safety of novel technologies must be verified, prior to their clinical application. Pathology has much to contribute towards this end. In this study, we compared the in vivo toxicity effects of poly- (lactic-co-glycolic acid) nanoparticles with and without chitosan shell. Both nanoparticle types were loaded with curcumin. The nanoparticles were assessed in vitro for potential cytotoxicity with cell viability studies. For the in vivo test, 36 adult Wistar rats were used, four of which were the control group. The remaining 32 were divided into 2 groups, each of which was administered differentially coated drug carriers: (A) nanoparticles without chitosan coating and (B) nanoparticles with chitosan coating. For both groups, the subcutaneous route was used for administration. Each group was further divided into 2 sub-groups of 8 animals each. The animals of the first sub-groups were sacrificed 24 h after the injection and those of the second on the 7th day. The control group was also divided into 2 subgroups of 2 animals each. At the appointed post-administrative date, the rats were sacrificed, and specimens from the brain, liver, kidneys, heart, stomach, lungs, and from the skin at the injection site were collected and studied histopathologically. The evaluation of both in vitro and in vivo testing shows that nanoparticles with chitosan have significantly less, if any, toxic effects compared to those without chitosan.

The protective effect of N-acetylcysteine against liposome and chitosan-induced cytotoxicity.

AIM: N-acetylcysteine (NAC) is an antioxidant used to moderate liposome and chitosan-induced cell cytotoxicity at their high concentrations. METHODS: Liposome and chitosan were prepared and characterised. The cytotoxicity effect of liposome with NAC-loaded liposome (liposome-NAC) and chitosan solution with chitosan solution containing NAC (chitosan-NAC) on the A549 cell line was compared. RESULTS: Particle size, zeta potential, and NAC drug release for liposome were 125.9 ± 8 nm, -34.7 ± 2.1 mV, and 51.1% ± 3%, respectively. Scanning electron microscope (SEM) and transmission electron microscope (TEM) indicated spherical shape of liposome. Encapsulation efficiency of liposome-NAC was 12% ± 0.98%. Particle size and zeta potential for chitosan solution were 361 ± 11.3 nm and 10.8 ± 1.52 mV. Stability storage study indicated good stability of chitosan and liposome. Cell viability of liposome-NAC and chitosan-NAC significantly was higher than liposome and chitosan at all four concentrations. CONCLUSIONS: NAC has a protective effect against liposome and chitosan-induced cell toxicity.

In vivo toxicity evaluation of tumor targeted glycol chitosan nanoparticles in healthy mice: repeated high-dose of glycol chitosan nanoparticles potentially induce cardiotoxicity.

BACKGROUND: Glycol chitosan nanoparticles (CNPs) have emerged as an effective drug delivery system for cancer diagnosis and treatment. Although they have great biocompatibility owing to biodegradable chemical structure and low immunogenicity, sufficient information on in vivo toxicity to understand the potential risks depending on the repeated high-dose have not been adequately studied. Herein, we report the results of in vivo toxicity evaluation for CNPs focused on the number and dose of administration in healthy mice to provide a toxicological guideline for a better clinical application of CNPs. RESULTS: The CNPs were prepared by conjugating hydrophilic glycol chitosan with hydrophobic 5ß-cholanic acid and the amphiphilic glycol chitosan-5ß-cholanic acid formed self-assembled nanoparticles with its concentration-dependent homogeneous size distributions (265.36-288.3 nm) in aqueous condition. In cell cultured system, they showed significantly high cellular uptake in breast cancer cells (4T1) and cardiomyocytes (H9C2) than in fibroblasts (L929) and macrophages (Raw264.7) in a dose- and time-dependent manners, resulting in severe necrotic cell death in H9C2 at a clinically relevant highly concentrated condition. In particular, when the high-dose (90 mg/kg) of CNPs were intravenously injected into the healthy mice, considerable amount was non-specifically accumulated in major organs (liver, lung, spleen, kidney and heart) after 6 h of injection and sustainably retained for 72 h. Finally, repeated high-dose of CNPs (90 mg/kg, three times) induced severe cardiotoxicity accompanying inflammatory responses, tissue damages, fibrotic changes and organ dysfunction. CONCLUSIONS: This study demonstrates that repeated high-dose CNPs induce severe cardiotoxicity in vivo. Through the series of toxicological assessments in the healthy mice, this study provides a toxicological guideline that may expedite the application of CNPs in the clinical settings.

