Lytic Polysaccharide Monooxygenase Activity of Tma12 Is Critical for Its Toxicity to Whitefly.

Lytic polysaccharide monooxygenases (LPMOs) are powerful redox enzymes that transform complex carbohydrates through oxidation and make them suitable for saccharification by canonical hydrolases. Due to this property, LPMOs are considered to be a valuable component of enzymatic consortia for industrial biorefineries. Tma12 is a fern entomotoxic protein that kills whitefly and has structural similarities with chitinolytic LPMO. However, its enzymatic activity is poorly understood. Studying the role of the LPMO-like activity in the insecticidal function of Tma12 can be of considerable importance. Our results show that Tma12 preferentially binds and digests ß-chitin. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis shows that the digestion of chitin produces chitin oligosaccharides of various lengths (DP2-DP7). The Michaelis constant (km) and catalytic constant (kcat) for hydrocoerulignone are 0.022 mM and 0.044 s-1, respectively. The attenuation of catalytic activity through diethylpyrocarbonate modification abolishes the insecticidal activity of the protein. Our findings reveal that (a) Tma12 is an active LPMO and (b) LPMO activity is indispensable for its function as a bioinsecticide.

Preparation, structure and activity of polysaccharide phosphate esters.

Polysaccharides have anti-virus, anti-cancer, anti-oxidation, immune regulation, hypoglycemia and other biological activities. Because of their safety, fewer side effects and other advantages, polysaccharides are considered as ideal raw materials in food and drugs. The biological activity of polysaccharides can be improved by structural modification (such as sulfation, carboxymethylation, phosphorylation, etc.), and even new biological activity can be generated. In this review, the recent advances in the phosphorylation of polysaccharides were reviewed from the perspectives of modification methods, structures, biological activities and structure-activity relationships.

Safety Assessment of Saccharide Esters as Used in Cosmetics.

This is a safety assessment of 40 saccharide ester ingredients as used in cosmetics. The saccharide esters are reported to function in cosmetics as emollients, skin-conditioning agents, fragrance ingredients, and emulsion stabilizers. The Expert Panel for Cosmetic Ingredient Safety (Panel) reviewed the relevant data for these ingredients. The Panel concluded that the saccharide esters are safe in cosmetics in the present practices of use and concentrations described in this safety assessment.

Semi-synthesis of hybrid ulvan-kappa-carrabiose polysaccharides and evaluation of their cytotoxic and anticoagulant effects.

In this study we described the synthesis of a hybrid polysaccharide harboring moieties of ulvan and kappa-carrabiose. Alkylamines (1,3-diaminopropane and 1,6-diaminohexane) were selectively inserted into ß-D-GlcAp and α-L-IdoAp units in the ulvan structure via an amide bond formation producing ulvan-amide derivatives F-DAP (N% = 1.77; Mw = 208 kg mol-1) and F-DAH (N% = 1.77; Mw = 202 kg mol-1), which were reacted with kappa-carrabiose via reductive amination to produce hybrid ulvan-kappa-carrabiose polysaccharides F-DAP-Kb (N% = 1.56; Mw = 206 kg mol-1) and F-DAH-Kb (N% = 1.16; Mw = 200 kg mol-1). All the ulvan derivatives were characterized by 1H and 13C NMR spectroscopy and did not show cytotoxicity against human dermal fibroblasts (HDFa) at the concentrations of 25, 100, and 500 µg mL-1, neither anticoagulant properties at the range of 10-150 µg mL-1. Therefore, the ulvan-amide derivatives and the hybrid ulvan-kappa-carrabiose polysaccharides showed good biocompatibility in vitro, presenting as worthy candidates for tailoring scaffolds for biomedical applications.

Does polysaccharide quaternization improve biological activity?

