Chitosan-Based Particulate Carriers: Structure, Production and Corresponding Controlled Release.

The state of the art in the use of chitosan (CS) for preparing particulate carriers for drug delivery applications is reviewed. After evidencing the scientific and commercial potentials of CS, the links between targeted controlled activity, the preparation process and the kinetics of release are detailed, focusing on two types of particulate carriers: matrix particles and capsules. More precisely, the relationship between the size/structure of CS-based particles as multifunctional delivery systems and drug release kinetics (models) is emphasized. The preparation method and conditions greatly influence particle structure and size, which affect release properties. Various techniques available for characterizing particle structural properties and size distribution are reviewed. CS particulate carriers with different structures can achieve various release patterns, including zero-order, multi-pulsed, and pulse-triggered. Mathematical models have an unavoidable role in understanding release mechanisms and their interrelationships. Moreover, models help identify the key structural characteristics, thus saving experimental time. Furthermore, by investigating the close relation between preparation process parameters and particulate structural characteristics as well as their effect on release properties, a novel "on-demand" strategy for the design of drug delivery devices may be developed. This reverse strategy involves designing the production process and the related particles' structure based on the targeted release pattern.

Effect of Silica Nanoparticles in Xanthan Gum Solutions: Evolution of Viscosity over Time.

The effect of silica nanoparticles (NP-SiO2) in xanthan gum (XG) solutions was investigated through the analysis of viscosity profiles. First, hydrocolloid XG solutions and hydrophilic NP-SiO2 suspensions were characterized individually through rheological measurements, with and without salt (NaCl). Then, nanofluids composed of XG and NP-SiO2 dispersed in water and brine were studied through two different aging tests. The addition of nanoparticles was shown to produce a slight effect on the viscosity of the fresh fluids (initial time), while a more remarkable effect was observed over time. In particular, it appears that the presence of NP-SiO2 stabilizes the polymer solution by maintaining its viscosity level in time, due to a delay in the movement of the molecule. Finally, characterization techniques such as confocal microscopy, capillary rheometry, and Zeta potential were implemented to analyze the XG/NP-SiO2 interaction. Intrinsic viscosity and relative viscosity were calculated to understand the molecular interactions. The presence of NP-SiO2 increases the hydrodynamic radius of the polymer, indicating attractive forces between these two components. Furthermore, dispersion of the nanoparticles in the polymeric solutions leads to aggregates of an average size smaller than 300 nm with a good colloidal stability due to the electrostatic attraction between XG and NP-SIO2. This study proves the existence of interactions between XG and NP-SiO2 in solution.

Chemical modification of waxy maize starch by esterification with saturated fatty acid chlorides: Synthesis, physicochemical and emulsifying properties.

In the current study, the physicochemical and emulsifying properties of modified waxy maize starch obtained through a new environmentally friendly method of esterification were evaluated. The starch modification was carried out in NaOH solution with different levels of octanoyl, myristoyl, and stearoyl chlorides. Increasing the fatty acid chlorides concentration led to the degree of substitution increment, while reaction efficiency and yield decreased. Based on fourier transform infrared spectroscopy results, the presence of two new bands of carbonyl (1740-1750 cm-1) and carboxyl (1570 cm-1) groups in the ester bond confirmed the successful starch esterification process. The level of 0.1 mL fatty acid chlorides/g of starch demonstrated the highest emulsifying properties. Upon esterification, the crystalline structure of amylopectin was destroyed, indicating no gelatinization features. Therefore, using the fatty acid chlorides in an alkaline condition could be suggested as a feasible way to modify waxy maize starch toward hydrophobicity increment with desirable properties.

Chitosan/carboxymethylcellulose-stabilized poly(lactide-co-glycolide) particles as bio-based drug delivery carriers.

Poly(lactide-co-glycolide) (PLGA) colloidal particles stabilized by complexes of two oppositely charged polysaccharides, chitosan (cationic, CS) and sodium carboxymethylcellulose (anionic, NaCMC), were fabricated. Dichloromethane containing dissolved PLGA was first emulsified in an aqueous phase containing mixtures of CS and NaCMC. Evaporation of dichloromethane from the resulting emulsion led to CS/NaCMC-covered-PLGA particles. CS and NaCMC contents affected the short-term stability of PLGA particles and also their intrinsic characteristics. The particles displayed pH-dependent characteristic. Zeta potential varied from +54 to -50 mV when pH was varied from 3 to 10. CS/NaCMC-covered-PLGA particles showed colloidal stability, over a wider pH range as compared to CS-covered-PLGA particles. Curcumin, a model hydrophobic drug, was encapsulated into the particles up to 10 wt% of PLGA. The CS/NaCMC-covered-PLGA particles loaded with curcumin showed delayed release in mildly acidic conditions and faster release in neutral and basic conditions. Cytotoxicity experiments were carried out with human colorectal carcinoma cells.