Evaluation of cytotoxicity, reactive oxygen species and nitrous oxide of nanochitosan from shrimp shell.

This work aims to synthesize, characterize and evaluate the biological activity of nanochitosan (NQ) prepared from shrimp, showing an innovative character and correlating with sustainable development, in promoting an alternative to the solid waste (shrimp) shell and a biological application of the novel nanomaterial. The NQ synthesis was carried out by the alkaline deacetylation process of chitin obtained of the demineralization, deproteinization and deodorization steps from shrimp shells. NQ was characterized by X-ray Powder Diffraction (XRD), Fourier Transform infrared spectroscopy (FTIR), Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDS), N2 porosimetry (BET/BJH methods), zeta potential (ZP) and zero charge point (pHZCP). To evaluate the safety profile was carried out the cytotoxicity, DCFHA and NO tests in 293T and HaCat cell lines. Regarding the cell viability, NQ did not show toxicity for the tested cell lines. In the evaluation of the ROS production and NO tests, there was no increase in the levels of free radicals and between the negative control, respectively. Therefore, NQ does not present cytotoxicity in the cell lines tested (10, 30, 100 and 300 µg mL-1), proposing new perspectives on the use of NQ as a potential nanomaterial for biomedical applications.

Role of mucoadhesive agent in ocular delivery of ganciclovir microemulsion: cytotoxicity evaluation in vitro and ex vivo.

PURPOSE: The aim of the present study was to investigate increase in delivery of drug upon formulation as mucoadhesive microemulsion system and further to investigate possibility of any cytotoxic effects using such formulation. MATERIAL AND METHODS: Considering hydrophilic and small molecular nature of the drug, it was attempted to be formulated as microemulsion, by using pseudo ternary phase diagram method. Thus, three types of microemulsions were prepared; oil in water, water in oil type and chitosan-coated microemulsion. These microemulsions were characterized for several physicochemical properties like size, zeta potential, Polydispersity index, and compared for in vitro cell viability and ex vivo corneal irritation study. RESULTS: All three microemulsions were quite stable, transparent and homogenous systems. They showed similar drug release pattern, but highest ex vivo goat corneal permeation was observed with Chitosan coated microemulsion when compared with ganciclovir solution. CONCLUSION: All microemulsions were found to be non-irritant in in vitro cell viability assay and ex vivo corneal irritation study, indicating the potential of using such systems for delivery of drug to eye.

Safety assessment of different unloaded polymeric nanocapsules in Caenorhabditis elegans.

Nano-sized drug delivery systems have been the subject of intense research in recent years because polymeric materials allow the absorption and release of active substances in a controlled manner. Despite the benefits, the safety of nanoparticulate systems is an aspect to be understood, particularly in vivo systems. Caenorhabditis elegans is a very useful alternative model for nanotoxicology and has been recently applied in this field. The aim of this study was to evaluate toxicological endpoints in C. elegans exposed to nanocapsules (NC) prepared with different coatings: polysorbate 80 (NCP80); polyethylene glycol (NCPEG), Eudragit® RS 100 (NCEUD) and chitosan (NCCS). Nanocapsules were prepared by nanoprecipitation method and showed acceptable physico-chemical characterization. Polyethylene glycol nanocapsules and chitosan nanocapsules increased worms lethality in a dose-dependent manner in acute exposure; polysorbate 80 nanocapsules, polyethylene glycol nanocpsules and chitonan nanocapsules also increased lethality following chronic exposure. Chitosan nanocapsules were the most toxic in all exposures, demonstrating toxicity even at low concentrations. Reproduction and body length were not affected by any of the nanocapsules exposures. The expression of superoxide dismutase showed that polysorbate 80 nanocapsules at the highest concentration slightly increased SOD-3::GFP expression. On the other hand, chitosan nanocapsules exposure blunted SOD-3 expression. This work demonstrates the toxicological differences between nanocapsule produced with different coatings and indicates higher safety for the use of eugragit nanocapsule in new formulations for future drug delivery and targeting systems.

Is nanoparaquat safer than bulk paraquat? An in vitro and in vivo evaluation.