The natural polysaccharides, due to their structural diversity, commonly present very distinct solubility and physical chemical properties and additionally have intrinsic biological activities that, gene-rally, reveal themselves in a light way. The chemical modification of the molecular structure can improve these parameters. In this review, original articles that approached the quaternization of polysaccharides for purposes of biological application were selected, without limitation of year of publication, in the databases Scopus, Web of Science and PubMed. The results obtained from the bibliographic survey indicate that the increase in positive charges caused by quaternization improves the interaction between modified polysaccharides and structures that have negative charges on their surface, such as the cell wall of microorganisms and some cells in the human body, such as the DNA. This greater interaction is reflected as an increase in the biological activity of all polysaccharides broached in this study. Another important data obtained was the fact that the chemical changes did not affect or irrelevantly affect the toxicity of almost all of the polysaccharides that were quaternized. Therefore, polysaccharide quaternization is a safe and effective way to obtain improvements in the biological behavior of these macromolecules.

Alginate-chitosan oligosaccharide-ZnO composite hydrogel for accelerating wound healing.

Moist, breathable and antibacterial microenvironment can promote cell proliferation and migration, which is beneficial to wound healing. Here, we fabricated a novel sodium alginate-chitosan oligosaccharide­zinc oxide (SA-COS-ZnO) composite hydrogel by spontaneous Schiff base reaction, using aldehydated sodium alginate (SA), chitosan oligosaccharide (COS), and zinc oxide (ZnO) nanoparticles, which can provide a moist and antibacterial environment for wound healing. The porosity and swelling degree of SA-COS-ZnO hydrogel are 80% and 150%, respectively, and its water vapor permeability is 682 g/m2/24h. The composite hydrogel showed good biocompatibility to blood cells, 3T3 cells, and 293T cells, and significant antibacterial activity against Escherichia coli, Staphylococcus aureus, Candida albicans, and Bacillus subtilis. Moreover, the hydrogel showed a promoting effect on wound healing in a rat scald model. The present study suggests that marine carbohydrates composite hydrogels are promising in wound care management.

Plant/algal polysaccharides extracted by microwave: A review on hypoglycemic, hypolipidemic, prebiotic, and immune-stimulatory effect.

Microwave-assisted extraction (MAE) is an emerging technology to obtain polysaccharides with an extensive spectrum of biological characteristics. In this study, the hypoglycemic, hypolipidemic, prebiotic, and immunomodulatory (e.g., antiinflammatory, anticoagulant, and phagocytic) effects of algal- and plant-derived polysaccharides rich in glucose, galactose, and mannose using MAE were comprehensively discussed. The in vitro and in vivo results showed that these bioactive macromolecules with the low digestibility rate could effectively alleviate the fatty acid-induced lipotoxicity, acute hemolysis, and dyslipidemia status. The optimally extracted glucomannan- and glucogalactan-containing polysaccharides revealed significant antidiabetic effects through inhibiting α-amylase and α-glucosidase, improving dynamic insulin sensitivity and secretion, and promoting pancreatic ß-cell proliferation. These bioactive macromolecules as prebiotics not only improve the digestibility in gastrointestinal tract but also reduce the survival rate of pathogens and tumor cells by activating macrophages and producing pro-inflammatory biomarkers and cytokines. They can effectively prevent gastrointestinal disorders and microbial infections without any toxicity.

Portulaca oleracea L. polysaccharides enhance the immune efficacy of dendritic cell vaccine for breast cancer.

Previous studies have reported that Portulaca oleracea L. polysaccharides (POL-P3b) is an immunoregulatory agent. However, few studies exist on POL-P3b as a novel immune adjuvant in combination with the DC vaccine for breast cancer treatment. In this work, a DC vaccine loaded with mouse 4T1 tumor cell antigen was prepared to evaluate the properties of POL-P3b in inducing the maturation and function of DC derived from mouse bone marrow, and then to investigate the effect of the DC vaccine combined with POL-P3b on breast cancer in vivo and in vitro. Morphological changes of DC were observed using scanning electron microscopy. Phenotypic and functional analyses of DC were detected by flow cytometry and allogeneic lymphocyte reaction. Cytokine levels in the DC culture supernatant were detected by ELISA. Western blotting analysis was used for the protein expression of TLR4, MyD88 and NF-κB. Apoptosis detection and protein expression of the tumor tissue were analyzed by TUNEL staining and immunohistochemistry, respectively. The security of POL-P3b was evaluated by the detection of hematological and blood biochemical indicators and pathological analysis for tissues. POL-P3b can induce DC activation and maturation, which is attributed to increasing the specific anti-tumor immune response, and the mechanism of action involved in the TLR4/MyD88/NF-κB signaling pathway. Experimental results in vivo further suggested that the administration of POL-P3b-treated antigen-primed DC achieved remarkable tumor growth inhibition through inducing apoptosis and enhancing immune responses. Moreover, the POL-P3b-treated DC vaccine was able to inhibit lung metastases. The results proved the feasibility of POL-P3b as an edible adjuvant of the DC vaccine for anti-breast cancer therapy.

