Acetic Acid-Modulated Room Temperature Synthesis of MIL-100 (Fe) Nanoparticles for Drug Delivery Applications.

Due to their flexible composition, large surface areas, versatile surface properties, and degradability, nanoscale metal organic frameworks (nano MOFs) are drawing significant attention in nanomedicine. In particular, iron trimesate MIL-100 (Fe) is studied extensively in the drug delivery field. Nanosized MIL-100 (Fe) are obtained mostly by microwave-assisted synthesis. Simpler, room-temperature (RT) synthesis methods attract growing interest and have scale-up potential. However, the preparation of RT MIL100 is still very challenging because of the high tendency of the nanoparticles to aggregate during their synthesis, purification and storage. To address this issue, we prepared RT MIL100 using acetic acid as a modulator and used non-toxic cyclodextrin-based coatings to ensure stability upon storage. Hydrodynamic diameters less than 100 nm were obtained after RT synthesis, however, ultrasonication was needed to disaggregate the nanoparticles after their purification by centrifugation. The model drug adenosine monophosphate (AMP) was successfully encapsulated in RT MIL100 obtained using acetic acid as a modulator. The coated RT MIL100 has CD-exhibited degradability, good colloidal stability, low cytotoxicity, as well as high drug payload efficiency. Further studies will focus on applications in the field of cancer therapy.

Potential of Exopolysaccharide from Porphyridium marinum to Contend with Bacterial Proliferation, Biofilm Formation, and Breast Cancer.

Exopolysaccharide (EPS) from marine microalgae are promising sources of a new generation of drugs. However, lot of them remain to be discovered and tested. In this study, EPS produced by Porphyridium marinum and its oligomers prepared by High Pressure Homogenizer have been tested for different biological activities, i.e., antibacterial, anti-fungal and antibiofilm activities on Candida albicans, as well as for their effects on the viability of murine breast cancer cells. Results have shown that all EPS samples present some biological activity. For antibacterial and antibiofilm activities, the native EPS exhibited a better efficiency with Minimum Inhibitory Concentration (MIC) from 62.5 µg/mL to 1000 µg/mL depending on the bacterial strain. For Candida albicans, the biofilm formation was reduced by about 90% by using only a 31.3 µg/mL concentration. Concerning breast cancer cells, lower molar masses fractions appeared to be more efficient, with a reduction of viability of up to 55%. Finally, analyses of polymers composition and viscosity measurements were conducted on all samples, in order to propose hypotheses involving the activities caused by the intrinsic properties of polymers.

Scaffolds Based on Collagen, Hyaluronan and Sericin with Potential Applications as Controlled Drug Delivery System.

Natural proteins have been extensively studied as matrices for tissue engineering, due to their excellent biocompatibility and biological properties associated with increasing cell proliferation. By generating complex materials, cell and tissue functions can be tailored to obtain a specific direction, according to the medical needs. The aim of this paper was to obtain scaffolds based on collagen, hyaluronan and sericin, with morphology and physical-chemical properties adequate for controlled drug delivery systems. In this aim various tests were performed: in vitro swelling and degradation studies, Fourier Transform Infrared spectroscopy (FT-IR), Scanning Electronic Microscopy (SEM) and thermogravimetric analysis. Loading and releasing of ibuprofen is also discussed. The results indicate that scaffolds based on collagen, hyaluronan and sericin have a porous structure, strength and stability adequate for skin tissue engineering. The obtained scaffolds swell, degrade and have controlled drug release properties in simulated biological fluids.

Carboxymethylpullulan Grafted with Aminoguaiacol: Synthesis, Characterization, and Assessment of Antibacterial and Antioxidant Properties.