Synthesis of Thermoresponsive Copolymers with Tunable UCST-Type Phase Transition Using Aqueous Photo-RAFT Polymerization.

Currently, the phase transition of aqueous binary systems containing thermoresponsive (co)polymers, and exhibiting upper critical solution temperature (UCST), is exclusively investigated in dilute solutions, which can limit the knowledge of their UCST-type phase transition. Herein, a photo-RAFT polymerization approach, using acrylamide (AAm) and acrylonitrile (AN) as monomer models, is used to prepare well-controlled poly(AAm-co-AN) copolymers "in situ" in highly concentrated dispersions (60 wt%). The impact of the copolymer concentration and the chemical composition (as a variation of AN fraction in the copolymers) on the cloud point temperature (TCP ) are investigated using turbidity measurements. Importantly, the results show that upon increasing the polymer concentration, a sharp increase of TCP up to a maximum point is observed, representing the UCST, before the decrease of TCP at higher polymer concentrations. Finally, a model equation is developed to fit the UCST values of poly(AAm-co-AN), which can be useful to design new poly(AAm-co-AN) copolymers with a desired UCST for a specific application.

Dextran-covered pH-sensitive oily core nanocapsules produced by interfacial Reversible Addition-Fragmentation chain transfer miniemulsion polymerization.

Aiming to prepare oily core pH-sensitive nanocapsules (NCs) for anticancer drugs delivery, the use of a dextran-based transurf (DexN3-τCTAγ) as both stabilizer and macromolecular chain transfer agent in methyl methacrylate/2-(diethylamino)ethyl methacrylate (MMA/DEAEMA) miniemulsion copolymerization was investigated. NCs of about 195 nm with an oily-core of Miglyol 810 (M810) and a dextran coverage covalently linked to the poly(MMA-co-DEAEMA) intern shell have been obtained. Compared to the non-sensitive PMMA-based NCs (prepared in a similar way), these novel objects were shown to swell in acidic media and to trigger Coumarin 1 release in physiological relevant pH range. As a starting point of NCs biological effects, cytotoxicity and NCs-proteins interactions studies were performed with both PMMA and poly(MMA-co-DEAEMA)-based NCs. Finally, free azide functions from dextran-based coverage were successfully exploited to attach fluorescent model dyes to NCs surface. The overall results suggest that this novel NCs platform could be potentially used as drug nanocarriers for intravenous injection.

Exploring the Contribution of Two Phosphorus-Based Groups to Polymer Flammability via Pyrolysis-Combustion Flow Calorimetry.

From a set of around 100 phosphorus-containing polymers tested in pyrolysis-combustion flow calorimetry, the contributions to flammability of two phosphorus-containing pendant groups (called 9,10-dihydro-9-oxa-10-phosphaphenanthrene-10-oxide (DOPO) and PO3) were calculated using an advanced method previously proposed and validated. The flammability properties include total heat release (THR) and heat release capacity (HRC) measured in standard conditions, i.e., anaerobic pyrolysis and complete combustion. The calculated contributions are in good agreement with the main modes of action of both phosphorus groups, i.e., flame inhibition for DOPO and char promotion for PO3. Moreover, the results provide first conclusions about the cooperative interaction between phosphorus and nitrogen, as well as the influence of the architecture of tested co-polymers.

First multi-reactive polysaccharide-based transurf to produce potentially biocompatible dextran-covered nanocapsules.

A multi-reactive polysaccharide-based transurf (acting both as macro-Chain Transfer Agent and stabilizer) was used to confine RAFT polymerization of methyl methacrylate (MMA) at the oil/water (o/w) miniemulsion interface. Dithiobenzoate groups and hydrophobic aliphatic side chains were introduced onto dextran, conferring it both transfer agent properties and ability to stabilize direct miniemulsion of MMA in the presence of a biocompatible oil, used as co-stabilizer. Because of their amphiphilic character, transurfs were initially adsorbed at the (o/w) interface and their reactive sites mediated RAFT polymerization via the R-group approach. PMMA-grafted dextran glycopolymers were consequently produced at the o/w interface, thus leading to dextran coverage/PMMA shell/oily core nanocapsules (NCs) as evidenced by Cryo-TEM analyses. The influence of dextran-based transurf chemistry and oil amount on MMA RAFT polymerization control was investigated. Positive preliminary results on NCs cytotoxicity suggest the potential of these objects for biomedical applications.