Toxicological evaluation of a glycan preparation from an enzymatic hydrolysis of Saccharomyces cerevisiae.

The aim of this paper was to provide a comprehensive toxicological and safety evaluation of a yeast cell wall preparation (YCWP) for use as an animal feed ingredient. The following toxicological assessments were carried out: the mutagenic activity was tested using the Ames' Test in five Salmonella typhimurium strains; clastogenic activity was investigated using the mammalian micronucleus test in Swiss ICO OF1 (IOPS Caw) mice; genotoxic activity was assessed using the in vitro mammalian chromosomal aberration test in human lymphocytes; acute oral toxicity was tested by administration of a single dose of 2000 mg/kg BW. Eye and skin irritation were assessed in rabbits according to OECD guidelines; skin sensitivity was established in guinea pigs by means of the Buehler test, while acute dermal and inhalation studies in rats were further completed, also according to OECD guidelines. All conducted tests were considered valid under the experimental conditions. No significant mutagenic activity or genotoxic activity was observed, and it was concluded that the test article did not induce any clastogenic effect. YCWP was found to be mildly irritating to the eye, slightly irritating to the skin but was found to be non-sensitizing in the guinea pig. The acute oral, dermal and inhalation studies did not yield any evidence of gross toxicity or pharmacological effects.

Exopolysaccharide from Porphyridium cruentum (purpureum) is Not Toxic and Stimulates Immune Response against Vibriosis: The Assessment Using Zebrafish and White Shrimp Litopenaeus vannamei.

Exopolysaccharides, or extracellular polysaccharides (EPS, sPS), represent a valuable metabolite compound synthesized from red microalgae. It is a non-toxic natural agent and can be applied as an immunostimulant. The toxicity test of exopolysaccharides from Porphyridium has been done in vivo using zebrafish (Danio rerio) embryonic model, or the ZET (zebrafish embryotoxicity test). The administration of extracellular polysaccharides or exopolysaccharides (EPS) from microalgae Porphyridium cruentum (synonym: P. purpureum) to shrimps Litopenaeus vannamei was investigated to determine the effect of this immunostimulant on their non-specific immune response and to test if this compound can be used as a protective agent for shrimps in relation to Vibrio infection. For immune response, exopolysaccharides were given to shrimps via the immersion method on day 1 and booster on day 8. Shrimp hemocytes were taken on day 1 (EPS administration), day 7 (no treatment), day 8 (EPS booster) and day 9 (Vibrio infection) and tested for their immune response on each treatment. The result shows that the EPS is not toxic, as represented by the normal embryonic development and the mortality data. In the Pacific white shrimps, an increase in the values of all immune parameters was shown, in line with the increasing EPS concentration, except for the differential hemocyte count (DHC). In detail, an increase was noted in total hemocytes (THC) value, phagocytotic activity (PA) and respiratory burst (RB) in line with the EPS concentration increase. These results and other previous studies indicate that EPS from Porphyridium is safe, enhances immune parameters in shrimp rapidly, and has the ability to act as an immunostimulant or an immunomodulator. It is a good modulator for the non-specific immune cells of Pacific white shrimps, and it can be used as a preventive agent against vibriosis.

Use of Zebrafish Embryo Assay to Evaluate Toxicity and Safety of Bioreactor-Grown Exopolysaccharides and Endopolysaccharides from European Ganoderma applanatum Mycelium for Future Aquaculture Applications.

