Study On The Structure Assembly And Antibacterial Mechanism Of Ag NP Loaded Chitosan-Based Layered Nanocomposites

Silver nanoparticle (Ag NP) is one of the most widely used nanophase materials, which iscommonly prepared via chemical reduction method. However, the additional reducing andstabilizing agents are harmful to human or enviroment in some extent, and they cannot beseparated completely from Ag NP, which limits its applications in medicine and biocatalysisareas. Therefore, it is urgent to find the green reducing and stabilizing agents and developefficient preparation method of Ag NP for nano metal industry. Chitosan is a naturalbiopolymer with many hydroxyl groups. The intermolecular and intramolecular hydrogenbonds in chitosan result in its independent space on the molecular level, which can provide agood template for the growth of nanoparticles. Due to the confinement effect of the layerspace, layered silicate such as montmorillonite is known as two dimension nano-reactor, andconsidered to be the perfect stability carrier for nanoparticles. Chitosan-based layered silicate(CLS) nanocomposites are synthesized by chitosan or its derivatives intercalation into theinterlayer of layered silicate under an external force, they are hybrid materials which cancombine the excellent capability of chitosan and silicate. At present, CLS nanocompositeshave been studied extensively, however, few researches are about the preparation of metalnanoparticles by using CLS nanocomposites as carrier.The objectives of the present study were to prepare chitosan derivatives, organic layeredsilicate, CLS nanocomposites and exfoliated Ag NP loaded CLS nanocomposites undermicrowave irradiation, and the intercalation mechanisms, antibacterial mechanisms andfunctional applications were investigated. The results are listed as follow:1. Rapid preparation of water-soluble chitosan derivatives xia microwave irradiationmethod and their properties(1) Rapid preparation of chitosan derivatives with various degree of substitutionWater-soluble chitosan derivatives such as quaternized chitosan (QCS), quaternizedcarboxymethyl chitosan (QCMC) and quaternized carboxymethyl chitosan oligosaccharides(QCMCO) were rapidly prepared under microwave irradiation in the aqueous solution. TheQCS obtained by microwave irradiation method had similar structure and higher degree ofsubstitution (DS) as compared to those obtained by traditional heating method. The degree ofsubstitution of carboxymethyl (DSCM) or quarternary ammonium (DSQ) groups of QCMC canbe controlled by changing the microwave time, microwave power and the dosage of modified agents, the highest DSCMand DSQof82%and48%respectively were obtained in theappropriate conditions.(3) Study on the properties of chitosan derivativesThe obtained QCS could flocculate CaCO3suspension. The flocculation behavior of QCSwas directly proportional to its DS and inversely proportional to the molecular weight, withthe best flocculation concentration of6mg/L.QCMCO had excellent oxidation resistance, which was positively proportional to itsconcentration and closely related to DSCMand DSQ. When the concentration of QCMCO wasonly5mg/mL, the scavenging rate on OH was63.6%, and the Fe²⁺chelating ability was81.98%.2. Rapid preparation of organic montmorillonite by microwave irradiation methodand their adsorption propertiesGemini-MMT (GMMT) and ester-MMT (EMMT) with large layer spacing were quicklyprepared by using new cationic surfactants of Gemini and Esterquat as modifier undermicrowave irradiation condition. With the saturated intercalation dosage of Gemini andEsterquat of0.5CEC and0.8CEC, the largest layer spacing of GMMT and EMMT was2.31nm and2.41nm respectively. In addition, the layer spacing of GMMT obtained by microwaveirradiation method (2.31nm) was larger than that by traditional heating method (2.23nm).Gemini was connected with MMT by electrostatic interaction, with combination ways ofintercalation, intercalation-adsorption and adsorption. However, even if the intercalation ofEsterquat was saturated, it was still combined with MMT by adsorption with the unchangedlayer spacing.The surface