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Chitosan for nanoparticles

Chitosan for nanoparticles

Rights and naniparticles Reprints and Chittosan. Fortified Hyperbranched PEGylated Chitosan-Based Common allergenic foods Composites for Chitoswn of Multiple Bacterial Allergy relief techniques. Chitosan-gadopentetic acid complex Common allergenic foods fod gadolinium neutron-capture therapy of cancer: Preparation by novel emulsion-droplet coalescence technique and characterization. Additionally, the possible interference of solvents was also assessed under the same testing conditions and as shown in Figure 4Fno stimulation of ROS production, as the fluorescence increase fold values were around 1.

Thank you fkr visiting nature. You flr using a browser version with Chktosan support for CSS. Citosan obtain Citosan best experience, we recommend you use a Common allergenic foods nanopparticles to date browser or turn off compatibility mode in Internet Explorer.

In the meantime, to ensure continued nanoparticlse, we are Common allergenic foods the site without styles and JavaScript. Naniparticles ionic gelation process for the synthesis of chitosan nanoparticles was carried out in microdroplet reactions.

The Common allergenic foods could be stopped nsnoparticles at different time points by cor dilution of the reaction mixture Chitosan for nanoparticles DI nannoparticles. Using nanoparticlew simple technique, the effect of temperature and reactant nanopagticles on the Enhancing creativity in athletics and distribution of the nanoparticles formed, as a function of time, could be investigated by DLS and SEM.

The concentration of nanopartifles, reaction temperature nsnoparticles well as naanoparticles, were found to Cyitosan and collectively determine dor size Thyroid Support Formulas nanoparticles and their distribution.

Chitosan nanoparticles are naanoparticles, relatively non-toxic, biodegradable, and cationic in nature 12. Thus, they are well nanoparticle in biomedical applications nanopartjcles as drug delivery 345.

Ionic nanpparticles is the most commonly forr method for Exercise and Nutrition Tips chitosan nanoparticles 6.

In this method, chitosan precursors are cross-linked using sodium tripolyphosphate Enhances mental efficiency. Even though ionic gelation nanopatricles a widely used method and factors Chiitosan the size and dispersivity of chitosan nanoparticles nanoparticlee as the concentration of reactants, temperature, forr, and the level of deacetylation are well known 7 our basic understanding of Cyitosan process at nanopatticles level is poor.

In the ionic gelation process, Chifosan crosslinks randomly oriented chitosan molecules, Collagen for Eye Health, in turn, are connected vor other similarly cross-linked moieties. Nanopagticles intra- and inter- molecular cross-linking is rather Chitoaan and leads to polydispersity in the nanoparticlws preparation.

Here, we hypothesised that using confined reaction volumes of microdroplets and at preset temperatures; it cor be possible to nanopaticles good control over the Chitossn of nanoparticles synthesis if the reactions Chitosan for nanoparticles be arrested nanopartkcles at desired time points.

Such an approach could enable time-lapsed capture of the synthesis process and provide us an opportunity to understand it at a more fundamental level. Thus, in our Chitossan experiments, synthesis reactions were Common allergenic foods at varying time Common allergenic foods to monitor the nucleation and growth of developing nanoparticles Common allergenic foods DLS and Boost career prospects. For this purpose a simple yet novel technique, nanopxrticles.

fast dilution of the reaction mixture with DI water was employed. In a separate nanparticles, we had determined that with a fold dilution, the reaction pH increased sharply from 2. Such a rise in pH could nanopartiicles a strong endothermic effect 8910resulting in extensive 90 de-protonation flr chitosan Dilution could also result in increased intra-molecular distances, reducing the possibility of molecular interaction.

The synthesis of chitosan nanoparticles tends to be arrested by DI water dilution owing to these reasons. It is evident from the data presented in Fig. The number of the hyphen should appear after nano nano-particles also increased with rising reaction temperature and time.

The number of nanoparticles was maximum 5. The narrowest size distribution of the fro was seen when the chitosan concentration was 0. It is clear from hCitosan data that an interplay of factors like temperature, time and concentration of reactants determines the size, distribution and the Chjtosan of particles formed.

a Simulation model of the droplet and the surrounding medium. b Simulation profile showing the variation of temperature and reactant concentration as a function of time. Dotted and continuous line represent temperature and concentration at the droplet periphery and the core respectively; nanopartcles Effect of temperature on the size distribution of chitosan nanoparticles chitosan concentration flr 0.

It was also seen that the magnitude of difference in the number of nanoparticles formed was highest when the chitosan concentration used was 0. Therefore, in further studies chitosan concentration was maintained at 0. The time-lapsed capture of the nanoparticles synthesis process was achieved by arresting the reactions at different time points and imaging the preparation by nanopaarticles electron microscopy SEM.

SEM images presented in Fig. Simultaneous and seamless nucleation and growth followed thereafter. The studies focused on understanding the heat and solute transport across the droplet.

Assigned reactant concentrations of the droplet and the medium were 0. The results of simulation studies are shown in Fig. At this nqnoparticles, the chitosan concentration at the droplet core was 0.

Najoparticles contradistinction, the chitosan concentration in the droplet periphery dropped instantaneously to 0. On the other hand, steep rise the in the droplet core temperature along with low molecular mobility favour faster reaction kinetics.

This study provides a critical insight into ionic gelation process of chitosan nanoparticles formation, caught in action, for the first time. The study shows that the concentration of reactants, temperature, and time, severally and collectively determine the fate of the synthesis process in terms of the size and distribution of particles formed.

Our simulation experiments point to a new concept, i. the quality of nanoparticles relies on the temperature dependent mechanistic pathway rather than the temporal endpoint.

Finally, these studies indicate that when it comes to obtaining superior quality nanopartifles nanoparticles, the secret lies at the core of the droplet! TPP and glacial acetic acid were purchased from Loba Chemie, India and Qualigens, India, respectively. Freshly prepared, deionized DI water pH 6.

Different sets of reactions were nanopatricles where chitosan and TPP concentrations were altered in the range of 0. The Chitosab of nanoparticles formed and their sizes were determined by dynamic light scattering DLS method nanopartticles Nanosight LM10 system Malvern, UK. The nanoparticles formed at different times were also visualized by scanning electron microscopy Zeiss EVO MA15, UK.

How to cite this article : Kamat, V. et al. Chitosan nanoparticles synthesis caught in action using microdroplet reactions. Wang, J. Recent advances of chitosan nanoparticles as drug carriers.

Google Scholar. Keawchaoon, L. Preparation, characterisation and in-vitro release study of carvacrol-loaded chitosan nanoparticles. Colloids and Surfaces B: Biointerfaces 84— Article CAS Google Scholar.

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Chitosan-based nanoparticles for tumor-targeted drug delivery. of Biological Macromolecules fr— Agnihotri, S. Recent advances on chitosan-based micro- and nanoparticles in drug delivery. Controlled Release5—28 Calvo, P.

Novel hydrophilic chitosan-polyethylene oxide naboparticles as protein carriers. Applied Polymer Science 63— Fan, W. Formation mechanism of monodisperse, low molecular weight chitosan nanoparticles by ionic technique. Colloids and Surfaces Fot Biointerfaces 9021—27 Mertins, O. Insights on the interactions of chitosan with phospholipid vesicles.

part i: Effect of polymer deprotonation. Nanoparticlss 29— Koukaras, E. Insight on the formation of chitosan nanoparticles through ionotropic gelation with tripolyphosphate.

Molecular Pharmaceutics 9— Bhumkar, D. Studies on effect of ph on cross-linking of chitosan hanoparticles sodium tripolyphosphate: A technical nanoparticlfs.

AAPS Pharm. Article Google Scholar. Rinaudo, M. Solubilization of chitosan in strong acidic medium. Analysis Characterization 5— Download references. gratefully acknowledges the fellowship from University Grants Commission UGCIndia. wishes to thank Director, ARI for support.

Nanobioscience, Agharkar Research Institute, GG Agarkar Road, Pune,India. You can also search for this author in PubMed Google Scholar. conceived the experiment sV. and D.

: Chitosan for nanoparticles

Chitosan nanoparticles preparation and applications | Environmental Chemistry Letters

Each of these diffraction peaks is a reflection of the hydrated crystalline structure and crystalline structure of anhydrous α-chitin This indicates the presence of a crystalline phase in the synthesized chitosan nanoparticles On the other hand, Olajire et al.

FTIR analysis is a powerful tool revealed various functional groups of organic compounds. The zeta potential value was used to estimate the surface charge and thus the stability of the synthesized nanoparticles.

Kheiri et al. Despite the fact that the suspension is physically stable, Muller et al. CNPs have a positive zeta potential, which indicates that they have a charge.

According to the findings of Khan et al. On the other hand, Qi et al. The differential scanning calorimeter, or DSC, is a frequently used thermal analytical tool that can assist in understanding the thermal behavior of polymers The DSC thermogram of CNPs showed two bands, which had typical polysaccharide thermal features.

The first was an endothermic wide band corresponding to polymeric dehydration ranged from The second thermal band was polymeric degradation, causing an exothermic band extending from to °C as shown in Fig.

Feyzioglu and Tornuk 94 reported that CNPs revealed an endothermic peak at Also, Vijayalakshmi et al. TGA is a thermal analysis technique that detects changes in chemical and physical characteristics of the materials as a function of growing temperature or as a function of time A thermogravimetric analyzer, model TGAH, was used to determine changes in the thermal characteristics of biosynthesized CNPs sample of about 6 mg.

The TGA of CNPs is characterized by the presence of five degradation stages Fig. These weight losses indicated partial thermal disintegration of CNPs. At heating temperature °C , the total loss was According to Sivakami et al.

On the other hand, Morsy et al. This loss is related to the evaporation of intra and inter-molecular moisture in the CNPs. When heated at °C, CNPs had a weight loss of The heat degradation of the chitosan backbone was responsible for the weight loss of Multi-drug resistant bacteria Acinetobacter baumannii complex was used to carry out the antibacterial activity tests of CNPs with concentrations of After incubation for 24 h, the inhibition zone diameter created by the well containing CNPs was recorded: 12, 16, 30 mm diameter, respectively.

The inhibition of bacterial growth increased as CNPs concentrations increased. Antibacterial activity of different concentrations of chitosan nanoparticles produced using Eucalyptus leaves extract against Acinetobacter baumannii.

The antibiotic resistance of A. baumannii complex is becoming increasingly serious. Colistin and Polymyxin, that target the cell membrane, are thought to be the final line of defense against drug-resistant bacteria, but they come with a lot of side effects, and drug resistance to these drugs is increasing gradually This study tried to control the growth of multi-drug resistant A.

baumannii complex using biosynthesized CNPs. To study the changes in the morphology of A. baumannii complex cells treated with CNPs. The control untreated cells of A. baumannii complex were represented in Fig. The cytoplasmic content of the bacterial cell was regularly distributed.

Compared with untreated cells, considerable morphological variations were detected in A. The damage in the cell membrane and the cytoplasm content leaked to the extracellular medium with increases in the periplasmic space black arrow head Fig. In addition, coagulated material was observed in the cytoplasm.

Figure 7 F Due to the loss of most cytoplasmic contents from the inner membrane, the outer membrane is enlarged and evacuated, resulting in complete membrane loss; ghost cells TEM examination of the effect of CNPs on multi-drug resistant A.

baumannii cells: A cells of untreated bacteria, B—F cells of bacteria treated with CNPs with different stages in damage and G mechanisms of antibacterial action of CNPs.

Several studies on chitosan nanoparticles revealed the stronger antibacterial activity of CNPs against Gram-negative and Gram-positive bacteria, Fig. Chitosan nanoparticles have the properties of chitosan as well as the benefits of nanoparticles. The unique properties of nanoparticles, such as their small size and quantum effects, can offer chitosan nanoparticles with higher capabilities.

This is due to the fact that the characteristics of bulk materials stay relatively constant regardless of volume; but, as their size reduces, the percentage of surface atoms increase, creating nanoparticles with some remarkable characteristics Avadi et al.

For a quantum-size impact, chitosan nanoparticles provide a stronger affinity towards bacteria cells. Chitosan nanoparticles are able to provide significant antimicrobial properties through various mechanisms.

Smaller molecules like potassium and phosphate seep out, followed by larger molecules like RNA and DNA etc. Chandrasekaran reported that chitosan nanoparticles have metallic ion chelation property which is a possible reason for its antimicrobial action; d Chitosan has the ability to create a polymer film on the cell surface, which acts as a barrier to oxygen and prevents nutrients from entering the cell, inhibiting aerobic bacterial growth Nanoparticles have a greater affinity to produce excess quantities of reactive oxygen species ROS.

Dizaj et al. Highly elevated amounts of reactive oxygen species ROS and other free radicals cause mitochondrial and endoplasmic reticulum disorder, along with severe damages to biomolecules, resulting in genotoxic effects.

In this study CNPs have been biologically synthesized and characterized, the CNPs obtained have small particle size with a regular spherical shape and positive surface charges, the antibacterial experiment indicated that the CNPs exhibited excellent antibacterial properties against Acinetobacter baumannii complex.

The biologically synthesized CNPs could be suitable for biological applications in medical treatments and food preservation. Nevertheless, CNP's mechanisms of action against bacteria have not yet been fully elucidated.

Therefore, investigations on the antibacterial mechanisms of CNPs and toxicological studies are necessary. Yin, Y. et al. Itaconic acid grafted carboxymethyl chitosan and its nanoparticles: Preparation, characterization and evaluation.

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Synthesis and physicochemical characterization of chitin derivatives. Rajam, M. Chitosan nanoparticles as a dual growth factor delivery system for tissue engineering applications. One prospective approach is to intensify the signal in the part of interest by conveying the appropriate enzyme.

For instance, horseradish peroxidase has been transported to tumours of xenograft via conjugation to tumour-specific monoclonal antibodies, and this has been utilized to oligomerize MR-definite ligands to attain an improved signal for imaging and detection of tumour [ 63 ].

Magnetic nanoparticles can be utilized for both advanced magnetic resonance imaging and applications of hyperthermia for progressive cancer management [ 63 ]. Iron oxide nanoparticles can be combined with methotrexate [ 64 ], paclitaxel [ 65 ], or other anticarcinogenic drugs [ 66 ] for theranostic therapeutic and diagnostic applications.

Nanoparticles of gold, quantum dots, and carbon nanotubes have also been customized and used for possible theranostic applications [ 67 ]. The manufacturing of medicine at the nanoscale level has been considered broadly. It is undoubtedly, the most progressive technology in the field of Np applications as of its possible benefits for instance the likelihood to alter properties such as bioavailability, solubility, diffusivity, medicine releasing profiles, and immunogenicity.

This can therefore lead to the progress and advancement of suitable administration ways, minimum toxicity, improved bio delivery, a small number of side effects, and expanded life cycle of drug [ 51 ].

Target transport is another significant part that uses nanomaterials as drug delivery systems and is classified into active and passive transport. In active positioning, moieties, for example peptides and antibodies are combined with system of drug delivery to connect them to the structures of receptor articulated at the target location.

In passive targeting, the prepared carrier compound of drug moves in the course of the blood flow. It is driven to the site of object by attraction or binding affected by properties like temperature, pH, shape and molecular site.

The chief targets in the human body are the receptors present on the cell membranes, antigens or proteins and lipid contents of the cell membrane and the surfaces [ 68 ].

The combination of diagnosis and treatment is described as theranostic and is being widely used for cancer management [ 69 , 70 ]. Theranostic nanoparticles can help in the diagnosis of the disease, recognizing the phase of the disease, reporting the location, and give information regarding the response of treatment.

Studies showed that the combination of alginate with folic acid-altered chitosan nanoparticles were efficient for revealing colorectal carcinoma cells using an illumination arbitrated mechanism based on the properties of nanoparticles to increase 5-aminolevulinic acid 5-ALA liberation in the lysosome of the cell [ 69 , 70 , 71 ].

Hyaluronic acid is one more biopolymeric material. This is a bio-friendly, negatively stimulated glycosaminoglycan, and is one of the most important components of the extracellular matrix [ 72 , 73 ]. Hyaluronic acid can combine with the CD44 glycoprotein receptor, which is frequently over-expressive in a variety of carcinogenic cells, using the receptor connecting interrelation.

Therefore, hyaluronic acid-altered nanoparticles are fascinating for their utilization in the diagnosis and treatment of carcinoma [ 74 , 75 , 76 ].

The perspective of this procedure was examined in both the live cells and in the laboratory. Amplified uptake of nanoparticles by cancer cells was detected by magnetic resonance imaging when an outer magnetic field was utilized [ 77 ].

After the intravenous administration of the nano-medium in three milligrams per kilogram concerning the free medicine rats, a huge ablation of the tumour was detected. After management, the tumours nearly vanished [ 77 , 78 ] made a nanoparticulate multipurpose complex system by encapsulating Fe 3 O 4 Np in dextran Np combined to redox-responsive chlorine 6 C6 for near-infrared and MR imaging.

Hong et al. produced glioma cells or theranostic nanoparticles of C6 mice. These particles consisted of gadolinium oxide nanoparticles covered with folic acid-combined dextran or paclitaxel. The bioprotective properties of dextran covering and the chemotherapeutic outcomes of paclitaxel on the C6 glioma cells were assessed by the MTT colorimetric assay.

The manufactured nanoparticles have been revealed to come in contact with C6 tumor cells by receptor-arbitrated endocytosis and offer improved contrast in MR concentration-reliant activity because of the paramagnetic property of the gadolinium nanoparticle.

Many nano-polymeric systems have been constructed and characterized based on both synthetic polymers and natural polymers having their drawbacks and advantages. Natural polymers such as alginate, chitosan, and hyaluronic acid have been studied for the fabrication of nanoparticle systems Table 2.

Despite progress in the drug delivery system, oral administration of the drug is still desired. Sithole et al. The review also elucidates the complexation of some natural polymers with selected synthetic chemicals to indicate few factors that have an impact on the preparation solubility, formation and stability of SSBC.

It also discusses specific significant structural and functional attributes or effects which are essential to be taken into consideration when an oral drug delivery system is developed [ 79 ].

Ahmad reported the rasagiline-encapsulated chitosan-coated poly lactic-co-glycolic acid PLGA nanoparticles RSG-CS-PLGA-NPS in a double emulsification-solvent evaporation technique.

The mean particle size, polydispersity index and encapsulation efficiency were Consequently, intranasal delivery of the drug showed significant enhancement of bioavailability in the brain [ 80 ]. Akilo et al. prepared the BCNU-Nano-co-Plex the bioactive agent loaded with chitosan, hydroxypropylmethylcellulose, pluronic F and polyaniline.

