Influence of Oxidation Degree on the Physicochemical Properties of Oxidized Inulin.

This paper reports the oxidation of inulin using varying ratios of sodium periodate and the characterization of the inulin polyaldehyde. The physicochemical properties of the inulin polyaldehyde (oxidized inulin) were characterized using different techniques including 1D NMR spectroscopy, 13C Nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), differential scanning calorimetric (DSC), ultraviolet-visible spectroscopy (UV), and scanning electron microscopy (SEM). The aldehyde peak was not very visible in the FTIR, because the aldehyde functional group exists in a masked form (hemiacetal). The thermal stability of the oxidized inulin decreased with the increasing oxidation degree. The smooth spherical shape of raw inulin was destructed due to the oxidation, as confirmed by the SEM result. The 1HNMR results show some new peaks from 4.8 to 5.0 as well as around 5.63 ppm. However, no aldehyde peak was found around 9.7 ppm. This can be attributed to the hemiacetal. The reaction of oxidized inulin with tert-butyl carbazate produced a carbazone conjugate. There was clear evidence of decreased peak intensity for the proton belonging to the hemiacetal group. This clearly shows that not all of the hemiacetal group can be reverted by carbazate. In conclusion, this work provides vital information as regards changes in the physicochemical properties of the oxidized inulin, which has direct implications when considering the further utilization of this biomaterial.

Doxorubicin-Loaded Delta Inulin Conjugates for Controlled and Targeted Drug Delivery: Development, Characterization, and In Vitro Evaluation.

Delta inulin, also known as microparticulate inulin (MPI), was modified by covalently attaching doxorubicin to its nanostructured surface for use as a targeted drug delivery vehicle. MPI is readily endocytosed by monocytes, macrophages, and dendritic cells and in this study, we sought to utilize this property to develop a system to target anti-cancer drugs to lymphoid organs. We investigated, therefore, whether MPI could be used as a vehicle to deliver doxorubicin selectively, thereby reducing the toxicity of this antibiotic anthracycline drug. Doxorubicin was covalently attached to the surface of MPI using an acid-labile linkage to enable pH-controlled release. The MPI-doxorubicin conjugate was characterized using FTIR and SEM, confirming covalent attachment and indicating doxorubicin coupling had no obvious impact on the physical nanostructure, integrity, and cellular uptake of the MPI particles. To simulate the stability of the MPI-doxorubicin in vivo, it was stored in artificial lysosomal fluid (ALF, pH 4.5). Although the MPI-doxorubicin particles were still visible after 165 days in ALF, 53% of glycosidic bonds in the inulin particles were hydrolyzed within 12 days in ALF, reflected by the release of free glucose into solution. By contrast, the fructosidic bonds were much more stable. Drug release studies of the MPI-doxorubicin in vitro, demonstrated a successful pH-dependent controlled release effect. Confocal laser scanning microscopy studies and flow cytometric analysis confirmed that when incubated with live cells, MPI-doxorubicin was efficiently internalized by immune cells. An assay of cell metabolic activity demonstrated that the MPI carrier alone had no toxic effects on RAW 264.7 murine monocyte/macrophage-like cells, but exhibited anti-cancer effects against HCT116 human colon cancer cells. MPI-doxorubicin had a greater anti-cancer cell effect than free doxorubicin, particularly when at lower concentrations, suggesting a drug-sparing effect. This study establishes that MPI can be successfully modified with doxorubicin for chemotherapeutic drug delivery.

Synthesis and Characterization of pH-Sensitive Inulin Conjugate of Isoniazid for Monocyte-Targeted Delivery.

