In vivo imaging of glycol chitosan-based nanogel biodistribution

The preclinical development of nanomedicines raises several challenges and requires a comprehensive characterization. Among them is the evaluation of the biodistribution following systemic administration. In previous work, the biocompatibility and in vitro targeting ability of a glycol chitosan (GC) based nanogel have been validated. In the present study, its biodistribution in the mice is assessed, using near-infrared (NIR) fluorescence imaging as a tool to track the nanogel over time, after intravenous administration. Rapid whole body biodistribution of both Cy5.5 labeled GC nanogel and free polymer is found at early times. It remains widespreadly distributed in the body at least up to 6 h postinjection and its concentration then decreases drastically after 24 h. Nanogel blood circulation half-life lies around 2 h with the free linear GC polymer presenting lower blood clearance rate. After 24 h, the blood NIR fluorescence intensity associated with both samples decreases to insignificant values. NIR imaging of the organs shows that the nanogel had a body clearance time of 48 h, because at this time point a weak signal of NIR fluorescence is observed only in the kidneys. Hereupon it can be concluded that the engineered GC nanogel has a fairly long blood circulation time, suitable for biomedical applications, namely, drug delivery, simultaneously allowing efficient and quick body clearance.

Immune cell impact of three differently coated lipid nanocapsules: pluronic, chitosan and polyethylene glycol.

Lipid nanocapsules (NCs) represent promising tools in clinical practice for diagnosis and therapy applications. However, the NC appropriate functionalization is essential to guarantee high biocompatibility and molecule loading ability. In any medical application, the immune system-impact of differently functionalized NCs still remains to be fully understood. A comprehensive study on the action exerted on human peripheral blood mononuclear cells (PBMCs) and major immune subpopulations by three different NC coatings: pluronic, chitosan and polyethylene glycol-polylactic acid (PEG) is reported. After a deep particle characterization, the uptake was assessed by flow-cytometry and confocal microscopy, focusing then on apoptosis, necrosis and proliferation impact in T cells and monocytes. Cell functionality by cell diameter variations, different activation marker analysis and cytokine assays were performed. We demonstrated that the NCs impact on the immune cell response is strongly correlated to their coating. Pluronic-NCs were able to induce immunomodulation of innate immunity inducing monocyte activations. Immunomodulation was observed in monocytes and T lymphocytes treated with Chitosan-NCs. Conversely, PEG-NCs were completely inert. These findings are of particular value towards a pre-selection of specific NC coatings depending on biomedical purposes for pre-clinical investigations; i.e. the immune-specific action of particular NC coating can be excellent for immunotherapy applications.

Chitosan-based nanogels for selective delivery of photosensitizers to macrophages and improved retention in and therapy of articular joints.

Macrophages play key roles in inflammatory disorders. Therefore, they are targets of treatments aiming at their local destruction in inflammation sites. However, injection of low molecular mass therapeutics, including photosensitizers, in inflamed joints results in their rapid efflux out of the joints, and poor therapeutic index. To improve selective uptake and increase retention of therapeutics in inflamed tissues, hydrophilic nanogels based on chitosan, of which surface was decorated with hyaluronate and which were loaded with one of three different anionic photosensitizers were developed. Optimal uptake of these functionalized nanogels by murine RAW 264.7 or human THP-1 macrophages as models was achieved after <4h incubation, whereas only negligible uptake by murine fibroblasts used as control cells was observed. The uptake by cells and the intracellular localization of the photosensitizers, of the fluorescein-tagged chitosan and of the rhodamine-tagged hyaluronate were confirmed by fluorescence microscopy. Photodynamic experiments revealed good cell photocytotoxicity of the photosensitizers entrapped in the nanogels. In a mouse model of rheumatoid arthritis, injection of free photosensitizers resulted in their rapid clearance from the joints, while nanogel-encapsulated photosensitizers were retained in the inflamed joints over a longer period of time. The photodynamic treatment of the inflamed joints resulted in a reduction of inflammation comparable to a standard corticoid treatment. Thus, hyaluronate-chitosan nanogels encapsulating therapeutic agents are promising materials for the targeted delivery to macrophages and long-term retention of therapeutics in leaky inflamed articular joints.

Controlled nerve growth factor release from multi-ply alginate/chitosan-based nerve conduits.