of GMMT and EMMT was hydrophobic, rough and fluffy. Therefore,bothGMMT and EMMT had excellent adsorption ability. The adsorption ability of GMMTincreased with increasing chain length of Gemini molecules, with the maximum actualadsorption capacity of48mg/g. The adsorption process of EMMT for triclosan was followedas the Lange Samuel isothermal adsorption equation, with the maximum theoreticaladsorption capacity of133mg/g.3. The effect of molecular parameters on the structure and properties of CLSnanocomposites (1) The effect of dosage on the intercalation processThe layer spacing of carboxymethyl quaternized chitosan/organic montmorillonite (QCOM)nanocomposites was proportional to the dosage of QCMC, when the DSCMand DSQof QCMCwas56.3%and74.6%respectively and its weight molecular weight (Mw) was2.9×105. Thelayer spacing of carboxymethyl quaternized chitosan oligosaccharide/organic rectorite(QCOOR) nanocomposites showed a firstly raised and then decreased trend with theincreasing dosage of QCMC, which the DSCMand DSQof QCMCO was88%and75%respectively and its Mwwas2×104.(2) The effect of DS on the intercalation processIn the QCOM nanocomposites which obtained by the QCMC with Mwof2.6×105³.0×105,the higher DSQcould promote the intercalation reaction, and extend the layer spacing ofQCOM nanocomposites. For example, when DSCMwas about30%, DSQincreased from29.6%to73.9%, the layer spacing of QCOM nanocomposites increased from3.70nm to4.50nm. But, the layer spacing of QCOM nanocomposites showed firstly increased and thendecreased trend with the increasing DSCMof QCMC. For example, when DSQwas about30%,DSQincreased from30.4%to85.2%, the layer spacing of QCOM nanocomposites increasedfrom3.70nm to4.22nm, and then decreased to3.79nm.In the QCOOR nanocomposites obtained by the QCMCO with Mwof8×103².0×104, theenhancemnt of the DSCMwas benefit for intercalation reaction, and getting larger layerspacing. For example, when DSQwas about45%, DSQincreased from23%to91%, the layerspacing of QCOOR nanocomposites increased from4.37nm to4.78nm. But DSQhad littleimpact on the layer spacing of QCOOR nanocomposites.(3) Preparation of exfoliated CLS nanocompositesThe exfoliated QCOM nanocomposites can be obtained when Mwof QCMC was2.6×105³.0×105, its DSCMand DSQwas53.6%and41.3%respectively, and the mass ratio ofQCMC to OMMT was8:1. But the exfoliated QCOOR nanocomposite was obtained whenMwof QCMCO was8×103².0×104, its DSCMand DSQwas88%and75%respectively, andthe mass ratio of QCMCO to OREC was4:1. Besides, the exfoliated quaternizedcarboxymethyl chitosan/rectorite (QCR) nanocomposite was obtained when Mwof QCMCwas8×103².0×104, its DSCMand DSQwas72%and80%respectively, and the quality ratio of QCMC to REC was above4:1.4. Study on the performance of CLS nanocomposites(1) Flocculation behavior of CLS nanocompositesQuaternized chitosan/organic montmorillonite (QOM) nanocomposites which combined theexcellent performance of QCS and OMMT showed good flocculation capacity. Theflocculation efficiency of QOM nanocomposite on CaCO3suspension was more than70%,when its dosage was0.005mg/L, which was only one-thousandth dosage of the traditionalflocculant such as cationic starch.(2) The controlled-release behavior of CLS crosslinked nanocomposite microsphereThe swelling behavior of quaternized carboxymethyl chitosan/organic montmorillonite(QCOM) nanocomposite (AQCOM) microsphere crosslinked with alginate was significantlyaffected by OMMT. With the mass ratios of QCMC to OMMT increased from1:1to8:1inQCOM nanocomposites, the swelling ratios of AQCOM microspheres was reached from44%to197%. The encapsulation efficiency of AQCOM microspheres increased with increasingcontent of QCMC and the layer spacing of QCOM nanocomposites. An appropriate dosage ofOMMT could be contributed to the controlled-release behavior of AQCOM microspheres, butexcessive levels of OMMT could destroy the behavior. In addition, the in vitro activecutaneous anaphylaxis test was carried out on guinea pigs, which revealed that AQCOMmicrosphere did not cause anaphylaxis.