The use of particles for monocyte-mediated delivery could be a more efficient strategy and approach to achieve intracellular targeting and delivery of antitubercular drugs to host macrophages. In this study, the potential of inulin microparticles to serve as a drug vehicle in the treatment of chronic tuberculosis using a monocytes-mediated drug targeting approach was evaluated. Isoniazid (INH) was conjugated to inulin via hydrazone linkage in order to obtain a pH-sensitive inulin-INH conjugate. The conjugate was then characterized using proton nuclear magnetic resonance (1HNMR), Fourier transform infrared spectroscopy (FTIR) as well as in vitro, cellular uptake and intracellular Mycobacterium tuberculosis (Mtb) antibacterial efficacy. The acid-labile hydrazone linkage conferred pH sensitivity to the inulin-INH conjugate with ~95, 77 and 65% of the drug released after 5 h at pH 4.5, 5.2, and 6.0 respectively. Cellular uptake studies confirm that RAW 264.7 monocytic cells efficiently internalized the inulin conjugates into endocytic compartments through endocytosis. The intracellular efficacy studies demonstrate that the inulin conjugates possess a dose-dependent targeting effect against Mtb-infected monocytes. This was through efficient internalization and cleavage of the hydrazone bond by the acidic environment of the lysosome, which subsequently released the isoniazid intracellularly to the Mtb reservoir. These results clearly suggest that inulin conjugates can serve as a pH-sensitive intracellular drug delivery system for TB treatment.

Preparation and Characterization of Oxidized Inulin Hydrogel for Controlled Drug Delivery.

Inulin-based hydrogels are useful carriers for the delivery of drugs in the colon-targeted system and in other biomedical applications. In this project, inulin hydrogels were fabricated by crosslinking oxidized inulin with adipic acid dihydrazide (AAD) without the use of a catalyst or initiator. The physicochemical properties of the obtained hydrogels were further characterized using different techniques, such as swelling experiments, in vitro drug release, degradation, and biocompatibility tests. The crosslinking was confirmed with Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), and differential scanning calorimetry (DSC). In vitro releases of 5-fluorouracil (5FU) from the various inulin hydrogels was enhanced in acidic conditions (pH 5) compared with physiological pH (pH 7.4). In addition, blank gels did not show any appreciable cytotoxicity, whereas 5FU-loaded hydrogels demonstrated efficacy against HCT116 colon cancer cells, which further confirms the potential use of these delivery platforms for direct targeting of 5-FU to the colon.

Review of polysaccharide particle-based functional drug delivery.

This review investigates the significant role polysaccharide particles play in functional drug delivery. The importance of these systems is due to the wide variety of polysaccharides and their natural source meaning that they can provide biocompatible and biodegradable systems with a range of both biological and chemical functionality valuable for drug delivery. This functionality includes protection and presentation of working therapeutics through avoidance of the reticuloendothelial system, stabilization of biomacromolecules and increasing the bioavailability of incorporated small molecule drugs. Transport of the therapeutic is also key to the utility of polysaccharide particles, moving drugs from the site of administration through mucosal binding and transport and using chemistry, size and receptor mediated drug targeting to specific tissues. This review also scrutinizes the methods of synthesizing and constructing functional polysaccharide particle drug delivery systems that maintain and extend the functionality of the natural polysaccharides.

Design and Characterization of Inulin Conjugate for Improved Intracellular and Targeted Delivery of Pyrazinoic Acid to Monocytes.

The propensity of monocytes to migrate into sites of mycobacterium tuberculosis (TB) infection and then become infected themselves makes them potential targets for delivery of drugs intracellularly to the tubercle bacilli reservoir. Conventional TB drugs are less effective because of poor intracellular delivery to this bacterial sanctuary. This study highlights the potential of using semicrystalline delta inulin particles that are readily internalised by monocytes for a monocyte-based drug delivery system. Pyrazinoic acid was successfully attached covalently to the delta inulin particles via a labile linker. The formation of new conjugate and amide bond was confirmed using zeta potential, Proton Nuclear Magnetic Resonance (1HNMR) and Fourier transform infrared spectroscopy (FTIR). Scanning electron microscopy (SEM) confirmed that no significant change in size after conjugation which is an important parameter for monocyte targeting. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) were used to establish the change in thermal properties. The analysis of in-vitro release demonstrated pH-triggered drug cleavage off the delta inulin particles that followed a first-order kinetic process. The efficient targeting ability of the conjugate for RAW 264.7 monocytic cells was supported by cellular uptake studies. Overall, our finding confirmed that semicrystalline delta inulin particles (MPI) can be modified covalently with drugs and such conjugates allow intracellular drug delivery and uptake into monocytes, making this system potentially useful for the treatment of TB.

