Chitosan as an Alternative to Oil-Based Materials for the Fabrication of Lab-on-a-Chip.

Given the growing importance of lab-on-a-chip in a number of fields, such as medical diagnosis or environmental analysis, the fact that the current fabrication process relies mainly on oil-based polymers raises an ecological concern. As an eco-responsible alternative, we presented, in this article, a manufacturing process for microfluidic devices from chitosan, a bio-sourced, biodegradable, and biocompatible polysaccharide. From chitosan powder, we produced thick and rigid films. To prevent their dissolution and reduce their swelling when in contact with aqueous solutions, we investigated a film neutralization step and characterized the mechanical and physical properties of the resulting films. On these neutralized chitosan films, we compared two micropatterning methods, i.e., hot embossing and mechanical micro-drilling, based on the resolution of microchannels from 100 µm to 1000 µm wide. Then, chitosan films with micro-drilled channels were bonded using a biocompatible dry photoresist on a glass slide or another neutralized chitosan film. Thanks to this protocol, the first functional chitosan microfluidic devices were prepared. While some steps of the fabrication process remain to be improved, these preliminary results pave the way toward a sustainable fabrication of lab-on-a-chip.

Comparative Evaluation of the In Vitro Cytotoxicity of a Series of Chitosans and Chitooligosaccharides Water-Soluble at Physiological pH.

Chitosans (CS) have been of great interest due to their properties and numerous applications. However, CS have poor solubility in neutral and basic media, which limits their use in these conditions. In contrast, chitooligosaccharides (COS) have better solubility in water and lower viscosity in aqueous solutions whilst maintaining interesting biological properties. CS and COS, unlike other sugars, are not single polymers with a defined structure but are groups of molecules with modifiable structural parameters, allowing the adaptation and optimization of their properties. The great versatility of CS and COS makes these molecules very attractive for different applications, such as cryopreservation. Here, we investigated the effect of the degree of polymerization (DP), degree of N-acetylation (DA) and concentration of a series of synthesized CS and COS, water-soluble at physiological pH, on their cytotoxicity in an L929 fibroblast cell culture. Our results demonstrated that CS and COS showed no sign of toxicity regarding cell viability at low concentrations (≤10 mg/mL), independently of their DP and DA, whereas a compromising effect on cell viability was observed at a high concentration (100 mg/mL).

Grafted chitosan thin films of various degrees of acetylation as a reusable platform for the investigation of biological interactions.

Surface treatment by adhesive polymers is a promising solution to immobilize and study bacteria cells through microscopic assays and, for example, control their growth or determine their susceptibility to antibiotic treatment. The stability of such functional films in wet conditions is crucial, as the film degradation would compromise a persistent use of the coated devices. In this work, low roughness chitosan thin films of degrees of acetylation (DA) ranging from 0.5 % to 49 % were chemically grafted onto silicon and glass substrates and we have demonstrated how the physicochemical properties of the surfaces and the bacterial response were DA-dependent. A fully deacetylated chitosan film presented an anhydrous crystalline structure while the hydrated crystalline allomorph was the preferred structure at higher DA. Moreover, their hydrophilicity increased at higher DA, leading to higher film swelling. Low DA chitosan-grafted substrate favored bacterial growth away from the surface and could be envisioned as bacteriostatic surfaces. Contrarily, an optimum of Escherichia coli adhesion was found for substrates modified with chitosan of DA = 35 %: these surfaces are adapted for the study of bacterial growth and antibiotic testing, with the possibility of reusing the substrates without affecting the grafted film - ideal for limiting single-use devices.

Preparation of highly stable and ultrasmooth chemically grafted thin films of chitosan.

