Maren Roman

Effect of Sulfate Groups from Sulfuric Acid Hydrolysis on the Thermal Degradation Behavior of Bacterial Cellulose

When used as fillers in polymer composites, the thermostability of cellulose crystals is important. Sulfate groups, introduced during hydrolysis with sulfuric acid, are suspected to diminish the thermostability. To elucidate the relationship between the hydrolysis conditions, the number of sulfate groups introduced, and the thermal degradation behavior of cellulose crystals, bacterial cellulose was hydrolyzed with sulfuric acid under different hydrolysis conditions. The number of sulfate groups in the crystals was determined by potentiometric titration. The thermal degradation behavior was investigated by thermogravimetric analysis. The sulfate group content increased with acid concentration, acid-to-cellulose ratio, and hydrolysis time. Even at low levels, the sulfate groups caused a significant decrease in degradation temperatures and an increase in char fraction confirming that the sulfate groups act as flame retardants. Profile analysis of the derivative thermogravimetric curves indicated thermal separation of the degradation reactions by the sulfate groups into low- and high-temperature processes. The Broido method was used to determine activation energies for the degradation processes. The activation energies were lower at larger amounts of sulfate groups suggesting a catalytic effect on the degradation reactions. For high thermostability in the crystals, low acid concentrations, small acid-to-cellulose ratios, and short hydrolysis times should be used.

Acid-catalyzed and solvolytic desulfation of H2SO4-hydrolyzed cellulose nanocrystals.

Cellulose nanocrystals (CNCs) prepared by H(2)SO(4) hydrolysis have sulfate groups on their surface, which have negative implications for some CNC applications. In this study, two desulfation methods were evaluated, and the properties of desulfated CNCs were compared to those of unsulfated CNCs, prepared by HCl hydrolysis. H(2)SO(4)-hydrolyzed CNCs from softwood sulfite pulp were subjected to either a mild acid hydrolytic desulfation or a solvolytic desulfation in dimethyl sulfoxide via the pyridinium salt. Removal of the sulfate groups was confirmed by conductometric titration and X-ray photoelectron spectroscopy. The effect of the desulfation procedure on the lateral crystallite dimensions was analyzed by X-ray diffraction. The extent of particle aggregation in the samples was assessed by atomic force microscopy and dynamic light scattering. The acid hydrolytic method achieved only partial desulfation and produced gradually decreasing sulfate contents upon successive repetition. The solvolytic method achieved nearly complete desulfation in a single step. The desulfated CNCs showed similar particle aggregation as the HCl-hydrolyzed CNCs, but the extent of aggregation was slightly less.

Synthesis and Cellular Uptake of Folic Acid-Conjugated Cellulose Nanocrystals for Cancer Targeting

Elongated nanoparticles have recently been shown to have distinct advantages over spherical ones in targeted drug delivery applications. In addition to their oblong geometry, their lack of cytotoxicity and numerous surface hydroxyl groups make cellulose nanocrystals (CNCs) promising drug delivery vectors. Herein we report the synthesis of folic acid-conjugated CNCs for the targeted delivery of chemotherapeutic agents to folate receptor-positive cancer cells. Folate receptor-mediated cellular binding/uptake of the conjugate was demonstrated on human (DBTRG-05MG, H4) and rat (C6) brain tumor cells. Folate receptor expression of the cells was verified by immunofluorescence staining. Cellular binding/uptake of the conjugate by DBTRG-05MG, H4, and C6 cells was 1452, 975, and 46 times higher, respectively, than that of nontargeted CNCs. The uptake mechanism was determined by preincubation of the cells with the uptake inhibitors chlorpromazine or genistein. DBTRG-05MG and C6 cells internalized the conjugate primarily via caveolae-mediated endocytosis, whereas H4 cells internalized the conjugate primarily via clathrin-mediated endocytosis.

