Olive Leaf Extracts from Three Italian Olive Cultivars Exposed to Drought Stress Differentially Protect Cells against Oxidative Stress.

Olive leaves are an abundant by-product of olive oil production. Olive leaf extracts (OLEs) are rich in polyphenols, which can be used for health benefits. As polyphenols are the main antioxidant molecules in plants, plants typically increase their polyphenol content when exposed to drought stress. However, the phenolic profile of OLEs can vary in relation to the origin and variety of the plant material. In this work, olive leaf extracts from three different Italian olive cultivars (Giarraffa, Leccino, and Maurino) both exposed and not exposed to drought stress were studied in terms of antioxidant properties and profile, intestinal permeation, and protection against oxidative stress of human umbilical vein endothelial cells (HUVECs), since HUVECs are considered a model to study a wide range of diseases. OLEs from stressed Maurino and Giarraffa plants showed the highest increase in antioxidant capacity compared to controls. The phenolic profile of Maurino' was mainly increased by water deficit, with a large increase in the compounds oleuropein and luteolin-7-O-rutinoside. All tested extracts exposed to a water deficit protected HUVECs against oxidative stress by reducing ROS production, and this effect was more pronounced in OLEs from Giarraffa and Maurino exposed to drought stress compared to all other extracts. Finally, OLE from the stressed Giarraffa group showed a higher apparent permeability of antioxidant molecules than that of Maurino.

Betaine- and L-Carnitine-Based Ionic Liquids as Solubilising and Stabilising Agents for the Formulation of Antimicrobial Eye Drops Containing Diacerein.

The therapeutic efficacy of topically administered drugs, however powerful, is largely affected by their bioavailability and, thus, ultimately, on their aqueous solubility and stability. The aim of this study was to evaluate the use of ionic liquids (ILs) as functional excipients to solubilise, stabilise, and prolong the ocular residence time of diacerein (DIA) in eye drop formulations. DIA is a poorly soluble and unstable anthraquinone prodrug, rapidly hydrolysed to rhein (Rhe), for the treatment of osteoarthritis. DIA has recently been evaluated as an antimicrobial agent for bacterial keratitis. Two ILs based on natural zwitterionic compounds were investigated: L-carnitine C6 alkyl ester bromide (Carn6), and betaine C6 alkyl ester bromide (Bet6). The stabilising, solubilising, and mucoadhesive properties of ILs were investigated, as well as their cytotoxicity to the murine fibroblast BALB/3T3 clone A31 cell line. Two IL-DIA-based eye drop formulations were prepared, and their efficacy against both Staphylococcus aureus and Pseudomonas aeruginosa was determined. Finally, the eye drops were administered in vivo on New Zealand albino rabbits, testing their tolerability as well as their elimination and degradation kinetics. Both Bet6 and Carn6 have good potential as functional excipients, showing solubilising, stabilising, mucoadhesive, and antimicrobial properties; their in vitro cytotoxicity and in vivo ocular tolerability pave the way for their future use in ophthalmic applications.

Bergamot Essential Oil: A Method for Introducing It in Solid Dosage Forms.

Bergamot essential oil (BEO) possess antimicrobial, antiproliferative, anti-inflammatory, analgesic, neuroprotective, and cardiovascular effects. However, it is rich in volatile compounds, e.g., limonene, that are susceptible to conversion and degradation reactions. The aim of this communication was to prepare a conjugate based on a quaternary ammonium chitosan derivative (QA-Ch) and methyl-ßCD (MCD), coded as BEO/QA-Ch-MCD, to encapsulate BEO in order to stabilize its volatile compounds, eliminate its unpleasant taste, and convert the oil in a solid dosage form. The obtained conjugate, BEO/QA-Ch-MCD, was highly soluble and had a percentage of extract association efficiency (AE %), in terms of polyphenols and limonene contents, of 22.0 ± 0.9 and 21.9 ± 1.2, respectively. Moreover, stability studies under UV stress in simulated gastric fluid showed that BEO/QA-Ch-MCD was more able to protect polyphenols and limonene from degradation compared to free BEO or BEO complexed with MCD (BEO/MCD). The complexation and subsequent lyophilization allowed the transformation of a liquid into a solid dosage form capable of eliminating the unpleasant taste of the orally administered oil and rendering the solid suitable to produce powders, granules, tablets, etc. These solid oral dosage forms, as they come into contact with physiological fluids, could generate nanosized agglomerates able to increase the stability of their active contents and, consequently, their bioavailability.