Paraquat (PQ) is an herbicide which has brought some health problems through the production of reactive oxygen species. The increasing interest in the novel formulation of agrochemicals has been aiming to provide safety for non-target organisms. Chitosan is a well-known non-toxic polymer, commonly used in preparing particles via ionotropic gelation. In this study, we prepared PQ nanoparticles (PQNPs) and evaluated their toxicity in vivo and in vitro. PQNPs were prepared and characterized in two forms, with and without the utilization of chitosan. Relative cell survival of PQNPs were studied against bulk PQ in HEK-293. Also, the acute lung injury of PQNP was assessed against treatment with acetylcysteine. Total antioxidant capacity (TAC), lipid peroxidation (LPO), total thiol groups (TTG), and hydroxyproline, along with histological changes were assessed in the lungs. The size, zeta potential, and polydispersity index of the optimum particles were about 157.7 ± 7.03, 22.25 ± 4.52, and 0.701, respectively. The encapsulation efficiency was 65.11 ± 10.45, and the loading percent of PQ was 58.57 ± 2.37. PQNPs showed an initial burst of PQ release followed by a zero-degree pattern. PQNPs displayed lower cell cytotoxicity compared to bulk PQ. LPO, TAC, TTG, and hydroxyproline levels in lungs generally showed more satisfying status in PQNPQs as well. The levels of oxidative status markers indicate lower oxidative damage in lungs and a more desirable response to acetylcysteine treatment, in line with histological changes. PQ loaded in chitosan-alginate particles offers safer characteristics compared with bulk PQ.

Marine polysaccharide laminarin embedded ZnO nanoparticles and their based chitosan capped ZnO nanocomposites: Synthesis, characterization and in vitro and in vivo toxicity assessment.

In the current scenario where more and more products containing nanomaterials are on the technological or pharmaceutical market, it is crucial to have a thorough knowledge of their toxicity before proposing possible applications. A proper analysis of the toxicity of the nanoproducts should include both in vitro and in vivo biological approaches and should consider that the synthesis and purification methods of nanomaterials may affect such toxicity. In the current work, the green synthesis of laminarin embedded ZnO nanoparticles (Lm-ZnO NPs) and their based chitosan capped ZnO nanocomposites (Ch-Lm-ZnO NCmps) is described for the first time. Furthermore, the evaluation of their in vitro cytotoxicity, phytotoxicity, and in vivo (Zebrafish embryo) toxicity was described. First, the green synthesized Lm-ZnO NPs and Ch-Lm-ZnO NCmps were fully physicochemically characterized. Lm-ZnO NPs were greatly agglomerated and had a spindle morphology ranging from 100 to 350 nm, while Ch-Lm-ZnO NCmps had irregular rod shape with flake-like structure clusters randomly aggregated with diverse sizes ranging from 20 to 250 nm. The in vitro cytotoxicity assessment of the green synthesized Lm-ZnO NPs and Ch-Lm-ZnO NCmps was carried out in normal human dermal fibroblasts (HDF) cells and human colon cancer (HT-29) cells by MTT assay. Lm-ZnO NPs and Ch-Lm-ZnO NCmps (0.1-500 µg/mL), significantly inhibited the viability of both cell lines, revealing dose-dependent cytotoxicity. Besides, the Lm-ZnO NPs and Ch-Lm-ZnO NCmps significantly affected seed germination and roots and shoots length of mung (Vigna radiata). Moreover, the zebrafish embryo toxicity of Lm-ZnO NPs and Ch-Lm-ZnO NCmps among the various concentrations used (0.1-500 µg/mL) caused deformities, increased mortality and decreased the survival rate of zebrafish embryo dose-dependently.

An innovative green synthesis approach of chitosan nanoparticles and their inhibitory activity against phytopathogenic Botrytis cinerea on strawberry leaves.

Green synthesis is a newly emerging field of nanobiotechnology that offers economic and environmental advantages over traditional chemical and physical protocols. Nontoxic, eco-friendly, and biosafe materials are used to implement sustainable processes. The current work proposes a new biological-based strategy for the biosynthesis of chitosan nanoparticles (CNPs) using Pelargonium graveolens leaves extract. The bioconversion process of CNPs was maximized using the response surface methodology. The best combination of the tested parameters that maximized the biosynthesis process was the incubation of plant extract with 1.08% chitosan at 50.38 °C for 57.53 min., yielding 9.82 ± 3 mg CNPs/mL. Investigation of CNPs by SEM, TEM, EDXS, zeta potential, FTIR, XRD, TGA, and DSC proved the bioconversion process's success. Furthermore, the antifungal activity of the biosynthesized CNPs was screened against a severe isolate of the phytopathogenic Botrytis cinerea. CNPs exerted efficient activity against the fungal growth. On strawberry leaves, 25 mg CNPs/mL reduced the symptoms of gray mold severity down to 3%. The higher concentration of CNPs (50 mg/mL) was found to have a reverse effect on the infected area compared with those of lower concentrations (12.5 and 25 mg CNPs/mL). Therefore, additional work is encouraged to reduce the harmful side effects of elevated CNPs concentrations.