Aminoguaiacol, the aminated derivative of guaiacol, a natural phenolic compound, was chemically grafted onto a polysaccharide (carboxymethylpullulan, CMP) in the presence of the activator agent 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDCI). The grafted polysaccharides were characterized by FTIR and 1H NMR spectroscopy to confirm and quantify the grafting. All polysaccharide derivatives (grafting rates of aminoguaiacol between 16% and 58%) were soluble in water. Their physicochemical properties were studied in a dilute regime and a semidilute regime by light scattering, fluorescence, and rheology, showing associative properties with peculiar polysoap behavior. The antibacterial activities of the synthesized products against Staphyloccocus aureus were assessed using a counting method. The antioxidant activities of the derivatives were also highlighted using the α,α-diphenyl-ß-picrylhydrazyl (DPPH) method. Finally, the cytotoxicity of the derivatives was studied with fibroblast cells and they showed a very good cytocompatibility. Such polymers could be used to replace chemical preservatives in food and cosmetic aqueous formulations.

Two methods for one-point anchoring of a linear polysaccharide on a gold surface.

Two strategies to achieve a one-point anchoring of a hydrolyzed pullulan (P9000) on a gold surface are compared. The first strategy consists of forming a self-assembled monolayer of a 6-amino-1-hexanethiol (AHT) and then achieving reductive amination on the surface between the aminated surface and the aldehyde of the polysaccharide reductive end sugar. The second consists of incorporating a thiol function at the extremity of the pullulan (via the same reductive amination), leading to P9000-AHT and then immobilizing it on gold by a spontaneous reaction between solid gold and thiol. The modified pullulan was characterized by NMR and size-exclusion chromatography coupled to a light-scattering detector. P9000-AHT appears to be in a disulfide dimer form in solution but recovers its unimer form with dithiothreitol (DTT) treatment. The comparison of the two strategies by contact angle and XPS revealed that the second strategy is more efficient for the pullulan one-point anchoring. P9000-AHT even in its dimer form is easily grafted onto the surface. The grafted polymer seems to be more in a coil conformation than in a rigid brush. Furthermore, QCM measurements highlighted that the second strategy leads to a grafting density of around 3.5 × 10(13) molecules·cm(-2) corresponding to a high surface coverage. The elaboration of a dense and oriented layer of polysaccharides covalently linked to a gold surface might enhance the use of such modified polysaccharides in various fields.

Hydrolysis of pullulan by entrapped pullulanase in Ca/alginate beads.

Pullulanase from Klebsiella pneumoniae was entrapped into calcium alginate beads. Its activity was estimated by the determination of number-average molar masses using two different methods: a colorimetric assay of reducing ends (REs) and a size-exclusion chromatography/multiangle light scattering/differential refractive index. The second method also provided weight-average molar masses of hydrolyzed pullulan and the quantity of maltotriose (DP3) and its multiples (DP6 and DP9) produced by the enzymatic treatment. The alginate beads showed a good retention of the loaded pullulanase (30%), and the system showed a downturn of hydrolysis kinetics in comparison with free pullulanase due to the limiting access of substrate-enzyme. On the contrary with the results obtained from free enzyme hydrolysis, for which a large distribution of pullulan fragments is observed during the treatment, the immobilized enzyme system has evidenced, during the enzymatic treatment, the coexistence of native or only slightly degraded pullulan chains together with maltotriose units. Complete hydrolysis of pullulan chains was achieved once diffused into the gel.

Purification of a low molecular weight fucoidan for SPECT molecular imaging of myocardial infarction.

Fucoidans constitute a large family of sulfated polysaccharides with several biochemical properties. A commercial fucoidan from brown algae, containing low molecular weight polysaccharidic species constituted of l-fucose, uronic acids and sulfate groups, was simply treated here with calcium acetate solution. This treatment led to a purified fraction with a yield of 45%. The physicochemical characterizations of the purified fucoidan using colorimetric assay, MALLS, dRI, FT-IR, NMR, exhibited molecular weight distributions and chemical profiles similar for both fucoidans whereas the sulfate and l-fucose contents increased by 16% and 71%, respectively. The biodistribution study in rat of both compounds labeled with 99mTc evidenced a predominant renal elimination of the purified fucoidan, but the crude fucoidan was mainly retained in liver and spleen. In rat myocardial ischemia-reperfusion, we then demonstrated the better efficiency of the purified fucoidan. This purified sulfated polysaccharide appears promising for the development of molecular imaging in acute coronary syndrome.