Comparing Metabolomic and Essential Oil Fingerprints of Citrus australasica F. Muell (Finger Lime) Varieties and Their In Vitro Antioxidant Activity.

Comparative chemical analyses among peel and pulp essential oils (EOs) and methanolic extracts of four Citrus australasica varieties (Red, Collette, Pink Ice, and Yellow Sunshine), and the hybrid Faustrime, were performed using GC-MS and UHPLC-DAD-HR-Orbitrap/ESI-MS. Peel and pulp extracts were also analysed for their in vitro antioxidant activity on a Balb/3T3 clone A31 mouse embryo fibroblast cell line. The results of peel and pulp EOs were mainly characterised by monoterpenes and sesquiterpenes, respectively. All peels displayed a higher total phenol content (TPC) than pulps, and consequently a greater antioxidant activity. Collette peels and Pink Ice pulps showed the highest amount of identified flavonoids (e.g., luteolin, isosakuranetin, and poncirin derivatives). Collette and Red peels were rich in anthocyanins (delphinidin and petunidin glycosides), exhibiting the maximum protective activity against induced oxidative damage. In conclusion, finger lime fruits are good sources of health-promoting phytocomplexes, with the Red, Collette, and Pink Ice varieties being the most promising.

Biomaterials for Regenerative Medicine in Italy: Brief State of the Art of the Principal Research Centers.

Regenerative medicine is the branch of medicine that effectively uses stem cell therapy and tissue engineering strategies to guide the healing or replacement of damaged tissues or organs. A crucial element is undoubtedly the biomaterial that guides biological events to restore tissue continuity. The polymers, natural or synthetic, find wide application thanks to their great adaptability. In fact, they can be used as principal components, coatings or vehicles to functionalize several biomaterials. There are many leading centers for the research and development of biomaterials in Italy. The aim of this review is to provide an overview of the current state of the art on polymer research for regenerative medicine purposes. The last five years of scientific production of the main Italian research centers has been screened to analyze the current advancement in tissue engineering in order to highlight inputs for the development of novel biomaterials and strategies.

Combination of Two Kinds of Medicated Microparticles Based on Hyaluronic Acid or Chitosan for a Wound Healing Spray Patch.

Olive leaves extract (OLE) has been extensively studied as antioxidant and antibiotic and these characteristics make it particularly interesting for use on wounds. For this reason, the aim of this study was to introduce OLE in microparticles (MP) of hyaluronic acid (MPHA-OLE) or chitosan (MPCs-OLE) to obtain a spray patch for the treatment of wounds in anatomical areas that are difficult to protect with traditional patches. The MP were characterized for particle size and ability to protect OLE from degradation, to absorb water from wound exudate, to control OLE release from MP. The MPHA and MPCs medicated or not and mixtures of the two types in different proportions were studied in vitro on fibroblasts by the scratch wound healing assay. The MP size was always less than 5 µm, and therefore, suitable for a spray patch. The MPCs-OLE could slow down the release of OLE therefore only about 60% of the polyphenols contained in it were released after 4 h. Both MPHA and MPCs could accelerate wound healing. A 50% MPHA-OLE-50% MPCs-OLE blend was the most suitable for accelerating wound healing. The MPHA-OLE-MPCs-OLE blends studied in this work were shown to have the characteristics suitable for a spray patch, thus giving a second life to the waste products of olive growers.

Impact of Peels Extracts from an Italian Ancient Tomato Variety Grown under Drought Stress Conditions on Vascular Related Dysfunction.