The release of bioactive agent demonstrated a Agotegaray et al. reported the MNPs magnetic nanoparticles consisting of magnetite functionalized with oleic acid and coated with the biopolymer chitosan and glutaraldehyde-cross-linked chitosan.

After 36 h, they observed the decrease in cell viability and concluded that improved biocompatibility of MNPs, resulting in better nano-systems for targeted drug delivery [ 82 ]. Dhanaraj et al. evaluated the chitosan nanoparticles containing methotrexate for the drug delivery system.

The nanoparticle was prepared by emulsion polymerization method using glutaraldehyde as the cross-linking agent. One of the samples showed the least particle size, optimum zeta potential range, moderate drug loading efficiency followed by sustained drug release over 48 h [ 83 ].

Farhadian et al. Drug loading DL , encapsulation efficiency EE and particle size were FTIR analysis showed the presence of hydrogen bonding and a few other intermolecular interactions. Liu et al. prepared the chitosan grafted halloysite nanotubes HNTs-g-CS. The study suggests that HNTs-g-CS are potential nanocarriers for drug delivery in cancer therapy, as curcumin loaded HNTs-g-CS increased apoptosis on EJ cells [ 85 ].

Shanmukhapuvvada and Vankayalapati developed hydrophilic polymers chitosan nanoparticles using the emulsification cross-linking method. One of their formulations exhibited release kinetics of Upadhyaya et al. synthesised O-carboxymethyl chitosan O-CMCS based nanocomposites NCs with nanostructured zinc oxide n-ZnO.

The drug release was and controlled in the initial phase and sustained in the later phase [ 87 ]. Taghizadeh et al. synthesised chitosan nanoparticles and chitosan beads as carriers for betamethasone and tetracycline using sodium citrate as the cross-linking agent.

They reported that the drug released from chitosan nanoparticles is lower than that released from chitosan beads [ 88 ]. There are many studies on the elaboration of chitosan-based nanoformulations which could be used in cancer treatment. Abbas et al.

This formulation showed great therapeutic improvements for drug delivery to tumours which are present in deep ling tissues [ 89 ]. The anti-metabolic compounds pyrazolopyrimidine and pyrazolopyridine thioglycosides were synthesized and encapsulated by chitosan nanoparticles to increase the anti-cancerous activity.

This nanoformulation was evaluated for its cytotoxicity against Huh-7 and Mcf-7 cells which are related to liver and breast cancer cells respectively. Genotoxic effects and a synergistic effect was conducted by cellular DNA fragmentation assay and simulated on CompuSyn software [ 90 ].

Almutairi et al. prepared the raloxifene-encapsulated hyaluronic acid-decorated chitosan nanoparticles by complexation. Bae et al. prepared the self-aggregates from deoxycholic acid-modified chitosan. These self-aggregates can form complexes with these self-aggregates. They may find potential applications as a delivery vehicle of genes and anti-cancer drugs placid DNA [ 92 ].

Deepa et al. evaluated the in-vitro efficacy of. The study suggests that chitosan nano-formulation would be an efficient approach for the release of cytarabine against solid tumours and might be a better [ 93 ]. Wu et al. synthesised hydroxycamptothecine nanoneedles integrated with an exterior thin layer of the methotrexate-chitosan conjugate, which is a dual drug, using co-precipitation in the aqueous phase.

They concluded that the emergence of a dual-drug delivery system which enhances the therapeutic performances in cancer treatment [ 94 ]. Li et al. Roy et al. encapsulated Fe3O4-bLf Fe 3 O 4 -saturated lactoferrin in alginate enclosed chitosan-coated calcium phosphate AEC-CP nanocarriers NCs.

Arunkumar et al. synthesised the composite injectable chitosan gel DZ-CGs comprising of doxorubicin-loaded zein nanoparticles DOX-SC ZNPs.

In vitro drug release profiles of composite DZ-CGs were found to be more controlled when compared to DOX-SC ZNPs.

Also, Composite DZ-CGs were more effective in killing cancer cells when compared to DOX-SC ZNPs [ 97 ]. Hwang et al. synthesised the hydrophobically modified glycol chitosan HGC nanoparticles loaded with the anticancer drug docetaxel DTX.

The DTX-HGC nanoparticles showed higher antitumor efficacy such as reduced tumour volume and increased the survival rate in A lung cancer cells [ 98 ].

Barbieri et al. prepared the nanoformulation based on phospholipid and chitosan, which efficiently loads tamoxifen by encapsulation method. The amount of drug permeated using the nano-formulation was increased from 1. This nano-formulation enhanced the non-metabolized drug passing through the rat intestinal tissue via paracellular transport [ 99 ].

Gomathi et al. fabricated the anticancer drug—letrozole® with chitosan nanoparticles using sodium tripolyphosphate as the crosslinking agent. The nano-formulation has biocompatible and hemocompatible properties which makes it an efficient pharmaceutical carrier for the anticancer drug letrozole [ ].

Jain and Banerjee compared the five different drug-carrier ratios of ciprofloxacin hydrochloride-loaded nanoparticles of albumin, gelatin, chitosan, and lipid [solid lipid nanoparticles SLNs ]. A drug-to-carrier ratio of 0. Their results suggest that chitosan nanoparticles and SLNs can act as promising carriers for sustained ciprofloxacin release in infective conditions [ ].

Khan et al. prepared temozolomide® loaded nano lipid-based chitosan hydrogel TMZNLCHG by encapsulation method. The study revealed the formulation of a non-invasive intranasal route for brain targeting as an alternative to another route for TMZ [ ]. Wang and Zhao optimized the preparation of anticancer drug—gefitinib® and chitosan protamine nanoparticles.

Koo et al. prepared the water-insoluble paclitaxel encapsulated into glycol chitosan nanoparticles with hydrotropic oligomers HO-CNPs. Paclitaxel-HO-CNPs showed higher therapeutic efficacy, compared to Abraxane®, a commercialized paclitaxel-formulation [ ].

Maya et al. prepared the O-carboxymethyl chitosan O-CMC nanoparticles, surface-conjugated with cetuximab Cet for targeted delivery of paclitaxel. They observed the Cet-Paklitaxel-O-CMC nanoparticles are a promising candidate for the targeted therapy of epidermal growth factor receptor EGFR overexpressing cancers [ ].

Al-Musawi et al. synthesised prepared chitosan-covered superparamagnetic iron oxide nanoparticles CS-SPION and applied them as a nano-carrier for loading of 5-FU CSFU-SPION.

FA-CSFU-SPION demonstrated sustained release of 5-FU at 37 °C in both phosphate and citrate buffer solutions using a reverse microemulsion technique.

There were no adverse outcomes reported for normal cells and observed that fluorescein isothiocyanate-labelled drug, has an effective entrance into a cancerous cell and stimulate cell death and apoptosis [ ].

Cavalli et al. prepared chitosan nanospheres with 5-FU by a combination of coacervation and emulsion droplet coalescence method.

Thus, nanospheres were effective in reducing tumour cell proliferation and were able to inhibit both HT29 and PC-3 adhesion to HUVEC after 48 h of treatment [ ]. Sahu et al. prepared 5-FU loaded biocompatible chitosan nanogels FCNGL using the ion gelation technique.

The pH-responsive character of nanogels triggered the release of 5-FU in an acidic environment, resulting in selective drug delivery, leading to sustained delivery of 5-FU for chemotherapy that can result in high efficacy, patient compliance and safety [ ].

The potential of intracorporeal chitosan-coated curcumin nanocrystals Chi-CUR-NC-4b were examined as a therapeutic application against endotoxemia-induced sepsis. The fabricated nanocrystals were assessed for pharmacokinetic and pharmacodynamic parameters.

Chi-CUR-NC 4b was ascertained to neutralise lipopolysaccharide LPS and increased plasma drug concentration with enhanced levels in the lungs and liver.

In vitro and in vivo pharmacodynamic studies implied that the defensive effects were mediated by the up-regulation of Nrf2 enhanced antioxidant activity, i. via elevated levels of Glutathione-S-transferase GST and Superoxide Dismutase SOD as well as the downregulation of nuclear factor kappa-light-chain-enhancer of activated B cells NF-kB.

NRF2 has been implicated in creating chemoresistance and has been linked to RAS driven cancer [ 16 , 17 ]. These effects lead to decreased cytokine secretion and decreased tissue injury resulting in enhanced survival in the murine model of LPS induced endotoxemia [ ].

Anitha et al. prepared the nanoformulation of curcumin using dextran sulphate and chitosan. The results showed the preferential killing of cancer cells compared to normal cells by the curcumin-loaded drug [ ].

Baghbani et al. The curcumin entrapment was Keerthikumarc et al. synthesised chitosan encapsulated curcumin nanoparticles by ionic gelation method. Chitosan nanoparticles formulations showed sustained release of the drug; also, in vitro cytotoxicity study showed high and long term anticancer efficacy in human oral cancer cell lines till 72 h [ ].

Rajan et al. synthesised curcumin nanoparticles loaded in chitosan biopolymer and bovine serum albumin. They observed that the selective drug targeting of colorectal carcinoma cells was effective when concentration was increased [ ]. Moreover, there are also studies on the testing of chitosan nanoparticles with plant extracts.

Shahiwala et al. synthesised the chitosan nanoparticles with alcoholic extract of Indigofera intricate— plant of potential antitumor properties. Almost a fold reduction in the extract concentration required to achieve the same anticancer activity when formulated as nanoparticles [ ].

Alipour et al. studied the sustained release of silibinin-loaded chitosan nanoparticles SCNP. George et al. studied the functionalised nanohybrid hydrogel using L-histidine HIS conjugated chitosan, phyto-synthesised zinc oxide nanoparticles ZNPs and dialdehyde cellulose DAC as a sustained drug delivery carrier for the polyphenol, plant-derived compounds—naringenin, quercetin and curcumin.

Anticancer studies towards A cells epidermoid carcinoma exhibited excellent cytotoxicity with a 15 to fold increase using the hybrid carrier, compared to the free polyphenol drugs [ ].

The chitosan nanoparticles are also tested for other groups of drugs. For example anti-inflammatory drugs. reported that the nanodevice consists of a magnetite core coated with chitosan Chit MNPs as a platform for diclofenac loading as a model drug and observed the marginal variation in the efficacy [ ].

Chaichanasak et al. prepared the chitosan-based nanoparticles with damnacanthal DAM. DAM increased the levels of the tumour suppressor non-steroidal anti-inflammatory drugs-activated gene 1 in the nucleus, therefore causing improved anticancer effects [ ].

There are also studies on antifungal and antibacterial drugs. Calvo et al. prepared the chitosan nanocapsule comprising tioconazole TIO and econazole ECO by encapsulation method.

The drug showed fungicidal activity against C. Albicans at non-toxic concentrations and reported it as the first step in the development of a pharmaceutical dosage for treating vaginal candidiasis [ ]. Abd Elsalam et al. proposed a novel chitosan-based nano-in-microparticles NIM , which acts as a combination therapy in the antibacterial platform.

PEGlyation PEG—polyethene glycol was done on chitosan, which increased its solubility in water. To treat multiple bacterial strains, the antibacterial activity of the PEG-CS was strengthened using immobilized silver nanoparticles and with dendritic polyamidoamine hyperbranches.

Ibuprofen encapsulated by montmorillonite nanoclay MMT was used as an anti-inflammatory drug. The developed drug showed good antibacterial activity against both aerobic and anaerobic bacteria resulting in treating multiple bacterial infections [ ].

Ciprofloxacin, a broad-spectrum antibiotic; a poorly soluble drug-loaded chitosan nanoparticle, was prepared for the therapeutics of various microbial infections.

The Fourier Transform Infrared Spectroscopy FTIR studies showed that there was zero interaction found between the drug ciprofloxacin and chitosan.

One of the formulations was found to have good entrapment efficacy, positive zeta potential value, and its size was from to nm [ ]. Manimekalai et al.

prepared the ceftriaxone sodium loaded chitosan nanoparticles using chitosan as a polymer and trisodium polyphosphate as a cross-linking agent. The chitosan nanoparticles developed was capable of sustained delivery of ceftriaxone sodium [ ].

Jamil et al. prepared the cefazolin loaded chitosan nanoparticles CSNPs by ionic gelation method. Kinetics study had demonstrated the excellent antimicrobial potential of cefazolin loaded CSNPs against multidrug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa [ ].

Moreover, Manuja et al. They concluded the ChQS-NPs are safe, less toxic and effective as compared to the conventional QS drug delivery [ ]. Among other drugs combined with chitosan nanoparticles, it is noteworthy to mention that are also studied antihypertensive, antidepressant and eye droop formulations.

Niaz et al. fabricated the antihypertensive AHT nano-carrier systems NCS encapsulating captopril, amlodipine and valsartan using chitosan CS polymer. They reported that the AHT nano-ceuticals of polymeric origin can improve the oral administration of currently available hydrophobic drugs while providing the extended-release function [ ].

Selvasudha and Koumaravelou prepared chitosan on simvastatin loaded nanoparticle. Better absorption was observed by reducing the lipid profile with several-fold reduced dose in the mouse model. Studies revealed possible synergistic functionalities of chitosan and the simvastatin as potential hypolipidaemic modality without any toxic manifestations [ ].

Dhayabaran et al. encapsulated antidepressant drugs with biopolymer chitosan. Synthetic drug venlafaxine and herbal extracts Hypericum perforamtum and Clitoria ternatea were encapsulated. They developed a strategy against depression by utilizing the potentials of Clitoria ternatea as a drug in nanomedicine [ ].

Yu et al. prepared water-soluble cerium oxide loaded glycol chitosan nanoparticle for the treatment of dry eye disease. The solubility of cerium in GC GCCNP increased to Concluded that GCCNP can be the potential drug in the form of eye drop for the treatment of dry eye [ ].

The performed scientific studies have provided promising results of chitosan nanoparticles in the anticancer drug delivery and oncological treatment Tables 3 , 4. Nevertheless, nowadays chitosan nanoparticles clinical applications for diagnosis and therapy of cancer has been discussed because of their minimal systemic toxicity both in vitro and some in vivo models and maximal cytotoxicity against cancer cells and tumours [ 33 , ].

Nano drug delivery systems based on chitosan nanoparticles have been developed for pre-clinical and clinical studies [ ]. Translation of novel nano-drug delivery systems from the bench to the bedside may require a collective approach.

Chitosan nanoparticles typically characterized by a positive surface charge and mucoadhesive capacities such that can adhere to mucus membranes and release the drug payload in a sustained release manner [ 33 ].

Due to such characteristics of chitosan nanoparticles their applications consist of per-oral delivery, ocular drug delivery, nasal drug delivery, pulmonary drug delivery, mucosal drug delivery, gene delivery, buccal drug delivery, vaccine delivery, vaginal drug delivery and cancer therapy [ ].

The clinical studies have shown that intravenous administration of chitosan-based nanocarriers for brain delivery and intranasal administration has been an alternative due to its mucoadhesive properties, improving the patient adhesion to therapy [ ] Table 2.

Various materials with different structural forms are conjugated with drugs to prepare nano-drug delivery systems. Considering recent approaches, the most commonly used drug delivery vehicles include liposomes [ ], nanoparticles ceramic, metallic and polymeric [ ], dendrimers [ ] and micelles [ ].

The self-assembled amphiphilic micelles based on chitosan and polycaprolactone were developed as carriers of paclitaxel to support its intestinal pharmacokinetic profile [ ].

Experimental results showed that chemical modification of chitosan nanoparticles can improve their use for therapy application [ ] and improve tumour targeting [ ] Table 2. Chitosan nanoparticles have shown anticancer activity in vitro and in vivo.

Xu et al. Also, Chitosan nanoparticles can be used to deliver siRNA targeting key components of tumor metabolism Due to their low or non-toxicity, chitosan nanoparticles and their derivatives can serve as a novel class of anti-cancer drug [ ] Table 2.

Chitosan nanoparticles can be used as carriers in the controlled drug delivery of doxorubicin, an anticancer drug used for the treatment of several tumours [ 18 ]. Doxorubicin can be toxic at some points and to protect patients from doxorubicin side effects were developed chitosan nanoparticles drug delivery system.

It is possible to encapsulate and deliver doxorubicin with reduced side effects. The chitosan oligosaccharide conjugated with biodegradable doxorubicin with farther high efficiency in the tumour growth suppression because of higher cellular uptake [ , ] Table 2.

Chitosan nanoparticles decorated with RGD peptides localize to the tumour vasculature and exert antiangiogenic effects [ ]. Another composition of chitosan nanoparticle was prepared by ionic crosslinking of N-trimethyl chitosan TMC with tripolyphosphate with a lower degree of quaternization and an increase in particle size, a decrease in zeta potential and a slower drug-release profile.

For example, ATP, a related derivative of triphosphate, is essential for life and use its encapsulation with chitosan nanoparticles can improve delivery and health effects. Such specific characteristics of N-trimethyl chitosan chloride nanoparticles can support the use of them as potential protein carriers in various modifications [ ].

Pre-clinical studies with chitosan and N,N, N-trimethyl chitosan nanoparticle encapsulation of Ocimum gratissimum essential oil exhibited antibacterial activity at a lower concentration for both Gram-negative and Gram-positive food pathogens.

In vitro cytotoxicity revealed the increased toxicity of N, N, N-trimethyl chitosan nanoparticle encapsulated in Ocimum gratissimum essential oil on MDA-MB breast cancer cell lines [ ].

Another collection approach of a nano drug delivery system based on a combination of chitosan nanoparticles with curcumin loaded dextran sulfate was studied regarding the promotion of curcumin anticancer activity.

In vitro cytotoxicity measurements demonstrated that curcumin loaded polymeric nanoparticles got significant therapeutic efficacy against colon HCT and breast MCF-7 cancer cells compared with free curcumin [ ] Table 2.

It was studied the use of chitosan nanoparticle for albumin delivery for its use as a plasma expander in critically ill patients and several other clinical applications mainly via intravenous infusion. Sustainable albumin release over time and high enzymatic stability from albumin-loaded nanoparticles were observed compared to the free albumin [ ].

The chitosan nanoparticles in the nano-system delivery in combination with hyaluronic acid can be a very promising injectable system for the controlled release of platelet-derived growth factor for tissue engineering applications, as well as for the treatment of ischemia-related diseases [ ] Table 2.

Pre-clinical studies based on development and in vitro and in vivo evaluation of chitosan nanoparticles based dry powder inhalation formulations of prothionamide revealed a dose in pulmonary administration, which will improve the management of tuberculosis [ ]. Hussain et al. had explored the histological and immunomodulatory actions of chitosan nanoparticle in the transport of hydrocortisone using chitosan nanoparticles against atopic dermatitis.

