Etherified pullulan-polyethylenimine based nanoscaffolds improved chemosensitivity of erlotinib on hypoxic cancer cells.

The current research endeavor aimed to accomplish hypoxia-responsive polyethyleneimine-conjugated carboxymethyl pullulan-based co-polymer (CMP-HA-NI-PEI-NBA) bearing nitroaromatic subunits to efficiently deliver erlotinib (ERL) to reverse its hypoxia-induced resistance in cancer cells. As compared to a control co-polymer (CMP-HA-MI-PEI-BA) devoid of hypoxia-sensitive moieties, this scaffold demonstrated a hypochromic shift in the UV spectra and rapid dismantling of its self-assembled architecture upon exposure to simulated hypoxic condition. The hypoxia-responsive co-polymer encapsulated ERL with desirable loading capacity (DEE, 63.05 ± 2.59%), causing attenuated drug crystallinity. The drug release rate of the scaffold under reducing condition was faster relative to that of non-reducing environment. Their cellular uptake occurred through an energy-dependent endocytic process, which could exploit its caveolae/lipid raft-mediated internalization pathway. The ERL-loaded scaffolds more efficiently induced apoptosis and suppressed the proliferation of drug-resistant hypoxic HeLa cells than the pristine ERL. Hence, this study presented a promising drug delivery nanoplatform to overcome hypoxia-evoked ERL resistance.

Pullulan films loading saffron extract encapsulated in nanoliposomes; preparation and characterization.

Nanoencapsulation of saffron extract (SE) components into the rapeseed lecithin nanoliposomes were performed by sonication of their aqueous dispersions as a green process. Dynamic light scattering (DLS) results exhibited that empty and SE loaded nanoliposomes (SENL) had average sizes in range of 118-138 nm, negative zeta potentials (-32.0 to -46.8 mV) and polydispersity index (PDI) less than 0.3 during storage for 28 days at 4 °C. Encapsulation efficiency of crocin was approximately 30%. The 70% of crocin released from SENLs within 5 h in PBS solution. Pullulan-based films were fabricated by incorporation of empty and SE loaded nanoliposomes into pullulan solution through casting method. The mechanical resistance and thermal stability of the films reduced by addition of nanoliposomes. FTIR and thermal characterizations indicated that SE was successfully encapsulated in the nanoliposomes and film matrix with high thermal stability. Incorporation of nanoliposomes enhanced the oxygen barrier properties of the films, while it didn't significantly affect the water vapor permeability (WVP) of the films. The obtained edible films or coatings can provide additional benefits due to unique flavor and color of saffron. In addition, the utilization of SE, can provide benefits for health-allegation from SE antioxidant capacity.

ZnI2-Directed Stereocontrolled α-Glucosylation.

Here we report a glucosylation strategy mediated by ZnI2, a cheap and mild Lewis acid, for the highly stereoselective construction of 1,2-cis-O-glycosidic linkages using easily accessible and common 4,6-O-tethered glucosyl donors. The versatility and effectiveness of the α-glucosylation strategy were demonstrated successfully with various acceptors, including complex alcohols. This approach demonstrates the feasibility of the modular synthesis of various α-glucans with both linear and branched backbone structures.

High-Solids, Solvent-Free Modification of Engineered Polysaccharides.

The nature-identical engineered polysaccharide α-(1,3) glucan, produced by the enzymatic polymerization of sucrose, was chemically modified by acylation with succinic anhydride. This modification reaction was initially performed at the micro scale in a TGA reactor to access a range of reaction conditions and to study the mechanism of the reaction. Subsequently, the best performing conditions were reproduced at the larger laboratory scale. The reaction products were characterized via coupled TGA/DSC analysis, FT-IR spectroscopy, solution viscosity and pH determination. The acylation path resulted in partially modifying the polysaccharide by altering its behavior in terms of thermal properties and solubility. The acylation in a solvent-free approach was found promising for the development of novel, potentially melt-processable and fully bio-based and biodegradable ester compounds.

Chitotriazolan (poly(ß(1-4)-2-(1H-1,2,3-triazol-1-yl)-2-deoxy-d-glucose)) derivatives: Synthesis, characterization, and evaluation of antibacterial activity.