Preparation, biocompatibility, and wound healing effects of O-carboxymethyl chitosan nonwoven fabrics in partial-thickness burn model.

This study was aimed at preparing O-carboxymethyl chitosan (CM-CTS) fabrics, and examining the wound healing effects on partial-thickness burn. The functional polysaccharides were produced from chitosan needle-punched nonwovens reacted with chloroacetic acid. Then the biocompatibility and biological functions were evaluated through fibroblast L-929 and SD rats. CM-CTS fabrics were obtained with elongation at break more than 42%, tensile strength reaching 0.65 N/mm2, and water vapor transmission rate about 2600 g/m2∙24 h. Moreover, CM-CTS fabrics could effectively promote the mouse L-929 migration in vitro. CM-CTS fabrics yielded satisfactory results in angiogenesis, collagen deposition, interleukin-6 content, transforming growth factor level and healing rate, which were superior to the positive control and model groups after rats suffering with partial-thickness burn. In conclusion, CM-CTS fabrics possessed proper mechanical properties, air permeability, favorable biocompatibility, acceleration on fibroblasts migration and healing capacity for partial-thickness burn injury, and owned good potential as high-quality wound dressing.

Precision-cut liver slices as a model for assess hepatic cellular response of chitosan-glutathione nanoparticles on cultures treated with zilpaterol and clenbuterol.

Zilpaterol and clenbuterol are two ß-adrenergic agonist drugs used in animal production. Both drugs have anabolic effects with advantages on carcass yield. Meanwhile, zilpaterol is approved for animal feed in authorized countries. Clenbuterol is a banned substance due to the risk of toxicity; however, it is still being used in unknown dose levels in many farm species. Therefore, the use and abuse of these substances should be closely monitored, considering the clenbuterol ability and the not proved yet of zilpaterol to produce reactive oxygen and nitrogen species. Regarding glutathione which is the main intracellular antioxidant plays detoxification functions on liver metabolism; in this work, it is our interest to know the capacity of chitosan-glutathione nanoparticles (CS/GSH-NP) as a complementary source of exogenous GSH to modify the oxide-reduction status on bovine precision-cut liver slice cultures (PCLS) exposed to clenbuterol and zilpaterol. A single drug assay was performed in first instance by adding clenbuterol, zilpaterol, chitosan nanoparticles (CS-NP), and CS/GSH-NP. Then combinate drug assay was carried out by testing clenbuterol and zilpaterol combined with CS-NP or CS/GSH-NP. The results showed that both ß-adrenergic agonists modify in a dose-dependent manner in oxide-reduction response through ROS generation. The activity or content of glutathione peroxidase activity, intracellular GSH, gamma glutamyl-transpeptidase, aspartate aminotrasnferase and alanine aminotrasnferase were modified. The exogenous GSH delivered by nanoparticles could be used to modulate these markers.

Highly resilient, biocompatible, and antibacterial carbon nanotube/hydroxybutyl chitosan sponge dressing for rapid and effective hemostasis.

Uncontrolled hemorrhage is the leading cause of trauma death. The development of safe and efficient hemostatic agents that can rapidly and effectively control bleeding is of great significance to rescue the injured. However, the mechanical, absorptive, and antibacterial properties of conventional two-dimensional hemostatic agents are not satisfactory. Herein, a series of effective three-dimensional hemostatic dressings (JWCNT/HBC sponges) are developed by chemical modification of joint-welded carbon nanotube (JWCNT) sponges with hydroxybutyl chitosan (HBC) for hemorrhage hemostasis. The JWCNT/HBC sponges exhibit high elasticity, porous structure, and suitable blood-absorption and blood-maintaining performance. Moreover, the introduction of HBC endows the JWCNT/HBC sponges with favorable blood compatibility and good antibacterial activity. The sponge treated with 0.5% HBC (JWCNT/0.5%HBC sponge) displays better antiseptic capability, faster blood clotting ability in vitro and shorter hemostasis time in vivo than the commercial gelatin sponge. The JWCNT/HBC sponges combine the advantages of JWCNT sponges and HBC in the adhesion and activation of platelets and red blood cells, thus becoming a good medical material for trauma hemostasis.