It was shown the significant capability of chitosan nanoparticles to minimize the severity of atopic dermatitis. Histological analysis revealed that chitosan nanoparticles inhibited the elastic fibres fragmentation and fibroblast infiltration.

Further, depicting their clinical importance in controlling the integrity of elastic connective tissues which makes such nanoparticles-based drug transport effective [ ] Table 2. Bupivacaine is a long-acting local anaesthetic that belongs to the amino-amide class which is widely used during surgical procedures and for postoperative pain.

Animals and in vivo studies such as infraorbital nerve blockade, local toxicity, and pharmacokinetics were used to discover the use of combination chitosan nanoparticles with bupivacaine.

Pre-clinical studies bupivacaine in chitosan nanoparticles revealed that encapsulation of bupivacaine prolongs the local anaesthetic effect after infraorbital nerve blockade and altered the pharmacokinetics after intrathecal injection [ ].

Currently in phase 3 clinical trials in the US and phase 2 clinical trials UK and EU is the chitosan-based nasal formulation of morphine RylomineTM [ ] Table 2.

Due to its biocompatibility, biodegradability and low toxicity, chitosan is widely recognized as a safe material in pharmaceutical nanotechnology.

Moreover, its versatile capabilities indicated this natural polymer and its nanoparticles as a viable vehicle in drug delivery. Once identified as an ideal drug carrier, chitosan has been exploited to design formulations for a large range of drug molecules including proteins, plasmid DNA, and oligonucleotides.

Production and clinical development of nanoparticles for gene delivery are discussed nowadays. Gene therapy is an auspicious strategy with intentionally altering the gene expression in pathological cells for the treatment of gene-associated human diseases. Its discussed role of chitosan nanoparticles as a very promising carrier for gene delivery due to high biocompatibility and close resemblance to the lipidic membranes, which facilitate their penetration into the cells [ ].

Furthermore, chitosan nanoparticles allow a controlled and, sometimes, site-specific delivery and are suitable to many routes of administration, especially for the non-invasive ones like oral, nasal, ocular and transdermal [ ]. The major advantage offered by chitosan-based nanoencapsulation is the ability to improve the dissolution rate of poorly soluble drugs thus increasing their bioavailability Fig.

This capability depends on the size of the particles as well as from the specific features of chitosan, which render this polymer an ideal drug carrier.

Chitosan is soluble in an aqueous solution but it possesses readily modifiable pH-responsive solubility. Generally, dissolution happens in dilute aqueous acid solutions, where the amino groups of chitosan become protonated.

However, many other factors contribute to controlling solution properties such as the distribution and number of acetyl groups along the chains, pH, the ionic concentration, the conditions of isolation and drying [ ].

Additionally, chitosan presents mucoadhesive and absorption-enhancing properties. The mucoadhesive nature of chitosan depends on electrostatic interaction between the positive charge on the ionizable protonated amine group and the negative charge on the mucosal surfaces.

These interactions trigger a reversible structural reorganization in the protein-associated tight junctions which opens the tight junctions between cells, allowing the drug to cross the mucosal cells [ ].

Mucoadhesion also extends the contact of the drug with the mucosal layer, and allow site-specific administration, in particular in those body site presenting specific mucosal surfaces such as buccal and nasal cavities.

Again, many factors can influence mucoadhesive properties such as the molecular weight, the flexibility of the chitosan chain, the electrostatic interaction, the availability of hydrogen bond formation, and the capacity of spreading into the mucus due to surface energy properties [ ].

Also, nanosized formulations are characterized by a large surface to volume ratios, which intensely strengthen the intrinsic properties of chitosan. Nanostructure of appropriate size and surface charge can improve drug penetration thus improving uptake through the cell membrane.

Therefore, nanosized carriers could effectively modulate pharmacokinetics, enhancing drug efficacy beside reduced toxicity [ ] and offer the possibility to deliver bioactive agents in a controlled and, sometimes, site-specific manner.

However, there are several challenges in the use of drug nanocarriers such as low drug encapsulation, premature release, poor permeability and instability, which could finally affect drug bioavailability. In particular, stability represents one of the most important factors regulating the efficiency of drug delivery systems, especially in the case of nanoparticles [ ].

As regards chitosan nanocarriers, instability could depend on degradation by digestive enzymes and pH variation throughout the gastrointestinal tract. Additionally, a surface charge strongly influences stability and distribution and limits there in vitro and in vivo application. Indeed, although positively charged particles are strongly attracted by negatively charged cell membranes leading to an efficient internalization in the cells, the interaction with serum components could lead to severe aggregation followed by a fast clearance from the circulatory system [ ].

Therefore, many attempts of tailoring the chitosan nanoparticles have been accomplished, aiming to confer improved stability against aggregation in biological settings. The most frequent strategy followed consists of hydrophilic modifications with molecules able to improve stability and solubility in slightly acid and neutral media such as β-cyclodextrin, succinic anhydride or PEG.

Besides, also surface decoration with hydrophilic polymers has been carried out in the attempt to contrast nanoparticles aggregation [ ]. However, changes in stability and aggregation of chitosan nanoparticles could also happen during storage. Different techniques of drying i.

Generally, nanopowder is easily re-dispersible, but occasionally aggregation or irreversible fusion of particles occurs making the redispersion more difficult.

In this regard, the addition of bioprotectants could reduce surface attraction maintaining the nanoparticles dispersed [ ]. Chitosan is a linear polysaccharide composed of D-glucosamine units deacetylated units and N-acetyl- d -glucosamine units β- 1—4 -connected.

Chitosan is deacetylated chitin Fig. Commercially chitosan is produced by deacetylation of chitin, a natural material, widespread in the world of exoskeletal crustaceans. It has some remarkable therapeutic properties such as blood coagulation, fat binding, heavy metal ion complexation, hemostatic action.

In addition to the degree of deacetylation for a given chitosan sample, the molecular weight of the macromolecule, which can vary between ,—, daltons, is also characteristic.

Chitin and chitosan are of high commercial interest due to their high nitrogen content 6. Both chitin and chitosan are biodegradable, biocompatible, non-toxic, non-allergenic and renewable biomaterials and find their application in fields such as medicine, perfumes and cosmetics, food industry and agriculture [ ].

Chitosan, due to the presence of the primary amine group in the sugar units form the polymeric structure, dissolves in dilute organic acids, but is insoluble in water, above pH 6—7 and in ordinary organic solvents. The solubility of chitinous substances is usually associated with the crystallinity of the sample.

Higher crystallinity suggests greater or increased molecular interactions between the polymer chains. A chitinous chemical can be dissolved only if these interactions are cancelled.

The intra- and intermolecular hydrogen bonds of the polymer chains are the major cause of these interactions and play an important role in the low solubility of these substances.

However, chemical modifications of chitosan result in derivatives that are water-soluble in a broader pH range, including in strongly basic environments.

The modifications consist of the introduction of ionic groups or substituents in the polymeric structure, which dissolves in polar solvents such as water through polar-polar interactions and determines the solubility of the macromolecule [ ].

The process of isolation of chitin begins in the marine food industry. One of the by-products of this process, such as carapace of radishes, shrimps, etc. Alternatively, if isolation of chitin is not desired, the sequence based on acid treatment may be reversed to produce chitosan directly. During the treatment with basic medium, concomitant hydrolysis of the acetamide groups of chitin takes place, the result being the formation of chitosan.

The physical properties of chitinous substances are governed by two factors: the degree of deacetylation and the molecular mass. The former has a direct impact on the secondary structure of the polymer chain and can influence and solubility of the polymer in organic or aqueous solvents.

It can also affect the chemical reactivity of the sample inhomogeneous processes [ ]. According to a selective nomenclature, chitinous substances that do not dissolve in dilute organic acids e. On the other hand, chitinous substances that dissolve in dilute aqueous acids are called chitosan.

A distribution of acetyl groups on the polymer structure results in homogeneous processing conditions and gives solubility of polymers in aqueous solutions of weak acids. Instead, under heterogeneous processing conditions, polymers are formed with distinct blocks of acetylated sugar residues and are not soluble in solvents.

The molecular weight of chitosan obtained at the end of the production process depends on the process parameters, time, temperature and HCl and NaOH concentration. The process parameters used in chitosan production are drastic and the cleavage of the chitin structure accompanies the process.

The degradation of the chitinous chain can be extended. In one preparation, a chitin sample with a molecular weight of 1. However, the charged nature of chitosan tends to form free aggregates and the differences in the degree of deacetylation for different chitosan samples require careful implementation of the constants [ ].

Many applications of any chemical, natural or synthetic, require chemical process ability. Thus, chitosan, a white powder, is difficult to handle due to the problems of solubility in neutral water, bases and organic solvents.

The pKa value of the primary amino groups in chitosan is 6. Even if chitosan and its derivatives are soluble at a pH lower than 6, most of its applications in the basic or neutral environment cannot be achieved [ ].

On the other hand, acidic solutions in which chitosan is soluble are not compatible with many applications, such as those in cosmetics, medicine and nutrition. There are two approaches in the literature on improving the solubility of chitosan at neutral pH. The first is the chemical derivatization of chitosan for example with substituents containing quaternary ammonium group, by carboxymethylation or sulfation so that the added substituent is hydrophilic.

Under the conditions of homogeneous processing, the obtained chitosan remains in solution after neutralization and no derivatization is required.

Some applications of chitosan use derivatized forms thereof and to improve the solubility it is necessary to introduce ionic groups in the polymeric structure [ ]. Traditionally, chitinous substances are used in rudimentary medicine and the treatment of wastewater.

In recent decades, these substances have found their applicability in various fields, from textile engineering to photography. Chitosan and its derivatives have attracted more interest than chitin, even though the latter has found its applicability in medicine, fibre, absorbable tissues and bandages.

It is interesting to note the resistance of chitinous substances to bile, pancreatic juice and urine, which leads to their use in surgery, but also the manufacture of human-made fibres for hard materials [ 95 ].

These substances may be subject to degradation with lysozyme, an enzyme found in nature and the human eye, and with chitinase. This has also led to the use of chitosan derivatives in the preparation of cleaning solutions for contact lenses to remove enzyme deposits [ ].

Chitosan has antimicrobial properties antibacterial and antifungal. Antibacterial action is rapid and eliminates bacteria within hours. Moreover, its derivatives are biodegradable and exhibit reduced toxicity in mammalian cells.

The antibacterial activity is associated with the length of the polymer chain and suggests a cooperative effect of the individual carbohydrate units. The antibacterial property of chitosan is useful in medicine, where it is used in the manufacture of surgical accessories such as gloves, bandages, etc.

It is also used to remove pathogens from water and as a food preservative by adding a layer to the outside of fruit and vegetable products [ ].

As chitosan is obtained by deacetylation usually not complete of chitin, studies related to the analytical characterization of chitin and chitosan are not without interest. As can be seen from the structures below, the two substances differ in the presence in the case of chitin and the only sporadic presence in the case of chitosan of the acetyl group grafted by the amino function.

Chitosan is immiscible with water. Some chitosan components contain hydroxyl group components, capable of intermolecular hydrogen bonds, due to the macromolecular character of the compound and due to the many intermolecular hydrogen bonds, even in the solid-state of the sample.

It is difficult to discuss the toxicity of this substance, because chitosan is a natural, non-toxic and biodegradable compound, widely used, due to its unique properties, in biotechnology, human and veterinary medicine, but also cosmetics.

Chitosan is widely regarded as being a non-toxic, biologically compatible polymer. It is approved for dietary applications in Japan and many countries from Europa and the FDA has approved it for use in wound dressings.

The modifications or degree of deacetylation DD made to chitosan could make it more or less toxic and any residual reactants should be carefully removed.

A synopsis of toxicity chitosan's reported is shown in Table 4. The toxicity of chitosan drug administration in animals was reported [ ]. For the reasons listed above, the analytical use of IR spectra was passed, in the spectral range — cm — 1 respectively — cm — 1 , in the transmittance form vs.

wave number. The bands are generally large due to the macromolecular character of the compound and due to the numerous intermolecular hydrogen bonds, manifested even in the solid-state of the sample. The absorption bands can be easily attributed to molecular fragments: the dominant band with a maximum at cm — 1 is due to the valence vibrations stretching, ν O—H and ν N—H of the O — H and N — H connections involved.

intense in hydrogen bonds. the band with maximum absorption at cm — 1 is due to the valence vibrations of the C — H connections. The series of bands between cm — 1 and cm — 1 are characteristic of the amide group the bands "amide I", … "amide VI".

Because this band is associated with the acetyl groups in the molecule, its use is warranted to specify the degree of deacetylation of a chitosan sample the more advanced the acetylation degree, the more intense this band is. To be able to use the intensity of the "amide I" band, the spectra obtained at different recordings must be standardized.

The normalization can be achieved by bringing by mathematical processing the intensity of the maximum band ν O—H and ν N—H to the value 1. According to the information studied the possible cases of toxicity may arise due to the chemical transformations to which chitosan is subjected, more precisely the Degree of deacetylation DD.

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Production of Chitosan Nanoparticles | Encyclopedia MDPI

Plasma μL was incubated with a volume of 50 μL of Chit NPs and Chit polymer suspensions two final concentrations: 0.

Then, samples were evaluated using Bio-TP LI PT and Bio-CK APTT kits Biolabo S. Platelet-rich plasma PRP was obtained from blood collected in sodium citrate tubes, and centrifuged at × g for 16 min.

Platelet-free plasma PFP was obtained after blood centrifugation at 2, × g for 10 min, followed by plasma centrifugation at 18, × g for 5 min. A volume of μL of PRP or μL of PFP were added to well plates and incubated for 5 min at 37°C. Then, 4 μL of Giemsa dye was added to each well and incubated for 5 min.

Finally, a dilution with saline solution was applied for platelet counting PC using a light microscope. Chit NPs were also incubated with PFP to evaluate the NPs interference in plasma. Results were expressed as mean ± standard error of the mean SEM.

Prism 6. Statistical significance was assessed using one-way ANOVA. The characterization of the polymers used and the nanoparticulate delivery system developed is critical to prevent erroneous interpretations of resultant immunotoxicity findings.

Different Chit characteristics can have different biological effects. Unfortunately, most studies addressing biological activity of Chit NPs lack the used polymer characterization, which also restricts comparisons among studies. The two Chit polymers used in this study were purified under endotoxin-free conditions to eliminate possible contaminants.

Since the purification process involves harsh conditions, namely heating the polymer suspension in NaOH 1 M, their DDA and MW were assessed before and after purification and the results presented in Table 1A. An important decrease from to 49 kDa is compatible with the fact that lower DDA Chit has higher enzymatic and acid hydrolysis degradation rate Kurita et al.

Table 1. Physicochemical characterization of Chit polymers and NPs. A Polymer molecular weight MW , deacetylation degree DDA , and size in acetate buffer and after resuspension in DMEM and RPMI at 37°C Mean ± SEM. Endotoxin contamination of pyrogen-free water was also evaluated for comparison.

Chit is soluble in acidic conditions, which is incompatible with cell culture as it leads to cell death. Particle size in acetate buffer and cell culture media is illustrated in Table 1A.

The mean average size of these particle suspensions was around μm in all situations. These NPs were isolated and concentrated in water. This result was important to calculate Chit NP concentration. After isolation and concentration, NP mean particle size, polydispersity index PDI , and zeta potential ζ were determined by DLS and ELS, respectively, and are summarized in Table 1B.

Results illustrate the effect of the different Chit on the NP characteristics. In fact, the same methodology, when applied to Chit polymers with different DDA and MW, resulted in NPs with different sizes.

These average particle sizes were illustrated by TEM and SEM analysis. The round shape of the NPs was the second conclusion inferred by observing the images Figures 1A,B of both techniques. Figure 1. Chit NP illustration by Electron Microscopy.

Due to the complexity of cell culture media, and the variability of their supplementation, results from NP colloidal system characterization in water are not transposable to in vitro conditions Moore et al. Chit NPs were therefore characterized in cell culture media to understand the changes that NPs experience during in vitro studies.

Even though the DLS methodology for size analysis in complex media such as cell culture medium has limitations, it can give us some insights about changes occurring to the different Chit NPs.

Most notably, the suspension of both Chit NPs in RPMI and DMEM resulted in increased PDI, meaning an increase of the size heterogeneity. This change induced by the adsorption of negatively charged proteins from the medium, to positively charged Chit residues, forms a protein corona, decreasing the suspension stability.

Under these conditions, the appearance of aggregates is inevitable which is part of the explanation for the PDI increment. In the water, the size of both NP was distributed over a single peak Figures 2A,D , while in cell culture media, there were at least three independent peaks Figures 2B,C,E,F.

We can hypothesize that the alterations observed in cell culture media size dispersion, including smaller and bigger size populations simultaneously, were induced not only by the presence of proteins, but also by the high ion content in comparison to water Moore et al.

Furthermore, as the media composition is different between RPMI and DMEM, the observed changes in the NP size distribution were not similar. A comparable phenomenon was described by Yang et al.

Figure 2. NP size distribution by DLS. The last step of characterization was related to endotoxin contamination. As previously mentioned, Chit polymers were purified by a method published by our group Lebre et al.

The method allows the obtainment of endotoxin-free chitosan, proved by two methods: Limulus Amebocyte Lysate LAL test and the absence of IL-6, secreted by dendritic cells DCs , cultured in the presence of chitosan. The chitosan does not induce IL-6 secretion by DCs and endotoxins do that stimulation.

Furthermore, for in vitro immunotoxicity studies, the NP production was performed under those conditions, to avoid endotoxin contamination, as the presence of these molecules can easily lead to false positive results. To assure that Chit purification and Chit NP production were successfully achieved, both Chit polymers and NPs as well as the pyrogen-free water and the TPP solution used for NP production, were submitted to LAL test.

Importantly, before establishing the methodology for endotoxin quantification with Pyrochrome® testing kit, all recommended tests to evaluate sample interference with LAL test were done to guarantee the suitability of the LAL test for Chit NPs, as described in the manufacturer's instructions.

The results were presented in Table 1C , and show that all tested samples were not significantly different from pyrogen-free water, the negative control, and all were far below 0. Thus, it was demonstrated that the process and conditions used to minimize the contamination and remove existent endotoxins during Chit purification and NP production was effective, and that Chit polymers and NPs used in immunotoxicity tests were indeed LPS-free, supporting the reliability of the results.

Nevertheless, these cells should be used carefully since their functional stability is not maintained at high passage number. Indeed, a recent article mentions the phenotype and functional characteristics to remain stable from passage 10 to 30 Roberts et al. The evaluation of the cytotoxic profile of Chit NPs and polymers was performed using the MTT metabolic activity assay, over a wide range of concentrations as illustrated in Figure 3.

