Angela Fabiano

Mucoadhesive nanoparticles made of thiolated quaternary chitosan crosslinked with hyaluronan

Mucoadhesive polymeric nanoparticles intended for drug transport across the gastrointestinal mucosa were prepared from quaternary ammonium-chitosan conjugates synthesised from reduced-MW chitosan (32 kDa). Conjugates contained pendant moieties of 2-4 adjacent diethyl-dimethylene-ammonium groups substituted on repeating units (26-55%). Conjugates were thiolated via amide bonds with thioglycolic acid to yield products with thiol content in the 35-87 μmol/g range. Nanoparticles with mean size in the 270-370 nm range and positive zeta-potential (+3.7 to +12.5 mV) resulted from ionotropic gelation of the thiolated conjugates with de-polymerised hyaluronic acid (470 kDa). The nanoparticles were fairly stable in size and thiol content and showed a significant mucoadhesivity, matching and even exceeding that of the constituent polymers. Nanoparticles were internalised by endothelial progenitor cells in direct relation to their surface charge intensity. Nanoparticle uptake significantly improved cell viability and resistance to oxidation. The lyophilised nanoparticles were re-dispersible and could make a manageable formulation for oral use.

Effect of different chitosan derivatives on in vitro scratch wound assay: A comparative study

Different strategies have been developed to make the wound-healing process faster and less painful. Recently, numerous studies demonstrated the ability of chitosan to accelerate wound healing. Aim of the present study has been to evaluate the effect of different chitosan derivatives to improve wound healing process. Quaternary ammonium-chitosan conjugates with low or high molecular weight (MW) and their thiolated derivatives effect were studied on human skin fibroblasts in terms of viability and migration (scratch wound assay). Results were compared both with basal medium (untreated cells) and with a positive control (chitosan chlorhydrate). After 24h both high and low MW chitosan derivatives were non-toxic up to 10 μg/ml. The concentration of 10 μg/ml was used for wound healing experiments. High-MW quaternary ammonium-chitosan conjugates bearing thiol groups on their chains were more effective in promoting cell migration than the non-thiolated conjugates and the chitosan chlorhydrate. Moreover, they significantly improve wound healing process compared to untreated cells. According to the present in vitro preliminary results, high MW thiolated quaternary ammonium-chitosan conjugates can be considered good candidates for the management of wounds.

Delivery of natural polyphenols by polymeric nanoparticles improves the resistance of endothelial progenitor cells to oxidative stress

PURPOSE Bone marrow-derived endothelial progenitor cells (EPCs) circulate into peripheral blood and significantly contribute to neo-vascularisation and re-endothelialisation as part of the process of vascular repair. Several studies have reported decreased EPC number in the presence of oxidative stress. Aim of this study was to evaluate the validity of mucoadhesive polymeric nanoparticles as a delivery system of natural products able to protect EPCs from oxidative stress. METHODS The total polyphenol content and antioxidant capacity of red grape seed extract (GSE) either pre-veraison (p-GSE) or ripe (r-GSE) were measured. Cell viability was evaluated by WST-1 assay. Nanoparticles were prepared by ionotropic crosslinking of two structurally different thiolated quaternary ammonium-chitosan conjugates. A hyaluronic acid solution, containing p-GSE or r-GSE, was added to a stirred solution of each of the two chitosan derivatives to obtain p- or r-GSE loaded nanoparticles (NP) of two types. RESULTS Both GSE types demonstrated strong antioxidant capacity. p-GSE showed a higher content in total polyphenols compared to r-GSE. NP size was in the 310-340 nm range, with 24 h stability, and nearly 100% encapsulation efficiency for both GSE types. NP were internalized by cells to an extent related directly with their surface charge intensity. GSE-NP uptake significantly improved cell viability and resistance to oxidation. CONCLUSIONS Nanotechnology has a great potential in nutraceutical delivery. The present results suggest that NP is a highly promising polyphenol carrier system particularly useful to protect EPCs from oxidative stress, thus improving their survival.

Mucoadhesive nano-sized supramolecular assemblies for improved pre-corneal drug residence time