A Novel Fabrication Approach for Multifunctional Graphene-based Thin Film Nano-composite Membranes with Enhanced Desalination and Antibacterial Characteristics.

A practical fabrication technique is presented to tackle the trade-off between the water flux and salt rejection of thin film composite (TFC) reverse osmosis (RO) membranes through controlled creation of a thinner active selective polyamide (PA) layer. The new thin film nano-composite (TFNC) RO membranes were synthesized with multifunctional poly tannic acid-functionalized graphene oxide nanosheets (pTA-f-GO) embedded in its PA thin active layer, which is produced through interfacial polymerization. The incorporation of pTA-f-GOL into the fabricated TFNC membranes resulted in a thinner PA layer with lower roughness and higher hydrophilicity compared to pristine membrane. These properties enhanced both the membrane water flux (improved by 40%) and salt rejection (increased by 8%) of the TFNC membrane. Furthermore, the incorporation of biocidal pTA-f-GO nanosheets into the PA active layer contributed to improving the antibacterial properties by 80%, compared to pristine membrane. The fabrication of the pTA-f-GO nanosheets embedded in the PA layer presented in this study is a very practical, scalable and generic process that can potentially be applied in different types of separation membranes resulting in less energy consumption, increased cost-efficiency and improved performance.

Single-Step Assembly of Multifunctional Poly(tannic acid)-Graphene Oxide Coating To Reduce Biofouling of Forward Osmosis Membranes.

Graphene oxide (GO) nanosheets have antibacterial properties that have been exploited as a biocidal agent used on desalination membrane surfaces in recent research. Nonetheless, improved strategies for efficient and stable attachment of GO nanosheets onto the membrane surface are still required for this idea to be commercially viable. To address this challenge, we adopted a novel, single-step surface modification approach using tannic acid cross-linked with polyethylene imine as a versatile platform to immobilize GO nanosheets to the surface of polyamide thin film composite forward osmosis (FO) membranes. An experimental design based on Taguchi's statistical method was applied to optimize the FO processing conditions in terms of water and reverse solute fluxes. Modified membranes were analyzed using water contact angle, adenosine triphosphate bioluminescence, total organic carbon, Fourier transform infrared spectroscopy, ζ potential, X-ray photoelectron spectroscopy, transmission electron microscopy, and atomic force microscopy. These results show that membranes were modified with a nanoscale (<10 nm), smooth, hydrophilic coating that, compared to pristine membranes, improved filtration and significantly mitigated biofouling by 33% due to its extraordinary, synergistic antibacterial properties (99.9%).

Physical characterization and in silico modeling of inulin polymer conformation during vaccine adjuvant particle formation.

This study combined physical data from synchrotron SAXS, FTIR and microscopy with in-silico molecular structure predictions and mathematical modeling to examine inulin adjuvant particle formation and structure. The results show that inulin polymer chains adopt swollen random coil in solution. As precipitation occurs from solution, interactions between the glucose end group of one chain and a fructose group of an adjacent chain help drive organized assembly, initially forming inulin ribbons with helical organization of the chains orthogonal to the long-axis of the ribbon. Subsequent aggregation of the ribbons results in the layered semicrystalline particles previously shown to act as potent vaccine adjuvants. γ-Inulin adjuvant particles consist of crystalline layers 8.5 nm thick comprising helically organized inulin chains orthogonal to the plane of the layer. These crystalline layers alternate with amorphous layers 2.4 nm thick, to give overall particle crystallinity of 78%.

Fine-Tuning the Surface of Forward Osmosis Membranes via Grafting Graphene Oxide: Performance Patterns and Biofouling Propensity.