Natural mycelial exopolysaccharide (EPS) and endopolysaccharide (ENS) extracted from bioreactor-cultivated European Ganoderma applanatum mushrooms are of potential high commercial value for both food and adjacent biopharmaceutical industries. In order to evaluate their potential toxicity for aquaculture application, both EPS (0.01-10 mg/mL) and ENS (0.01-10 mg/mL) extracts were tested for Zebrafish Embryo Toxicity (ZFET); early development effects on Zebrafish Embryos (ZE) were also analyzed between 24 and 120 h post-fertilization (HPF). Both EPS and ENS are considered non-toxic with LC50 of 1.41 mg/mL and 0.87 mg/mL respectively. Both EPS and ENS did not delay hatching and teratogenic defect towards ZE with <1.0 mg/mL, respectively. No significant changes in the ZE heart rate were detected following treatment with the two compounds tested (EPS: 0.01-10 mg/mL: 176.44 ± 0.77 beats/min and ENS: 0.01-10 mg/mL: 148.44 ± 17.75 beats/min) compared to normal ZE (120-180 beats/min). These initial findings support future pre-clinical trials in adult fish models with view to safely using EPS and ENS as potential feed supplements for supplements for development of the aquaculture industry.

Characterization and Hepatoprotections of Ganoderma lucidum Polysaccharides against Multiple Organ Dysfunction Syndrome in Mice.

BACKGROUND: The liver is one of the most commonly affected organs in multiple organ dysfunction syndrome (MODS). In recent years, there have been many studies on Ganoderma lucidum polysaccharides (GLP), but the role of GLP in MODS is still unclear. The purpose of this work was to explore the antioxidant, anti-inflammatory, and protective effects of GLP on the liver in MODS model mice. METHODS: The characteristic properties of GLP were processed by physicochemical analysis. The MODS models were successfully established with intraperitoneal injection of zymosan in Kunming strain mice. The antioxidant, anti-inflammatory, and hepatoprotective effects of GLP were processed both in vitro and in vivo by evaluating the oxidative parameters, inflammatory factors, and liver pathological observations. RESULTS: The characterization analysis revealed that GLP was a pyranose mainly composed of glucose with the molecular weights (Mw) of 8309 Da. The experimental results proved that GLP had potential hepatoprotection possibly by improving the antioxidant status (scavenging excessive oxygen radicals, increasing the antioxidant enzyme activities, and reducing the lipid peroxide), alleviating the inflammatory response (reducing the inflammatory factor levels), and guaranteeing the liver functions. CONCLUSIONS: This research suggested that GLP had the potential to be developed as a natural medicine for the treatment of multiple organ failure.

Preparation and characterization of folic acid conjugated sulfated polysaccharides on NK cell activation and cellular uptake in HeLa cells.

In this study, the sulfated polysaccharide (SP) of Codium fragile was conjugated to folic acid (SP-FA). FT-IR and 1H NMR techniques revealed the occurrence of esterification reaction between the hydroxyl group of SP and the γ-carboxyl group of FA that confirming the SP-FA conjugation. SP and SP-FA did not show any direct toxicity on NK cells and HeLa cells. However, the treatment of SP and SP-FA enhance the NK cells cytotoxicity against HeLa cells by the upregulation of IFN-γ, TNF-α, perforin, and Granzyme-B. Moreover, NK cells activation was stimulated through NF-кB and MAPK pathways. The binding capacity studies exposed the targeting ability of HeLa cells by folate receptor (FR) which was assessed by a confocal quantitative image cytometer analysis. These results indicate that SP-FA could be used as selective drug delivery systems for targeting FR-overexpressed cancer cells with less toxicity.

Thermoresponsive polysaccharides with tunable thermoresponsive properties via functionalisation with alkylamide groups.