Here we describe the first synthesis of a new type of polysaccharides derived from chitosan. In these structures, the 2-amino group on the pyranose ring was quantitively replaced by an aromatic 1,2,3-triazole moiety. The 2-amino group of chitosan and di-TBDMS chitosan was converted into an azide by diazo transfer reaction. The chitosan azide and TBDMS-chitosan azide were poorly soluble but could be fully converted to triazoles by "copper-catalysed Huisgen cycloaddition" in DMF or DMSO. The reaction could be done with different alkynes but derivatives lacking cationic or anionic groups were poorly soluble or insoluble in tested aqueous and organic solvents. Derivatives with N,N-dimethylaminomethyl, N,N,N-trimethylammoniummethyl, sulfonmethyl, and phosphomethyl groups linked to the 4-position of the triazole moiety were soluble in water at neutral or basic conditions and could be analyzed by 1H, 13C APT, COSY, and HSQC NMR. The quaternized cationic chitotriazolan's had high activity against S. aureus and E. coli, whereas the anionic chitotriazolan's lacked activity.

A novel maltoheptaose-based sugar ester having excellent emulsifying properties and optimization of its lipase-catalyzed synthesis.

A novel maltoheptaose-palmitate ester (G7-PA) was synthesized and investigated for emulsion properties. First of all, the optimal conditions for lipase-catalyzed G7-PA synthesis, which were 0.2 of the G7/PA molar ratio, 33.5 U of immobilized CALB per 1 g of PA in 10% DMSO, were determined by response surface methodology. G7-PA was compared with the commercial sucrose-PA (S-PA) in terms of emulsion-forming ability and stability at extreme conditions. At the 0.1% surfactant concentration, G7-PA emulsion exhibited a droplet distribution similar to the 0.2% surfactant condition, while S-PA emulsion was quickly destabilized. G7-PA showed better emulsifying properties than the S-PA at the acidic condition (pH 3). Flocculation and phase separation was observed at the S-PA emulsion, but the G7-PA emulsion was stable for 7-day. In thermostability tests, G7-PA and S-PA both were stable up to the boiling temperature. Conclusively, G7-PA exhibits excellent properties as a biosurfactant in O/W emulsion compared with S-PA.

Comparative Studies on Regioselectivity of α- and ß-Linked Glucan Tosylation.

Alpha- and beta-linked 1,3-glucans have been subjected to conversion with p-toluenesulfonic acid (tosyl) chloride and triethylamine under homogeneous reaction conditions in N,N-dimethyl acetamide/LiCl. Samples with a degree of substitution of tosyl groups (DSTs) of up to 1.91 were prepared by applying 5 mol reagent per mole repeating unit. Hence, the reactivity of α-1,3-glucan is comparable with cellulose and starch, while the ß-1,3-linked glucan curdlan is less reactive. The samples dissolve in aprotic dipolar media independent of the DSTs and possess a solubility in less polar solvents that depends on the DSTs. NMR studies on the tosyl glucans and of the peracylated derivatives showed a preferred tosylation of position 2 of the repeating unit. However, the selectivity is less pronounced compared with starch. It could be concluded that the α-configurated glycosidic bond directs tosyl groups towards position 2.

Cationic hydrogels prepared from regioselectively azidated (1→3)-α-d-glucan via crosslinking and amination: Physical and adsorption properties.

Cationic hydrogels with amino groups were successfully prepared using (1→3)-α-d-glucan synthesized by glucosyltransferase J (GtfJ) cloned from Streptococcus salivarius through a three-step reaction: (i) Azido groups were regioselectively introduced at the C6 position of (1→3)-α-d-glucan by a bromination-azidation process (degree of substitution 0.94), (ii) Azido groups were partially crosslinked with 1,8-nonadiyne via a copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction, (iii) Azido groups that were unused for crosslinking were reduced to amino groups by sodium borohydride (NaBH4). The introduction of amino groups was confirmed quantitatively and qualitatively by elemental, Fourier transform infrared (FT-IR), and nuclear magnetic resonance (NMR) analyses. These cationic hydrogels showed a specific adsorption ability for bovine serum albumin (BSA) over a wide pH range of 4.5-8.0 due to their high pH values at the point of zero charge (pHpzc 8.80-8.92).

Cationised dextran and pullulan modified with diethyl aminoethyl methacrylate for gene delivery in cancer cells.