Polyelectrolyte complexes of chitosan and hyaluronic acid or carboxymethylpullulan and their aminoguaiacol derivatives with biological activities as potential drug delivery systems.

Polyelectrolyte complexes (PECs) were elaborated from chitosan as cationic polymer and carboxy-methylpullulan (CMP), hyaluronic acid (HA) and their derivatives grafted with aminoguaiacol (G) with different degrees of substitution (DSGA) with the aim of obtaining nanogels for drug delivery. For each couple of polysaccharides, the charge ratios giving the smaller size with the lower PDI were selected to produce PECs. CMP_CHIT and CMP-G_CHIT PECs had smaller sizes (220-280 nm) than HA_CHIT and HA-G_CHIT PECs (280-390 nm). PECs were stable at 4 °C during 28 days at pH 5. In phosphate buffer saline (PBS) at pH 7.4, at 4 °C, a better stability of PECs based on CMP-G derivatives was observed. The hydrophobic associations between aminoguaiacol groups (highlighted by measurements of pyrene fluorescence) led to a better PECs' stabilization in PBS. The PECs' antioxidant and antibacterial activities were demonstrated and related to the DSGA. Diclofenac and curcumin were used as drug models: their loading reached 260 and 53 µg/mg PEC, respectively. The release of diclofenac in PBS at 37 °C followed a quasi-Fickian diffusion mechanism with release constant between 0.88 and 1.04 h-1. The curcumin release followed a slow linear increase in PBS/EtOH (60/40 V/V) with an effect of DSGA.

Injectable polyoxazoline grafted hyaluronic acid thermoresponsive hydrogels for biomedical applications.

Injectable thermosensitive hydrogels based on hyaluronic acid (HA) grafted with lower critical solution temperature (LCST) polyoxazoline (copolymers of poly(isopropyl-co-butyl oxazoline)) or P(iPrOx-co-BuOx) have been elaborated with tunable solution/gel temperature transitions and gel state elastic modulus. A suitable HA-g-P(iPrOx-co-BuOx-67/33)-0.10 sample with an iPrOx/BuOx ratio of 67/33, a polymerization degree (DP) of 25, a substitution degree (DS) of 10%, and displaying thermally induced gelling character with elastic (G') and viscous (G'') moduli crossover points at 25 °C and a G' at 37 °C around 80 Pa has been chosen for medical application. Hydrogels obtained with HA-g-P(iPrOx-co-BuOx-67/33)-0.10 exhibited high stability at 37 °C and excellent injectability properties with full and quick reversibility. The incorporation of a secondary network (HA), until 35 wt%, into the thermosensitive hydrogel also demonstrated very good stability and injectability.

Exopolysaccharide from marine microalgae belonging to the Glossomastix genus: fragile gel behavior and suspension stability.

With the aim to find new polysaccharides of rheological interest with innovated properties, rhamnofucans produced as exopolysaccharides (EPS) in a photobioreactor (PBR) and an airlift bioreactor (ABR) by the marine microalgae Glossomastix sp. RCC3707 and RCC3688 were fully studied. Chemical characterizations have been conducted (UHPLC - MS HR). Analyses by size-exclusion chromatography (SEC) coupled online with a multiangle light scattering detector (MALS) and a differential refractive index detector showed the presence of large structures with molar masses higher than 106 g.mol-1. The rheological studies of these EPS solutions, conducted at different concentrations and salinities, have evidenced interesting and rare behavior characteristic of weak and fragile hydrogels i.e. gel behavior with very low elastic moduli (between 10-2 and 10 Pa) and yield stresses (between 10-2 and 2 Pa) according to the EPS source, concentration, and salinity. These results were confirmed by diffusing wave spectroscopy. Finally, as one of potential application, solutions of EPS from Glossomastix sp. have evidenced very good properties as anti-settling stabilizers, using microcrystalline cellulose particles as model, studied by multiple light scattering (MLS) with utilization in cosmetic or food industry. Compared to alginate solution with same viscosity for which sedimentation is observed over few hours, microalgae EPS leads to a stable suspension over few days.