Encapsulation of monomyristin into polymeric nanoparticles improved its in vitro antiproliferative activity against cervical cancer cells.

The cytotoxicity of monomyristin (MM), a monoacylglycerol, was investigated against cervical cancer cells (HeLa) and two normal cells (Vero and endometrial epithelial cells). MM exhibited cytotoxicity specifically to HeLa cells and not against normal cells except at the highest investigated doses (> 500 µg/mL). MM was showed to increase apoptotic dead cells by intrinsic mitochondrial pathway. To overcome the poor water solubility of MM and increase its efficacy against HeLa cells, MM was encapsulated into dextran-covered polylactide (PLA) nanoparticles (NPs). NPs comprised a PLA core which encapsulated MM and a superficial layer of dextran loops which was used for conjugating a protein, transferrin (Tf), known to be overexpressed on cancer cells' surface. Encapsulation of MM into NPs increased its cytotoxicity against HeLa cells at lower doses of MM than free MM. Additionally, the presence of conjugated Tf further increased the cytotoxicity of MM against HeLa cells as compared to non-conjugated NPs. Remarkably, both conjugated and non-conjugated MM loaded NPs were safe to normal cells (Vero and endometrial).

Stability of a biodegradable microcarrier surface: physically adsorbed versus chemically linked shells.

Mesenchymal stem cells (MSCs) have gained increasing interest for tissue engineering and cellular therapy. MSC expansion on microcarriers (MCs) in stirred bioreactors has emerged as an attractive method for their scaled up production. Some MCs have been developed based on polyesters as a hydrophobic biodegradable core. However, most of these MCs are formulated by an emulsion/organic solvent evaporation (E/E) process using poly(vinyl alcohol) as a shell steric stabilizer, which is biocompatible but not degradable in vivo. Moreover, in most of these MCs, the polymer shell is only physically adsorbed at the particle surface. To the best of our knowledge, no study deals with the stability of such a shell when the MCs are in contact with competitive surfactants or with proteins contained in the culture medium. In this study, fully in vivo bioresorbable dextran-covered polylactide-based MCs were formulated using an E/E process, which allowed to control their surface chemistry. Different dextran derivatives with alkyne or ammonium groups were firstly synthesised. Then, on the one hand, some MCs (non-clicked MCs) were formulated with a physically adsorbed polysaccharide shell onto the core. On the other hand, the polysaccharide shell was linked to the core via in situ CuAAC click-chemistry carried out during the E/E process (clicked MCs). The stability of such coverage was first studied in the presence of competitive surfactants (sodium dodecyl sulfate-SDS, or proteins contained in the culture medium) using nanoparticles (NPs) exhibiting the same chemical composition (core/shell) as MCs. The results revealed the total desorption of the dextran shell for non-clicked NPs after treatment with SDS or the culture medium, while this shell desorption was greatly decreased for clicked NPs. A qualitative study of this shell stability was finally carried out on MCs formulated using a new fluorescent dextran-based surfactant. The results were in agreement with those observed for NPs, and showed that non-clicked MCs are characterized by poor shell stability in contact with a competitive surfactant, which could be quite an issue during MSC expansion. In contrast, clicked MCs possess better shell stability, which allow a better control of the MC surface chemistry, especially during cell culture.

Nanoparticulate delivery systems for alkyl gallates: Influence of the elaboration process on particle characteristics, drug encapsulation and in-vitro release.

PLA nanoparticles loaded with n-alkyl gallates (AGs) were prepared either by nanoprecipitation (NP) or by O/W emulsion/solvent evaporation (E/SE). A nonionic hydrophobically modified polysaccharide was used for surface coverage and for ensuring colloidal stability. Different parameters were systematically assessed to enhance the drug incorporation, with the aim of obtaining monomodal and narrow particle size distributions. The nanoparticles were characterized by 1H NMR, transmission electron microscopy (TEM) and laser light scattering granulometry. The colloidal stability of suspensions was evaluated after incubation in NaCl solutions and was maintained up to 1M NaCl. The mean particle diameter and the width of size distribution were found very similar for both processes (slightly lower diameters when using E/SE) with various drug loadings. The amount of encapsulated AG by E/SE was about twice that encapsulated by NP. The in-vitro release of AG was evaluated under sink conditions and no burst effect was observed. Release curves were successfully modeled using the Fick diffusion model with a constant diffusion coefficient and assuming non-swellable particles. Diffusion coefficients of AG loaded in nanoparticles prepared by NP were higher than those found in nanoparticles elaborated by E/SE.