Figure 3. Cell viability studies in RAW D Evaluation of the cell viability resultant from the incubation of RAW The reduction of the reagent MTT by cells leads to the generation of insoluble crystals of formazan that once dissolved in DMSO generate a purple signal van Meerloo et al.

Since it is a colorimetric assay, and although the cell medium with the testing sample was aspirated before solubilizing the formazan crystals, NP interferences with the readout were tested to validate the assay Figure 3C.

As it is possible to observe, the measured absorbance Abs was not increased by the presence of the NPs or polymer suspension. Additionally, to guarantee that the cell viability results were only related with the NP and polymers, and not with the solvents, the supernatants collected from the NPs last washing step with water, as well as the acetate buffer used to disperse the polymers, were also tested using MTT assay Figure 3D.

Results showed that the solvents did not cause any decrease in cell viability. Reactive oxygen species ROS are unstable molecules that easily react with other molecules and may cause damage to DNA, RNA, proteins and ultimately lead to cell death, when accumulated Schieber and Chandel, To evaluate the effect of Chit polymers and Chit NPs on ROS production by RAW The increase in ROS production was concentration dependent, however, for the concentration range tested, the effect was not as high as LPS-induced ROS production.

Importantly, all tested conditions did not induce cellular death as confirmed by the MTT assay performed at the end of each experiment Figure S1A. In order to have a more complete picture, studies were conducted to evaluate that the polymers and NPs would not play an inhibitory role in the production of ROS by cells stimulated with LPS.

Results in Figure 4B show that no inhibitory effect was observed for any of the tested samples. Figure 4. Immunotoxicity assays in RAW All assays were performed with non-cytotoxic concentrations of NPs, polymers and controls evaluated by MTT assay after every experiment.

For the experiment, test samples were incubated with RAW For the experiment, LPS and test samples were co-incubated with RAW Negative control C— was not co-incubated with LPS. Data are presented as mean ± SEM. The possibility of having the nanoparticles interfering with the methods should not be ruled out, leading to false positives or false negatives.

So, to evaluate the interference of Chit NPs and Chit polymers in the fluorescence readouts, the ROS production assay was performed without cells and at the highest polymer and NPs concentrations. The values obtained for test samples were similar to the medium alone Figure 4E , meaning that they do not interfere with ROS measurement.

Additionally, the possible interference of solvents was also assessed under the same testing conditions and as shown in Figure 4F , no stimulation of ROS production, as the fluorescence increase fold values were around 1.

NO is an important inflammatory mediator released by macrophages during inflammation, being one of the main cytostatic, cytotoxic, and pro-apoptotic mechanisms of the immune response Bosca et al.

NO production by RAW Again, all test samples were sterile and endotoxin-free in order to prevent false positive results, and used in adequate concentrations that did not affect cell viability Cell viability study in Supplementary Figures 1C,D.

With the aim to evaluate whether one of the polymers or Chit NPs would be able to induce cells to produce NO, samples were incubated with the RAW None of the Chit NPs or polymer concentrations tested induced NO production.

The results shown in Figure 4D indicate that there was a slight but significant inhibitory effect on LPS-induced NO production, at all concentrations tested when compared to the LPS control.

Since the Chit and Chit NP concentrations tested did not induce significant reduction in cell viability Supplementary Figure 1D we can exclude the hypothesis that it was a consequence of cellular death. For all NPs, the possible interference with optical detection methods is a hypothesis that should be tested before doing the test itself.

So, similar to ROS assay, the NO assay was performed in the presence of the test samples, without cells and the results were presented on Figure 4G.

The solvent of the Chit NPs suspension or the chitosan polymer suspension were evaluated to understand if they also had an effect on NO production Figure 4H. No interferences were observed in the readout, and the solvents were not able to induce NO production.

An additional control was performed for NO production assay, to evaluate whether Chit and Chit NPs, due to their cationic charge, could be adsorbing NO at their surface, reducing the amount of NO quantified.

Such phenomenon would provide an explanation for the NO production inhibition observed. To evaluate this hypothesis, we performed the NO calibration curve in the presence and absence of Chit NPs and polymers Figure 4I. As shown, the NO curves are all overlapping, meaning no interferences from Chit NPs and Chit polymers were observed.

PBMCs are a good model to study immune responses, since they secrete regulatory and pro-inflammatory cytokines and chemokines in the human body. In vitro cell viability experiments give an indication of a particle cytotoxic profile that may be observed in vivo.

Similar to RAW The results depicted in Figure 5A showed that Chit NPs were more cytotoxic than the respective polymers. Figure 5. Cell viability studies in PBMCs and assay interference evaluation. This difference was further confirmed with the PI assay, where the cell membrane integrity rather than the metabolic activity was evaluated Figure 5B.

To note, Chit NP and polymer highest concentrations tested during cell viability assessment in both RAW In our case, these concentrations were needed to correctly calculate the IC As explained for the RAW The absorbance readout showed no interference for formulations equal Abs values and the resultant cell viability following solvent incubation with PBMCs during 24 h showed comparable cell viability to the control.

Cytokines participate in many physiological processes, mostly in the regulation of immune and inflammatory responses Ai et al. Interleukin-6 IL-6 is a pleiotropic cytokine inflammatory and anti-inflammatory properties able to modulate the activity of immune cells Wang et al.

Tumor necrosis factor-α TNF-α is a pro-inflammatory cytokine released from macrophages or activated T cells which plays a crucial role in many immune and inflammatory processes, such as proliferation, apoptosis, and cell survival Cai et al. Results showed that neither Chit NPs nor Chit polymers stimulated the production of IL-6 and TNF-α, as no differences were found before and after incubation with test samples.

Importantly, the use of positive controls such as LPS and Con A, give us an indication of the cell function regarding the cytokine we are analyzing. Notably, both positive controls significantly increased the cytokine secretion in PBMCs.

Figure 6. Cytokine secretion in PBMCs and interference evaluation. Cytokine quantification was performed by ELISA. Dotted lines represent the original calibration curve in ELISA diluent.

Additionally, since chitosan's positive charge favors cytokine adsorption, the possible interference of Chit NPs in cytokine quantification was tested. For that, Chit NPs suspended in cell culture medium were incubated with known concentrations of each cytokine calibration curve for 24 h, then centrifuged and supernatant cytokine content similarly quantified by ELISA.

The percentage of cytokine quantification in cell culture medium incubated with the nanoparticles in comparison to cell culture medium without nanoparticles, can reveal if the cytokines adsorbed to the NPs, preventing their quantification. Thus, we can assume that the absence of TNF-α and for IL-6 production upon stimulation with Chit NPs and polymers was indeed due to the lack of the samples' ability to stimulate the cells, which strengthens the conclusion that they do not induce a pro-inflammatory cytokine response, at least when produced under endotoxin-free conditions.

Hemolysis is characterized by the rupture of red blood cells RBCs and the release of their contents, ultimately leading to anemia, jaundice and renal failure Dobrovolskaia et al. All materials entering the blood get in contact with RBCs and so the evaluation of the hemolytic ability of the biomaterials is of utmost importance.

Chit NPs and polymers hemolytic activity was evaluated following a 3 h incubation at 37°C with RBCs. Triton X was used as the hemolytic agent whose effect is possible to observe by the red color of the supernatant after centrifugation of the experiment tube 1 and 2 Figure 7B.

Although no hemolytic activity was induced by Chit NPs and polymers, solvents were tested as well as the NP interference with the assay readout. As depicted in Figure 7C , the NPs had no interference in the absorbance measurements and Figure 7D illustrates that no hemolysis was induced by the NPs or solvents of the suspensions of the NPs or polymers.

Figure 7. Hemolysis assay. A Hemolytic activity of Chit polymers and NPs in human blood after 3 h incubation at 37°C. C Evaluation of the Chit NP and Chit polymers interferences with the absorbance readout, without blood.

D Evaluation of the hemolysis resultant from the incubation of NPs solvent and polymers solvent in human blood after 3 h incubation at 37°C.

The plasma coagulation cascade is responsible for blood clotting and consists of a series of protein interactions Laloy et al. To evaluate the effect of Chit NPs and polymer samples on plasma coagulation time, two concentrations 0.

In this assay, both blood coagulation pathways, the activated partial thromboplastin time APTT and the prothrombin time PT were separately tested Figure 8A. Figure 8. Coagulation assay. A Effect of Chit NPs and polymers at 0. The two coagulation pathways, APTT and PT, were separately tested.

APTT reference range of values is 20—40 s and for PT is 11—14 s. B Controls of interferences of NPs and polymers solvents with the coagulation times assay. The results showed that Chit NPs and polymers at 0.

NPs suspension solvent and polymer suspension solvent acetate buffer was also tested to discard any method interference and no effect was observed in plasma coagulation Figure 8B. Platelets play an important role not only in hemostasis but also in immune and inflammatory responses Golebiewska and Poole, Homeostatic imbalance as a result of platelet function alterations affect primary hemostasis and can result in thrombotic or hemorrhagic disorders Golebiewska and Poole, Therefore, it is important to study Chit NPs interactions with platelet function.

To assess platelet aggregation, a cytometer is frequently used to count the platelets, however, by this method the interference of NPs, due to their size have to be taken into account.

To evaluate the interference of Chit NPs with the platelet count, Chit NPs were incubated with platelet-free plasma PFP and visualized under the light microscope. Results showed that Chit NPs, most likely in the form of aggregates, were possibly counted as platelets, which invalidated the use of such method.

To overcome this setback and assess platelet aggregation, the experiment was performed by counting platelets manually under a microscope, using a Neubauer chamber.

Results from microscopy observation were summarized in Figures 9A,B. Figure 9. A Representative images of platelet aggregation assay stained with Giemsa dye.

Untreated platelet free plasma PFP is represented in image 1 and untreated platelet rich plasma PRP is represented in image 2.

B Quantification of the platelet aggregation effect. Platelet count is presented as the final average of a minimum of three donors ± SD. The Figure 9A -1 clearly shows the absence of platelets typical from PFP, while plenty of platelets were observed in PRP, with no signs of aggregates Figure 9A When platelets were incubated with calcium chloride, we observed the formation of fibrins, a sign of platelet aggregation Figure 9A Similarly, collagen also induced platelet aggregation, but in this case no fibrins were observed Figure 9A When analyzing both types of Chit NPs incubated with PFP we can see their tendency to form NPs aggregates, which were hypothetically the cause of the observed interference in the cytometry technique Figures 9A -5,7.

Nevertheless, under microscopic observation, these aggregates were not misinterpreted as platelets. Besides that, platelet aggregation was observed.

A hot topic in the nanomedicine field are polymeric NPs, which are engineered to either interact or not with the immune system. In the early stages of the development of a nanotechnology-based medicine, when the drug is to be encapsulated into NPs, the first question to be considered is, whether it is supposed that the new nanomedicine, in addition to its main pharmacological action, also acts on the immune system.

This kind of approach is part of the SbD. Particularly, in the case of chitosan, as it is a set of polymers with different MW and DDA [quality attributes QA ], it is important to understand if there are differences between them, regarding possible interactions with the immune system.

For Chit NPs, in addition to polymer QA, NP characteristics, like size and zeta potential or shape can also be important. Therefore, physicochemical characteristics PCC of the polymers and NP might influence their immunological properties, and therefore a thorough characterization of both is very important to supplement the immunotoxicity studies and to draw meaningful conclusions Crist et al.

The lack of an exhaustive characterization may preclude the correct interpretation of results and may lead to misinterpretations hindering the establishment of trends regarding how Chit NP PCC influence the immune response.

Additionally, one of the most important challenges encountered in in vitro immunotoxicity tests for NPs is related to their unique physicochemical properties.

These can interfere with the established tests, originally developed for testing conventional chemicals Dobrovolskaia and McNeil, Such interference depends both, on the NPs tested and the in vitro assay and can lead to false-positive or false-negative results Dobrovolskaia and McNeil, Lastly, in order to achieve a correct result interpretation, it is important to identify the presence of biological contaminants in the NP preparation Dobrovolskaia and McNeil, The main biological contamination in in vitro assays, even when working under sterile conditions, are endotoxins, which may lead to inflammatory responses Dobrovolskaia and McNeil, The present case study intends to provide a systematic analysis of the effects of Chit NPs and respective Chit polymers on different biological outcomes commonly tested under the immunotoxicity scope, considering, as most important the effect of DDA and MW, without neglecting possible interferences and contaminants.

In detail, as literature suggests, we found that Chit NPs appear to be more cytotoxic than the respective Chit polymers from which they were derived.

On the other hand, when the polymers were assembled into NPs, the same range of Chit concentrations induced a concentration dependent reduction in cell viability. CNPs were used to provide a prolonged release of hair growth agent, minoxidil sulphate, into hair follicles without dermal exposure 4.

Chitosan nanoparticles were also used as an additive in antimicrobial textiles for healthcare 5. Chitosan nanoparticles were also used for herbicide delivery for weed eradication 6 , in insecticide 7 , nanofertilizer for balanced nutrition of plants 8 and fungicide treatment 9.

Chitosan nanoparticles show effective antimicrobial activity against medical pathogens Escherichia coli , Klebsiella pneumoniae , Pseudomonas aeruginosa and Staphylococcus aureus The use of chemical and physical methods has many disadvantages which are the use of high-pressure, energy, temperature, toxic chemicals and the large particles size 11 , 12 , A self-assembled chitosan nanoparticles were prepared in the range — nm Nguyen et al.

Van et al. According to the findings of Ha et al. Ghormade et al. As a result, there is a critical need to develop environmentally safe strategies for nanoparticles synthesis with ultrafine size.

Green approaches were utilized to produce ultrafine nanoparticles with a size of less than nm, which is a crucial characteristic for a great number of applications in which the specific surface area plays a role Microorganisms such as bacteria 19 and fungi 20 were used for the biosynthesis of nanoparticles.

Additionally, secondary metabolites found in plant leaves extracts were used as a reducing agent for nanoparticles biosynthesis It is claimed that biological agents act as stabilizers, reducers, or both during the nanoparticle formation process Eucalyptus family, Myrtaceae is one of the most widely planted genera on the world Besides essential oils the Eucalyptus genus contains; flavonoids eucalyptin 5-hydroxy-7, 4'-dimethoxy-6, 8- dimethylflavone , triterpenes ursolic acid , long chain ketones tritriacontane, dione and its 4-hydroxy equivalent , glycosides, acylphloroglucinol derivatives and a combination of many different chemical entities.

The leaf waxes are an illustration of the variety of compounds found in Eucalyptus. Antimicrobial drug resistance has progressively developed over the past several decades and is one of the most important challenges since many microbial infections are getting more resistant to currently marketed antimicrobial medications 24 , 25 , Due to the increasing incidence of pathogenic multidrug-resistant bacteria, the pharmaceutical industry has an urgent need for more rational approaches for the discovery of innovative medications Acinetobacter baumannii strains are a common pathogen that can cause severe nosocomial infections acquired in hospitals, particularly in intensive care units.

These infections can include bacteremia, pneumonia, and urinary tract infections 28 , skin infections and soft-tissue in patients with burn injuries. Additionally, strains of Acinetobacter baumannii have the ability to create a biofilm, which is one of the major bacterial pathogens.

This is due to the fact that biofilms are resistant to multiple classes of antibiotics including tetracyclines, carbapenems, aminoglycosides, fluoroquinolones, and other extended-spectrum β-lactams 29 , Consequently, it is vital to find novel strategies to avoid and treat infections caused by biofilm forming Acinetobacter baumannii strains.

Biosynthesis of chitosan nanoparticles is affected by various conditions such as temperature, pH, incubation time and chitosan concentration.

The statistical design, including the response surface methodology, is efficient for optimization operational parameters. The response surface methodology RSM is a set of mathematical and statistical methods for building models, designing experiments, and finding the optimum conditions for optimizing the reaction conditions.

There are several advantages for using RSM that includes suitability for multiple factors experiments, less experiment numbers, finding of the best suitable conditions and studying the interaction between the factors 31 , 32 , In the previous studies, the mean size of CNPs synthesized by ionic gelation, nano spray dryer and self-assembly varied between and nm 8 , 14 , 15 , Bekmukhametova et al.

The chitosan nanoparticles with sizes ranging from 10 to 80 nm shown potential for nanomedicine, biomedical engineering, industrial, and pharmaceutical fields Therefore, there is a critical need to develop safe strategies for CNPs biosynthesis with ultrafine size for biomedical applications.

In the present study, an extract of Eucalyptus globulus Labill leaves was used to produce ultrafine CNPs with a size range between 6. This is a crucial characteristic for many applications where the specific surface area is important.

This study was mainly focused on the green synthesis of chitosan nanoparticles from chitosan solution by using Eucalyptus globulus Labill leaves extract. The characterizations of the biosynthesized nanoparticles were also performed and the antibacterial activity of the CNPs were evaluated against biofilm forming Acinetobacter baumannii as a test strain.

Fresh Eucalyptus globulus Labill leaves Supplementary Fig. Permission was obtained for collection of leaves. The plant was kindly identified by Prof.

Mohamed Fathy Azzazy, head of Surveys of Natural Resources Department, Environmental Studies and Research Institute, University of Sadat City, Egypt. The voucher specimen Eucalyptus globulus Labill has been deposited at the herbarium of Environmental Studies and Research Institute, at University of Sadat City, Egypt.

The Eucalyptus globulus Labill leaves were collected according to institutional, national, and international guidelines and legislation. The plant leaves were rinsed three times with tap water, followed by a final washing with distilled water to remove any remaining dirt, then chopped into appropriate pieces.

For the biosynthesis of chitosan, the Eucalyptus globulus Labill leaves extract was prepared by mixing 25 g of chopped leaves with one hundred milliliters of distilled water, boiling for ten minutes, and filtering through filter paper.

The pH was adjusted to 4. To ensure that the chitosan was entirely dissolved in the solution, it was stirred for twenty-four hours. CNPs were obtained by shaking the mixture at rpm for 60 min at 50 °C.

After incubation, the CNPs suspension was centrifuged at 10,× g for ten minutes, then freeze dried. Box—Behnken experimental design 36 is a response surface method that could be used to obtain maximum response and to observe the interactions among the process factors and the response.

The following second-order polynomial equation is used to fit the RSM experimental results using the response surface regression approach:. The Box—Behnken design was created by Design-Expert software Version 7. Multiple regression analysis was performed on the experimental data to determine the analysis of variance ANOVA , to determine P ˗value, F -value, and confidence levels.