Thermo-Gelling Dendronized Chitosans as Biomimetic Scaffolds for Corneal Tissue Engineering.

Biomimetic scaffolds with transparent, biocompatible, and in situ-forming properties are highly desirable for corneal tissue engineering, which can deeply fill corneal stromal defects with irregular shapes and support tissue regeneration. We here engineer a novel class of corneal scaffolds from oligoethylene glycol (OEG)-based dendronized chitosans (DCs), whose aqueous solutions show intriguing sol-gel transitions triggered by physiological temperature, resulting in highly transparent hydrogels. Gelling points of these hydrogels can be easily tuned, and furthermore, their mechanical strengths can be significantly enhanced when injected into PBS at 37 °C instead of pure water. In vitro tests indicate that these DC hydrogels exhibit excellent biocompatibility and can promote proliferation and migration of keratocyte. When applied in the rabbit eyes with corneal stromal defects, in situ formed DC hydrogels play a positive effect for new tissue regeneration. Overall, this thermo-gelling DCs possess appealing features as corneal tissue substitutes with their excellent biocompatibility and unprecedented thermoresponsiveness.

A comparative study on chitosan nanoparticle synthesis methodologies for application in aquaculture through toxicity studies.

Chitosan nanoparticles (CSNPs) have been recently used for various applications in aquaculture, especially as drug carriers. The aim of this study was to synthesise and investigate a superlative method of CSNP synthesis for application in aquaculture through aquaculture-based toxicology screening methods. Two different methods were analysed: the first a direct ionic gelation method (A) and the other involving a low-molecular-weight chitosan microparticle intermediate method (B). Dynamic light scattering characterisation revealed that the CSNP particle sizes were 192.7 ± 11.8 and 22.9 nm from methods A and B, respectively. The LC50 values for brine shrimp toxicity were found to be 1.51 and 0.02 ppt in 24 h for methods A and B, respectively. Acute toxicity studies in Litopenaeus vannamei rendered LC50 values of 3235.94 and 2884.03 ppt in 24 h for methods A and B, respectively. Zebrafish toxicity studies revealed mortality rates of 21.67% and 55% at 20 mg/L concentration for methods A and B, respectively, with an increased expression of intracellular reactive oxygen species in method B. From these findings, it can be concluded that a comparatively reduced toxicity of CSNPs derived from ionic gelation method makes it more appropriate for application in aquaculture.

Cell Coding Arrays Based on Fluorescent Glycan Nanoparticles for Cell Line Identification and Cell Contamination Evaluation.

Cell lines are applied on a large scale in the field of biomedicine, but they are susceptible to issues such as misidentification and cross-contamination. This situation is becoming worse over time due to the rapid growth of the biomedical field, and thus there is an urgent need for a more effective strategy to address the problem. As described herein, a cell coding method is established based on two types of uniform and stable glycan nanoparticles that are synthesized using the graft-copolymerization-induced self-assembly (GISA) method, which further exhibit distinct fluorescent properties due to elaborate modification with fluorescent labeling molecules. The different affinity between each nanoparticle and various cell lines results in clearly distinguishable differences in their endocytosis degrees, thus resulting in distinct characteristic fluorescence intensities. Through flow cytometry measurements, the specific signals of each cell sample can be recorded and turned into a map divided into different regions by statistical processing. Using this sensing array strategy, we have successfully identified six human cell lines, including one normal type and five tumor types. Moreover, cell contamination evaluation of different cell lines with HeLa cells as the contaminant in a semiquantitative analysis has also been successfully achieved. Notably, the whole process of nanoparticle fabrication and fluorescent testing is facile and the results are highly reliable.

Chitosan Augments Tetramycin against Soft Rot in Kiwifruit and Enhances Its Improvement for Kiwifruit Growth, Quality and Aroma.

