Characterization of Gd loaded chitosan-TPP nanohydrogels by a multi-technique approach combining dynamic light scattering (DLS), asymetrical flow-field-flow-fractionation (AF4) and atomic force microscopy (AFM) and design of positive contrast agents for molecular resonance imaging (MRI).

Chitosan CS-tripolyphosphate TPP/hyaluronic acid HA nanohydrogels loaded with gadolinium chelates (GdDOTA ⊂ CS-TPP/HA NGs) synthesized by ionic gelation were designed for lymph node (LN) MRI. In order to be efficiently drained to LNs, nanogels (NGs) needed to exhibit a diameter ϕ < 100 nm. For that, formulation parameters were tuned, using (i) CS of two different molecular weights (51 and 37 kDa) and (ii) variable CS/TPP ratio (2 < CS/TPP < 8). Characterization of NG size distribution by dynamic light scattering (DLS) and asymetrical flow-field-flow-fractionation (AF4) showed discrepancies since DLS diameters were consistently above 200 nm while AF4 showed individual nano-objects with ϕ < 100 nm. Such a difference could be correlated to the presence of aggregates inherent to ionic gelation. This point was clarified by atomic force microscopy (AFM) in liquid mode which highlighted the main presence of individual nano-objects in nanosuspensions. Thus, combination of DLS, AF4 and AFM provided a more precise characterization of GdDOTA ⊂ CS-TPP/HA nanohydrogels which, in turn, allowed to select formulations leading to NGs of suitable mean sizes showing good MRI efficiency and negligible toxicity.

A new magnetic resonance imaging contrast agent loaded into poly(lacide-co-glycolide) nanoparticles for long-term detection of tumors.

The incorporation of a lipophilic Gd chelate (GdDO3A-C12) in biocompatible PLGA poly(D, L-lactide-co-glycolide) nanoparticles was explored as an approach to increase the relaxivity of contrast agents for magnetic resonance imaging. By nanoprecipitation, it was possible to obtain PEGylated gadolinium nanoparticles (mean diameter of 155 nm) with high Gd loading (1.1 × 10(4) Gd centers per nanoparticle). The corresponding GdDO3AC12 ⊂ NPs nanoparticles exhibited an enhanced relaxivity (up to sixfold greater than DOTAREM® at 40 MHz) because the nanoparticle framework constrained the lipophilic Gd chelate motion and favorably impacted the Gd chelate rotational correlation time. T1-weighted imaging at 3 T on phantoms showed enhanced contrast for the GdDO3AC12 ⊂ NPs. Importantly, Gd chelate leakage was almost nonexistent, which suggested that these GdDO3AC12 ⊂ NPs could be useful for long-term MRI detection.

Vectorization of copper complexes via biocompatible and biodegradable PLGA nanoparticles.

A double emulsion-solvent diffusion approach with fully biocompatible materials was used to encapsulate copper complexes within biodegradable nanoparticles, for which the release kinetics profiles have highlighted their potential use for a prolonged circulating administration.

Evaluation of mTHPC-loaded PLGA nanoparticles for in vitro photodynamic therapy on C6 glioma cell line.

Photodynamic therapy (PDT) is a very attractive strategy to complement or replace common cancer treatments such as radiotherapy, surgery, and chemotherapy. Some molecules have shown their efficiency as photosensitizers (PS), still many issues have to be solved such as the inherent cytotoxicity of the PS or its hydrophobic properties causing limitation in their solubility, leading to side effects. In this study, the encapsulation of an approved PS, the meso-tetra hydroxyphenylchlorine (mTHPC, Foscan®) within biocompatible and biodegradable poly(D, l-lactide-co-glycolide) acid (PLGA) NPs prepared by the nanoprecipitation method was studied. The mTHPC-loaded NPs (mTHPC ⊂ PLGA NPs) were analyzed by UV-vis spectroscopy to determine the efficiency of mTHPC encapsulation, and by dynamic light scattering (DLS) and atomic force microscopy (AFM) to determine mTHPC ⊂ PLGA NPs sizes, morphologies and surface charges. The longitudinal follow-up of mTHPC release from the NPs indicated that 50% of the encapsulated PS was retained within the NP matrix after a period of five days. Finally, the cytotoxicity and the phototoxicity of the mTHPC ⊂ PLGA NPs were determined in murine C6 glioma cell lines and compared to the ones of mTHPC alone. The studies showed a strong decrease of mTHPC cytotoxicity and an increase of mTHPC photo-cytotoxicity when mTHPC was encapsulated. In order to have a better insight of the underlying cellular mechanisms that governed cell death after mTHPC ⊂ PLGA NPs incubation and irradiation, annexin V staining tests were performed. The results indicated that apoptosis was the main cell death mechanism.

Hydroxyethylstarch microcapsules: a preliminary study for tumor immunotherapy application.