Formation and Properties of Chitosan−Cellulose Nanocrystal Polyelectrolyte−Macroion Complexes for Drug Delivery Applications

This study examines a novel polyelectrolyte-macroion complex (PMC) between chitosan, a cationic polysaccharide, and cellulose nanocrystals (CNCs), anionic, cylindrical nanoparticles, for potential applications in drug delivery. CNCs were prepared by H(2)SO(4) hydrolysis of wood pulp. The formation of PMCs was monitored by turbidimetric titration. In titrations of a chitosan solution with a CNC suspension, the turbidity reached a plateau, but it had a maximum and then decreased when the direction of titration was reversed. PMC particles were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, dynamic light scattering, and laser Doppler electrophoresis. The particles were composed primarily of CNCs and ranged in size from a few hundred nanometers to several micrometers, depending on the cellulose/chitosan ratio. Particles formed at amino/sulfate group molar ratios >1 were nearly spherical in shape and positively charged, whereas particles formed at ratios <1 had well-defined nonspherical shapes and were negatively charged.

CYTOTOXICITY AND CELLULAR UPTAKE OF CELLULOSE NANOCRYSTALS

There is growing evidence that filamentous nanoparticles offer advantages over spherical ones in drug delivery applications. The purpose of this study was to assess the potential of rod-like, plant-derived cellulose nanocrystals (CNCs) for nanomedical uses. Besides a nonspherical morphology, their facile bioconjugation, surface hydrophilicity and small size render CNCs promising drug carriers. The cytotoxicity of CNCs against nine different cell lines (HBMEC, bEnd.3, RAW 264.7, MCF-10A, MDA-MB-231, MDA-MB-468, KB, PC-3 and C6) was determined by MTT and LDH assay. CNCs showed no cytotoxic effects against any of these cell lines in the concentration range and exposure time studied (0–50 μg/mL and 48 h, respectively). Cellular uptake of fluorescein-5′-isothiocyanate-labeled CNCs by these cell lines, quantified with a fluorescence microplate reader, was minimal. The lack of cytotoxicity and the low nonspecific cellular uptake support our hypothesis that CNCs are good candidates for nanomedical applications.

Equilibrium Water Contents of Cellulose Films Determined via Solvent Exchange and Quartz Crystal Microbalance with Dissipation Monitoring

Model cellulose surfaces have attracted increasing attention for studying interactions with cell wall matrix polymers and as substrates for enzymatic degradation studies. Quartz crystal microbalance with dissipation monitoring (QCM-D) solvent exchange studies showed that the water content of regenerated cellulose (RC) films was proportional to the film thickness (d) and was consistent with about five water molecules per anhydroglucose unit. Sulfated nanocrystalline cellulose (SNC) and desulfated nanocrystalline cellulose (DNC) films had comparable water contents and contained about five times more water than RC films. A cellulase mixture served as a probe for studies of substrate accessibility and degradation. Cellulase adsorption onto RC films was independent of d, whereas degradation times increased with d. However, adsorption onto SNC and DNC films increased with d, whereas cellulase degradation times for DNC films were independent of studied d. Enhanced access to guest molecules for SNC and DNC films revealed they are more porous than RC films.

Effects of sulfate groups on the adsorption and activity of cellulases on cellulose substrates.

Pretreatment of lignocellulosic biomass with sulfuric acid may leave sulfate groups on its surface that may hinder its biochemical conversion. This study investigates the effects of sulfate groups on cellulase adsorption onto cellulose substrates and the enzymatic hydrolysis of these substrates. Substrates with different sulfate group densities were prepared from H2SO4- and HCl-hydrolyzed and partially and fully desulfated cellulose nanocrystals. Adsorption onto and hydrolysis of the substrates was analyzed by quartz crystal microbalance with dissipation monitoring (QCM-D). The surface roughness of the substrates, measured by atomic force microscopy, increased with decreasing sulfate group density, but their surface accessibilities, measured by QCM-D H2O/D2O exchange experiments, were similar. The adsorption of cellulose binding domains onto sulfated substrates decreased with increasing sulfate group density, but the adsorption of cellulases increased. The rate of hydrolysis of sulfated substrates decreased with increasing sulfate group density. The results indicated an inhibitory effect of sulfate groups on the enzymatic hydrolysis of cellulose, possibly due to nonproductive binding of the cellulases onto the substrates through electrostatic interactions instead of their cellulose binding domains.