Molecular size characterization and kinetics studies on hydrolysis of pullulan by pullulanase in an entangled alginate medium.

The behavior of a hydrolytic enzyme (pullulanase) toward its substrate (pullulan) in the presence of a nonsubstrate (alginate), both below and above the critical entanglement concentration (C*), was studied. The hydrolysis kinetics were studied with the enzyme and alginate concentrations varied using two main methods: a colorimetric assay of the reducing extremities (RE), which allowed the number-average molar masses (Mn) of the oligosaccharides to be determined, and size exclusion chromatography with on-line, multiangle light scattering, viscometer, and differential refractive index detectors, which allowed the average molar masses, Mn and Mw, of the oligosaccharides during hydrolysis to be determined. Free pullulanase acts via an "endo" process. The presence of alginate slows the hydrolysis kinetics, particularly when the alginate concentration is greater than the C*. These results were confirmed by the evolution of the kinetic parameters (KM, Vmax) obtained via isothermal titration calorimetry (ITC). The amount of oligosaccharides produced is not dependent on the alginate concentration, and the endo enzyme behavior is not modified by the entanglement in the medium. These observations were also confirmed by ITC analysis in the presence of degraded alginate (without entanglement). Our results correlated with the substrate diffusion in entangled media. The pullulanase reaction in the presence of alginate is shown to be diffusion-dependent.

Lyophilization for Formulation Optimization of Drug-Loaded Thermoresponsive Polyelectrolyte Complex Nanogels from Functionalized Hyaluronic Acid.

The lyophilization of nanogels is practical not only for their long-term conservation but also for adjusting their concentration and dispersant type during reconstitution for different applications. However, lyophilization strategies must be adapted to each kind of nanoformulation in order to minimize aggregation after reconstitution. In this work, the effects of formulation aspects (i.e., charge ratio, polymer concentration, thermoresponsive grafts, polycation type, cryoprotectant type, and concentration) on particle integrity after lyophilization and reconstitution for different types of polyelectrolyte complex nanogels (PEC-NGs) from hyaluronic acid (HA) were investigated. The main objective was to find the best approach for freeze-drying thermoresponsive PEC-NGs from Jeffamine-M-2005-functionalized HA, which has recently been developed as a potential platform for drug delivery. It was found that freeze-drying PEC-NG suspensions prepared at a relatively low polymer concentration of 0.2 g.L-1 with 0.2% (m/v) trehalose as a cryoprotectant allow the homogeneous redispersion of PEC-NGs when concentrated at 1 g.L-1 upon reconstitution in PBS without important aggregation (i.e., average particle size remaining under 350 nm), which could be applied to concentrate curcumin (CUR)-loaded PEC-NGs for optimizing CUR content. The thermoresponsive release of CUR from such concentrated PEC-NGs was also reverified, which showed a minor effect of freeze-drying on the drug release profile.

Thermoresponsive nanogels based on polyelectrolyte complexes between polycations and functionalized hyaluronic acid.

A novel kind of thermoresponsive polyelectrolyte complex-based nanogels (PEC-NGs) was elaborated by mixing hyaluronic acid (HA) functionalized with Jeffamine® M-2005 (M2005, a thermoresponsive amine-terminated polyether) and diethylaminoethyl dextran (DEAE-D) or poly-l-lysine (PLL) in water. The presence of M2005 grafts led to PEC-NGs with larger particle size, lower net surface charge and thermoresponsiveness, namely shrinkage with increasing hydrophobicity at higher temperature. Both M2005 grafts and replacing DEAE-D with PLL as polycation allowed PEC-NGs to have higher stability against salinity and better encapsulation of curcumin, most probably through intraparticle hydrophobic interactions, whereas interparticle hydrophobic interactions may facilitate particle aggregation over time. Curcumin encapsulation can be optimized by applying higher temperature during the complexation. Enzymatic degradability of PEC-NGs was also verified through particle size evolution in the presence of hyaluronidase. These results provide new insights into the physicochemical aspect of such systems as promising nanocarriers for drug delivery.