The coefficient of determination R 2 and adjusted R 2. The concentration of the biosynthesized CNPs suspension was reached to 0. The diluted CNPs suspension was first homogenized in a high-speed homogenizer at a speed of 13, rpm for 10 min before the analysis, after which it was kept in an ultrasonic bath.

The sample was analyzed thrice. XRD is one of the most essential tools for characterizing the structural features of CNPs. The generator was running at 10 kV with a current of 30 mA. The thermal properties of CNPs were investigated using differential scanning calorimetric DSC analysis at the Central Laboratory, City of Scientific Research and Technological Applications, Alexandria, Egypt.

Freeze-drying sample of approximately 3. The temperatures used during the scan ranged between 25 to °C. CNPs sample was analyzed using TGAH Thermogravimetric analyzer on a sample of approximately 6 mg. The FTIR spectroscopy investigation has been carried out in order to investigate the surface characteristics of CNPs.

For surface characteristics investigation, sample of CNPs was ground with KBr pellets. The Shimadzu FTIR S spectrophotometer was used to take the measurements for the CNPs' FTIR spectrum. The scanning range was between and cm —1 with a 1 cm —1 resolution.

Scanning electron microscopy SEM investigation was used to detect the surface morphology, size and shape of CNPs. Sample of CNPs coated with gold by using a sputter coater SPI-Module.

Energy dispersive X-ray Spectroscopy EDX , which obtained using TEM, is often used for determining a sample's elemental composition. Antibacterial activity was tested against multidrug-resistant Acinetobacter baumannii bacteria isolated from clinical specimens and kindly provided by Mabaret El Asafra Hospital, Alexandria, Egypt.

A stock culture of A. baumannii was grown on nutrient agar medium then incubated at 30 °C for 24 h before being stored at 4 °C until use. The well-diffusion method was used to test the ability of the biosynthesized CNPs to inhibit the growth of multidrug-resistant Acinetobacter baumannii bacteria using swab inoculation assay method.

Bacterial suspension was prepared according to the method of Amini Tapouk et al. In sterilized Petri dishes, 50 mL of nutrient agar medium was poured and allowed to solidify. After solidifying, the nutrient agar plate surfaces were inoculated by spreading a volume of the bacterial suspension over the nutrient agar plate surface.

Then, wells with a diameter of 6 mm were punched aseptically, and a volume of µL of the CNPs were introduced into the well.

Petri plates were incubated at 37 °C for 24 h. Following the incubation period, the plates were examined to determine whether or not inhibition zones had developed around the wells. The diameter of the inhibition zone surrounding the well, including the diameter of the well, was measured in millimeters.

The bacterial cells were cultured in nutrient broth medium. After incubation time, the bacterial cells were harvested by centrifuging at × g for 10 min and then suspended them in water. The bacterial cells suspended in water were treated with CNPs and incubated at 37 °C for an hour.

The residues of treated cells were collected and fixed with formalin-glutaraldehyde fixative in 0. The residues of treated cells were then embedded in a polymerizing resin before being sectioned to a thickness of 90 Angstroms. Sections should be stained with uranyl acetate for 5 min on the grid cobber, followed by 2 min of lead citrate staining Transmission electron microscopy JEOL-JSM PLUS, Alexandria, Egypt was used to investigate the effect of CNPs on the cell morphology of the multidrug resistant A.

Many different approaches have been utilized in the synthesis of chitosan nanoparticles. The stability and safety of the CNPs, in addition to the particle size, are factors that should be considered when choosing an acceptable preparation process In this study, E.

citriodora leaves extract was used for CNPs biosynthesis. The optical characteristics of CNPs revealed a single peak, and the highest absorbance was measured at a wavelength of nm.

A Three vials contain 1, Chitosan solution; 2, Eucalyptus globulus Labill leaves extract; 3, biosynthesized chitosan nanoparticles.

B UV—vis spectrum of chitosan nanoparticles biosynthesised using Eucalyptus globulus Labill leaves extract. In order to achieve optimal conditions for biosynthesis of chitosan nanoparticles, the Box-Behnken design BBD was used.

A total of 29 runs were used to optimize the levels of the selected variables, with 5 runs runs 15, 17, 21, 25, and 27 at the middle level center point runs. As indicated in Table 1 , the minimum production of CNPs was 3.

On the other hand, the maximum production of CNPs was 9. Table 1 displays both the actual and predicted results of the biosynthesized CNPs. It can be demonstrated that the experimental results of the biosynthesized CNPs slightly differ from the predicted results. The analysis of variance ANOVA is shown in Table 2.

The determination coefficient R 2 was used to check the model's fit. The R 2 values are a measure of the amount of variance in response values that can be explained by the experimental variables and the interactions between them The estimated value of R 2 was 0.

The R 2 value is typically in the range of 0 and 1. When the determination coefficient R 2 value is close to 1, the applied design is more effective in predicting the response When the R 2 value of the regression model is closest to one, it means that the model predicted values are nearly close to the actual values The adjusted R 2 value was 0.

It has been established that the regression model whose R 2 value is more than 0. Table 2 shows the F -values and P -values. P- values were calculated to assess the significance of each coefficient and the degree of the mutual interactions between various parameters.

As the P -values decreased, the significance of the corresponding coefficient increased. In addition, process factors whose confidence levels were greater than or equal to 95 percent and whose P -values were less than or equal to 0. The model's F -value was P -value less than 0. A, B, C, and D had F -values of 5.

Signs of the coefficients were applied to interpret the data positive or negative effect on the response On the basis of the calculated coefficients Table 2 , linear effects B, D , mutual interactions AC, BC and CD , quadratic effects A 2 , B 2 , C 2 and D 2 exerted negative effects on the chitosan nanoparticles biosynthesis.

The presence of a positive coefficient indicates that there is a synergistic impact being produced by the interactions between two variables. The adequate precision value measures the signal-to-noise ratio; a signal-to-noise ratio greater than four is ideal and demonstrates the model's accuracy The current model has an adequate precision value of The model showed mean, standard deviation and C.

Table 3 show Box—Behnken design fit summary results of CNPs biosynthesis using Eucalyptus globulus Labill leaves extract. The fit summary used to choose the adequate model for CNPs biosynthesis using Eucalyptus globulus Labill leaves extract linear, 2 factors interactions 2FI or quadratic model.

On the basis of the significance of the model terms and the insignificance of the lack of fit tests, the appropriate model is chosen. Furthermore, the model summary statistics for CNPs biosynthesis quadratic model revealed the minimum standard deviation of 0.

In order to explore the relationship between independent and dependent variables, a polynomial equation of the second order was used.

This equation was used to determine the optimum levels of initial pH A , incubation time B , chitosan concentration C , and temperature D , as well as the highest CNPs biosynthesis that corresponded to these optimum levels. By using multiple regression analysis to the collected experimental results, the following second-order polynomial equation defining the predicted CNPs biosynthesis Y regarding the independent variables A, B, C, and D was obtained:.

In which Y is the predicted CNPs biosynthesis, A is the value of the initial pH level, B is the incubation time value, C is the chitosan concentration value and D is value of temperature. The three-dimensional response surface graphs were generated for determination of the pairwise interaction effects between the independent variables initial pH level, incubation time, chitosan concentration and temperature as well as the optimal levels of the variables for maximum CNPs biosynthesis Fig.

Kamat et al. Three-dimensional surface plots of CNPs biosynthesis using Eucalyptus globulus Labill leaves extract Fig. Three-dimensional surface plot for chitosan nanoparticles biosynthesis using Eucalyptus globulus Labill leaves extract.

The 3D surface graphs Fig. The plots reveal that the CNPs biosynthesis increased by the increasing of initial pH level. Maximum CNPs biosynthesis was obtained toward the center point of initial pH level around 4. Further increase or decrease led to the decrease in the CNPs biosynthesis.

Sathiyabama et al. The plots reveal that the CNPs biosynthesis increased by the increasing of incubation period. Maximum CNPs biosynthesis was obtained toward the center point of incubation period around Our findings are in agreement with those of El-Naggar et al. graveolens leaves extract was estimated to be 9.

Oliveira et al. Saifful and Shahidan 52 reported that the extension of incubation time to 18 h produced a greater average size of nanoparticles compared with the shorter time 2 h. On the other hand, Fig. Maximum CNPs biosynthesis 9.

Further increase led to the decrease in the CNPs biosynthesis. Vaezifar et al. Handani et al. Mahmoud et al.

Figure 2 C,E depicts the three-dimensional response surface plots as function of temperature on the CNPs biosynthesis when interacting with the other variables: initial pH level and incubation period; respectively.

The plots reveal that the CNPs biosynthesis increased as temperature increased to the optimal level. The synthesis of nanoparticles decreases as the temperature increases. The normal probability plot NPP of the residuals is an important graphical technique represents the residual distribution to check the model's adequacy In the normal probability of the experimental residuals, as shown in Fig.

The residuals are the variation between the predicted CNPs biosynthesis values by the theoretical model and the experimental values of the CNPs biosynthesis. A small residual value indicates that the model prediction is accurate and the model was well fitted with the experimental results.

A Normal probability plot of internally studentized residuals, B Box—Cox plot of model transformation, C plot of predicted versus residuals, and D plot of internally studentized actual values versus predicted values of for chitosan nanoparticles biosynthesis using Eucalyptus globulus Labill leaves extract.

Figure 3 B represents the Box—Cox plot generated by the model transformation for the biosynthesis of chitosan nanoparticles. As shown in Fig. The model is in the optimal zone because the blue line of the current Lambda lies between the two vertical red lines.

This indicates that the model fits the obtained experimental results well and that no data transformation is required In Fig. As shown in the graph, the residuals were scattered in a random pattern all around the zero line. The residuals were scattered equally and randomly above and below the zero line, indicating that the residuals had a constant variance and supporting the model's precision Figure 3 D depicts a plot of predicted versus actual chitosan nanoparticles biosynthesis.

Figure 3 D shows all points were collected along the diagonal line, indicating a significant correlation between the theoretical values that were predicted by the model and the actual results of the chitosan nanoparticles biosynthesis which confirms the accuracy of the model The purpose of the desirability function and experimental design was to determine the optimal predicted conditions for maximizing the response.

The desirability function values ranged from 0 undesirable to 1 desirable The desirability function value is often determined mathematically prior to experimental validation of the optimization process In this study, the predicted values obtained for the tested variables were as the following: incubation time The maximum predicted value of biosynthesized CNPs was Figure 4 A—D depicts an investigation of the morphology of biosynthesized chitosan nanoparticles using SEM and TEM, respectively.

The morphology of all nanoparticles was relatively homogeneous, with a quite consistent particle size distribution and spherical in shape. The SEM analysis indicates spherical particles with a smooth surface. While, TEM analysis of the obtained biosynthesized chitosan nanoparticles reveals particles ranging in size between 6.

Comparing this study to earlier ones, the biosynthesized CNPs made from an aqueous extract of fresh Eucalyptus globulus Labill leaves have the smallest particle sizes. The SEM micrograph of chitosan nanoparticles in Wardani et al. Using FE-SEM, Khanmohammadi et al. According to the findings of Van et al.

Bodnar et al. In Dudhani et al. Zhang et al. A,B SEM micrographs, C,D TEM micrograph of chitosan nanoparticles biosynthesised using Eucalyptus globulus Labill leaves extract.

The EDX spectrum analysis of the biosynthesized chitosan nanoparticles detected the presence of: carbon C , oxygen O and nitrogen N , as main elements in chitosan nanoparticles as shown in Fig.

A EDX, B XRD, C FTIR, D zeta potential, E DSC and F TGA analyses of chitosan nanoparticles biosynthesised using Eucalyptus globulus Labill leaves extract. An X-ray diffraction pattern was used to recognize the crystal phases of the materials.

X-ray diffraction was used to detect the crystallinity of CNPs as shown in Fig. The XRD pattern of the dried CNPs was recorded at angles within the range of 10°—40° 2θ with time per step s, generator tension of 30 kV, generator current of 10 mA, and temperature of The XRD pattern of CNPs sample showed three distinctive peaks at 2 θ which were at The crystalline structure of chitosan nanoparticles was demonstrated by XRD patterns that displayed strong peak at angle of Similar results were obtained by Rasaee et al.

In the XRD diffraction patterns of the chitosan and chitosan nanoparticles, the diffraction peaks at 2 θ of Each of these diffraction peaks is a reflection of the hydrated crystalline structure and crystalline structure of anhydrous α-chitin This indicates the presence of a crystalline phase in the synthesized chitosan nanoparticles On the other hand, Olajire et al.

FTIR analysis is a powerful tool revealed various functional groups of organic compounds. The zeta potential value was used to estimate the surface charge and thus the stability of the synthesized nanoparticles.

Kheiri et al. Despite the fact that the suspension is physically stable, Muller et al. CNPs have a positive zeta potential, which indicates that they have a charge. According to the findings of Khan et al. On the other hand, Qi et al. The differential scanning calorimeter, or DSC, is a frequently used thermal analytical tool that can assist in understanding the thermal behavior of polymers The DSC thermogram of CNPs showed two bands, which had typical polysaccharide thermal features.

The first was an endothermic wide band corresponding to polymeric dehydration ranged from The second thermal band was polymeric degradation, causing an exothermic band extending from to °C as shown in Fig.

Feyzioglu and Tornuk 94 reported that CNPs revealed an endothermic peak at Also, Vijayalakshmi et al. TGA is a thermal analysis technique that detects changes in chemical and physical characteristics of the materials as a function of growing temperature or as a function of time A thermogravimetric analyzer, model TGAH, was used to determine changes in the thermal characteristics of biosynthesized CNPs sample of about 6 mg.

The TGA of CNPs is characterized by the presence of five degradation stages Fig. These weight losses indicated partial thermal disintegration of CNPs. At heating temperature °C , the total loss was According to Sivakami et al. On the other hand, Morsy et al.

This loss is related to the evaporation of intra and inter-molecular moisture in the CNPs. When heated at °C, CNPs had a weight loss of The heat degradation of the chitosan backbone was responsible for the weight loss of Multi-drug resistant bacteria Acinetobacter baumannii complex was used to carry out the antibacterial activity tests of CNPs with concentrations of After incubation for 24 h, the inhibition zone diameter created by the well containing CNPs was recorded: 12, 16, 30 mm diameter, respectively.

The inhibition of bacterial growth increased as CNPs concentrations increased. Antibacterial activity of different concentrations of chitosan nanoparticles produced using Eucalyptus leaves extract against Acinetobacter baumannii. The antibiotic resistance of A.

baumannii complex is becoming increasingly serious. Colistin and Polymyxin, that target the cell membrane, are thought to be the final line of defense against drug-resistant bacteria, but they come with a lot of side effects, and drug resistance to these drugs is increasing gradually This study tried to control the growth of multi-drug resistant A.

baumannii complex using biosynthesized CNPs. To study the changes in the morphology of A. baumannii complex cells treated with CNPs. The control untreated cells of A. baumannii complex were represented in Fig.

The cytoplasmic content of the bacterial cell was regularly distributed. Compared with untreated cells, considerable morphological variations were detected in A.

The damage in the cell membrane and the cytoplasm content leaked to the extracellular medium with increases in the periplasmic space black arrow head Fig. AAPS Pharm. Article Google Scholar. Rinaudo, M. Solubilization of chitosan in strong acidic medium.

Analysis Characterization 5 , — Download references. gratefully acknowledges the fellowship from University Grants Commission UGC , India.

wishes to thank Director, ARI for support. Nanobioscience, Agharkar Research Institute, GG Agarkar Road, Pune, , India. You can also search for this author in PubMed Google Scholar. conceived the experiment s , V. and D. conducted the experiment s , D. and K. analyzed the results and co-wrote the manuscript.

All authors reviewed the manuscript. Correspondence to Dhananjay Bodas or Kishore Paknikar. This work is licensed under a Creative Commons Attribution 4. Reprints and permissions.

Kamat, V. Sci Rep 6 , Download citation. Received : 22 October Accepted : 10 February Published : 29 February Anyone you share the following link with will be able to read this content:.

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Abstract The ionic gelation process for the synthesis of chitosan nanoparticles was carried out in microdroplet reactions. Introduction Chitosan nanoparticles are biocompatible, relatively non-toxic, biodegradable, and cationic in nature 1 , 2.

Results and Discussion It is evident from the data presented in Fig. Full size image. Figure 2: SEM of chitosan nanoparticles captured at varying time points and temperatures. Figure 3. Additional Information How to cite this article : Kamat, V. References Wang, J. Google Scholar Keawchaoon, L.

Article CAS Google Scholar Patel, T. Article CAS Google Scholar Prabaharan, M. Article CAS Google Scholar Agnihotri, S. Article CAS Google Scholar Calvo, P. Article CAS Google Scholar Fan, W. Article CAS Google Scholar Mertins, O.

Article CAS Google Scholar Koukaras, E. Article CAS Google Scholar Bhumkar, D. Article Google Scholar Rinaudo, M. Article CAS Google Scholar Download references.

Chitosan Nanoparticles: Shedding Light on Immunotoxicity and Hemocompatibility

Moreover, there are also studies on the testing of chitosan nanoparticles with plant extracts. Shahiwala et al. synthesised the chitosan nanoparticles with alcoholic extract of Indigofera intricate— plant of potential antitumor properties.

Almost a fold reduction in the extract concentration required to achieve the same anticancer activity when formulated as nanoparticles [ ]. Alipour et al. studied the sustained release of silibinin-loaded chitosan nanoparticles SCNP.

George et al. studied the functionalised nanohybrid hydrogel using L-histidine HIS conjugated chitosan, phyto-synthesised zinc oxide nanoparticles ZNPs and dialdehyde cellulose DAC as a sustained drug delivery carrier for the polyphenol, plant-derived compounds—naringenin, quercetin and curcumin.

Anticancer studies towards A cells epidermoid carcinoma exhibited excellent cytotoxicity with a 15 to fold increase using the hybrid carrier, compared to the free polyphenol drugs [ ].

The chitosan nanoparticles are also tested for other groups of drugs. For example anti-inflammatory drugs. reported that the nanodevice consists of a magnetite core coated with chitosan Chit MNPs as a platform for diclofenac loading as a model drug and observed the marginal variation in the efficacy [ ].

Chaichanasak et al. prepared the chitosan-based nanoparticles with damnacanthal DAM. DAM increased the levels of the tumour suppressor non-steroidal anti-inflammatory drugs-activated gene 1 in the nucleus, therefore causing improved anticancer effects [ ]. There are also studies on antifungal and antibacterial drugs.

Calvo et al. prepared the chitosan nanocapsule comprising tioconazole TIO and econazole ECO by encapsulation method. The drug showed fungicidal activity against C. Albicans at non-toxic concentrations and reported it as the first step in the development of a pharmaceutical dosage for treating vaginal candidiasis [ ].