Amphiphilic Polysaccharides Acting both as Stabilizers and Surface Modifiers during Emulsification in Microfluidic Flow-Focusing Junction.

A continuous emulsion/solvent diffusion process was designed for the preparation of polysaccharide-covered poly(d,l-lactide) (PLA) microparticles. The emulsification step was carried out in a flow-focusing junction where ethyl acetate containing dissolved PLA was dispersed into an aqueous solution of hydrophobically modified dextran. It was demonstrated that poly(dimethylsiloxane) devices could be used for oil-in-water emulsion preparation provided that the microfluidic devices were preconditioned by simply circulating the aqueous phase containing the amphiphilic polysaccharide during a sufficient time (30 h). The adsorption of the polymers at the surface of the channel walls permitted the wetting by the aqueous phase with a hydrophilic character maintained at least throughout 2 months. The preconditioning time was significantly reduced by pretreating the microfluidic device with piranha solution and KOH solution during 15 min each before the circulation of the aqueous solution of dextran derivative. Dextran-covered PLA microparticle aqueous suspensions were produced with well-controlled size distribution. The suspensions could be lyophilized and reconstituted by retrieving the initial size distribution without adding any cryoprotectant. The reported procedure was used for preparing octyl gallate-loaded PLA microparticles.

Dilational rheology of oil/water interfaces covered by amphiphilic polysaccharides derived from dextran.

This work studied the adsorption at dodecane/water interface of amphiphilic polysaccharides derived from dextran (a nonionic bacterial polysaccharide) by random attachment of phenoxy groups along the chains (between 10 and 20 attached phenoxy groups per 100 glucose repeat units). The long-time kinetics of interfacial tension decrease was satisfactorily described assuming diffusion-limited adsorption of hydrophobic units (over 4h). Dilational rheology of dodecane/water interface was studied for the first time with that kind of amphiphilic polysaccharides and evidenced a significant elastic component. For all dextran derivatives, experimental results were conveniently described using Lucassen-van den Tempel model which assumed diffusion-limited of surface active species. The characteristic frequency increased with the number of attached phenoxy groups and its order of magnitude (10-3-10-2rad.s-1) was consistent with estimations based on the previous model. Experimental results were compared to those obtained with commercial stabilizers like Pluronics (L64, P105, F68 and F127) and Tween 80.

Lipase-catalyzed synthesis of sucrose monoester: Increased productivity by combining enzyme pretreatment and non-aqueous biphasic medium.

Sucrose monocaprate was synthesized by carrying out a lipase-catalyzed transesterification in a non-aqueous biphasic medium. Vinyl caprate was mechanically dispersed into a solution of sucrose in DMSO. The use of DMSO allowed increasing sucrose concentration up to 0.7M (in DMSO). The denaturing effect of DMSO on lipase was avoided by pretreatment of lipase by pH adjustment in the presence of crown ether. This pretreatment maintained a significant catalytic activity which led to 0.2M sucrose monoester within 1h at 50°C, which represented higher productivity than already reported. Detailed structural characterization revealed that only monoester was recovered and the 2-O-acylated sucrose monocaprate was the major isomer in the final product.

First multi-reactive dextran-based inisurf for atom transfer radical polymerization in miniemulsion.

A multi-reactive polysaccharide-based inisurf (acting both as initiator and stabilizer) has been designed for the first time from dextran with the aim of preparing dextran-covered nanoparticles with covalent linkage between core and coverage. This inisurf was used for polymerizing butyl acrylate in miniemulsion by AGET-ATRP. Both hydrophobic phenoxy groups and initiator groups (bromoisobutyryl ester) were introduced within hydrophilic dextran chain, conferring it amphiphilic and macroinitiator characters. Amphiphilic properties of dextran inisurfs have been evidenced as well as their ability to stabilize the direct miniemulsion of n-butyl acrylate. After optimization of polymerization conditions with model studies, assays were successfully realized with dextran-based inisurfs. Because of their amphiphilic character, inisurfs migrated at oil/water interface and initiated polymerization from bromoisobutyryl ester groups. Therefore graft copolymers were produced at oil/water interface, due to the multifunctional character of these inisurfs and constituted the particle inner core with covalent links to the dextran coverage.

Toward one-pot lipase-catalyzed synthesis of poly(ε-caprolactone) particles in aqueous dispersion.