Hybrid systems combining liposomes and entangled hyaluronic acid chains: Influence of liposome surface and drug encapsulation on the microstructure.

Mixtures of hyaluronic acid (HA) with liposomes lead to hybrid colloid-polymer systems with a great interest in drug delivery. However, little is known about their microstructure. Small angle neutron scattering (SANS) is a valuable tool to characterize these systems in the semi-dilute entangled regime (1.5% HA) at high liposome concentration (80 mM lipids). The objective was to elucidate the influence of liposome surface (neutral, cationic, anionic or anionic PEGylated), drug encapsulation and HA concentration in a buffer mimicking biological fluids (37 °C). First, liposomes were characterized by SANS, cryo-electron microscopy, and dynamic light scattering and HA by SANS, size exclusion chromatography, and rheology. Secondly, HA-liposome mixtures were studied by SANS. In HA, liposomes kept their integrity. Anionic and PEGylated liposomes were in close contact within dense clusters with an amorphous organization. The center-to-center distance between liposomes corresponded to twice their diameter. A depletion mechanism could explain these findings. Encapsulation of a corticoid did not modify this organization. Cationic liposomes formed less dense aggregates and were better dispersed due to their complexation with HA. Liposome surface governed the interactions and microstructure of these hybrid systems.

Microgels Based on Carboxymethylpullulan Grafted with Ferulic Acid Obtained by Enzymatic Crosslinking in Emulsion for Drug Delivery Systems.

Carboxymethylpullulan (CMP) grafted with ferulic acid (FA) is crosslinked with laccase by the reverse water-in-oil emulsion technique (with sunflower oil) to obtain microgels with size from 40 to 200 µm. It is demonstrated that laccase activity and dispersion time have an impact on microgels' size. Fluorescence spectroscopy of different probes (e.g., pyrene, Nile red, and curcumin) shows the nonpolar characteristics of hydrophobic microdomains formed by the FA moieties and its dimers forming the crosslinking nodes. Encapsulation and release of curcumin or lidocaine used as drug models are studied in different buffers. Curcumin is well encapsulated but retained in microgels, while lidocaine is released at 65-70% in 2 h and 30 min in buffer simulating the gastrointestinal tract and at 75-85% in 1 h in acetate buffer pH 5.6 or phosphate-buffered saline (PBS) pH 6.9.

Hyaluronic Acid Functionalization with Jeffamine® M2005: A Comparison of the Thermo-Responsiveness Properties of the Hydrogel Obtained through Two Different Synthesis Routes.

Hyaluronic acid (HA) of different molar masses (respectively 38,000, 140,000 and 1,200,000 g.mol-1) have been functionalized with a commercial poly(etheramine), Jeffamine® M2005, in order to devise physical thermo-responsive hydrogels. Two routes have been studied, involving the use of either water for the first one or of N,N'-Dimethylformamide (DMF), a polar aprotic solvent, for the second one. In the case of the water route, the reaction was performed using a mixture of N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling reagents. The reaction was optimized while making sure no free M2005 remained in the final material, leading to M2005 grafting degrees of about 4%, which enabled the formation of hydrogels by increasing the temperature. In the case of the organic solvent route, propylphosphonic anhydride T3P® was used as a coupling reagent in DMF, resulting in a M2005 grafting degree of around 8% with better thermo-responsive properties of HA-g-M2005 compared to those obtained when the reaction was performed in water. However, the reaction systematically led to covalent cross-linking in the case of the HA, with the highest starting molar masses resulting in a very different rheological behaviour and with higher gel strength retaining thermo-responsive behaviour but being only poorly soluble in water.

Characterization of dextran particle size: How frit-inlet asymmetrical flow field-flow fractionation (FI-AF4) coupled online with dynamic light scattering (DLS) leads to enhanced size distribution.