The objective of this work was to prepare microcapsules which would allow protection and slow release of antigens used for melanoma immunotherapy treatment. Hydroxyethylstarch (HES) microcapsules were prepared using interfacial cross-linking with terephthaloyl chloride (TC). They were characterized with respect to morphology (microscopy) and size (in the 4-15 microm range). Bovine serum albumin (BSA) was used as model protein for loading and release studies. Microcapsules were loaded with solutions at different protein concentrations (0.5-5%). The maximum loading efficiency (20%) was observed with the concentration of 2.5%, which allowed a loading capacity near 100%. Confocal laser scanning microscopy (CLSM) visualization showed that BSA was entrapped within the microcapsules and not only associated to their outer surface. BSA-release studies showed a 20% BSA release within 30 min while 80% remained entrapped in the microcapsules for 4 days. Microcapsules were degraded by alpha-amylase and addition of esterase to alpha-amylase enhanced slightly their degradation. In vitro studies on melanoma cells showed that HES microcapsules were non-toxic. Preliminary in vivo studies demonstrated that microcapsules were biodegradable after intraperitoneal injection (i.p.). The observation of peritoneal wash showed a complete degradation within 7 days, indicating a possible application as an in vivo drug delivery system especially to enhance the presentation of antigens.

Fourier transform infrared spectroscopic studies of cross-linked human serum albumin microcapsules. 3. Influence of terephthaloyl chloride concentration on spectra and correlation with microcapsule morphology and size.

Microcapsules were prepared from human serum albumin (HSA) by interfacial cross-linking with terephthaloyl chloride (TC). TC concentrations were increased from 0.5 to 5% w/v, while pH (9.8) and reaction time (30 min) were kept constant. Fourier transform infrared (FT-IR) spectra of lyophilized microcapsules were compared. Correlations were established with microcapsule morphology and size. The results were compared with those of previous studies exploring pH or reaction time and with those of parallel determinations of microcapsule free amino groups. With 0.5% TC, decreases of the ester-assigned 1724-cm-1 band area and of the carboxylate-assigned 1394-cm-1 band area were observed compared with pure HSA. This phenomenon was attributed to a removal of contaminating lipids of HSA. Increasing TC concentration resulted in a progressive increase of the band areas at 1724 cm-1 (esters) and 1795 cm-1 (anhydrides), in a further decrease of the 1394-cm-1 band area (carboxylates), and in marked alterations of teh 1340-1080-cm-1 region. These changes, which revealed the progressive acylation of hydroxy and carboxylate groups of HSA, were accompanied by an increase of the 1624-cm-1 band area (beta-sheet), reflecting interchain H-bonding due to cross-linking. As observed in the previous studies of pH and reaction time, important spectral changes corresponded to low values of -NH2 content, to a decrease in microcapsule mean size (from > 30 to < 15 microns), and to modifications of the membrane surface (made rough).

Fourier transform infrared spectroscopic studies of human serum albumin microcapsules prepared by interfacial cross-linking with terephthaloylchloride: influence of polycondensation pH on spectra and relation with microcapsule morphology and size.

Fourier transform infrared (FT-IR) spectroscopic studies were performed on microcapsules prepared through interfacial cross-linking of human serum albumin (HSA) with terephthaloylchloride at various pH values (5.9 to 11). Correlations were established with morphology and size of microcapsules. Increasing polycondensation pH resulted notably in a progressive increase of peaks at 1795 and 1724 cm-1, assigned to anhydride and ester; respectively, in a decrease of the carboxylate-assigned 1394 cm-1 peak, and in alterations of the 1340-1080-cm-1 region. These spectral changes were most pronounced from pH 9 and were shown to correspond to smaller-sized microcapsules (mean size decreased from 30-40 microns to less than 15 microns) with rough surfaces. Further soaking of highly cross-linked microcapsules in a pH 7.5 buffer resulted in the disappearance of the 1795 cm-1 peak, with a concurrent increase of the 1394 cm-1 peak and a decrease of the 1724 cm-1 peak. These changes, attributed to complete breaking of anhydride and partial hydrolysis of esters, were accompanied by an unwrinkling of the microcapsule membrane, then made smooth, and a significant increase in size. Treating microcapsules with hydroxylamine under alkaline conditions allowed complete reversal of the spectral alterations assigned to anhydride and ester formation. A comparable (slightly higher) increase in size was observed with microcapsules which exhibited smooth surfaces and a low density.

Fourier-transform infrared spectroscopic studies of cross-linked human serum albumin microcapsules. 2. Influence of reaction time on spectra and correlation with microcapsule morphology and size.