(3) The antibacterial behavior of CLS nanocompositesThe antibacterial activity of quaternized carboxymethyl chitosan oligosaccharides/rectorite(QCOR) nanocomposites was better than that of QCMCO. The Gram-positive bacteria andGram-negative bacteria which treated by QCOR nanocomposite had following phenomenon:the surface was collapse and deformation, the bacterial cell walls even ruptured and the cellcontents seep away, while the fungal spores burst and their normal physiological activity wasinhibited. In addition, the inhibitory effect of QCOR nanocomposite on the Gram-positivebacteria was better than that of Gram-negative bacteria and Fungi.5. Study on the structural assembly and antibacterial mechanism of Ag NP loadedCLS nanocomposites(1) Preparation of Ag NP loaded chitosan-based nanocomposites by Tollen method The spherical Ag NP with uniform size was quickly obtained under microwave irradiationby utilizing the reducing capacity of carboxymethyl groups and quaternary ammonium groups.The elevating reaction temperature or prolonging the reaction time was conducive to theformation of Ag NP. The preparation activation energy of carboxymethyl chitosan (CMC),QCS and QCMC was69.7,62.8and103.7kJ/mol, respectively. Compared withcarboxymethyl groups, quaternary ammonium groups are more beneficial to prepare Ag NP.The silver content of CMC-Ag, QCS-Ag and QCMC-Ag was0.67‰,4.85‰and5.57‰respectively, and particle size of Ag NPs was mainly in the range of60-80nm,40-60nm and5-12nm, respectively. The results of FT-IR and NMR revealed that not all the carboxymethylgroups and quaternary ammonium groups were reducted for synthesizing Ag NP, and theremaining chitosan chain might form networks to wrap Ag NP and prevent their reunion.TEM micrographs showed that Ag NP was in mainly spherical, and few square or bar form. Inaddition, the thermal stability of Ag NP loaded chitosan-based composites was higher thanthat of chitosan derivatives.(2) The assembly mechanism of Ag NP loaded CLS compositesWhen the mass ratio of initial quaternized chitosan to Ag+increased from100mg:0.1mmol to100mg:1mmol, the silver content in Ag NP loaded quaternizedchitosan/montmorillonite (QMAg) nanocomposites and Ag NP loaded quaternizedchitosan/organic montmorillonite (QOMAg) nanocomposites increased from0.07‰to14.14‰and0.61‰to33.21‰, respectively. And when the dose ratio of clay increased from5mg to20mg, the silver content in Ag NP loaded quaternized carboxymethylchitosan/rectorite (QCRAg) nanocomposites and Ag NP loaded quaternized chitosan/organicrectorite (QCORAg) nanocomposites increased from0.16‰to7.75‰and0.91‰to12.04‰,respectively.The surfactant in the organic clay did not take part in the chemical reaction of Ag NPpreparation, but it could increase the yield of Ag NP. TEM micrographs showed that Ag NPmaintained spherical in drying Ag NP loaded CLS nanocomposites, with uniformity size anddispersion, and the exfoliated silicate layers were evenly distributed in chitosan derivativesmatrix as the growth template of Ag NP. Ag NP loaded CLS nanocomposites showedexcellent thermal stability, which enhanced with increasing amount of Ag+and clay. (3) The antimicrobial mechanism of Ag NP loaded CLS nanocompositesQMAg and QOMAg nanocomposites had excellent antibacterial activity which wasincreased with increasing Ag NP content, and the minimum inhibitory concentration (MIC)was0.0005%and0.00001%(wt.) respectively, which was only1/2000and1/200time thanQM and QOM nanocomposites. The antimicrobial course may be the following: firstly, MMTwith large specific surface area had the capacity of adsorption and immobilization onmictobes, hydrophobic groups in QCS and Gemini can also be associated with the cell wall oflipoprotein, lipopolysaccharide and phospholipids and other lipophilic compounds, whichresulted in better adsorption and immobilization on bacteria; secondly, quaternary ammoniumgroups in QCS and Gemini can form complexes with cell surface, thereby the permeability ofcell membrane was changed, cell membrane of normal physiological activity was disrupted;thirdly, Ag NP can be associated with bacterial cell wall and cytoplasm containing S, Pcompound, which can affect cell infiltration and split, resulting in the death of bacteria.