In this study, the co-application of chitosan and tetramycin against kiwifruit soft rot and its effects on the disease resistance, growth, quality and aroma of kiwifruit were investigated. The results show that chitosan could effectively enhance tetramycin against soft rot of kiwifruit with the field control efficacy of 85.33% for spraying chitosan 100 time + 0.3% tetramycin AS 5000-time dilution liquid, which was higher than 80.99% for 0.3% tetramycin AS 5000-time dilution liquid and significantly (p < 0.01) higher than 40.66% for chitosan 100-time dilution liquid. Chitosan could significantly (p < 0.05) improve the promoting effects of tetramycin on total phenolics, total flavonoids, SOD activity of kiwifruit compared to tetramycin during storage for 0-28 days and enhance the disease resistance of kiwifruit. Moreover, the co-application of chitosan and tetramycin was more effective than tetramycin or chitosan alone in enhancing fruit growth, improving fruit quality and increasing fruit aroma. This study highlights that chitosan can be used as an adjuvant to enhance tetramycin against soft rot of kiwifruit and promote tetramycin's improvement for the single fruit volume and weight, vitamin C, soluble sugar, soluble solid, dry matter, soluble protein, titratable acidity and aroma of kiwifruit.

Bioactive chitosan biguanidine-based injectable hydrogels as a novel BMP-2 and VEGF carrier for osteogenesis of dental pulp stem cells.

Nowadays, vascularization and mineralization of bone defects is the main bottleneck in the bone regeneration field that is needed to be overcome and developed. Here, we prepared novel in-situ formed injectable hydrogels based on chitosan biguanidine and carboxymethylcellulose loaded with vascular endothelial growth factor (VEGF) and recombinant Bone morphogenetic protein 2 (BMP-2) and studied its influence on osteoblastic differentiation of dental pulp stem cells (DPSCs). The sequential release behavior of the VEGF and BMP-2 from hydrogels adjusted with the pattern of normal human bone growth. MTT assay exhibited that these hydrogels were non-toxic and significantly increased DPSCs proliferation. The Real-time PCR and Western blot analysis on CG11/BMP2-VEGF showed significantly higher gene and protein expression of ALP, COL1α1, and OCN. These results were confirmed by mineralization assay by Alizarin Red staining and Alkaline phosphatase enzyme activity. Based on these evaluations, these hydrogel holds potential as an injectable bone tissue engineering platform.

The polyaminosaccharide-based buffers as a new type of zwitterionic buffering macromolecules for biochemical applications.

A new type of biocompatible buffers based on zwitterionic polyaminosaccharides is reported. The carboxy- and amino-groups containing carboxymethyl chitosan (CM-CS) was synthesized and reacted with hydrochloric/acetic acid resulting in CM-CS-HCl and CM-CS-HAc buffers with buffering capacity of 20.6 and 15.2 mM/pH. The new buffers were comprehensively characterized for their physicochemical properties and checked on enzymatic reactions of acetylcholinesterase (AChE) and alkaline phosphatase (ALP). Their performance was compared to the phosphate and Tris buffers. The chloride-free, CM-CS-HAc demonstrated excellent buffering activity with Michaelis constants of 0.50 and 1.00 mM and maximum reaction rates of 5.62 and 2.26 µmol/min/mL for AChE and ALP reactions, respectively. Toxicity studies on stress-sensitive bioreporter bacteria verified nontoxicity of CM-CS-HAc. Zwitterionic polyaminosaccharides overcome drawbacks of monomeric buffers, such as interference with enzyme active sites, cell membrane injury and purification difficulties. Therefore, they may become the next generation of effective buffers for biological and biochemical applications.

Chitosan - Rosmarinic acid conjugates with antioxidant, anti-inflammatory and photoprotective properties.

Rosmarinic acid is an attractive candidate for skin applications because of its antioxidant, anti-inflammatory, and photoprotective functions, however, its poor bioavailability hampers its therapeutic outcome. In this context, synthesis of polymer conjugates is an alternative to enlarge its applications. This work describes the synthesis of novel water-soluble chitosan - rosmarinic acid conjugates (CSRA) that have great potential for skin applications. Chitosan was functionalized with different contents of rosmarinic acid as confirmed by ATR-FTIR, 1H NMR and UV spectroscopies. CSRA conjugates presented three-fold radical scavenger capacity compared to the free phenolic compound. Films were prepared by solvent-casting procedure and the biological activity of the lixiviates was studied in vitro. Results revealed that lixiviates reduced activation of inflamed macrophages, improved antibacterial capacity against E. coli with respect to native chitosan and free rosmarinic acid, and also attenuated UVB-induced cellular damage and reactive oxygen species production in fibroblasts and keratinocytes.