This work describes the synthesis and characterisation of cationised dextran and pullulan modified with diethyl aminoethyl methacrylate (DEAEM) for gene delivery in cancer cells. To dextran and pullulan, PEI was conjugated to impart cationicity. These cationised polysaccharides were then modified with DEAEM monomer via Michael addition reaction and synthesised four different derivatives viz DPD I, DPD II, PPD I and PPD II. These vectors form nanocomplexes with DNA exhibiting positive zeta potential. These nanoplexes are cytocompatible in C6, HeLa and L929 cells. Transfection efficiency of these vectors was evaluated using p53 plasmid which demonstrated good transfection in cancer cells (C6 and HeLa) alone. Biodistribution studies of DPD II and PPD II in BALB/c mice shows its tendency to accumulate in liver tissue and not in any vital organs like brain, lungs and heart. In addition, these derivatives also exhibit good renal clearance.

Reagent controlled stereoselective synthesis of teichoic acid α-(1,2)-glucans.

The stereoselective construction of 1,2-cis-glycosidic linkages is key in the assembly of biologically relevant glycans, but remains a synthetic challenge. Reagent-controlled glycosylation methodologies, in which external nucleophiles are employed to modulate the reactivity of the glycosylation system, have become powerful means for the construction of 1,2-cis-glycosidic linkages. Here we establish that nucleophilic additives can support the construction of α-1,2-glucans, and apply our findings in the construction of a d-alanine kojibiose functionalized glycerol phosphate teichoic acid fragment. This latter molecule can be found in the cell wall of the opportunistic Gram-positive bacterium, Enterococcus faecalis and represents a structural element that can possibly be used in the development of therapeutic vaccines and diagnostic tools.

Safe-by-Design of Glucan Nanoparticles: Size Matters When Assessing the Immunotoxicity.

Glucan (from Alcaligenes faecalis) is a polymer composed of ß-1,3-linked glucose residues, and it has been addressed in different medical fields, namely in nanotechnology, as a vaccine or a drug delivery system. However, due to their small size, nanomaterials may present new risks and uncertainties. Thus, this work aims to describe the production of glucan nanoparticles (NPs) with two different sizes, and to evaluate the influence of the NPs size on immunotoxicity. Results showed that, immediately after production, glucan NPs presented average sizes of 129.7 ± 2.5 and 355.4 ± 41.0 nm. Glucan NPs of 130 nm presented greater ability to decrease human peripheral blood mononuclear cells and macrophage viability and to induce reactive oxygen species production than glucan NPs of 355 nm. Both NP sizes caused hemolysis and induced a higher metabolic activity in lymphocytes, although the concentration required to observe such effect was lower for the 130 nm glucan NPs. Regarding pro-inflammatory cytokines, only the larger glucan NPs (355 nm) were able to induce the secretion of IL-6 and TNF-α, probably due to their recognition by dectin-1. This higher immunomodulatory effect of the larger NPs was also observed in its ability to stimulate the production of nitric oxide (NO) and IL-1ß. On the contrary, a small amount of Glu 130 NPs inhibited NO production. In conclusion, on the safe-by-design of glucan NPs, the size of the particles should be an important critical quality attribute to guarantee the safety and effectiveness of the nanomedicine.

Solubilization of poorly water-soluble bioactive molecules in neutral aqueous media by complexation with renatured ß-1,3-1,6-glucan nanoparticles.

The design of scaffolds for solubilizing/dispersing poorly water-soluble bioactive molecules in neutral aqueous media is a major challenge of functional food, pharmaceuticals, and cosmetics development, as highlighted by the plethora of corresponding solubilization/dispersion strategies. Herein, renatured ß-1,3-1,6-glucan (r-glucan) nanoparticles prepared by neutralization of alkali-denatured ß-1,3-1,6-glucan and subsequent centrifugation are used as a host to disperse water-insoluble bioactive molecules (curcumin, all-trans-retinoic acid, and rebamipide) by simple mixing of host and guest solutions. Curcumin in the r-glucan cavity is found to be stacked in the form of J-aggregates and twisted along the helix, and is demonstrated to be retained for significantly longer than curcumin in the corresponding γ-cyclodextrin (γ-CD) complex. Specifically, curcumin incorporated in γ-CD is released within 5.5 hours, whereas that in the r-glucan complex is released very slowly, with 12% of curcumin in the latter complex retained after 31-day incubation at 37°C. Thus, inclusion protocol simplicity and slow release ability make r-glucan nanoparticles a potential carrier scaffold for various applications.

Synthesis of cationic quaternized pullulan derivatives for miRNA delivery.