Chitosan-coated surfaces are of great interest for biomedical applications (antibacterial coatings, implants, would healing, single-cell microfluidics…). However, one major limitation of chitosan-based systems is the high solubility of the polymer under acidic aqueous conditions. Herein, we describe a simple procedure to prepare extremely smooth and stable chitosan coatings. In detail, chitosan films with a low degree of N-acetylation and of thicknesses varying from 40 nm to 10 µm were grafted onto epoxy-functionalized silicon wafers via an optimized water-temperature treatment (WTT). The formation of a grafted chitosan network insoluble in acidic aqueous media (pH 3.5) was evidenced and the films were stable for at least 2 days at pH 3.5. The film morphology and the swelling behavior were characterized by atomic force microscopy (AFM) and neutron reflectivity, which showed that the film roughness was extremely low. The physical cross-linking of the films was demonstrated using infrared spectroscopy, dynamic mechanical analysis (DMA) and wide-angle X-ray scattering (WAXS). Finally, we show that the swelling behavior of such films was largely influenced by the environmental conditions, such as the pH or ionic strength of the solution.

Topical reinforcement of the cervical mucus barrier to sperm.

Close to half of the world's pregnancies are still unplanned, reflecting a clear unmet need in contraception. Ideally, a contraceptive would provide the high efficacy of hormonal treatments, without systemic side effects. Here, we studied topical reinforcement of the cervical mucus by chitosan mucoadhesive polymers as a form of female contraceptive. Chitosans larger than 7 kDa effectively cross-linked human ovulatory cervical mucus to prevent sperm penetration in vitro. We then demonstrated in vivo using the ewe as a model that vaginal gels containing chitosan could stop ram sperm at the entrance of the cervical canal and prevent them from reaching the uterus, whereas the same gels without chitosan did not substantially limit sperm migration. Chitosan did not affect sperm motility in vitro or in vivo, suggesting reinforcement of the mucus physical barrier as the primary mechanism of action. The chitosan formulations did not damage or irritate the ewe vaginal epithelium, in contrast to nonoxynol-9 spermicide. The demonstration that cervical mucus can be reinforced topically to create an effective barrier to sperm may therefore form the technological basis for muco-cervical barrier contraceptives with the potential to become an alternative to hormonal contraceptives.

Regiospecific Grafting of Chitosan Oligomers Brushes onto Silicon Wafers.

The functionalization of surfaces using chitosan oligomers is of great interest for a wide range of applications in biomaterial and biomedical fields, as chitosan oligomers can provide various functional properties including biocompatibility, wetting, adhesion, and antibacterial activity. In this study, an innovative process for the regiospecific chemical grafting of reducing-end-modified chitosan oligomers brushes onto silicon wafers is described. Chitosan oligomers (COS) with well-defined structural parameters (average DP ~19 and DA ~0%) and bearing a 2,5-anhydro-d-mannofuranose (amf) unit at the reducing end were obtained via nitrous acid depolymerization of chitosan. After a silanization step where silicon wafers were modified with aromatic amine derivatives, grafting conditions were studied to optimize the reductive amination between aldehydes of amf-terminated COS and aromatic amines of silicon wafers. Functionalized surfaces were fully characterized by AFM, ATR-FTIR, ellipsometry, contact angle measurement, and ToF-SIMS techniques. Smooth surfaces were obtained with a COS layer about 3 nm thick and contact angle values between 72° and 76°. Furthermore, it was shown that the addition of the reducing agent NaBH3CN could positively improve the COS grafting density and/or led to a better stability of the covalent grafting to hydrolysis. Finally, this study also showed that this grafting process is also efficient for chitosan oligomers of higher DA (i.e., ~21%).

Reducing-End Functionalization of 2,5-Anhydro-d-mannofuranose-Linked Chitooligosaccharides by Dioxyamine: Synthesis and Characterization.

The nitrous acid depolymerization of chitosan enables the synthesis of singular chitosan oligosaccharides (COS) since their reducing-end unit is composed of 2,5-anhydro-d-mannofuranose (amf). In the present study, we describe a chemical method for the reducing-end conjugation of COS-amf by the commercially available dioxyamine O,O'-1,3-propanediylbishydroxylamine in high mass yields. The chemical structure of resulting dioxyamine-linked COS-amf synthesized by both oximation and reductive amination ways were fully characterized by 1H- and 13C-NMR spectroscopies and MALDI-TOF mass spectrometry. The coupling of chemically attractive linkers such as dioxyamines at the reducing end of COS-amf forms a relevant strategy for the development of advanced functional COS-based conjugates.