The delivery kinetics of growth factors has been suggested to play an important role in the regeneration of peripheral nerves following axotomy. In this context, we designed a nerve conduit (NC) with adjustable release kinetics of nerve growth factor (NGF). A multi-ply system was designed where NC consisting of a polyelectrolyte alginate/chitosan complex was coated with layers of poly(lactide-co-glycolide) (PLGA) to control the release of embedded NGF. Prior to assessing the in vitro NGF release from NC, various release test media, with and without stabilizers for NGF, were evaluated to ensure adequate quantification of NGF by ELISA. Citrate (pH 5.0) and acetate (pH 5.5) buffered saline solutions containing 0.05% Tween 20 yielded the most reliable results for ELISA active NGF. The in vitro release experiments revealed that the best results in terms of reproducibility and release control were achieved when the NGF was embedded between two PLGA layers and the ends of the NC tightly sealed by the PLGA coatings. The release kinetics could be efficiently adjusted by accommodating NGF at different radial locations within the NC. A sustained release of bioactive NGF in the low nanogram per day range was obtained for at least 15days. In conclusion, the developed multi-ply NGF loaded NC is considered a suitable candidate for future implantation studies to gain insight into the relationship between local growth factor availability and nerve regeneration.

Cell Response to the Exposure to Chitosan-TPP//Alginate Nanogels.

Hydrophilic nanocarriers formed by electrostatic interaction of chitosan with oppositely charged macromolecules have a high potential as vectors in biomedical and pharmaceutical applications. However, comprehensive information about the fate of such nanomaterials in biological environment is lacking. We used chitosan from both animal and fungal sources to form well-characterized chitosan-pentasodium triphosphate (TPP)//alginate nanogels suitable for comparative studies. Upon exposure of human colon cancer cells (HT29 and CaCo2), breast cancer cells (MDA-MB-231 and MCF-7), glioblastoma cells (LN229), lung cancer cells (A549), and brain-derived endothelial cells (HCEC) to chitosan-(TPP)//alginate nanogels, cell type-, nanogel dosage-, and exposure time-dependent responses are observed. Comparing chitosan-TPP//alginate nanogels prepared from either animal or fungal source in terms of nanogel formation, cell uptake, reactive oxygen species production, and metabolic cell activity, no significant differences become obvious. The results identify fungal chitosan as an alternative to animal chitosan in particular if biomedical/pharmaceutical applications are intended.

In vitro antitumor activity of methotrexate via pH-sensitive chitosan nanoparticles

Nanoparticles with pH-sensitive behavior may enhance the success of chemotherapy in many cancers by efficient intracellular drug delivery. Here, we investigated the effect of a bioactive surfactant with pH-sensitive properties on the antitumor activity and intracellular behavior of methotrexate-loaded chitosan nanoparticles (MTX-CS-NPs). NPs were prepared using a modified ionotropic complexation process, in which was included the surfactant derived from Nα,Nε-dioctanoyl lysine with an inorganic lithium counterion. The pH-sensitive behavior of NPs allowed accelerated release of MTX in an acidic medium, as well as membrane-lytic pH-dependent activity, which facilitated the cytosolic delivery of endocytosed materials. Moreover, our results clearly proved that MTX-CSNPs were more active against the tumor HeLa and MCF-7 cell lines than the free drug. The feasibilty of using NPs to target acidic tumor extracellular pH was also shown, as cytotoxicity against cancer cells was greater in a mildly acidic environment. Finally, the combined physicochemical and pH-sensitive properties of NPs generally allowed the entrapped drug to induce greater cell cycle arrest and apoptotic effects. Therefore, our overall results suggest that pH-sensitive MTX-CS-NPs could be potentially useful as a carrier system for tumor and intracellular drug delivery in cancer therapy.

A comparative study between crosslinked sodium hyaluronate (healaflow glaucoma implant) and high molecular weight sodium hyaluronate (healon 5) as subconjunctival spacers injected into failed filtration blebs during needling with MMC