Pullulan is a natural polysaccharide of potential interest for biomedical applications due to its non-toxic, non-immunogenic and biodegradable properties. The aim of this work was to synthesize cationic pullulan derivatives able to form complexes with microRNAs (miRNAs) driven by electrostatic interaction (polyplexes). Quaternized ammonium groups were linked to pullulan backbone by adding the reactive glycidyltrimethylammonium chloride (GTMAC). The presence of these cationic groups within the pullulan was confirmed by elemental analysis, Fourier-transform infrared spectroscopy (FTIR) and proton nuclear magnetic resonance (1H NMR). The alkylated pullulan was able to interact with miRNA and form stable polyplexes that were characterized regarding size, zeta potential and morphology. The presence of miRNA was confirmed by agarose gel electrophoresis and UV spectrophotometry. In vitro tests on human umbilical vein endothelial cells did not show any cytotoxicity after 1 day of incubation with nanosized polyplexes up to 200 µg/mL. QA-pullulan was able to promote miRNA delivery inside cells as demonstrated by fluorescence microscopy images of labelled miRNA. In conclusion, the formation of polyplexes using cationic derivatives of pullulan with miRNA provided an easy and versatile method for polysaccharide nanoparticle production in aqueous media and could be a new promising platform for gene delivery.

Simple and dual cross-linked chitosan millicapsules as a particulate support for cell culture.

The chitosan hydrochloride (Cs·HCl) was obtained as a polymer soluble in physiological solutions to be used as potential support for safely cell culture or cell encapsulation. Viability tests showed that concentrations between 0.16 and 5 mg/mL of Cs·HCl were not toxic for the HEK293 cells. In parallel, aldehyde-functionalized pullulan (Pul-CHO) was synthesized as the macromolecular cross-linker. Cs·HCl was dissolved in 0.9% NaCl and injected (INJECTOMAT SEP 21S PLUS) through a needle to obtain small droplets in a sodium tripolyphosphate solution in the absence and presence of 0.1% (w/v) Pul-CHO. Simple and dual cross-linked millicapsules were obtained with pore size ranging from 50 µm to 5 µm, respectively. FITC-Dextran with molecular weights of 4000 and 70,000 g/mol was encapsulated during microcapsule synthesis as macromolecular models to check the permeability of Cs millicapsules. The results show that FITC-Dextran 4000 and 70,000 diffuses quickly from simple cross-linked millicapsules while dual cross-linked millicapsules release slowly both FITC-dextrans. Microscopy experiments show that HEK 293 cells adhere to the surface of millicapsules. Taken together the data reveal that Cs millicapsules allow the cell growth on their surface, and thus, they offer new perspectives for cell encapsulation strategy.

Synthesis of amphiphilic pullulan-graft-poly(ε-caprolactone) via click chemistry.

Chemical modification of natural polymers has been commonly employed for the development of new bio-based materials, aiming at adjusting specific properties such as solubility, biodegradability, thermal stability and mechanical behavior. Among all natural polymers, polysaccharides are promising materials, in which biodegradability, processability and bioreactivity make them suitable for biomedical applications. In this context, this work describes the synthesis and characterization of a novel amphiphilic pullulan-g-poly(ε-caprolactone) (Pull-g-PCL) graft copolymer. In a first step, pullulan was chemically modified with 2-bromopropionyl bromide to obtain bromo-functionalized pullulan (PullBr). Then, this precursor was modified with sodium azide, leading to azide pullulan (PullN3). In parallel, propargyl-terminated poly(ε-caprolactone) was prepared via ring-opening polymerization (ROP). These preliminary steps involved the synthesis of azide and alkyne compounds, capable of being linked together via alkyne-azide cycloaddition reaction catalyzed by copper (Cu (I)), which leads to Pull-g-PCL. The chemical structures of the polymers were assessed by Proton Nuclear Magnetic Resonance (1H NMR) and Fourier Transform Infrared (FTIR).

Paramylon and synthesis of its ionic derivatives: Applications as pharmaceutical tablet disintegrants and as colloid flocculants.

Paramylon, a high molecular weight polysaccharide, is a linear and unbranched (1 → 3)-ß-d-glucan. Despite its numerous biological benefits, the poor aqueous solubility of crystalline paramylon is a drawback that has hampered some of its applications. In an effort to make this biomaterial amenable to practical uses, cationic and anionic paramylon derivatives were obtained. The degrees of substitution of both products were determined. The products were characterized by FT-IR spectrocopy, ESI mass spectrometry, 1H, 13C and 1H-13C NMR and SEM microscopy. These techniques confirmed the success of the substitution reactions. 1H NMR analysis was used to develop alternative methods for an approximate estimation of the degree of substitution. 1H-13C HSQC NMR spectra were assigned for both derivatives. New applications of native, cationic and anionic paramylon were found. Native paramylon showed similar performance as pharmaceutical tablet disintegrant than sodium croscarmellose. Cationic paramylon behavior as colloid flocculant was comparable with commercial cationic polyacrylamides. The anionic derivative could eventually be used in the formulation of matrix controlled release systems or as a suspending agent.