Abstract Context: Mucoadhesive nanoparticles were compared with non-aggregated constituent polymers for effect on pre-corneal residence of dexamethasone phosphate (DP) or met-enkephalin (ME), administered by eye-drops to rabbits. Objective: Deepening the knowledge of ophthalmic nanoparticulate systems in terms of ability to prolong pre-corneal residence. Materials and methods: Medicated nanoparticles resulted from gelation of quaternary ammonium–chitosan conjugate or its thiolated derivative with hyaluronan in the presence of drug. Particles were analyzed by light scattering. Dialysis removed non-encapsulated drug, dynamic dialysis measured drug–polymer interactions, and lyophilization-stabilized product. Dispersions were regenerated from lyophilized products. Also solutions of non-thiolated or thiolated chitosan derivative were administered. Mean drug residence time (MRT) in tears was determined by collecting samples from lower marginal tear strip of albino rabbits using capillaries. Results and discussion: Nanoparticle size of regenerated dispersions was 400–430 nm (DP-systems), 360–370 nm (ME-systems); the drug content was 2.5 mg/mL (DP) or 0.3 mg/mL (ME). The MRT for DP nanoparticles from non-thiolated derivative was higher than that for non-aggregated polymer, due to stronger concurrent interactions of positively charged nanoparticles with ocular surface and drug. Thiolated polymer nanoparticles and non-aggregated parent polymer, both interacting weakly with DP, showed similar MRT values. The MRT of ME could only be enhanced by protecting drug from enzymatic hydrolysis. This was done by nanoparticle systems, whereas non-aggregated polymers were ineffective. Conclusion: Developing a nanoparticle system rather than a solution of mucoadhesive polymer, for prolonging pre-corneal residence, is convenient, provided nanoparticles interact strongly with both ocular surface and drug, or protect drug from metabolic degradation.

Thermosensitive hydrogel based on chitosan and its derivatives containing medicated nanoparticles for transcorneal administration of 5-fluorouracil

A thermosensitive ophthalmic hydrogel (TSOH) – fluid at 4°C (instillation temperature), semisolid at 35°C (eye temperature), which coupled the dosing accuracy and administration ease of eyedrops with the increased ocular bioavailability of a hydrogel – was prepared by gelling a chitosan hydrochloride (ChHCl) solution (27.8 mg/mL) medicated with 1.25 mg/mL 5-fluorouracil (5-FU) with β-glycerophosphate 0.8 mg/mL. Polymer mixtures, where Ch was partially (10%, 15%, or 20%) replaced by quaternary ammonium–chitosan conjugates (QA-Ch) or thiolated derivatives thereof, were also used to modulate 5-FU-release properties of TSOH. Also, Ch-based nanoparticles (NPs; size after lyophilization and redispersion 341.5±15.2 nm, polydispersity 0.315±0.45, ζ-potential 10.21 mV) medicated with 1.25 mg/mL 5-FU prepared by ionotropic cross-linking of Ch with hyaluronan were introduced into TSOH. The 5-FU binding by TSOH polymers in the sol state was maximum with plain Ch (31.4%) and tended to decrease with increasing QA presence in polymer mixture. 5-FU release from TSOH with or without NPs was diffusion-controlled and linear in √t. The different TSOH polymers were compared on a diffusivity basis by comparing the slopes of √t plots. These showed a general decrease with NP-containing TSOH, which was the most marked with the TSOH, where Ch was 20% replaced by the derivative QA-Ch50. This formulation and that not containing NP were instilled in rabbits and the 5-FU transcorneal penetration was measured by analyzing the aqueous humor. Both TSOH solutions increased the area under the curve (0–8 hours) 3.5 times compared with the plain eyedrops, but maximum concentration for the NP-free TSOH was about 0.65 µg/mL, followed by a slow decline, while the NP-containing one showed a plateau (0.25–0.3 µg/mL) in a time interval of 0.5–7 hours. This is ascribed to the ability of this TSOH to control drug release to a zero order and that of NPs to be internalized by corneal cells.

Mucoadhesivity and release properties of quaternary ammonium–chitosan conjugates and their nanoparticulate supramolecular aggregates: An NMR investigation

Selective relaxation rate measurements effectively proved the affinity of dexamethasone 21-phosphate disodium salt for quaternary ammonium-chitosan conjugates, their thiolated derivatives and the corresponding nanostructured aggregates. Affinity was also probed by dynamic dialysis. The release profile of dexamethasone loaded nanoparticles was defined by quantitative NMR and interrupted dialysis experiments, and mucoadhesivity of empty nanoparticles was effectively probed by selective relaxation rate measurements.