Polysaccharides have been used widely in many industries, from food technology and mining to cosmetics and biomedical applications. Over recent years there has been growing interest in the development of responsive polysaccharides with unique and switchable properties, particularly systems that display lower-critical solution temperatures (LCSTs). Therefore, in this study we aimed to investigate a novel strategy that would allow the conversion of non-responsive polysaccharides into thermoresponsive polysaccharides with tuneable LCSTs. Through the functionalisation of dextran with alkylamide groups (isopropyl amide, diethyl amide, piperidinyl and diisobutyl amide) using a carbodiimide coupling approach in conjunction with amic acid derivatives, we prepared a library of novel dextrans with various degrees of substitution (DS), which were characterised via nuclear magnetic resonance (NMR) spectroscopy and gel permeation chromatography (GPC). The alkylamide-functionalised dextrans were found to have good solubility in aqueous solutions, with the exception of those having a high DS of large hydrophobic substituents. Determination of the thermoresponsive characteristics of the polymer solutions via UV-vis spectroscopy revealed that the LCST of the alkylamide-functionalised dextrans was highly dependent on the type of alkylamide group and the DS and could be tuned over a large range (5-35 °C). Above the LCST, all of the thermoresponsive alkylamide-functionalised dextrans formed colloidal dispersions with particles sizes ranging from 400 -600 nm, as determined by dynamic light scattering (DLS). In addition, the polymers were found to exhibit a fast and reversible phase transition in solution with narrow hysteresis (∼ 1-5 °C). Finally, the injectability and biocompatibility of the novel thermoresponsive dextrans was confirmed in vivo via subcutaneous and intracranial ventricle injections, with no local or systemic toxicity noted over a 14 d period. Overall, the alkylamide-functionalised dextrans display interesting thermoresponsive properties and trends that may make them useful in biomedical applications, such as drug-delivery.

Dynamic Covalent Chemistry Enables Reconfigurable All-Polysaccharide Nanogels.

Dynamic covalent bonds are established upon molecular recognition of sugar derivatives by boronic acid molecules. These reversible links can be used in a cross-linking method to fabricate polymer-based responsive nanosystems. Herein, the design of the first dynamic nanogels made entirely of polysaccharides (PS) is reported. Based on PS chains alternately modified with phenyl boronic acid groups and sugar moieties, these colloids self-assemble in physiological conditions and combine the biocompatible nature of their PS backbone with the reconfiguration capacities of their cross-linking chemistry. These dynamic nanogels are easily prepared, stable for a long time, pH responsive, and efficiently internalized by cancer cells.

Fucoidan Induces Apoptosis of HT-29 Cells via the Activation of DR4 and Mitochondrial Pathway.

Fucoidan has a variety of pharmacological activities, but the understanding of the mechanism of fucoidan-induced apoptosis of colorectal cancer cells remains limited. The results of the present study demonstrated that the JNK signaling pathway is involved in the activation of apoptosis in colorectal cancer-derived HT-29 cells, and fucoidan induces apoptosis by activation of the DR4 at the transcriptional and protein levels. The survival rate of HT-29 cells was approximately 40% in the presence of 800 µg/mL of fucoidan, but was increased to 70% after DR4 was silenced by siRNA. Additionally, fucoidan has been shown to reduce the mitochondrial membrane potential and destroy the integrity of mitochondrial membrane. In the presence of an inhibitor of cytochrome C inhibitor and DR4 siRNA or the presence of cytochrome C inhibitor only, the cell survival rate was significantly higher than when cells were treated with DR4 siRNA only. These data indicate that both the DR4 and the mitochondrial pathways contribute to fucoidan-induced apoptosis of HT-29 cells, and the extrinsic pathway is upstream of the intrinsic pathway. In conclusion, the current work identified the mechanism of fucoidan-induced apoptosis and provided a novel theoretical basis for the future development of clinical applications of fucoidan as a drug.

Polysaccharide enabled biogenic fabrication of pH sensing fluorescent gold nanoclusters as a biocompatible tumor imaging probe.