Graphene oxide (GO) nanosheets were attached to the polyamide selective layer of thin film composite (TFC) forward osmosis (FO) membranes through a poly L-Lysine (PLL) intermediary using either layer-by-layer or hybrid (H) grafting strategies. Fourier transform infrared spectroscopy, zeta potential, and thermogravimetric analysis confirmed the successful attachment of GO/PLL, the surface modification enhancing both the hydrophilicity and smoothness of the membrane's surface demonstrated by water contact angle, atomic force microscopy, and transmission electron microscopy. The biofouling resistance of the FO membranes determined using an adenosine triphosphate bioluminescence test showed a 99% reduction in surviving bacteria for GO/PLL-H modified membranes compared to pristine membrane. This antibiofouling property of the GO/PLL-H modified membrane was reflected in reduced flux decline compared to all other samples when filtering brackish water under biofouling conditions. Further, the high density and tightly bound GO nanosheets using the hybrid modification reduced the reverse solute flux compared to the pristine, which reflects improved membrane selectivity. These results illustrate that the GO/PLL-H modification is a valuable addition to improve the performance of FO TFC membranes.

Inulin crystal initiation via a glucose-fructose cross-link of adjacent polymer chains: atomic force microscopy and static molecular modelling.

Semi-crystalline microparticles of inulin (MPI) have clinical utility as potent human vaccine adjuvants but their relevant surface structure and crystal assembly remain undefined. We show inulin crystal surfaces to resemble multi-layered, discoid radial spherulites resulting from very rapid formation of complex tertiary structures, implying directed crystal initiation. Physical and in silico molecular modelling of unit cells confirm steric feasibility of initiation by hydrogen-bonded cross-linking of terminal glucose to a fructose of another chain, mimicking bonding in sucrose crystals. A strong, chelate-like dual H-bond is proposed to compel the known antiparallel alignment of inulin chains. Such cross-linking would require one extra fructose per chain in the native inulin crystal, as observed. Completion of five H-bonded internal ring-domains would 'lock in' each new 6-fructose structural unit of each antiparallel helix pair to create a new isoform. All known properties of inulin isoforms follow readily from these concepts.

Inulin isoforms differ by repeated additions of one crystal unit cell.

Inulin isoforms, especially delta inulin, are important biologically as immune activators and clinically as vaccine adjuvants. In exploring action mechanisms, we previously found regular increments in thermal properties of the seven-member inulin isoform series that suggested regular additions of some energetic structural unit. Because the previous isolates carried additional longer chains that masked defining ranges, these were contrasted with new isoform isolates comprising only inulin chain lengths defining that isoform. The new series began with 19 fructose units per chain (alpha-1 inulin), increasing regularly by 6 fructose units per isoform. Thus the 'energetic unit' equates to 6 fructose residues per chain. All isoforms showed indistinguishable X-ray diffraction patterns that were also identical with known inulin crystals. We conclude that an 'energetic unit' equates to one helix turn of 6 fructose units per chain as found in one unit cell of the inulin crystal. Each isoform chain comprised progressively more helix turns plus one additional fructose and glucose residues per chain.

Chiral self-assembly of designed amphiphiles: influences on aggregate morphology.

A series of novel amphiphiles were designed for self-assembly into chiral morphologies, the amphiphiles consisting of a glutamic acid (Glu) headgroup connected through an 11-carbon alkoxy chain to a diphenyldiazenyl (Azo) group and terminated with a variable length alkyl chain (R-Azo-11-Glu, where R denotes the number of carbons in the distal chain). TEM imaging of amphiphile aggregates self-assembled from heated, methanolic, aqueous solution showed that chiral order, expressed as twisted ribbons, helical ribbons, and helically based nanotubes, increased progressively up to a distal chain length containing eight carbons, and then decreased with further increases in distal chain length. TEM and CD showed that the chiral aggregations of single enantiomers were influenced by the molecular chirality of the headgroup. However, the assembly of D,L-10-Azo-11-Glu into nanotubes demonstrated that chiral symmetry breaking effected by the azo group was also relevant to the chiral organization of the amphiphiles. The chiral order of aggregate morphologies was additionally affected by the temperature and solvent composition of assembly in a manner correlated to the mechanism driving assembly; i.e., D,L-10-Azo-11-Glu was sensitive to the temperature of assembly but less so to solvent composition, while L-14-Azo-11-Glu was sensitive to solvent composition and not to temperature. FTIR and UV-vis spectroscopic investigations into the organization of the head and azo groups, in chiral and achiral structures, illustrated that a balance of the influences of the hydrophilic and hydrophobic components on self-assembly was required for the optimization of the chiral organization of the self-assembled structures.