Purpose: To compare the efficacy and safety of a slowly resorbable glaucoma implant, Healaflow (HF) and Healon 5 (H5) as spacers injected into failing filtration blebs at the time of bleb needlings, augmented with MMC Methods: Eighty-four glaucoma patients with filtration bleb failure following glaucoma surgery (deep sclerectomy/ trabeculectomy) underwent bleb needling using either HF (n=44) or H5 (n=40) as an intrableb viscoelastic spacer. All needlings were augmented by MMC and performed in the operating room, by a single surgeon. The choice between HF and H5 was randomised. Postoperative data for IOP, number of glaucoma medications (GMs), antimetabolite injections (AMIs), bleb morphology and all complications were recorded at day 1 (D1), weeks 1 (W1), 4 (W4), month 6 (M6) and last visit. Success was defined as IOP≤21mmHg and 20% reduction in IOP from baseline Results: Age was comparable between groups (HF vs H5; 66 vs 72 years; p=0.13*). There were no differences in mean postoperative IOP or GMs at any time point between groups (mean IOP = HF vs H5; prior to needling=19.8mmHg vs 18.7mmHg, p=0.48*; D1=10.4mmHg vs 10.2mmHg, p=0.85*; W1=12.3mmHg vs 13.8mmHg, p=0.33*; W4=15.7mmHg vs 15.1mmHg, p=0.69*; M6=14.3mmHg vs 13.8mmHg, p=0.69*. Mean GMs= HF vs H5; prior to needling=1.7 vs 1.7, p=0.96*; M6=0.9 vs 1.1, p=0.66*). Success rates were comparable between HF and H5 groups (74% vs 71%). Requirements for AMIs were similar between groups; post filtration surgery and prior to needling (mean AMIs 1.54 vs 1.65 p=0.82*) and within the first six months following needling ( 0.77 vs 0.65 p=0.70*). Complication rates were infrequent in both groups (9%, HF; 3%, H5) * two-sample t-test Conclusions: The early to mid-term success rates of needlings using intrableb spacers were high. However there were no differences observed between the use of crosslinked and high molecular weight sodium hyaluronate

Optimized synthesis of O-carboxymethyl-N,N,N-trimethyl chitosan.

We present here the synthesis of a highly O-carboxymethylated chitosan derivative. First, an improved protocol for the two-step synthesis of N-trimethyl chitosan (TMC) from chitosan was developed, yielding a maximum degree of quaternization (DQ) of up to 46.6%. Successively, the chitosan derivative O-carboxymethyl-N-trimethyl chitosan (CMTMC) was synthesized from the TMC obtained by applying an optimized synthesis pathway. In contrast to previous reports, the optimized protocol was shown to yield very high rates (>85%) of O-carboxymethylation of CMTMC, as shown by (1)H NMR and heteronuclear single quantum correlation ((1)H-(13)C HSQC). Finally, in vitro cytocompatibility (viability >80%) of the polymer was demonstrated using human fibroblasts.

Peptide-decorated chitosan derivatives enhance fibroblast adhesion and proliferation in wound healing.

RGD peptide sequences are known to regulate cellular activities by interacting with α5β1, αvβ5 and αvβ3 integrin, which contributes to the wound healing process. In this study, RGDC peptide was immobilized onto chitosan derivative 1,6-diaminohexane-O-carboxymethyl-N,N,N-trimethyl chitosan (DAH-CMTMC) to display RGDC-promoting adhesion for enhanced wound healing. The efficiency of N-methylation, O-carboxymethylation and spacer grafting was quantitatively and qualitatively analyzed by (1)H NMR and FTIR, yielding 0.38 degree of substitution for N-methylation and >0.85 for O-carboxymethylation. The glass transition temperatures for chitosan derivatives were also studied. Peptide immobilization was achieved through sulfhydryl groups using sulfosuccinimidyl (4-iodoacetyl)amino-benzoate (sulfo-SIAB method). RGDC immobilized peptide onto DAH-CMTMC was found to be about 15.3μg/mg of chitosan derivative by amino acid analysis (AAA). The significant increase of human dermal fibroblast (HDF) viability in vitro over 7 days suggests that RGDC-functionalized chitosan may lead to enhanced wound healing (viability >140%). Moreover, bio-adhesion and proliferation assays confirmed that coatings of RGDC-functionalized chitosan derivatives exhibit in vitro wound healing properties by enhancing fibroblast proliferation and adhesion. These results showed that RGDC peptide-functionalized chitosan provides an optimal environment for fibroblast adhesion and proliferation.

Microesferas de quitosana reticuladas com tripolifosfato utilizadas para remoção da acidez, ferro(III) e manganês(II) de águas contaminadas pela mineração de carvão

Considerable attention has been paid to chitosan and derivatives as efficient adsorbents of pollutants such as metal ions and dyes in aqueous medium. Nevertheless, no report can be found on the remedial actions of chitosan microspheres crosslinked with tripolyphosphate to control acidity, iron (III) and manganese (II) contents in wastewaters from coal mining. In this work, chitosan microspheres crosslinked with tripolyphosphate were used for the neutralization of acidity and removal of Fe (III) and Mn (II) from coal mining wastewaters. The study involved static and dinamic methods. The neutralization capacity of the surface of the static system was 395 mmol of H3O+ per kilogram of microspheres, higher than that of the dynamic one (223 mmol kg-1). The removal of Fe(III) in wastewater was of 100% and that of Mn(II) was 90%.