Abd Elsalam et al. proposed a novel chitosan-based nano-in-microparticles NIM , which acts as a combination therapy in the antibacterial platform. PEGlyation PEG—polyethene glycol was done on chitosan, which increased its solubility in water. To treat multiple bacterial strains, the antibacterial activity of the PEG-CS was strengthened using immobilized silver nanoparticles and with dendritic polyamidoamine hyperbranches.

Ibuprofen encapsulated by montmorillonite nanoclay MMT was used as an anti-inflammatory drug. The developed drug showed good antibacterial activity against both aerobic and anaerobic bacteria resulting in treating multiple bacterial infections [ ].

Ciprofloxacin, a broad-spectrum antibiotic; a poorly soluble drug-loaded chitosan nanoparticle, was prepared for the therapeutics of various microbial infections. The Fourier Transform Infrared Spectroscopy FTIR studies showed that there was zero interaction found between the drug ciprofloxacin and chitosan.

One of the formulations was found to have good entrapment efficacy, positive zeta potential value, and its size was from to nm [ ]. Manimekalai et al. prepared the ceftriaxone sodium loaded chitosan nanoparticles using chitosan as a polymer and trisodium polyphosphate as a cross-linking agent.

The chitosan nanoparticles developed was capable of sustained delivery of ceftriaxone sodium [ ]. Jamil et al. prepared the cefazolin loaded chitosan nanoparticles CSNPs by ionic gelation method. Kinetics study had demonstrated the excellent antimicrobial potential of cefazolin loaded CSNPs against multidrug-resistant Klebsiella pneumoniae, Pseudomonas aeruginosa [ ].

Moreover, Manuja et al. They concluded the ChQS-NPs are safe, less toxic and effective as compared to the conventional QS drug delivery [ ]. Among other drugs combined with chitosan nanoparticles, it is noteworthy to mention that are also studied antihypertensive, antidepressant and eye droop formulations.

Niaz et al. fabricated the antihypertensive AHT nano-carrier systems NCS encapsulating captopril, amlodipine and valsartan using chitosan CS polymer.

They reported that the AHT nano-ceuticals of polymeric origin can improve the oral administration of currently available hydrophobic drugs while providing the extended-release function [ ].

Selvasudha and Koumaravelou prepared chitosan on simvastatin loaded nanoparticle. Better absorption was observed by reducing the lipid profile with several-fold reduced dose in the mouse model.

Studies revealed possible synergistic functionalities of chitosan and the simvastatin as potential hypolipidaemic modality without any toxic manifestations [ ]. Dhayabaran et al. encapsulated antidepressant drugs with biopolymer chitosan. Synthetic drug venlafaxine and herbal extracts Hypericum perforamtum and Clitoria ternatea were encapsulated.

They developed a strategy against depression by utilizing the potentials of Clitoria ternatea as a drug in nanomedicine [ ]. Yu et al. prepared water-soluble cerium oxide loaded glycol chitosan nanoparticle for the treatment of dry eye disease. The solubility of cerium in GC GCCNP increased to Concluded that GCCNP can be the potential drug in the form of eye drop for the treatment of dry eye [ ].

The performed scientific studies have provided promising results of chitosan nanoparticles in the anticancer drug delivery and oncological treatment Tables 3 , 4. Nevertheless, nowadays chitosan nanoparticles clinical applications for diagnosis and therapy of cancer has been discussed because of their minimal systemic toxicity both in vitro and some in vivo models and maximal cytotoxicity against cancer cells and tumours [ 33 , ].

Nano drug delivery systems based on chitosan nanoparticles have been developed for pre-clinical and clinical studies [ ]. Translation of novel nano-drug delivery systems from the bench to the bedside may require a collective approach.

Chitosan nanoparticles typically characterized by a positive surface charge and mucoadhesive capacities such that can adhere to mucus membranes and release the drug payload in a sustained release manner [ 33 ].

Due to such characteristics of chitosan nanoparticles their applications consist of per-oral delivery, ocular drug delivery, nasal drug delivery, pulmonary drug delivery, mucosal drug delivery, gene delivery, buccal drug delivery, vaccine delivery, vaginal drug delivery and cancer therapy [ ].

The clinical studies have shown that intravenous administration of chitosan-based nanocarriers for brain delivery and intranasal administration has been an alternative due to its mucoadhesive properties, improving the patient adhesion to therapy [ ] Table 2.

Various materials with different structural forms are conjugated with drugs to prepare nano-drug delivery systems. Considering recent approaches, the most commonly used drug delivery vehicles include liposomes [ ], nanoparticles ceramic, metallic and polymeric [ ], dendrimers [ ] and micelles [ ].

The self-assembled amphiphilic micelles based on chitosan and polycaprolactone were developed as carriers of paclitaxel to support its intestinal pharmacokinetic profile [ ]. Experimental results showed that chemical modification of chitosan nanoparticles can improve their use for therapy application [ ] and improve tumour targeting [ ] Table 2.

Chitosan nanoparticles have shown anticancer activity in vitro and in vivo. Xu et al. Also, Chitosan nanoparticles can be used to deliver siRNA targeting key components of tumor metabolism Due to their low or non-toxicity, chitosan nanoparticles and their derivatives can serve as a novel class of anti-cancer drug [ ] Table 2.

Chitosan nanoparticles can be used as carriers in the controlled drug delivery of doxorubicin, an anticancer drug used for the treatment of several tumours [ 18 ]. Doxorubicin can be toxic at some points and to protect patients from doxorubicin side effects were developed chitosan nanoparticles drug delivery system.

It is possible to encapsulate and deliver doxorubicin with reduced side effects. The chitosan oligosaccharide conjugated with biodegradable doxorubicin with farther high efficiency in the tumour growth suppression because of higher cellular uptake [ , ] Table 2.

Chitosan nanoparticles decorated with RGD peptides localize to the tumour vasculature and exert antiangiogenic effects [ ]. Another composition of chitosan nanoparticle was prepared by ionic crosslinking of N-trimethyl chitosan TMC with tripolyphosphate with a lower degree of quaternization and an increase in particle size, a decrease in zeta potential and a slower drug-release profile.

For example, ATP, a related derivative of triphosphate, is essential for life and use its encapsulation with chitosan nanoparticles can improve delivery and health effects.

Such specific characteristics of N-trimethyl chitosan chloride nanoparticles can support the use of them as potential protein carriers in various modifications [ ].

Pre-clinical studies with chitosan and N,N, N-trimethyl chitosan nanoparticle encapsulation of Ocimum gratissimum essential oil exhibited antibacterial activity at a lower concentration for both Gram-negative and Gram-positive food pathogens.

In vitro cytotoxicity revealed the increased toxicity of N, N, N-trimethyl chitosan nanoparticle encapsulated in Ocimum gratissimum essential oil on MDA-MB breast cancer cell lines [ ].

Another collection approach of a nano drug delivery system based on a combination of chitosan nanoparticles with curcumin loaded dextran sulfate was studied regarding the promotion of curcumin anticancer activity. In vitro cytotoxicity measurements demonstrated that curcumin loaded polymeric nanoparticles got significant therapeutic efficacy against colon HCT and breast MCF-7 cancer cells compared with free curcumin [ ] Table 2.

It was studied the use of chitosan nanoparticle for albumin delivery for its use as a plasma expander in critically ill patients and several other clinical applications mainly via intravenous infusion.

Sustainable albumin release over time and high enzymatic stability from albumin-loaded nanoparticles were observed compared to the free albumin [ ]. The chitosan nanoparticles in the nano-system delivery in combination with hyaluronic acid can be a very promising injectable system for the controlled release of platelet-derived growth factor for tissue engineering applications, as well as for the treatment of ischemia-related diseases [ ] Table 2.

Pre-clinical studies based on development and in vitro and in vivo evaluation of chitosan nanoparticles based dry powder inhalation formulations of prothionamide revealed a dose in pulmonary administration, which will improve the management of tuberculosis [ ]. Hussain et al. had explored the histological and immunomodulatory actions of chitosan nanoparticle in the transport of hydrocortisone using chitosan nanoparticles against atopic dermatitis.

It was shown the significant capability of chitosan nanoparticles to minimize the severity of atopic dermatitis. Histological analysis revealed that chitosan nanoparticles inhibited the elastic fibres fragmentation and fibroblast infiltration. Further, depicting their clinical importance in controlling the integrity of elastic connective tissues which makes such nanoparticles-based drug transport effective [ ] Table 2.

Bupivacaine is a long-acting local anaesthetic that belongs to the amino-amide class which is widely used during surgical procedures and for postoperative pain. Animals and in vivo studies such as infraorbital nerve blockade, local toxicity, and pharmacokinetics were used to discover the use of combination chitosan nanoparticles with bupivacaine.

Pre-clinical studies bupivacaine in chitosan nanoparticles revealed that encapsulation of bupivacaine prolongs the local anaesthetic effect after infraorbital nerve blockade and altered the pharmacokinetics after intrathecal injection [ ]. Currently in phase 3 clinical trials in the US and phase 2 clinical trials UK and EU is the chitosan-based nasal formulation of morphine RylomineTM [ ] Table 2.

Due to its biocompatibility, biodegradability and low toxicity, chitosan is widely recognized as a safe material in pharmaceutical nanotechnology. Moreover, its versatile capabilities indicated this natural polymer and its nanoparticles as a viable vehicle in drug delivery.

Once identified as an ideal drug carrier, chitosan has been exploited to design formulations for a large range of drug molecules including proteins, plasmid DNA, and oligonucleotides. Production and clinical development of nanoparticles for gene delivery are discussed nowadays.

Gene therapy is an auspicious strategy with intentionally altering the gene expression in pathological cells for the treatment of gene-associated human diseases. Its discussed role of chitosan nanoparticles as a very promising carrier for gene delivery due to high biocompatibility and close resemblance to the lipidic membranes, which facilitate their penetration into the cells [ ].

Furthermore, chitosan nanoparticles allow a controlled and, sometimes, site-specific delivery and are suitable to many routes of administration, especially for the non-invasive ones like oral, nasal, ocular and transdermal [ ].

The major advantage offered by chitosan-based nanoencapsulation is the ability to improve the dissolution rate of poorly soluble drugs thus increasing their bioavailability Fig.

This capability depends on the size of the particles as well as from the specific features of chitosan, which render this polymer an ideal drug carrier.

Chitosan is soluble in an aqueous solution but it possesses readily modifiable pH-responsive solubility. Generally, dissolution happens in dilute aqueous acid solutions, where the amino groups of chitosan become protonated.

However, many other factors contribute to controlling solution properties such as the distribution and number of acetyl groups along the chains, pH, the ionic concentration, the conditions of isolation and drying [ ]. Additionally, chitosan presents mucoadhesive and absorption-enhancing properties.

The mucoadhesive nature of chitosan depends on electrostatic interaction between the positive charge on the ionizable protonated amine group and the negative charge on the mucosal surfaces. These interactions trigger a reversible structural reorganization in the protein-associated tight junctions which opens the tight junctions between cells, allowing the drug to cross the mucosal cells [ ].

Mucoadhesion also extends the contact of the drug with the mucosal layer, and allow site-specific administration, in particular in those body site presenting specific mucosal surfaces such as buccal and nasal cavities.

Again, many factors can influence mucoadhesive properties such as the molecular weight, the flexibility of the chitosan chain, the electrostatic interaction, the availability of hydrogen bond formation, and the capacity of spreading into the mucus due to surface energy properties [ ].

Also, nanosized formulations are characterized by a large surface to volume ratios, which intensely strengthen the intrinsic properties of chitosan. Nanostructure of appropriate size and surface charge can improve drug penetration thus improving uptake through the cell membrane.

Therefore, nanosized carriers could effectively modulate pharmacokinetics, enhancing drug efficacy beside reduced toxicity [ ] and offer the possibility to deliver bioactive agents in a controlled and, sometimes, site-specific manner.

However, there are several challenges in the use of drug nanocarriers such as low drug encapsulation, premature release, poor permeability and instability, which could finally affect drug bioavailability.

In particular, stability represents one of the most important factors regulating the efficiency of drug delivery systems, especially in the case of nanoparticles [ ]. As regards chitosan nanocarriers, instability could depend on degradation by digestive enzymes and pH variation throughout the gastrointestinal tract.

Additionally, a surface charge strongly influences stability and distribution and limits there in vitro and in vivo application. Indeed, although positively charged particles are strongly attracted by negatively charged cell membranes leading to an efficient internalization in the cells, the interaction with serum components could lead to severe aggregation followed by a fast clearance from the circulatory system [ ].

Therefore, many attempts of tailoring the chitosan nanoparticles have been accomplished, aiming to confer improved stability against aggregation in biological settings.

The most frequent strategy followed consists of hydrophilic modifications with molecules able to improve stability and solubility in slightly acid and neutral media such as β-cyclodextrin, succinic anhydride or PEG.

Besides, also surface decoration with hydrophilic polymers has been carried out in the attempt to contrast nanoparticles aggregation [ ]. However, changes in stability and aggregation of chitosan nanoparticles could also happen during storage.

Different techniques of drying i. Generally, nanopowder is easily re-dispersible, but occasionally aggregation or irreversible fusion of particles occurs making the redispersion more difficult. In this regard, the addition of bioprotectants could reduce surface attraction maintaining the nanoparticles dispersed [ ].

Chitosan is a linear polysaccharide composed of D-glucosamine units deacetylated units and N-acetyl- d -glucosamine units β- 1—4 -connected.

Chitosan is deacetylated chitin Fig. Commercially chitosan is produced by deacetylation of chitin, a natural material, widespread in the world of exoskeletal crustaceans. It has some remarkable therapeutic properties such as blood coagulation, fat binding, heavy metal ion complexation, hemostatic action.

In addition to the degree of deacetylation for a given chitosan sample, the molecular weight of the macromolecule, which can vary between ,—, daltons, is also characteristic.

Chitin and chitosan are of high commercial interest due to their high nitrogen content 6. Both chitin and chitosan are biodegradable, biocompatible, non-toxic, non-allergenic and renewable biomaterials and find their application in fields such as medicine, perfumes and cosmetics, food industry and agriculture [ ].

Chitosan, due to the presence of the primary amine group in the sugar units form the polymeric structure, dissolves in dilute organic acids, but is insoluble in water, above pH 6—7 and in ordinary organic solvents.

The solubility of chitinous substances is usually associated with the crystallinity of the sample. Higher crystallinity suggests greater or increased molecular interactions between the polymer chains. A chitinous chemical can be dissolved only if these interactions are cancelled.

The intra- and intermolecular hydrogen bonds of the polymer chains are the major cause of these interactions and play an important role in the low solubility of these substances. However, chemical modifications of chitosan result in derivatives that are water-soluble in a broader pH range, including in strongly basic environments.

The modifications consist of the introduction of ionic groups or substituents in the polymeric structure, which dissolves in polar solvents such as water through polar-polar interactions and determines the solubility of the macromolecule [ ].

The process of isolation of chitin begins in the marine food industry. One of the by-products of this process, such as carapace of radishes, shrimps, etc. Alternatively, if isolation of chitin is not desired, the sequence based on acid treatment may be reversed to produce chitosan directly. During the treatment with basic medium, concomitant hydrolysis of the acetamide groups of chitin takes place, the result being the formation of chitosan.

The physical properties of chitinous substances are governed by two factors: the degree of deacetylation and the molecular mass. The former has a direct impact on the secondary structure of the polymer chain and can influence and solubility of the polymer in organic or aqueous solvents.

It can also affect the chemical reactivity of the sample inhomogeneous processes [ ]. According to a selective nomenclature, chitinous substances that do not dissolve in dilute organic acids e.

On the other hand, chitinous substances that dissolve in dilute aqueous acids are called chitosan. A distribution of acetyl groups on the polymer structure results in homogeneous processing conditions and gives solubility of polymers in aqueous solutions of weak acids.

Instead, under heterogeneous processing conditions, polymers are formed with distinct blocks of acetylated sugar residues and are not soluble in solvents.

The molecular weight of chitosan obtained at the end of the production process depends on the process parameters, time, temperature and HCl and NaOH concentration. The process parameters used in chitosan production are drastic and the cleavage of the chitin structure accompanies the process.

The degradation of the chitinous chain can be extended. In one preparation, a chitin sample with a molecular weight of 1. However, the charged nature of chitosan tends to form free aggregates and the differences in the degree of deacetylation for different chitosan samples require careful implementation of the constants [ ].

Many applications of any chemical, natural or synthetic, require chemical process ability. Thus, chitosan, a white powder, is difficult to handle due to the problems of solubility in neutral water, bases and organic solvents.

The pKa value of the primary amino groups in chitosan is 6. Even if chitosan and its derivatives are soluble at a pH lower than 6, most of its applications in the basic or neutral environment cannot be achieved [ ].

On the other hand, acidic solutions in which chitosan is soluble are not compatible with many applications, such as those in cosmetics, medicine and nutrition.

There are two approaches in the literature on improving the solubility of chitosan at neutral pH. The first is the chemical derivatization of chitosan for example with substituents containing quaternary ammonium group, by carboxymethylation or sulfation so that the added substituent is hydrophilic.

Under the conditions of homogeneous processing, the obtained chitosan remains in solution after neutralization and no derivatization is required. Some applications of chitosan use derivatized forms thereof and to improve the solubility it is necessary to introduce ionic groups in the polymeric structure [ ].

Traditionally, chitinous substances are used in rudimentary medicine and the treatment of wastewater. In recent decades, these substances have found their applicability in various fields, from textile engineering to photography. Chitosan and its derivatives have attracted more interest than chitin, even though the latter has found its applicability in medicine, fibre, absorbable tissues and bandages.

It is interesting to note the resistance of chitinous substances to bile, pancreatic juice and urine, which leads to their use in surgery, but also the manufacture of human-made fibres for hard materials [ 95 ].

These substances may be subject to degradation with lysozyme, an enzyme found in nature and the human eye, and with chitinase.

This has also led to the use of chitosan derivatives in the preparation of cleaning solutions for contact lenses to remove enzyme deposits [ ]. Chitosan has antimicrobial properties antibacterial and antifungal.

Antibacterial action is rapid and eliminates bacteria within hours. Moreover, its derivatives are biodegradable and exhibit reduced toxicity in mammalian cells. The antibacterial activity is associated with the length of the polymer chain and suggests a cooperative effect of the individual carbohydrate units.

The antibacterial property of chitosan is useful in medicine, where it is used in the manufacture of surgical accessories such as gloves, bandages, etc. It is also used to remove pathogens from water and as a food preservative by adding a layer to the outside of fruit and vegetable products [ ].