Microcapsules were prepared from human serum albumin (HSA) through interfacial cross-linking with terephthaloyl chloride (TC). Reaction times were increased from 2 to 60 min, while pH (9.8) and TC concentration (2.5% w/v) were kept constant. Fourier-transform infrared (FT-IR) spectra of lyophilized microcapsules were compared. Correlations were established with microcapsule morphology and size, as had been done in a previous study exploring the effect of increasing pH values. Microcapsules obtained after 2 min had to be considered separately. Minor alterations were observed in the spectrum as compared with pure HSA. They consisted of a decrease of the ester-assigned 1724-cm-1 band and of the carboxylate-assigned 1394-cm-1 band, attributed to a removal of contaminating lipids of HSA, and an increase of the 1624-cm-1 band, attributed to interchain H bonding following acylation of the NH2 groups. Prolonging the reaction time resulted in a progressive increase of the bands at 1724 (esters), 1795 (anhydrides), and 1624 cm-1 (beta-sheet), in a further decrease of the 1394-cm-1 band (carboxylates), and in marked alterations of the 1340-1080-cm-1 region. These important changes, which appeared after 5 min, reflect the progressive acylation of the hydroxy and carboxylate groups of HSA. As in the previous series of pH-based assays, important spectral changes were shown to correspond to a decrease in microcapsule mean size (from 32 to < 15 microns) and in important modifications of the membrane surface, made rough.

Cross-linked beta-cyclodextrin microcapsules. II. Retarding effect on drug release through semi-permeable membranes.

Microcapsules were prepared by interfacial cross-linking of beta-cyclodextrins (beta-CD) with terephthaloylchloride (TC) as described previously. Complexation assays were conducted with propranolol HCl. After 1 h incubation of 50 mg lyophilized microcapsules in 10 ml propranolol solution, the amounts of fixed drug were 507.5+/-8.6 micromol and 811.2+/-16.0 micromol per g lyophilized microcapsules with 1 mM and 2 mM solutions, respectively. A dialysis experiment was then performed. After 1 h incubation of microcapsules (10 or 50 mg) in 10 ml of 2 mM propranolol solution, the suspension was dialysed against a phosphate buffer pH 7.4 at 37 degrees C. The drug diffusion was all the more retarded that the amount of added beta-CD microcapsules was higher. Finally, double microcapsules were prepared using a suspension of beta-CD microcapsules (10-100 mg) in a solution of methylene blue in an acetate buffer pH 7.4. After adding human serum albumin (HSA), the suspension was emulsified in cyclohexane and double microcapsules were obtained by cross-linking the HSA with TC. In vitro release studies showed that the incorporation of beta-CD microcapsules resulted in a decrease in release rate of methylene blue, the decrease being related to the amount of encapsulated beta-CD microcapsules. The study then suggests interesting applications of beta-CD microcapsules for modulating the release rate of drugs through semi-permeable membranes.

Cross-linked beta-cyclodextrin microcapsules: preparation and properties.

Microcapsules were prepared by interfacial cross-linking of beta-cyclodextrins (beta-CD) with terephthaloyl chloride (TC). Batches were prepared from beta-CD solutions in 1 M NaOH, using 5% TC and a 30 min reaction time. Microcapsules were studied with respect to morphology (microscopy), size (laser diffraction technique) and, for selected batches, IR spectroscopy, determination of beta-CD content (polarimetry after alkaline dissolution of microcapsules) and complexing properties, evaluated using p-nitrophenol (pNP) as the guest molecule. Well-formed microcapsules were obtained from 5, 7.5, and 10% beta-CD solutions. The mean size of all batches was in the 10-35 microm range. The IR spectrum showed bands at 1724, 1280 and 731 cm(-1), reflecting the formation of esters. The beta-CD contents were 46, 56-58 or 60-66% for batches prepared from 5, 7.5 or 10% beta-CD solutions, respectively. The experiments conducted with 1 mM pNP showed a rapid complexation reaching a maximum within 1 h. When incubating 50 mg lyophilized microcapsules in 10 ml pNP solution, the maximal fixation (97.8 micromol/g microcapsules) was observed for small-sized particles ( approximately 11 microm) prepared from a 7.5% beta-CD solution. The method then appears as a simple and rapid procedure to provide stable microcapsules, having an interesting guest-binding ability.

[Microcapsules with modulable properties made with cross-linked proteins and polysaccharides]. / Microcapsules à propriétés modulables formées de protéines et de polysaccharides coréticulés.

A cross-linking process was applied to mixtures of a protein (gelatin A or B) and a polysaccharide. Mixed-walled microcapsules were then obtained, which exhibited properties different from those of microcapsules prepared from the protein alone. They were shown to be more resistant to enzymatic lysis and this effect depended on the ratio of the polysaccharide, on its nature and on the cross-linking pH. Very hydrophilic microcapsules were obtained through cross-linking of gelatin admixed with alginate or carboxymethylcellulose. The addition of alginate to gelatin resulted in a slower release of encapsulated pilocarpine.