Preparation, properties, and structural characterization of ß-glucan/pullulan blend films.

This study investigates the physico-mechanical and structural properties of ß-glucan (BG)/pullulan (PUL) composite edible films successfully prepared with 0-0.3 g of BG. Results demonstrated that BG addition significantly increases the elongation at break (p < 0.05), tensile strength, and water dissolution time of the resulting films. The transparency of the 0.2PUL:0.1BG film and the oxygen barrier property of the 0.15PUL:0.15BG film decreased remarkably compared with those of the plain films (0.3PUL:0BG and 0PUL:0.3BG) and other composite films (p < 0.05). FTIR indicated hydrogen bonding interactions between PUL and BG molecules, and microstructural observations showed that aggregated BG is homogeneously dispersed in the PUL continuous matrix. Among the films tested, the thermal stability of the 0.15PUL:0.15BG film was the best. A PUL:BG mixing ratio of 0.15:0.15 is thus suggested to provide the best film properties. This research offers an alternative method to improve PUL-based edible films.

Enzymatic synthesis and characterization of maltoheptaose-based sugar esters.

In this study, maltoheptaose (G7)-based sugar esters were synthesized from maltoheptaose and fatty acids (C10-C16) using a commercial lipase. With the exception of dimethyl sulfoxide (DMSO; 76.4%, w/v), G7 showed only limited solubility in organic solvents. Among the fatty acids, palmitic acid (PA) was the best substrate for G7-based ester formation. G7-PA ester was successfully synthesized as the monoester structure exclusively in 10% DMSO of t-butanol with a 22% conversion yield. NMR and enzymatic analyses of the purified monoester product revealed that the ester bond in the G7 was located at C-6 of the glucose at the reducing end. The G7-PA monoester showed the melting temperature at 56.3 °C that was 6.5 °C lower than that of the free PA and exhibited a different endothermic pattern from the free G7. The G7-PA monoester exhibited excellent emulsifier potential with more even droplet size distribution compared with the commercial sucrose esters for an oil-in-water emulsion system.

Synthesis of highly branched α-glucans with different structures using GH13 and GH57 glycogen branching enzymes.

Glycogen branching enzymes (GBEs) convert starch into branched α-glucan polymers. To explore if the amylose content of substrates effects the structure of the branched α-glucans, mixtures of amylose and amylopectin were converted by four thermophilic GBEs. The degree of branching and molecular weight of the products increased with an increasing percentage of amylose with the GH57 GBEs of Thermus thermophilus and Thermococcus kodakarensis, and the GH13 GBEs of Rhodothermus marinus and Petrotoga mobilis. The only exception is that the degree of branching of the Petrotoga mobilis GBE products is not influenced by the amylose content. A second difference is the relatively high hydrolytic activity of two GH57 GBEs, while the two GH13 GBEs have almost no hydrolytic activity. Moreover, the two GH13 GBEs synthesize branched α-glucans with a narrow molecular weight distribution, while the two GH57 GBEs products consist of two or three molecular weight fractions.

Pullulan dialdehyde crosslinked gelatin hydrogels with high strength for biomedical applications.

Herein the construction of a strong gelatin hydrogel is presented by using pullulan dialdehyde (PDA) as a macromolecular crosslinker. The resultant PDA crosslinked gelatin hydrogels (G-PDA) exhibit extremely high mechanical strength, manifested in the achieved optimal compressive stress of 5.80 MPa at 80% strain, which is up to 152 times higher than pure gelatin hydrogel. The G-PDA were characterized by Fourier transform infrared (FTIR) spectroscopy and scanning electron microscopy (SEM). The extent of crosslinking was determined by ninhydrin assay. The results suggested that the synergistic effect of dual-crosslinking, which is composed of short- and long-range covalent crosslinking and thermoreversible physical crosslinking, may played a key role in enhancing the load-bearing capacity of ensuing hydrogels. The swelling and enzymatic degradation of G-PDA are gradually limited with increasing PDA concentration. The result from MTT assay demonstrated that G-PDA is non-cytotoxic against MC3T3 cells, regardless of the concentrations of PDA.