As chitosan is obtained by deacetylation usually not complete of chitin, studies related to the analytical characterization of chitin and chitosan are not without interest.

As can be seen from the structures below, the two substances differ in the presence in the case of chitin and the only sporadic presence in the case of chitosan of the acetyl group grafted by the amino function.

Chitosan is immiscible with water. Some chitosan components contain hydroxyl group components, capable of intermolecular hydrogen bonds, due to the macromolecular character of the compound and due to the many intermolecular hydrogen bonds, even in the solid-state of the sample. It is difficult to discuss the toxicity of this substance, because chitosan is a natural, non-toxic and biodegradable compound, widely used, due to its unique properties, in biotechnology, human and veterinary medicine, but also cosmetics.

Chitosan is widely regarded as being a non-toxic, biologically compatible polymer. It is approved for dietary applications in Japan and many countries from Europa and the FDA has approved it for use in wound dressings.

The modifications or degree of deacetylation DD made to chitosan could make it more or less toxic and any residual reactants should be carefully removed. A synopsis of toxicity chitosan's reported is shown in Table 4. The toxicity of chitosan drug administration in animals was reported [ ].

For the reasons listed above, the analytical use of IR spectra was passed, in the spectral range — cm — 1 respectively — cm — 1 , in the transmittance form vs. wave number. The bands are generally large due to the macromolecular character of the compound and due to the numerous intermolecular hydrogen bonds, manifested even in the solid-state of the sample.

The absorption bands can be easily attributed to molecular fragments: the dominant band with a maximum at cm — 1 is due to the valence vibrations stretching, ν O—H and ν N—H of the O — H and N — H connections involved.

intense in hydrogen bonds. the band with maximum absorption at cm — 1 is due to the valence vibrations of the C — H connections. The series of bands between cm — 1 and cm — 1 are characteristic of the amide group the bands "amide I", … "amide VI". Because this band is associated with the acetyl groups in the molecule, its use is warranted to specify the degree of deacetylation of a chitosan sample the more advanced the acetylation degree, the more intense this band is.

To be able to use the intensity of the "amide I" band, the spectra obtained at different recordings must be standardized. The normalization can be achieved by bringing by mathematical processing the intensity of the maximum band ν O—H and ν N—H to the value 1.

According to the information studied the possible cases of toxicity may arise due to the chemical transformations to which chitosan is subjected, more precisely the Degree of deacetylation DD. The importance of nanotechnology, in the target delivery of drugs using nanotechnologies and its application for the discovery and development of new oncological drugs are topics of great importance.

The latest studies on chitosan-based nanomaterials have shown the high utility of this polymer for modern drug delivery. The physical properties and non-toxicity of chitosan and chitosan derivatives make it an ideal material for the creation of chitosan-based nanomaterials and their use in nanomedicine especially in oncological treatment.

The special focus of the studies carried out so far has been on the development of drugs against tumor cells. The requirements of chitosan for its use in nanomedicine—drug formulations provide many new solutions and applications in the development of modern medicine.

The use of chitosan for the construction of nanoparticles is very important in this case. Chitosan-based nanoparticles can be used for the delivery of active ingredients, such as drugs or natural products, by diverse routes of administration such as oral and parenteral delivery.

Nowadays chitosan nanoparticles have become of great interest for nanomedicine, biomedical engineering and the development of new therapeutic drug release systems. They improved bioavailability, increased specificity and sensitivity, and reduced pharmacological toxicity of studied drugs.

Currently, cancer disorders are one of the most important global problems. Our review provides the most important information on the effectiveness of nanomedicine in oncological treatment. The scientific studies give special attention to recent advances in chitosan nano-delivery for cancer treatment.

The combinations of chitosan-based nanomaterials with such oncological drugs as doxorubicin, paclitaxel, rapamycin, lactoferrin, tamoxifen, docetaxel, letrozole, gefitinib and 5-fluorouracil, were studied.

The researches reveal good outcomes. The use of chitosan nanomaterials in drugs used in oncological treatment have shown enhancement of drug delivery to tumours and improving the cytotoxicity effect on cancer cell lines. Additionally, studies on the use of chitosan-based nanomaterials in combination with plant-derived secondary metabolites like curcumin, silibinin and polyphenols also have provided promising results.

Moreover, the application of chitosan-based nanomaterials in the discovery and development of e. antibacterial, anti-inflammatory, antidepressant and antihypertensive formulations which could be used in the treatment of other diseases, was tested.

The performed studies have revealed that chitosan-based nanomaterials showed significant enhancement of drug bioavailability drug loading efficiency, drug-releasing capacity and drug encapsulation efficiency.

The latest advantages of chitosan nanoparticles applications in nanomedicine are supported also by pre-clinical and clinical studies. Zlatian OM, Comanescu MV, Rosu AF, Rosu L, Cruce M, Gaman AE, Calina CD, Sfredel V.

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Kalpana Nagpal , Shailendra Kumar Singh , Dina Nath Mishra Author information. Kalpana Nagpal Division of Pharmaceutics, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology Shailendra Kumar Singh Division of Pharmaceutics, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology Dina Nath Mishra Division of Pharmaceutics, Department of Pharmaceutical Sciences, Guru Jambheshwar University of Science and Technology.

Corresponding author. Keywords: chitosan , nanoparticle , ionotropic gelation , solvent evaporation , complex coacervation. JOURNAL FREE ACCESS. Published: November 01, Received: April 12, Available on J-STAGE: November 01, Accepted: July 16, Advance online publication: - Revised: -.

Download PDF K Download citation RIS compatible with EndNote, Reference Manager, ProCite, RefWorks. Article overview. References Chitosan, due to its molecular structure, can be dissolved well within a variety of solvents and a variety of biologics, such as acids like formic and lactic acid.

Chitosan is a polysaccharide that is derived from chitin that is composed of an alkaline deacetylated monomer of glucosamine and an acetylated monomor glucosamine and binding through β-1,4 glycosidic and hydrogen bonds. There are various mechanisms for chitosan nanoparticle synthesis.

Emulsion droplet coalescence involves the formulation of chitosan nanoparticles by creating two stable emulsions with liquid paraffin by adding one with a stabilizer and another with sodium hydroxide again containing a stabilizer.

This mixture of the two emulsions can be used to form nanoparticles. Reverse miscellisation involves taking an organic solvent lipophilic surfactant and adding chitosan with a drug and cross-linker like glutaraldehyde. The nanoparticles are then extracted.

Desolvation includes preparing chitosan solution and adding a precipitate with a stabilizing solution and precipitate such as acetone. Due to the insolubility of chitosan , the precipitate begins to form through the elimination of the liquid surrounding chitosan.

A crosslinker such as glutaraldehyde can be added to formulate the nanoparticles [3] [26]. Chitosan -based solution is developed in the oil face and translated into stabilized liquid. A crosslinker such as glutaraldehyde can then be used to derive chitosan nanoparticles.

Nanoprecipitation refers to using chitosan and dissolving it within a solvent and then having a pump to differentiate the dispersing phase and with tween 80, derive nanoparticles from the dispersing phase. Spray drying involves taking chitosan and adding it to the solvent acetic acid solution.

The solution will then be atomized. These droplets will be mixed with drying gas and after further evaporation , nanoparticles can be derived [3] [29]. Poly acrylic acid refers to acrylic acid that is polymerized. Poly acrylic acid is also known to have a neutral pH, have beneficial crosslinking properties, due to the charge properties of the side changes and poly acrylic acid being anionic [1] [11] [12] [13] [21] [22] 1,11—13,21, Poly acrylic acid is known to have good biocompatibility with chitosan , particularly with the amine groups -NH 2 [30].

An alternative method for the fabrication of chitosan nanoparticles includes the inclusion of polymerized groups of chitosan Figure 2. This methodology can allow for the improvement of the chitosan cross-linking mechanism and improve overall drug release profiles for drugs such as amoxicillin and meloxicam.

Ionic gelation with radical polymerization takes in a chitosan solution after through the addition of an acid monomer , the chitosan changes from the anion of an acrylic monomer. The nanoparticles are then derived after being self-settled overnight, and the unreacted monomer is removed.

This is the main method for the formulation of poly acrylic acid based chitosan nanoparticles. Figure 2 Procedure way for the formation of chitosan poly acrylic acid nanoparticles.

Adopted from Saberi et al, Biomedical applications of chitosan -based nanoparticles range from cancer treatment to regenerative medicine and tissue engineering to inflammatory diseases to diabetic treatment to the treatment of cerebral diseases, cardiovascular diseases , infectious diseases , and even for vaccine delivery.

Figure 3 Advantages of chitosan nanoparticles.

Frontiers | Chitosan Nanoparticles: Shedding Light on Immunotoxicity and Hemocompatibility

Lu et al. The epidermal cells of tobacco leave treated with oligochitosan showed an increase in the levels of intracellular H 2 O 2 , NO, and increased activity of phenylalanine ammonia-lyase PAL indicating induction of plant defense response against TMV Table 1. ChNPs can directly interact with the cellular membrane of microorganisms due to their unique physicochemical properties and can easily permeate into the cytoplasm.

The direct mode of action of ChNPs against fungi includes inhibition of spore germination, germ tube elongation, mycelial growth, and cell lysis. Benhamou [ 69 ] conducted ultrastructural studies and reported that chitosan induces numerous structural and morphological changes leading to distorted hyphae.

This can be explained as the chitosan particles are polycationic in nature, it allows alteration in membrane permeability and cytoplasmic aggregations. As a result, the activity of enzymes involved in the synthesis and assembly of cell wall polymers are dwindled.

The antibacterial effect includes disruption of the bacterial cell wall, cellular membrane, loss of external appendages, such as flagella, finally, leading to cell lysis. None of the studies have proved ChNPs to inactivate viruses and viroids.

Most of the studies reported inhibition of virus replication, multiplication, and spread by chitosan. However, against pests and pathogens, ChNPs operate via an indirect mechanism, such as induction of host resistance.

Chitosan molecule is generally used as an elicitor rather than an antimicrobial agent in plant disease control.

It can be recognized by the plant PRRs and can trigger a cascade of defense responses. ChNPs can agglutinate around the penetration sites of the pathogen after its application on plant tissues and has two major effects.

The first effect includes isolation of the penetration site from healthy tissues by forming a physical barrier that prevents further spread of the pathogen. Around the isolated zone, several biochemical changes occur that lead to the elicitation of hypersensitive response, accumulation of H 2 O 2 and other free radicals, which lead to cell wall fortification and induction of systemic acquired resistance.

The second effect includes the initiation of wound healing process by binding with various materials Figure 2. Schematic representation of the mechanism of action of chitosan nanoparticles on plants.

Nano-chitosan imparts an eustress effect on seedling germination and plant growth parameters, such as plant height, shoot length, root length, and biomass content, which have been confirmed through a series of studies.

The study conducted on the effect of nano-chitosan on Phaseolus vulgaris L. under salt stress conditions revealed that 0. Also, significant increase in M. I, Chl. a, Chl. b, proline, catalase, carotenoids, and antioxidant enzymes were observed [ 72 ]. Chitosan is a naturally occurring miracle compound having enthralling antimicrobial and eliciting properties.

Nano-chitosan is gaining attention nowadays due to its greater efficacy and biosafety. Chitosan nanomaterials can be used in varied ways for plant disease management, thereby preserving crop quality and yield. In recent years, several findings have been gathered indicating nano-chitosan as a potential plant health material.

However, more studies need to be channelized to unveil the exact mode of action of nano-chitosan specific to the pathosystem. Incorporation of chitosan nanomaterials into integrated pest management practices by devising suitable incorporation techniques need to be pursued.

The biopolymer-based nanomaterials need extensive exploration owing to their multifunctional properties and diverse mechanisms. In the coming years, the use of nano-chitosan for combating biotic and abiotic stresses and transport of agrochemicals would be a promising discipline for utility in sustainable agriculture.

Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3. Edited by Brajesh Kumar. Open access peer-reviewed chapter Chitosan Nanoparticle: Synthesis, Characterization, and Use as Plant Health Materials Written By Pranab Dutta, Arti Kumari and Madhusmita Mahanta.

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Impact of this chapter. Abstract Chitosan is a naturally occurring biopolymer having multifaceted applications in agriculture, medicine, food industry, and cosmetics.

Keywords chitosan nanoparticles biopolymer green synthesis characterization plant protection. Introduction Agriculture is a primary activity upon which the economic status of a country relies. Chitosan nanomaterial Application Target pathogen Effect Reference Chitosan nanocomposite with T.

asperellum Antifungal Rhizoctonia solani, Fusarium oxysporum and Sclerotium rolfsii. Reduction in mycelial growth [ 5 ] Chitosan NPs Antifungal A. alternata , M. phaseolina and R. solani Reduction in mycelial growth [ 51 ] Chitosan Antifungal Alternaria kikuchiana Tanaka and Physalospora piricola Inhibition of spore germination, germ tube elongation, and mycelial growth [ 48 ] Rhodamine-labeled chitosan Antifungal F.

oxysporum Inhibition of mycelial growth, cell membrane permealization, and lysis [ 49 ] Chitosan NPs Antifungal and plant growth promotion Penicillium expansum Reduction in natural and artificial infections. oryzae Reduction in infection [ 60 ] Oligochitosan Induction of resistance in plants against TMV Tobacco Mosaic Virus Increase in the levels of intracellular H 2 O 2 , NO, and phenylalanine ammonia-lyase PAL [ 65 ].

Table 1. Antimicrobial properties of chitosan-based nanomaterials. References 1. Global burden of crop loss. Choudhary RC, Kumaraswamy RV, Kumari S, Pal A, Raliya R, Biswas P, et al. Synthesis, characterization, and application of chitosan nanomaterials loaded with zinc and copper for plant growth and protection.

In: Prasad R, Kumar M, Kumar V, editors. Nanotechnology: An Agricultural Paradigm. Singapore: Springer; Ali A, Ahmed S. A review on chitosan and its nanocomposites in drug delivery. International Journal of Biological Macromolecules. Saharan V, Pal A. Chitosan Based Nanomaterials in Plant Growth and Protection.

New Delhi: Springer; Boruah S, Dutta P. Fungus mediated biogenic synthesis and characterization of chitosan nanoparticles and its combine effect with Trichoderma asperellum against Fusarium oxysporum , Sclerotium rolfsii and Rhizoctonia solani.

Indian Phytopathology. DOI: Perera UMSP, Rajapakse N. Chitosan nanoparticles: Preparation, characterization, and applications. In: Seafood Processing By-products. New York: Springer; Sivashankari PR, Prabaharan M.

Deacetylation modification techniques of chitin and chitosan. In: Chitosan Based Biomaterials. India: Woodhead Publishing; Oh JW, Chun SC, Chandrasekaran M. Preparation and in vitro characterization of chitosan nanoparticles and their broad-spectrum antifungal action compared to antibacterial activities against phytopathogens of tomato.

El-Naggar NEA, Saber WI, Zweil AM, Bashir SI. An innovative green synthesis approach of chitosan nanoparticles and their inhibitory activity against phytopathogenic Botrytis cinerea on strawberry leaves. Scientific Reports. Malerba M, Cerana R. Chitosan effects on plant systems. International Journal of Molecular Sciences.

Knežević-Jugović Z, Petronijević Ž, Šmelcerović A. Chitin and chitosan from microorganisms. In: Chitin, Chitosan, Oligosaccharides and Their Derivatives: Biological Activities and Applications Boca Raton: CRC Press; Yanat M, Schroën K. Preparation methods and applications of chitosan nanoparticles; with an outlook toward reinforcement of biodegradable packaging.

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Hernandez-Lauzardo AN, Bautista-Baños S, Velazquez-Del Valle MG, Méndez-Montealvo MG, Sánchez-Rivera MM, Bello-Perez LA. Antifungal effects of chitosan with different molecular weights on in vitro development of Rhizopus stolonifer Ehrenb.

Carbohydrate Polymers. Anusuya S, Sathiyabama M. Effect of Chitosan on Rhizome Rot Disease of Turmeric Caused by Pythium aphanidermatum.

International Scholarly Research Notices; Van SN, Minh HD, Anh DN. Study on chitosan nanoparticles on biophysical characteristics and growth of Robusta coffee in green house.

Biocatalysis and Agricultural Biotechnology. Mao S, Sun W, Kissel T. Chitosan-based formulations for delivery of DNA and siRNA. Advanced Drug Delivery Reviews. Iriti M, Varoni EM. Chitosan-induced antiviral activity and innate immunity in plants. Environmental Science and Pollution Research.

Das A, Dutta P. Antifungal activity of biogenically synthesized silver and gold nanoparticles against sheath blight of rice. Journal of Nanoscience and Nanotechnology. Dutta P, Kaman P, Kaushik H, Boruah S. Biotechnological and nanotechnological approaches for better plant health management.

Trends in Biosciences. Kaman PK, Dutta P. Nanocentric plant health management with special reference to silver. International Journal of Current Microbiology and Applied Sciences.

Kaman PK, Boruah S, Kaushik H, Dutta P. Effect of biosynthesized silver nanoparticles on morphophysiology of host. International Journal of Botany and Research. Goswami R, Bhattacharyya A, Dutta P. Nanotechnological approach for management of anthracnose and crown rot diseases of banana.

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leaf extract against Corcyra cephalonia S. The characterization of the polymers used and the nanoparticulate delivery system developed is critical to prevent erroneous interpretations of resultant immunotoxicity findings.

Different Chit characteristics can have different biological effects. Unfortunately, most studies addressing biological activity of Chit NPs lack the used polymer characterization, which also restricts comparisons among studies. The two Chit polymers used in this study were purified under endotoxin-free conditions to eliminate possible contaminants.

Since the purification process involves harsh conditions, namely heating the polymer suspension in NaOH 1 M, their DDA and MW were assessed before and after purification and the results presented in Table 1A. An important decrease from to 49 kDa is compatible with the fact that lower DDA Chit has higher enzymatic and acid hydrolysis degradation rate Kurita et al.

Table 1. Physicochemical characterization of Chit polymers and NPs. A Polymer molecular weight MW , deacetylation degree DDA , and size in acetate buffer and after resuspension in DMEM and RPMI at 37°C Mean ± SEM.

Endotoxin contamination of pyrogen-free water was also evaluated for comparison. Chit is soluble in acidic conditions, which is incompatible with cell culture as it leads to cell death.

Particle size in acetate buffer and cell culture media is illustrated in Table 1A. The mean average size of these particle suspensions was around μm in all situations.

These NPs were isolated and concentrated in water. This result was important to calculate Chit NP concentration. After isolation and concentration, NP mean particle size, polydispersity index PDI , and zeta potential ζ were determined by DLS and ELS, respectively, and are summarized in Table 1B.