Sucrosomial® iron absorption studied by in vitro and ex-vivo models

ABSTRACT This paper presents a comparative evaluation of different oral ferric iron formulations for ability to retain Fe3+ in simulated gastric fluid (SGF), be internalized by cells lining intestinal epithelium, and cross it to reach the bloodstream. In all formulations iron was ferric pyrophosphate, the excipients were different types and fractions of lecithin plus sucrose esters of fatty acids matrix (Sideral® RM; PRT1; PRT2) or lecithin without sucrester (SUN). Dissolution kinetics of formulations in SGF was studied by USP method. The ability of the formulations to promote iron intestinal absorption was evaluated by the Caco‐2 cell model, measuring cellular ferritin content, and by the excised rat intestine model, yielding apparent permeability parameters (Papp). All formulations limited iron release in SGF to ≤10%. Sideral® RM was by far the most absorbed by Caco‐2, as ferritin content was in the order: Sideral® RM ≫ PRT2 > PRT1 > SUN > control. The Fe3+ crossing the intestinal barrier was in part reduced to Fe2+ by epithelial enzymes, in part it was carried by formulation rearrangement into nano‐structures able to protect it from reduction and apt for internalization by epithelium cells. Papp parameters were in the order: Sideral® RM ≫ PRT1 > PRT2 > SUN = control. Relevance of transepithelial Fe2+carrier, DMT‐1, to Fe3+ transport was ruled out using a DMT‐1 inhibitor. In conclusion, Sideral® RM retains iron in SGF, and is the most suitable for Fe3+ internalization by Caco‐2 cells, Fe3+ protection from enzymatic reduction and promotion of Fe3+ absorption across intestinal epithelium, non‐mediated by DMT‐1. Graphical Abstract Figure. No Caption available.

About the impact of water movement on the permeation behaviour of nanoparticles in mucus

The purpose of this study was to establish a method to evaluate the diffusion behaviour of nanoparticles (NP) in mucus taking also the water movement into account. For this purpose, NP based on different chitosan derivatives, either thiolated or not, and marked with fluorescein diacetate were prepared by ionotropic gelation with hyaluronan. NP size and polydispersity were in the respective intervals 363.5±33.3-385.7±36.5nm, and 0.35±0.11-0.39±0.10. An in vitro study of water-assisted NP transport through mucus was realized by filling the barrel of a syringe kept in vertical position, tip down, with mucus. Then a bottom-to-surface PBS flow across the mucus layer was realized by connecting the tip of the syringe to the bottom of a vertical cylindrical vessel by a flexible tubing, filling the vessel with PBS, level with the surface of the mucus layer in the syringe, and dripping PBS into the vessel without causing any phase mixing. Although the mucoadhesive NP interact more strongly with the mucus, yet they are able to overcome this barrier with the aid of the water movement from lumen to epithelium. This new method promises to be more predictive of in vivo NP transport across the mucus than already reported methods, as it takes into account the water movement and regulates its contribution to the physiologic value.

A water-soluble, mucoadhesive quaternary ammonium chitosan-methyl-β-cyclodextrin conjugate forming inclusion complexes with dexamethasone

AbstractThe ocular bioavailability of lipophilic drugs, such as dexamethasone, depends on both drug water solubility and mucoadhesion/permeation. Cyclodextrins and chitosan are frequently employed to either improve drug solubility or prolong drug contact onto mucosae, respectively. Although the covalent conjugation of cyclodextrin and chitosan brings to mucoadhesive drug complexes, their water solubility is restricted to acidic pHs. This paper describes a straightforward grafting of methyl-β-cyclodextrin (MCD) on quaternary ammonium chitosan (QA-Ch60), mediated by hexamethylene diisocyanate. The resulting product is a water-soluble chitosan derivative, having a 10-atom long spacer between the quaternized chitosan and the cyclodextrin. The derivative is capable of complexing the model drug dexamethasone and stable complexes were also observed for the lyophilized products. Furthermore, the conjugate preserves the mucoadhesive properties typical of quaternized chitosan and its safety as solubilizing excipient for ophthalmic applications was preliminary assessed by in vitro cytotoxicity evaluations. Taken as a whole, the observed features appear promising for future processing of the developed product into 3D solid forms, such as controlled drug delivery systems, films or drug eluting medical devices.

Ex Vivo and in Vivo Study of Sucrosomial® Iron Intestinal Absorption and Bioavailability

The present study aimed to demonstrate that Sideral® RM (SRM, Sucrosomial® Raw Material Iron) is transported across the excised intestine via a biological mechanism, and to investigate the effect that this transport route may produce on oral iron absorption, which is expected to reduce the gastrointestinal (GI) side effects caused by the bioavailability of non-absorbed iron. Excised rat intestine was exposed to fluorescein isothiocyanate (FITC)-labeled SRM in Ussing chambers followed by confocal laser scanning microscopy to look for the presence of fluorescein-tagged vesicles of the FITC-labeled SRM. To identify FITC-labeled SRM internalizing cells, an immunofluorescence analysis for macrophages and M cells was performed using specific antibodies. Microscopy analysis revealed the presence of fluorescein positive particulate structures in tissues treated with FITC-labeled SRM. These structures do not disintegrate during transit, and concentrate in macrophage cells. Iron bioavailability was assessed by determining the time-course of Fe3+ plasma levels. As references, iron contents in liver, spleen, and bone marrow were determined in healthy rats treated by gavage with SRM or ferric pyrophosphate salt (FP). SRM significantly increased both area under the curve (AUC) and clearance maxima (Cmax) compared to FP, thus increasing iron bioavailability (AUCrel = 1.8). This led to increased iron availability in the bone marrow at 5 h after single dose gavage.