In situ synthesis of Fe3O4 nanoparticles coated by chito-oligosaccharides: physico-chemical characterizations and cytotoxicity evaluation for biomedical applications.

Fe3O4 nanoparticles coated with chito-oligosaccharides (COS) were prepared in situ by a simple co-precipitation method through a mixing of iron ions (Fe3+ and Fe2+) and COS aqueous solutions followed by precipitation with ammonia. The impact of COS with different degree of polymerization (DP 10, 24 and 45) and degree of N-acetylation (DA) âˆ¼ 24% and 50% (exhibiting high solubility) on the synthesis and physical properties of the coated magnetic nanoparticles was evaluated. Several advantages were found when the magnetic nanoparticles were prepared in the presence of the studied COS, such as: preparation of functionalized magnetic nanoparticles with narrower size distributions and, consequently, higher saturation magnetization (an increase of up to 22%); and an expressive increasing in the concentration of COS-coated magnetic nanoparticles (up to twice) in the cell viability test in comparison with pure Fe3O4 nanoparticles. Furthermore, among the analyzed samples, the magnetic nanoparticles coated by COS with DA âˆ¼ 50% present a higher cytocompatibility. Our results allow envisioning various biomedical applications, valorizing the use of coated-magnetic nanoparticles for magnetic-field assisted drug delivery, enzyme or cell immobilization, or as a marker for specific cell tracking, among others.

Assessment of Oligo-Chitosan Biocompatibility toward Human Spermatozoa.

The many interesting properties of chitosan polysaccharides have prompted their extensive use as biomaterial building blocks, for instance as antimicrobial coatings, tissue engineering scaffolds, and drug delivery vehicles. The translation of these chitosan-based systems to the clinic still requires a deeper understanding of their safety profiles. For instance, the widespread claim that chitosans are spermicidal is supported by little to no data. Herein, we thoroughly investigate whether chitosan oligomer (CO) molecules can impact the functional and structural features of human spermatozoa. By using a large number of primary sperm cell samples and by isolating the effect of chitosan from the effect of sperm dissolution buffer, we provide the first realistic and complete picture of the effect of chitosans on sperms. We found that CO binds to cell surfaces or/and is internalized by cells and affected the average path velocity of the spermatozoa, in a dose-dependent manner. However, CO did not affect the progressive motility, motility, or sperm morphology, nor did it cause loss of plasma membrane integrity, reactive oxygen species production, or DNA damage. A decrease in spermatozoa adenosine triphosphate levels, which was especially significant at higher CO concentrations, points to possible interference of CO with mitochondrial functions or the glycolysis processes. With this first complete and in-depth look at the spermicidal activities of chitosans, we complement the complex picture of the safety profile of chitosans and inform on further use of chitosans in biomedical applications.

Water-Soluble 2,5-Anhydro-d-mannofuranose Chain End Chitosan Oligomers of a Very Low Molecular Weight: Synthesis and Characterization.

Five chitosans with different degrees of N-acetylation (DAs), molar masses, and origins were depolymerized by nitrous acid treatment in acidic media, leading to water-soluble 2,5-anhydro-d-mannose chain end oligomers with DPn < 20. The kinetics of the reaction was studied, and the best work conditions were found to be 3 h reaction at 50 °C. It was shown that the DPn of oligomers only depends on the quantity of NaNO2 involved. Molar masses or DAs do not have an impact on the depolymerization process when targeting oligomers with less than 20 units. This depolymerization process also leads to free 2,5-anhydromannofuranose (AMF) that might react with the free amines of obtained oligomers to form imines. This reaction is pH-dependent and in acidic condition leads to the formation of 5-hydromethyl-2-furfural (HMF). At the end, the oligomers were purified by dialysis to get rid of most of the free AMF (<5%) and other residual salts and appeared to have no acute toxicity.