Results illustrate the effect of the different Chit on the NP characteristics. In fact, the same methodology, when applied to Chit polymers with different DDA and MW, resulted in NPs with different sizes.

These average particle sizes were illustrated by TEM and SEM analysis. The round shape of the NPs was the second conclusion inferred by observing the images Figures 1A,B of both techniques.

Figure 1. Chit NP illustration by Electron Microscopy. Due to the complexity of cell culture media, and the variability of their supplementation, results from NP colloidal system characterization in water are not transposable to in vitro conditions Moore et al.

Chit NPs were therefore characterized in cell culture media to understand the changes that NPs experience during in vitro studies. Even though the DLS methodology for size analysis in complex media such as cell culture medium has limitations, it can give us some insights about changes occurring to the different Chit NPs.

Most notably, the suspension of both Chit NPs in RPMI and DMEM resulted in increased PDI, meaning an increase of the size heterogeneity. This change induced by the adsorption of negatively charged proteins from the medium, to positively charged Chit residues, forms a protein corona, decreasing the suspension stability.

Under these conditions, the appearance of aggregates is inevitable which is part of the explanation for the PDI increment. In the water, the size of both NP was distributed over a single peak Figures 2A,D , while in cell culture media, there were at least three independent peaks Figures 2B,C,E,F.

We can hypothesize that the alterations observed in cell culture media size dispersion, including smaller and bigger size populations simultaneously, were induced not only by the presence of proteins, but also by the high ion content in comparison to water Moore et al.

Furthermore, as the media composition is different between RPMI and DMEM, the observed changes in the NP size distribution were not similar. A comparable phenomenon was described by Yang et al.

Figure 2. NP size distribution by DLS. The last step of characterization was related to endotoxin contamination. As previously mentioned, Chit polymers were purified by a method published by our group Lebre et al. The method allows the obtainment of endotoxin-free chitosan, proved by two methods: Limulus Amebocyte Lysate LAL test and the absence of IL-6, secreted by dendritic cells DCs , cultured in the presence of chitosan.

The chitosan does not induce IL-6 secretion by DCs and endotoxins do that stimulation. Furthermore, for in vitro immunotoxicity studies, the NP production was performed under those conditions, to avoid endotoxin contamination, as the presence of these molecules can easily lead to false positive results.

To assure that Chit purification and Chit NP production were successfully achieved, both Chit polymers and NPs as well as the pyrogen-free water and the TPP solution used for NP production, were submitted to LAL test.

Importantly, before establishing the methodology for endotoxin quantification with Pyrochrome® testing kit, all recommended tests to evaluate sample interference with LAL test were done to guarantee the suitability of the LAL test for Chit NPs, as described in the manufacturer's instructions.

The results were presented in Table 1C , and show that all tested samples were not significantly different from pyrogen-free water, the negative control, and all were far below 0.

Thus, it was demonstrated that the process and conditions used to minimize the contamination and remove existent endotoxins during Chit purification and NP production was effective, and that Chit polymers and NPs used in immunotoxicity tests were indeed LPS-free, supporting the reliability of the results.

Nevertheless, these cells should be used carefully since their functional stability is not maintained at high passage number. Indeed, a recent article mentions the phenotype and functional characteristics to remain stable from passage 10 to 30 Roberts et al.

The evaluation of the cytotoxic profile of Chit NPs and polymers was performed using the MTT metabolic activity assay, over a wide range of concentrations as illustrated in Figure 3.

Figure 3. Cell viability studies in RAW D Evaluation of the cell viability resultant from the incubation of RAW The reduction of the reagent MTT by cells leads to the generation of insoluble crystals of formazan that once dissolved in DMSO generate a purple signal van Meerloo et al.

Since it is a colorimetric assay, and although the cell medium with the testing sample was aspirated before solubilizing the formazan crystals, NP interferences with the readout were tested to validate the assay Figure 3C. As it is possible to observe, the measured absorbance Abs was not increased by the presence of the NPs or polymer suspension.

Additionally, to guarantee that the cell viability results were only related with the NP and polymers, and not with the solvents, the supernatants collected from the NPs last washing step with water, as well as the acetate buffer used to disperse the polymers, were also tested using MTT assay Figure 3D.

Results showed that the solvents did not cause any decrease in cell viability. Reactive oxygen species ROS are unstable molecules that easily react with other molecules and may cause damage to DNA, RNA, proteins and ultimately lead to cell death, when accumulated Schieber and Chandel, To evaluate the effect of Chit polymers and Chit NPs on ROS production by RAW The increase in ROS production was concentration dependent, however, for the concentration range tested, the effect was not as high as LPS-induced ROS production.

Importantly, all tested conditions did not induce cellular death as confirmed by the MTT assay performed at the end of each experiment Figure S1A. In order to have a more complete picture, studies were conducted to evaluate that the polymers and NPs would not play an inhibitory role in the production of ROS by cells stimulated with LPS.

Results in Figure 4B show that no inhibitory effect was observed for any of the tested samples. Figure 4. Immunotoxicity assays in RAW All assays were performed with non-cytotoxic concentrations of NPs, polymers and controls evaluated by MTT assay after every experiment.

For the experiment, test samples were incubated with RAW For the experiment, LPS and test samples were co-incubated with RAW Negative control C— was not co-incubated with LPS. Data are presented as mean ± SEM. The possibility of having the nanoparticles interfering with the methods should not be ruled out, leading to false positives or false negatives.

So, to evaluate the interference of Chit NPs and Chit polymers in the fluorescence readouts, the ROS production assay was performed without cells and at the highest polymer and NPs concentrations.

The values obtained for test samples were similar to the medium alone Figure 4E , meaning that they do not interfere with ROS measurement. Additionally, the possible interference of solvents was also assessed under the same testing conditions and as shown in Figure 4F , no stimulation of ROS production, as the fluorescence increase fold values were around 1.

NO is an important inflammatory mediator released by macrophages during inflammation, being one of the main cytostatic, cytotoxic, and pro-apoptotic mechanisms of the immune response Bosca et al. NO production by RAW Again, all test samples were sterile and endotoxin-free in order to prevent false positive results, and used in adequate concentrations that did not affect cell viability Cell viability study in Supplementary Figures 1C,D.

With the aim to evaluate whether one of the polymers or Chit NPs would be able to induce cells to produce NO, samples were incubated with the RAW None of the Chit NPs or polymer concentrations tested induced NO production.

The results shown in Figure 4D indicate that there was a slight but significant inhibitory effect on LPS-induced NO production, at all concentrations tested when compared to the LPS control. Since the Chit and Chit NP concentrations tested did not induce significant reduction in cell viability Supplementary Figure 1D we can exclude the hypothesis that it was a consequence of cellular death.

For all NPs, the possible interference with optical detection methods is a hypothesis that should be tested before doing the test itself.

So, similar to ROS assay, the NO assay was performed in the presence of the test samples, without cells and the results were presented on Figure 4G.

The solvent of the Chit NPs suspension or the chitosan polymer suspension were evaluated to understand if they also had an effect on NO production Figure 4H. No interferences were observed in the readout, and the solvents were not able to induce NO production.

An additional control was performed for NO production assay, to evaluate whether Chit and Chit NPs, due to their cationic charge, could be adsorbing NO at their surface, reducing the amount of NO quantified.

Such phenomenon would provide an explanation for the NO production inhibition observed. To evaluate this hypothesis, we performed the NO calibration curve in the presence and absence of Chit NPs and polymers Figure 4I.

As shown, the NO curves are all overlapping, meaning no interferences from Chit NPs and Chit polymers were observed. PBMCs are a good model to study immune responses, since they secrete regulatory and pro-inflammatory cytokines and chemokines in the human body. In vitro cell viability experiments give an indication of a particle cytotoxic profile that may be observed in vivo.

Similar to RAW The results depicted in Figure 5A showed that Chit NPs were more cytotoxic than the respective polymers. Figure 5. Cell viability studies in PBMCs and assay interference evaluation.

This difference was further confirmed with the PI assay, where the cell membrane integrity rather than the metabolic activity was evaluated Figure 5B. To note, Chit NP and polymer highest concentrations tested during cell viability assessment in both RAW In our case, these concentrations were needed to correctly calculate the IC As explained for the RAW The absorbance readout showed no interference for formulations equal Abs values and the resultant cell viability following solvent incubation with PBMCs during 24 h showed comparable cell viability to the control.

Cytokines participate in many physiological processes, mostly in the regulation of immune and inflammatory responses Ai et al. Interleukin-6 IL-6 is a pleiotropic cytokine inflammatory and anti-inflammatory properties able to modulate the activity of immune cells Wang et al.

Tumor necrosis factor-α TNF-α is a pro-inflammatory cytokine released from macrophages or activated T cells which plays a crucial role in many immune and inflammatory processes, such as proliferation, apoptosis, and cell survival Cai et al.

Results showed that neither Chit NPs nor Chit polymers stimulated the production of IL-6 and TNF-α, as no differences were found before and after incubation with test samples.

Importantly, the use of positive controls such as LPS and Con A, give us an indication of the cell function regarding the cytokine we are analyzing. Notably, both positive controls significantly increased the cytokine secretion in PBMCs. Figure 6. Cytokine secretion in PBMCs and interference evaluation.

Cytokine quantification was performed by ELISA. Dotted lines represent the original calibration curve in ELISA diluent. Additionally, since chitosan's positive charge favors cytokine adsorption, the possible interference of Chit NPs in cytokine quantification was tested.

For that, Chit NPs suspended in cell culture medium were incubated with known concentrations of each cytokine calibration curve for 24 h, then centrifuged and supernatant cytokine content similarly quantified by ELISA. The percentage of cytokine quantification in cell culture medium incubated with the nanoparticles in comparison to cell culture medium without nanoparticles, can reveal if the cytokines adsorbed to the NPs, preventing their quantification.

Thus, we can assume that the absence of TNF-α and for IL-6 production upon stimulation with Chit NPs and polymers was indeed due to the lack of the samples' ability to stimulate the cells, which strengthens the conclusion that they do not induce a pro-inflammatory cytokine response, at least when produced under endotoxin-free conditions.

Hemolysis is characterized by the rupture of red blood cells RBCs and the release of their contents, ultimately leading to anemia, jaundice and renal failure Dobrovolskaia et al.

All materials entering the blood get in contact with RBCs and so the evaluation of the hemolytic ability of the biomaterials is of utmost importance. Chit NPs and polymers hemolytic activity was evaluated following a 3 h incubation at 37°C with RBCs.

Triton X was used as the hemolytic agent whose effect is possible to observe by the red color of the supernatant after centrifugation of the experiment tube 1 and 2 Figure 7B.

Although no hemolytic activity was induced by Chit NPs and polymers, solvents were tested as well as the NP interference with the assay readout. As depicted in Figure 7C , the NPs had no interference in the absorbance measurements and Figure 7D illustrates that no hemolysis was induced by the NPs or solvents of the suspensions of the NPs or polymers.

Figure 7. Hemolysis assay. A Hemolytic activity of Chit polymers and NPs in human blood after 3 h incubation at 37°C.

C Evaluation of the Chit NP and Chit polymers interferences with the absorbance readout, without blood. D Evaluation of the hemolysis resultant from the incubation of NPs solvent and polymers solvent in human blood after 3 h incubation at 37°C. The plasma coagulation cascade is responsible for blood clotting and consists of a series of protein interactions Laloy et al.

To evaluate the effect of Chit NPs and polymer samples on plasma coagulation time, two concentrations 0. In this assay, both blood coagulation pathways, the activated partial thromboplastin time APTT and the prothrombin time PT were separately tested Figure 8A.

Figure 8. Coagulation assay. A Effect of Chit NPs and polymers at 0. The two coagulation pathways, APTT and PT, were separately tested. APTT reference range of values is 20—40 s and for PT is 11—14 s.

B Controls of interferences of NPs and polymers solvents with the coagulation times assay. The results showed that Chit NPs and polymers at 0. NPs suspension solvent and polymer suspension solvent acetate buffer was also tested to discard any method interference and no effect was observed in plasma coagulation Figure 8B.

Platelets play an important role not only in hemostasis but also in immune and inflammatory responses Golebiewska and Poole, Homeostatic imbalance as a result of platelet function alterations affect primary hemostasis and can result in thrombotic or hemorrhagic disorders Golebiewska and Poole, Therefore, it is important to study Chit NPs interactions with platelet function.

To assess platelet aggregation, a cytometer is frequently used to count the platelets, however, by this method the interference of NPs, due to their size have to be taken into account.

To evaluate the interference of Chit NPs with the platelet count, Chit NPs were incubated with platelet-free plasma PFP and visualized under the light microscope. Results showed that Chit NPs, most likely in the form of aggregates, were possibly counted as platelets, which invalidated the use of such method.

To overcome this setback and assess platelet aggregation, the experiment was performed by counting platelets manually under a microscope, using a Neubauer chamber.

Results from microscopy observation were summarized in Figures 9A,B. Figure 9. A Representative images of platelet aggregation assay stained with Giemsa dye. Untreated platelet free plasma PFP is represented in image 1 and untreated platelet rich plasma PRP is represented in image 2.

B Quantification of the platelet aggregation effect. Platelet count is presented as the final average of a minimum of three donors ± SD.

The Figure 9A -1 clearly shows the absence of platelets typical from PFP, while plenty of platelets were observed in PRP, with no signs of aggregates Figure 9A When platelets were incubated with calcium chloride, we observed the formation of fibrins, a sign of platelet aggregation Figure 9A Similarly, collagen also induced platelet aggregation, but in this case no fibrins were observed Figure 9A When analyzing both types of Chit NPs incubated with PFP we can see their tendency to form NPs aggregates, which were hypothetically the cause of the observed interference in the cytometry technique Figures 9A -5,7.

Nevertheless, under microscopic observation, these aggregates were not misinterpreted as platelets. Besides that, platelet aggregation was observed. A hot topic in the nanomedicine field are polymeric NPs, which are engineered to either interact or not with the immune system.

In the early stages of the development of a nanotechnology-based medicine, when the drug is to be encapsulated into NPs, the first question to be considered is, whether it is supposed that the new nanomedicine, in addition to its main pharmacological action, also acts on the immune system.

This kind of approach is part of the SbD. Particularly, in the case of chitosan, as it is a set of polymers with different MW and DDA [quality attributes QA ], it is important to understand if there are differences between them, regarding possible interactions with the immune system. For Chit NPs, in addition to polymer QA, NP characteristics, like size and zeta potential or shape can also be important.

Therefore, physicochemical characteristics PCC of the polymers and NP might influence their immunological properties, and therefore a thorough characterization of both is very important to supplement the immunotoxicity studies and to draw meaningful conclusions Crist et al.

The lack of an exhaustive characterization may preclude the correct interpretation of results and may lead to misinterpretations hindering the establishment of trends regarding how Chit NP PCC influence the immune response. Additionally, one of the most important challenges encountered in in vitro immunotoxicity tests for NPs is related to their unique physicochemical properties.

These can interfere with the established tests, originally developed for testing conventional chemicals Dobrovolskaia and McNeil, Such interference depends both, on the NPs tested and the in vitro assay and can lead to false-positive or false-negative results Dobrovolskaia and McNeil, Lastly, in order to achieve a correct result interpretation, it is important to identify the presence of biological contaminants in the NP preparation Dobrovolskaia and McNeil, The main biological contamination in in vitro assays, even when working under sterile conditions, are endotoxins, which may lead to inflammatory responses Dobrovolskaia and McNeil, The present case study intends to provide a systematic analysis of the effects of Chit NPs and respective Chit polymers on different biological outcomes commonly tested under the immunotoxicity scope, considering, as most important the effect of DDA and MW, without neglecting possible interferences and contaminants.

In detail, as literature suggests, we found that Chit NPs appear to be more cytotoxic than the respective Chit polymers from which they were derived. On the other hand, when the polymers were assembled into NPs, the same range of Chit concentrations induced a concentration dependent reduction in cell viability.

Another important result we found was that PBMCs isolated from human blood were more sensitive to the NPs than RAW To discuss these results some aspects must be analyzed. To begin with, the cell culture media were different for PBMCs and RAW The physicochemical characterization of the NPs in water stock suspension is important, but their characterization when dispersed in the medium used for in vitro assays can provide further evidence.

Moreover, the NPs size analysis in cell culture media resulted in very high PDI. Therefore, the most noteworthy size differences occurred in RPMI, which could explain the different cell viability profile between the NPs in PBMCs.

Literature review showed several contradictory results regarding Chit NPs effect on cellular ROS production. One study suggested that Chit NPs had an inhibitory activity Bor et al. Concerning the polymer, same conflicting results were also found Arora et al.

From our case study, we concluded that, despite no significant differences were found in the cytotoxic profile of both NPs in RAW Thus, the effect was dependent on the type of Chit polymer: Chit with the lowest DDA induced ROS production.

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Introduction Study on chitosan nanoparticles on biophysical characteristics and growth of Robusta coffee in green house. carotovora and X. de Paz LEC, Resin A, Howard KA, Sutherland DS, Wejse PL Antimicrobial effect of chitosan nanoparticles on streptococcus mutans biofilms. Figure 2 Procedure way for the formation of chitosan poly acrylic acid nanoparticles. Three of the in vitro studies investigated poly lactic-co-glycolid acid PLGA nanoparticles coated with chitosan. Article type Paper.
Chitosan -poly acrylic Chitoaan Common allergenic foods a composite that Anti-aging skincare been Chitpsan used to create chitosan-poly acrylic nanopartickes nanoparticles. Tor which already features strong biodegradability and biocompatibility nature Cnitosan be merged Chitosna polyacrylic acid Common allergenic foods Chiosan hybrid nanoparticles that allow for greater Chitosan for nanoparticles Chitosxn as well Common allergenic foods promote Chitosan for nanoparticles biocompatibility and homeostasis nature of chitosan Cyitosan acrylic acid complex. Research on nanoparticles and their chitosan nanoparticles grew in popularity in the early s. Chitosan, due to its molecular structure, can be dissolved well within a variety of solvents and a variety of biologics, such as acids like formic and lactic acid. Chitosan is a polysaccharide that is derived from chitin that is composed of an alkaline deacetylated monomer of glucosamine and an acetylated monomor glucosamine and binding through β-1,4 glycosidic and hydrogen bonds. There are various mechanisms for chitosan nanoparticle synthesis. Emulsion droplet coalescence involves the formulation of chitosan nanoparticles by creating two stable emulsions with liquid paraffin by adding one with a stabilizer and another with sodium hydroxide again containing a stabilizer.

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