The polysaccharide inulin is characterized by an extensive series of periodic isoforms with varying biological actions.

In studying the molecular basis for the potent immune activity of previously described gamma and delta inulin particles and to assist in production of inulin adjuvants under Good Manufacturing Practice, we identified five new inulin isoforms, bringing the total to seven plus the amorphous form. These isoforms comprise the step-wise inulin developmental series amorphous → alpha-1 (AI-1) → alpha-2 (AI-2) → gamma (GI) → delta (DI) → zeta (ZI) → epsilon (EI) → omega (OI) in which each higher isoform can be made either by precipitating dissolved inulin or by direct conversion from its precursor, both cases using regularly increasing temperatures. At higher temperatures, the shorter inulin polymer chains are released from the particle and so the key difference between isoforms is that each higher isoform comprises longer polymer chains than its precursor. An increasing trend of degree of polymerization is confirmed by end-group analysis using (1)H nuclear magnetic resonance spectroscopy. Inulin isoforms were characterized by the critical temperatures of abrupt phase-shifts (solubilizations or precipitations) in water suspensions. Such (aqueous) "melting" or "freezing" points are diagnostic and occur in strikingly periodic steps reflecting quantal increases in noncovalent bonding strength and increments in average polymer lengths. The (dry) melting points as measured by modulated differential scanning calorimetry similarly increase in regular steps. We conclude that the isoforms differ in repeated increments of a precisely repeating structural element. Each isoform has a different spectrum of biological activities and we show the higher inulin isoforms to be more potent alternative complement pathway activators.

Chiral self-assembly of designed amphiphiles: optimization for nanotube formation.

Four amphiphiles with L-aspartic acid headgroups (Asp) and a diphenyldiazenyl group (Azo) contained within the hydrophobic tails were designed and synthesized for self-assembly into helically based nanotubes. The amphiphiles of the form R'-{4-[(4-alkylphenyl)diazenyl]phenoxy}alkanoyl-L-aspartic acid (where R' is 10 or 11) varied only in alkyl chain lengths either side of the azo group, having 4, 7, or 10 carbon distal chains and 10 or 11 carbon proximal chains (R-Azo-R'-Asp, where R denotes the number of carbons in the distal chain and R' denotes the number of carbons in the proximal chain). Despite the molecular similarities, distinct differences were identified in the chiral order of the structures self-assembled from hot methanolic aqueous solutions using microscopy and spectroscopic analyses. This was reflected in dominant thermodynamic aggregate morphologies that ranged from amorphous material for 10-Azo-10-Asp, through twisted ribbons (196 ± 49 nm pitch) for 7-Azo-11-Asp, to the desired helically based nanotubes for 4- and 7-Azo-10-Asp (81 ± 11 and 76 ± 6 nm diameters, respectively). Another key variable in the self-assembly of the amphiphiles was the use of a second method to precipitate aggregates from solution at room temperature. This method enabled the isolation of thermodynamically unstable and key transitional structures. Helical ribbons were precursor structures to the nanotubes formed from 4- and 7-Azo-10-Asp as well as the wide, flattened nanotube structures (587 ± 85 nm width) found for 4-Azo-10-Asp. Overall, the results highlighted the interplay of influence of the headgroup and the hydrophobic tail on self-assembly, providing a basis for future rational design of self-assembling amphiphiles.