Ultrasound-Assisted Extraction Optimization of Phenolic Compounds from Citrus latifolia Waste for Chitosan Bioactive Nanoparticles Development.

Bioactive Phenols-loaded chitosan nanoparticles (PL-CNps) were developed by ionic gelation from Persian lemon (Citrus latifolia) waste (PLW) and chitosan nanoparticles. Response Surface Methodology (RSM) was used to determine the optimal Ultrasound-Assisted Extraction (UAE) conditions for the total phenolic compounds (TPC) recovery from PLW (58.13 mg GAE/g dw), evaluating the ethanol concentration, extraction time, amplitude, and solid/liquid ratio. Eight compounds expressed as mg/g dry weight (dw) were identified by ultra-performance liquid chromatography coupled photo diode array (UPLC-PDA) analysis: eriocitrin (20.71 ± 0.09), diosmin (18.59 ± 0.13), hesperidin (7.30 ± 0.04), sinapic acid (3.67 ± 0.04), catechin (2.92 ± 0.05), coumaric acid (2.86 ± 0.01), neohesperidin (1.63 ± 0.00), and naringenin (0.44 ± 0.00). The PL-CNps presented size of 232.7 nm, polydispersity index of 0.182, Z potential of -3.8 mV, and encapsulation efficiency of 81.16%. The results indicated that a synergic effect between phenolic compounds from PLW and chitosan nanoparticles was observed in antioxidant and antibacterial activity, according to Limpel's equation. Such results indicate that PLW in such bioprocesses shows excellent potential as substrates for the production of value-added compounds with a special application for the food industry.

Effects of Chain Length of Chitosan Oligosaccharides on Solution Properties and Complexation with siRNA.

In the context of gene delivery, chitosan has been widely used as a safe and effective polycation to complex DNA, RNA and more recently, siRNA. However, much less attention has been paid to chitosan oligosaccharides (COS) despite their biological properties. This study proposed to carry out a physicochemical study of COS varying in degree of polymerization (DP) from 5 to 50, both from the point of view of the solution properties and the complexing behavior with siRNA. The main parameters studied as a function of DP were the apparent pKa, the solubility versus pH, the binding affinity with siRNA and the colloidal properties of complexes. Some parameters, like the pKa or the binding enthalpy with siRNA, showed a marked transition from DP 5 to DP 13, suggesting that electrostatic properties of COS vary considerably in this range of DP. The colloidal properties of siRNA/COS complexes were affected in a different way by the COS chain length. In particular, COS of relatively high DP (≥50) were required to form small complex particles with good stability.

Reducing-end "clickable" functionalizations of chitosan oligomers for the synthesis of chitosan-based diblock copolymers.

Chitooligosaccharides (COS) produced by nitrous acid depolymerization of chitosan are unique chitosan oligomers due to the presence of the 2,5-anhydro-d-mannofuranose (amf) unit at their reducing end. In this work, we focused on the reductive amination and the oximation of the amf aldehyde group towards various functionalized anilines, hydrazides and O-hydroxylamines. The aim of this work was to synthesize new COS-based building blocks functionalized at their reducing end by different "clickable" chemical groups such as alkene, alkyne, azide, hydrazide and thiol. Targeted functionalized COS were synthesized in excellent mass yields and fully characterized by NMR spectroscopy and MALDI-TOF mass spectrometry. Our results showed these functionalizations are quantitative, versatile and can be easily performed in mild reaction conditions. Finally, these COS-based building blocks could be useful intermediates for the development of advanced functional COS-based conjugates, as illustrated in this work by the synthesis of new COS-poly(ethylene glycol) (PEG) diblock copolymers.

Reinforcing Mucus Barrier Properties with Low Molar Mass Chitosans.