[The effect of cross-linked protein microcapsules on cell cultures]. / Effet de microcapsules de protéines réticulées sur des cultures de cellules.

Microcapsules (diameter range: 5 to 100 microns) prepared through interfacial cross-linking of proteins with terephthaloylchloride exhibited a cytotoxic effect on L 1210 cell cultures. IC50 was: 0.86 mg/ml +/- 0.24 for microcapsules prepared from human serum albumin (AT microcapsules) and 0.63 mg/ml +/- 0.05 for those obtained from egg white lysozyme (LT microcapsules). With K 562 cells IC50 were 0.42 +/- 0.11 mg/ml (AT microcapsules), 0.06 mg/ml (LT microcapsules). An increase in the cytotoxicity was observed when reducing the size of the microcapsules and when increasing the reaction pH or the terephthaloylchloride concentration, or the relative concentration of microcapsules vs cells. On the contrary, the cytotoxic effect decreased, when prolonging the cross-linking time. The activity was not affected when the microcapsules were washed with toluene or with an alkaline solute. The cytotoxic effect, which appears for relatively high doses, apparently involves a contact between the microcapsules and the cells and seems to be related with the degree of cross-linking of the constitutive protein.

Development and physicochemical characterization of copper complexes-loaded PLGA nanoparticles.

PLGA nanoparticles were prepared via a modified W/O/W emulsion solvent diffusion process, in which all formulation components were fully biocompatible and biodegradable. Different independent processing parameters were systematically studied. Nanoparticles were characterized by DLS (particle size, polydispersity, zeta-potential) and TEM/AFM (surface morphology). An optimized formulation was used to encapsulate copper complexes of cyclen and DOTA as potential PET imaging agents. Results showed that the predominant formulation factors appeared to be the lactide-to-glycolide (L:G) ratio of PLGA, the nature of the diffusion phase, and the presence of hydroxyl ions in the first-emulsion aqueous phase. By regulating those 3 parameters, PLGA nanoparticles were prepared with very good preparation yields (>95%), a size less than 200 nm and a polydispersity index less than 0.1. TEM pictures showed nanoparticles with a narrow size distribution, a spherical shape and a smooth surface. The optimized formulation allowed to encapsulate Cu-cyclen and Cu-DOTA complexes with an encapsulation efficiency between 20% and 25%.

Mixed-wall microcapsules made of cross-linked proteins and polysaccharides: preparation and properties.

Microcapsules were prepared through an interfacial cross-linking process using terephthaloylchloride and applied to mixtures of a protein (human serum albumin or gelatin) and a polysaccharide. Their properties were compared with those of microcapsules prepared from the protein alone. Morphological characteristics of mixed-walled microcapsules were often modified, as seen by light and electron microscopy. Otherwise, they appeared to be more resistant to digestive media: they were gastroresistant, and their degradation time in pancreatin was prolonged upon raising the amount of polysaccharide. Moreover, the lysis time was shown to depend on the nature of the polysaccharide: microcapsules prepared from acidic polysaccharides at pH 9.8 were hydrolyzed faster. Lastly, the resistance increased upon decreasing the polymers/acylchloride ratio, or upon raising the reaction pH. Encapsulation assays were carried out with sodium salicylate, which was incorporated with a high efficiency. Mixed-walled microcapsules allowed a prolonged release of the tracer in vitro. As compared with protein microcapsules, the release profiles of batches prepared with hydroxyethylstarch exhibited only slight modifications of the initial part of the curve, while a significant burst effect was observed with carboxymethylcellulose-containing microcapsules.

Polyhydroxamic microcapsules prepared from proteins: a novel type of chelating microcapsules.

Microcapsules were prepared from three proteins, namely human serum albumin (HSA), bovine fibrinogen and ovalbumin, by an interfacial crosslinking process using terephthaloylchloride. They were further treated with alkaline hydroxylamine in order to disrupt ester and anhydride bonds in the walls. All microcapsules survived the treatment. They exhibited a significant increase in size and became sensitive to trypsin. The hydroxylamine treatment also resulted in attachment of hydroxamic groups to the membrane, making the microcapsules capable of iron binding. These properties were evaluated after soaking microcapsules in a 140 mumol/l ferric solution and determination of iron in the supernantant. Lower amounts of iron were found to be complexed by HSA microcapsules (mean value: 29.3 mumol iron/g microcapsule dry weight) as compared with fibrinogen and ovalbumin microcapsules (43.7 and 44.9 mumol/g, respectively). Microcapsule chelating properties were further improved by esterification of the free carboxyl groups of the membrane with benzyl alcohol or ethanol using a carbodiimide, prior to the hydroxylamine treatment. Comparable values of iron binding were obtained from esterified and hydroxylamine-treated batches prepared from the three proteins (about 50 mumol iron/g).