Ultrathin chitin films for nanocomposites and biosensors.

Chitin is the second most abundant biopolymer and insight into its natural synthesis, enzymatic degradation, and chemical interactions with other biopolymers is important for bioengineering with this renewable resource. This work is the first report of smooth, homogeneous, ultrathin chitin films, opening the door to surface studies of binding interactions, adsorption kinetics, and enzymatic degradation. The chitin films were formed by spincoating trimethylsilyl chitin onto gold or silica substrates, followed by regeneration to a chitin film. Infrared and X-ray photoelectron spectroscopy, X-ray diffraction, ellipsometry, and atomic force microscopy were used to confirm the formation of smooth, homogeneous, and amorphous chitin thin films. Quartz crystal microbalance with dissipation monitoring (QCM-D) solvent exchange experiments showed these films swelled with 49% water by mass. The utility of these chitin films as biosensors was evident from QCM-D and surface plasmon resonance studies that revealed the adsorption of a bovine serum albumin monolayer.

Folate Conjugated Cellulose Nanocrystals Potentiate Irreversible Electroporation-induced Cytotoxicity for the Selective Treatment of Cancer Cells

Cellulose nanocrystals are rod-shaped, crystalline nanoparticles that have shown prom-ise in a number of industrial applications for their unique chemical and physical properties. However, investigations of their abilities in the biomedical field are limited. The goal of this study is to show the potential use of folic acid-conjugated cellulose nanocrystals in the potentiation of irreversible electroporation-induced cell death in folate receptor (FR)-positive cancers. We optimized key pulse parameters including pulse duration, intensity, and incubation time with nanoparticles prior to electroporation. FR-positive cancer cells, KB and MDA-MB-468, were preincubated with cellulose nanocrystals (CNCs) conjugated with the targeting molecule folic acid (FA), 10 and 20 min respectively, prior to application of the optimized pulse electric field (PEF), 600 and 500 V/cm respectively. We have shown cellulose nanocrystals’ ability to potentiate a new technique for tumor ablation, irreversible electroporation. Pre-incubation with FA-conjugated CNCs (CNC-FA) has shown a significant increase in cytotoxicity induced by irreversible electroporation in FR-positive cancer cells, KB and MDA-MB-468. Non-targeted CNCs (CNC-COOH) did not potentiate IRE when preincubated at the same parameters as previously stated in these cell types. In addition, CNC-FA did not potentiate irreversible electroporation-induced cytotoxicity in a FR-negative cancer cell type, A549. Without changing irreversible electroporation parameters it is possible to increase the cytotoxic effect on FR-positive cancer cells by exploiting the specific binding of FA to the FR, while not causing further damage to FR-negative tissue.

Surface-initiated dehydrogenative polymerization of monolignols: a quartz crystal microbalance with dissipation monitoring and atomic force microscopy study.

This work highlights a real-time and label-free method to monitor the dehydrogenative polymerization of monolignols initiated by horseradish peroxidase (HRP) physically immobilized on surfaces using a quartz crystal microbalance with dissipation monitoring (QCM-D). The dehydrogenative polymer (DHP) films are expected to provide good model substrates for studying ligninolytic enzymes. The HRP was adsorbed onto gold or silica surfaces or onto and within porous desulfated nanocrystalline cellulose films from an aqueous solution. Surface-immobilized HRP retained its activity and selectivity for monolignols as coniferyl and p-coumaryl alcohol underwent dehydrogenative polymerization in the presence of hydrogen peroxide, whereas sinapyl alcohol polymerization required the addition of a nucleophile. The morphologies of the DHP layers on the surfaces were investigated via atomic force microscopy (AFM). Data from QCM-D and AFM showed that the surface-immobilized HRP-initiated dehydrogenative polymerization of monolignols was greatly affected by the support surface, monolignol concentration, hydrogen peroxide concentration, and temperature.