Adsorção de arsênio(V) pela quitosana ferro - III reticulada

The removal of As(V) by a crosslinked iron(III)-chitosan adsorbent was evaluated under various conditions. The adsorption capacity of CH-FeCL was around 54 mg/g of As(V). The kinetics of adsorption obeys a pseudo-first-order model with rate constants equal to 0.022, 0.028, and 0.033 min-1 at 15, 25 and 35 ºC respectively. Adsorption data were well described by the Langmuir model, although they could be modeled also by the Langmuir-Freundlich equation. The maximum adsorption capacity, calculated with the Langmuir model, was 127 mg g-1 of As(V). The inhibition by competing anions is dependant on their kind and valence.

Preparação e caracterização de blendas de quitosana/poli(álcool vinílico) reticuladas quimicamente com glutaraldeído para aplicação em engenharia de tecido

In this study, novel Chitosan/PVA based films were chemically crosslinked by glutaraldehyde, under pH=(4,00 ±0,05), in order to achieve structures tailored for wound tissue engineering applications. Both precursors and developed films were characterized by FTIR, SEM and XRD in order to determine the presence of chemicals groups and nanostructural order, respectively. The results have shown that the glutaraldehyde crosslinking have altered the crystallinity of pure chitosan and the increase on the C=N bands and simultaneous decrease on NH2 bands suggested that Chitosan/GA crosslinking has preference to occur in carbon-2 of the saccharide ring by the Schiff's base reaction. Also, FTIR spectroscopy clearly showed that crosslinking has also taken place with blends of PVA and chitosan. The mechanical properties presented high degree dependence with on the increase of the content of chitosan and glutaraldehyde. The results have indicated that, by controlling the ratio [PVA]/[chitosan] in the blends and the extent of chemical crosslinking, it was possible to tailor the hybrid network produced aiming to obtain properties of interest for the specific application.

Imobilização da lacase em micropartículas de quitosana obtidas por spray drying e usadas na construção de biossensores

Biosensors based on laccase immobilized on microparticles of chitosan crosslinked with tripolyphosphate (biosensor I) and glyoxal (biosensor II) obtained by spray drying for the determinations of rutin in pharmaceutical formulations were developed. Under optimized operational conditions (pH 4.0, frequency of 30 Hz, pulse amplitude of 40 mV and scan increment of 2.0 mV) two analytical curves were obtained for both biosensors showing a detection limit of 6.2x10-8 mol L-1 for biosensor (I) and 2.0x10-8 mol L-1 for biosensor (II). The recovery of rutin from pharmaceutical sample ranged from 90.7 to 105.0% and the lifetime of these biosensors were 4 months (at least 400 determinations).

Elimination of dyes from aqueous solutions using iron oxides and chitosan as adsorbents: a comparative study

This work investigates the adsorption of Alizarin, Eriochrome Blue Black R and Fluorescein using chitosan, goethite and magnetite as adsorbents. For Alizarin, the best adsorbent is chitosan with a Langmuir parameter of 15.8 mmol dye/g adsorbent. For Eriochrome Blue Black R only 1.94 mmol dye/g chitosan is adsorbed. Langmuir parameters for the Alizarin adsorption on both iron oxides display one or two orders of magnitude lower than for chitosan and two orders of magnitude lower in the case of Eriochrome Blue Black R. Fluorescein does not adsorb in appreciable amounts on chitosan and it presents the lower affinity on the iron oxides.

Removal of phenol and conventional pollutants from aqueous effluent by chitosan and chitin

The present study deals with phenol adsorption on chitin and chitosan and removal of contaminants from wastewater of a petroleum refinery. The adsorption kinetic data were best fitted to first- and second-order models for chitosan and chitin, respectively. The results of adsorption isotherms showed Langmuir model more appropriately described than a Freundlich model for both adsorbents. The adsorption capacity was 1.96 and 1.26 mg/g for chitin and chitosan, respectively. Maximum removal of phenol was about 70-80% (flow rate: 1.5 mL/min, bed height: 18.5 cm, and 30 mg/L of phenol. Wastewater treatment with chitin in a fixed-bed system showed reductions of about 52 and 92% for COD and oil and greases, and for chitosan 65 and 67%, respectively. The results show improvement of the effluent quality after treatment with chitin and chitosan.