The mucus gel covers the wet epithelia that forms the inner lining of the body. It constitutes our first line of defense protecting the body from infections and other deleterious molecules. Failure of the mucus barrier can lead to the inflammation of the mucosa such as in inflammatory bowel diseases. Unfortunately, there are no effective strategies that reinforce the mucus barrier properties to recover or enhance its ability to protect the epithelium. Herein, we describe a mucus engineering approach that addresses this issue where we physically cross-link the mucus gel with low molar mass chitosan variants to reinforce its barrier functions. We tested the effect of these chitosans on mucus using in-lab purified porcine gastric mucins, which mimic the native properties of mucus, and on mucus-secreting HT29-MTX epithelial cell cultures. We found that the lowest molar mass chitosan variant (degree of polymerization of 8) diffuses deep into the mucus gels while physically cross-linking the mucin polymers, whereas the higher molar mass chitosan variants (degree of polymerization of 52 and 100) interact only superficially. The complexation resulted in a tighter mucin polymer mesh that slowed the diffusion of dextran polymers and of the cholera toxin B subunit protein through the mucus gels. These results uncover a new use for low molar mass mucoadhesive polymers such as chitosans as noncytotoxic mucosal barrier enhancers that could be valuable in the prevention and treatment of mucosal diseases.

Extensively deacetylated high molecular weight chitosan from the multistep ultrasound-assisted deacetylation of beta-chitin.

High intensity ultrasound irradiation was used to convert beta-chitin (BCHt) into chitosan (CHs). Typically, beta-chitin was suspended in 40% (w/w) aqueous sodium hydroxide at a ratio 1/10 (gmL(-1)) and then submitted to ultrasound-assisted deacetylation (USAD) during 50min at 60°C and a fixed irradiation surface intensity (52.6Wcm(-2)). Hydrogen nuclear magnetic resonance spectroscopy and capillary viscometry were used to determine the average degree of acetylation (DA‾) and viscosity average degree of polymerization (DPv‾), respectively, of the parent beta-chitin (DA‾=80.7%; DPv‾=6865) and USAD chitosans. A first USAD reaction resulted in chitosan CHs1 (DA‾=36.7%; DPv‾=5838). Chitosans CHs2 (DA‾=15.0%; DPv‾=5128) and CHs3 (DA‾=4.3%; DPv‾=4889) resulted after repeating the USAD procedure to CHs1 consecutively once and twice, respectively. Size-exclusion chromatography analyzes allowed the determination of the weight average molecular weight (Mw‾) and dispersity (Ð) of CHs1 (Mw‾=1,260,000gmol(-1); Ð=1.4), CHs2 (Mw‾=1,137,000gmol(-1); Ð=1.4) and CHs3 (Mw‾=912,000gmol(-1); Ð=1.3). Such results revealed that, thanks to the action of high intensity ultrasound irradiation, the USAD process allowed the preparation of unusually high molecular weight, randomly deacetylated chitosan, an important breakthrough to the development of new high grade chitosan-based materials displaying superior mechanical properties.

Enzymatic hydrolysis of chitin pretreated by rapid depressurization from supercritical 1,1,1,2-tetrafluoroethane toward highly acetylated oligosaccharides.

The hydrolysis of chitin treated under supercritical conditions was successfully carried out using chitinases obtained by an optimized fermentation of the fungus Lecanicillium lecanii. The biopolymer was subjected to a pretreatment based on suspension in supercritical 1,1,1,2-tetrafluoroethane (scR134a), which possesses a critical temperature and pressure of 101°C and 40bar, respectively, followed by rapid depressurization to atmospheric pressure and further fibrillation. This methodology was compared to control untreated chitins and chitin subjected to steam explosion showing improved production of reducing sugars (0.18mg/mL), enzymatic hydrolysis and high acetylation (FA of 0.45) in products with degrees of polymerization between 2 and 5.

Fine microstructure of processed chitosan nanofibril networks preserving directional packing and high molecular weight.