Accurate determinations of particle size and particle size distribution (PSD) are essential to achieve the clinical translation of medical nanoparticles (NPs). Herein, dextran-based NPs produced via a water-in-oil emulsification/crosslinking process and developed as nanomedicines were studied. NPs were first characterized using traditional batch-mode techniques as dynamic light scattering (DLS) and laser diffraction. In a second step, their analysis by frit-inlet asymmetrical flow field-flow fractionation (FI-AF4) was explored. The major parameters of the AF4 procedure, namely, crossflow, detector flow, crossflow decay programming and relaxation time were set up. The sizes of the particle fractions eluted under optimized conditions were measured using DLS as an online detector. We demonstrate that FI-AF4 is a powerful method to characterize dextran-NPs in the 200 nm -1 µm range. It provided a more realistic and comprehensive picture of PSD, revealing its heterogenous character and clearly showing the ratio of different populations in the sample, while batch-mode light scattering techniques only detected the biggest particle sizes.

Porous nanoparticles with engineered shells release their drug cargo in cancer cells.

Highly porous nanoscale metal-organic frameworks (nanoMOFs) attract growing interest as drug nanocarriers. However, engineering "stealth" nanoMOFs with poly(ethylene glycol) (PEG) coatings remains a main challenge. Here we address the goal of coating nanoMOFs with biodegradable shells using novel cyclodextrin (CD)-based oligomers with a bulky structure to avoid their penetration inside the open nanoMOF porosity. The PEG chains were grafted by click chemistry onto the CDs which were further crosslinked by citric acid. Advantageously, the oligomers' free citrate units allowed their spontaneous anchoring onto the nanoMOFs by complexation with the iron sites in the top layers. Up to 31 wt% oligomers could be firmly attached by simple incubation with the nanoMOFs in an aqueous medium. Moreover, the anticancer drug doxorubicin (DOX) was successfully entrapped in the core-shell nanoMOFs with loadings up to 41 wt%. High resolution STEM (HR-STEM) showed that the organized crystalline structures were preserved. Remarkably, at the highest loadings, DOX was poorly released out of the nanoMOFs at pH 7.4 (<2% in 2 days). In contrast, around 80% of DOX was released out at pH 4.5 of artificial lysosomal fluid in 24 h. Confocal microscopy investigations showed that the DOX-loaded nanoMOFs penetrated inside Hela cancer cell together with their PEG shells. There, they released the DOX cargo which further diffused inside the nucleus to eradicate the cancer cells.

Biomimetic hydrogel by enzymatic crosslinking of pullulan grafted with ferulic acid.

A novel eco-friendly two-step synthesis process of neutral pullulan (PUL)-ferulic acid (FA) conjugates was reported in this work. Ferulic acid was first transformed to activated ferulate-imidazolide using N,N'-carbonyldiimidazole (CDI), a green activated reagent. Issued product was then reacted with pullulan. PUL-FA derivatives were characterized by FTIR and 1H NMR leading to substitution degrees (DS) between 0.02 and 0.1 (mol FA per mol PUL repeat unit). The study in dilute regime indicated an associative behavior with the presence of aggregate structures in solution due to the hydrophobic interactions between the grafted FA onto polysaccharide backbones. Laccase from Trametes versicolor was then used to crosslink polysaccharide chains to obtain biomimetic PUL-FA hydrogels. Gelling's kinetics were analyzed with rheology in dynamic mode showing the impact of laccase amount, DS and concentration. Mechanical and swelling properties appear related only to DS and concentration of PUL-FA products.

Gd(DOTA)-grafted submicronic polysaccharide-based particles functionalized with fucoidan as potential MR contrast agent able to target human activated platelets.

Early detection of thrombotic events remains a big medical challenge. Dextran-based submicronic particles bearing Gd(DOTA) groups and functionalized with fucoidan have been produced via a simple and green water-in-oil emulsification/co-crosslinking process. Their capacity to bind to human activated platelets was evidenced in vitro as well as their cytocompatibility with human endothelial cells. The presence of Gd(DOTA) moieties was confirmed by elemental analysis and total reflection X-ray fluorescence (TRXF) spectrometry. Detailed characterization of particles was performed in terms of size distribution, morphology, and relaxation rates. In particular, longitudinal and transversal proton relaxivities were respectively 1.7 and 5.0 times higher than those of DOTAREM. This study highlights their potential as an MRI diagnostic platform for atherothrombosis.