The preparation of polyester particles using enzyme-catalyzed (lipase from Candida antarctica B, CALB) ring-opening polymerization of ε-caprolactone (ε-CL) in aqueous dispersion was demonstrated for the first time. Immobilization of CALB enabled a significant increase of the number-average degree of polymerization of ε-CL oligomers (up to 38) as compared to dissolved CALB (8 at the maximum). The nature and amount of lipase, as well as the nature of the support material were identified as key parameters controlling ring-opening polymerization of ε-CL in aqueous dispersion. In addition, the involvement of solubilized monomers in polymerization elementary reactions was demonstrated and the consequences on oligomers average length were detailed. An overall mechanism of lipase-catalyzed ε-CL polymerization in aqueous dispersion taking into account the colloidal nature of reaction medium was proposed on the basis of experimental results.

Synthesis of water-soluble and water-insoluble amphiphilic derivatives of dextran in organic medium.

Hydrophobically modified dextrans were prepared by reacting native polysaccharide with 1,2-epoxydodecane in dimethylsulfoxide. Epoxide oligomerization was shown to occur as a secondary reaction when hydroxide ions were used as base catalysts. By adjusting the amount of epoxide in the feed, dextran derivatives with degrees of substitution (DS) between 0% and 164% were obtained. Polymers with DS above 100% were readily soluble in organic solvents like tetrahydrofuran, dioxane and water-saturated chloroform and dichloromethane. Their solution properties in organic solvent were characterized by capillary viscometry. Water-soluble derivatives were compared to other amphiphilic dextrans obtained using a heterogeneous modification in aqueous medium. The effect of modification conditions on substitution pattern was evidenced.

Polysaccharide-covered nanoparticles with improved shell stability using click-chemistry strategies.

Dextran-covered PLA nanoparticles have been formulated by two strategies. On one hand, dextran-g-PLA copolymers have been synthesized by click-chemistry between azide-multifunctionalized dextran (DexN3) and alkyne end-functionalized PLA chains (α-alkyne PLA); then nanoprecipitated without any additional surfactants. On the other hand, DexN3 exhibiting surfactant properties have been emulsified with unfunctionalized or α-alkyne PLA, which are dissolved in organic phase with or without CuBr. Depending on the o/w emulsion/evaporation process experimental conditions, dextran-g-PLA copolymers have been produced in situ, by click chemistry at the liquid/liquid interface during the emulsification step. Whatever the process, biodegradable core/shell polymeric nanoparticles have been obtained, then characterized. Colloidal stability of these nanoparticles in the presence of NaCl or SDS has been studied. While the physically adsorbed polysaccharide based shell has been displaced by SDS, the covalently-linked polysaccharide based shell ensures a permanent stability, even in the presence of SDS.

Lipase-catalyzed synthesis of hydrophobically modified dextrans: activity and regioselectivity of lipase from Candida rugosa.

Vinyl decanoate-modified dextran macromolecules (DexT40-VD) were synthesized in dimethyl sulfoxide at 50°C using lipase AY from Candida rugosa for catalyzing transesterification between polysaccharide and vinyl fatty esters. The extent of dextran modification (quantified by the molar ratio of attached alkyl tails to sugar repeat units) with native-, pH-adjusted-, 18-crown-6 ether pretreated pH-adjusted-, and stepwise addition of pretreated lipase AY yielded <3%, 49%, 64% and 96% modified dextran respectively. Lipase AY accelerated the transesterification of DexT40 from 2- to 63-fold higher than the non-catalyzed system. This procedure was extended to other acyl donors showing that modification pattern exhibited regioselectivity depending on acyl donor structure. Regioselectivity equaled between 2- and 3-OH with saturated fatty acyl donors. The 2-OH was favored for unsaturated fatty acyl donors, while sterically hindered acyl donors oriented modification toward 3-OH position. DexT40-VD at 96% modification was a water-insoluble polymer forming 150nm diameter nanoparticles in water which can be used as drug carrier systems.

Effect of sporulation conditions on the resistance of Bacillus subtilis spores to heat and high pressure.

Bacillus subtilis(B. subtilis) cells were placed in various environmental conditions to study the effects of aeration, water activity of the medium, temperature, pH, and calcium content on spore formation and the resulting properties. Modification of the sporulation conditions lengthened the growth period of B. subtilis and its sporulation. In some cases, it reduced the final spore concentration. The sporulation conditions significantly affected the spore properties, including germination capacity and resistance to heat treatment in water (30 min at 97°C) or to high pressure (60 min at 350 MPa and 40°C). The relationship between the modifications of these spore properties and the change in the spore structure induced by different sporulation conditions is also considered. According to this study, sporulation conditions must be carefully taken into account during settling sterilization processes applied in the food industry.