A biocompatible natural polysaccharide (PSP001) isolated from the fruit rind of Punica granatum was conjugated with L-cysteine (Y) to be used as a skeleton for the fabrication of fluorescent gold nanoclusters (AuNCs) represented as PSP-Y-AuNCs. With an average size of ~ 6 nm, PSP-Y-AuNCs demonstrated high quantum yield (31%), with a pH-sensitive fluorescence emission behavior. An emission maximum of 520 nm was obtained at acidic pH, which was blue shifted with increasing pH. This feature provides the possibilities for accurate ratiometric pH imaging. The PSP-Y-AuNCs not only demonstrated excellent biocompatibility with cancer cells and isolated peripheral lymphocytes and red blood cells but also demonstrated to be an active molecular imaging probe with appealing cellular uptake efficiency. The investigations with BALB/c mice further confirmed the non-toxic nature and in vivo imaging potential of the AuNCs. Estimation of the bio-distribution on solid tumor bearing syngeneic murine models revealed a tumor-targeted enhanced fluorescence emission pattern which is attributed to the pH responsive fluorescence behavior and the acidic microenvironment of the tumor. These findings were further confirmed with an impressive tumor accumulation pattern displayed in a xenograft of human cancer bearing nude mice. On account of their impressive biocompatibility and photophysical features, PSP-Y-AuNCs can exploited for the real-time fluorescence imaging of cancer tissues. Graphical abstract Fluorescent gold nanoclusters (PSP-Y-AuNCs) fabricated using a non-toxic natural polysaccharide (PSP001) demonstrated pH sensitive fluorescence emission pattern. The increased fluorescence readouts at acidic conditions and excellent biocompatibility made the PSP-Y-AuNCs an appealing candidate for in vivo tumor imaging applications.

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

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

Cytotoxic and pharmacokinetic studies of Indian seaweed polysaccharides for formulating raindrop synbiotic candy.

Gut microbiome evidenced as the assembling mode of action facilitates the relationship of environmental factors (such as diet and lifestyle) with colorectal cancer. The cytotoxic and anticancer studies of the enzymatically extracted polysaccharides from selected Indian seaweeds (such as S. wightii, E. compressa, and A. spicifera) on Raw 264.7 macrophage and HT-29 human colon cancer cell line were investigated. E. compressa showed nitric oxide production up to a concentration of 6.99 ± 0.05 µM. The polysaccharide extract of seaweed (PES), A. spicifera (100 µg/ml) had shown the highest in-vitro cytotoxicity effect on HT-29 cells up to 52.13 ± 1.4%. Absorption, distribution, metabolism and excretion (ADME) predictions were performed for exploring the possibility of anti-cancer drug development. The formulated synbiotic candy exhibited post storage survivability of probiotic species L. plantarum NCIM 2083 up to 107 CFU/ml until three weeks and it could be an aesthetic functional food for treating colon cancer.

Analysis of In Vitro Cytotoxicity of Carbohydrate-Based Materials Used for Dissolvable Microneedle Arrays.

PURPOSE: Dissolvable microneedle arrays (MNAs) can be used to realize enhanced transdermal and intradermal drug delivery. Dissolvable MNAs are fabricated from biocompatible and water-soluble base polymers, and the biocargo to be delivered is integrated with the base polymer when forming the MNAs. The base polymer is selected to provide mechanical strength, desired dissolution characteristics, and compatibility with the biocargo. However, to satisfy regulatory requirements and be utilized in clinical applications, cytotoxicity of the base polymers should also be thoroughly characterized. This study systematically investigated the cytotoxicity of several important carbohydrate-based base polymers used for production of MNAs, including carboxymethyl cellulose (CMC), maltodextrin (MD), trehalose (Treh), glucose (Gluc), and hyaluronic acid (HA). METHODS: Each material was evaluated using in vitro cell-culture methods on relevant mouse and human cells, including MPEK-BL6 mouse keratinocytes, NIH-3T3 mouse fibroblasts, HaCaT human keratinocytes, and NHDF human fibroblasts. A common laboratory cell line, human embryonic kidney cells HEK-293, was also used to allow comparisons to various cytotoxicity studies in the literature. Dissolvable MNA materials were evaluated at concentrations ranging from 3 mg/mL to 80 mg/mL. RESULTS: Qualitative and quantitative analyses of cytotoxicity were performed using optical microscopy, confocal fluorescence microscopy, and flow cytometry-based assays for cell morphology, viability, necrosis and apoptosis. Results from different methods consistently demonstrated negligible in vitro cytotoxicity of carboxymethyl cellulose, maltodextrin, trehalose and hyaluronic acid. Glucose was observed to be toxic to cells at concentrations higher than 50 mg/mL. CONCLUSIONS: It is concluded that CMC, MD, Treh, HA, and glucose (at low concentrations) do not pose challenges in terms of cytotoxicity, and thus, are good candidates as MNA materials for creating clinically-relevant and well-tolerated biodissolvable MNAs.