Crystalline chitosan nanofibril networks were prepared, preserving the native structural packing and the polymer high molecular weight. The fine microstructure of the nanomaterial, obtained by mild hydrolysis of chitosan (CHI), was characterized by using synchrotron small- and wide-angle X-ray scattering (SAXS and WAXS), transmission electron microscopy (TEM) and electron diffraction. Hydrolysis of chitosan yielded a network of crystalline nanofibrils, containing both allomorphs of chitosan: hydrated and anhydrous. The comparison of WAXS data in transmission and reflection mode revealed the preferential orientation of the CHI crystals when subjected to mechanical compression constrains. The results are in agreement with the existence of a network nanostructure containing fiber-like crystals with the principal axis parallel to the polymer chain axis. The evolution of the CHI allomorphic composition with temperature was studied to further elucidate the mechanism of structural transitions occurring during CHI nanofibril network processing.

Access to tetra-N-acetyl-chitopentaose by chemical N-acetylation of glucosamine pentamer.

Nowadays, the easy access of tetra-N-acetyl-chitopentaose and its counterparts is highly interesting since such chemical compounds are precursors of biological signal molecules with a strong agro-economic impact. The chemical synthesis of tetra-N-acetyl-chitopentaose by controlled N-acetylation of the glucosamine pentamer hydrochloride under mild conditions is described herein. A systematic study on the influence of the different parameters involved in this reaction, such as the solvent, the acetylating agent, and the base used for the deprotonation of ammonium groups of the starting material was carried out. The characterization of final reaction products by HPLC and MALDI-TOF mass spectrometry showed that each of these parameters affects differently the acetylation reaction. Whereas the solvent plays an important role in the N- or O-acetylation selectivity, the acetylating agent and the base were found to influence both the degree of N-acetylation and the distribution of the partially N-acetylated derivatives in the product mixtures. Based on these results, optimized reaction conditions have been established allowing tetra-N-acetyl-chitopentaose to be synthesized in a one-pot deprotonation/N-acetylation of the glucosamine pentamer hydrochloride in a moderate yield (ca 30%).

Activity of chitin deacetylase from Colletotrichum gloeosporioides on chitinous substrates.

Production of chitin deacetylases from the phytopathogenic fungus Colletotrichum gloeosporioides was successfully achieved by submerged fermentation. The highest specific activity of 0.018 U mg(-1) of protein was obtained after 96 h of cultivation at pH 6 and 28°C. Two bands with molecular weights of 35 kDa and 170 kDa determined with SDS-PAGE displayed deacetylase activities as detected in the zymograms. Reacetylated commercial chitosan (52% acetylation degree) was used as substrate for the extracellular crude extract in order to estimate the kinetic parameters of acetate production as undirected deacetylation measurement. The highest acetate production of 12.8 µmol mL(-1) was obtained using 7.5 mg mL(-1) of substrate. The produced enzyme from C. gloeosporioides achieved up to 25% deacetylation of a chitin substrate (hydrolyzed biological chitin) having 80% degree of acetylation, MW of 102×10(3) g mol(-1) and a crystallinity index of ca. 60%.

Structural characterization of chitin and chitosan obtained by biological and chemical methods.

Chitin production was biologically achieved by lactic acid fermentation (LAF) of shrimp waste (Litopenaeus vannameii) in a packed bed column reactor with maximal percentages of demineralization (D(MIN)) and deproteinization (D(PROT)) after 96 h of 92 and 94%, respectively. This procedure also afforded high free astaxanthin recovery with up to 2400 µg per gram of silage. Chitin product was also obtained from the shrimp waste by a chemical method using acid and alkali for comparison. The biologically obtained chitin (BIO-C) showed higher M(w) (1200 kDa) and crystallinity index (I(CR)) (86%) than the chemically extracted chitin (CH-C). A multistep freeze-pump-thaw (FPT) methodology was applied to obtain medium M(w) chitosan (400 kDa) with degree of acetylation (DA) ca. 10% from BIO-C, which was higher than that from CH-C. Additionally, I(CR) values showed the preservation of crystalline chitin structure in BIO-C derivatives at low DA (40-25%). Moreover, the FPT deacetylation of the attained BIO-C produced chitosans with bloc copolymer structure inherited from a coarse chitin crystalline morphology. Therefore, our LAF method combined with FPT proved to be an affective biological method to avoid excessive depolymerization and loss of crystallinity during chitosan production, which offers new perspective applications for this material.