BACKGROUND: Tomato by-products contain a great variety of biologically active substances and represent a significant source of natural antioxidant supplements of the human diet. The aim of the work was to compare the antioxidant properties of a by-product from an ancient Tuscan tomato variety, Rosso di Pitigliano (RED), obtained by growing plants in normal conditions (-Ctr) or in drought stress conditions (-Ds) for their beneficial effects on vascular related dysfunction. METHODS: The antioxidant activity and total polyphenol content (TPC) were measured. The identification of bioactive compounds of tomato peel was performed by HPLC. HUVEC were pre-treated with different TPC of RED-Ctr or RED-Ds, then stressed with H2O2. Cell viability, ROS production and CAT, SOD and GPx activities were evaluated. Permeation of antioxidant molecules contained in RED across excised rat intestine was also studied. RESULTS: RED-Ds tomato peel extract possessed higher TPC than compared to RED-Ctr (361.32 ± 7.204 mg vs. 152.46 ± 1.568 mg GAE/100 g fresh weight). All extracts were non-cytotoxic. Two hour pre-treatment with 5 µg GAE/mL from RED-Ctr or RED-Ds showed protection from H2O2-induced oxidative stress and significantly reduced ROS production raising SOD and CAT activity (* p < 0.05 and ** p < 0.005 vs. H2O2, respectively). The permeation of antioxidant molecules contained in RED-Ctr or RED-Ds across excised rat intestine was high with non-significant difference between the two RED types (41.9 ± 9.6% vs. 26.6 ± 7.8%). CONCLUSIONS: RED-Ds tomato peel extract represents a good source of bioactive molecules, which protects HUVECs from oxidative stress at low concentration.

Antioxidant Effect of Cocoa By-Product and Cherry Polyphenol Extracts: A Comparative Study.

BACKGROUND: Recent studies have highlighted the importance of cherry and cocoa extracts consumption to protect cells from oxidative stress, paying particular attention to cocoa by-products. This study aims to investigate the protective effect of cocoa husk extract (CHE) and cherry extracts (CE) against ROS-induced oxidative stress in Human Umbilical Vein Endothelial Cells (HUVECs). METHODS: CE and CHE had antioxidant activity characterized by total polyphenols content (TPC). HUVECs were treated for 2 h and 24 h with increasing TPC concentrations of CE and CHE (5-10-25-50-100 µg Gallic Acid Equivalent (GAE)/mL) and then with H2O2 for 1 h. Cell viability and ROS production were evaluated. CE and CHE polyphenols permeability on excised rat intestine were also studied. RESULTS: CE and CHE showed a similar antioxidant activity (2.5 ± 0.01 mmol Fe2+/100 g FW (fresh weight) and 2.19 ± 0.09 mmol Fe2+/100 g FW, respectively, p > 0.05) whereas CHE had a higher TPC (7105.0 ± 96.9 mg GAE/100 g FW) than CE (402.5 ± 8.4 mg GAE/100 g), p < 0.05. The in vitro viability assay showed that both extracts were non-cytotoxic. CHE resulted in protection against ROS at lower concentrations than CE. CHE showed a 2-fold higher apparent permeability compared to CE. CONCLUSIONS: CHE represents a high-value antioxidant source, which is interesting for the food and pharmaceutical industries.

Antioxidant and Anti-Inflammatory Properties of Cherry Extract: Nanosystems-Based Strategies to Improve Endothelial Function and Intestinal Absorption.

Cherry fruit has a high content in flavonoids. These are important diet components protecting against oxidative stress, inflammation, and endothelial dysfunction, which are all involved in the pathogenesis of atherosclerosis, which is the major cause of cardiovascular diseases (CVD). Since the seasonal availability of fresh fruit is limited, research has been focused on cherry extract (CE), which also possesses a high nutraceutical potential. Many clinical studies have demonstrated the nutraceutical efficacy of fresh cherries, but only a few studies on CE antioxidant and anti-inflammatory activities have been carried out. Here, the results concerning the antioxidant and anti-inflammatory activities of CE are reviewed. These were obtained by an in vitro model based on Human Umbilical Vein Endothelial Cells (HUVEC). To clarify the CE mechanism of action, cells were stressed to induce inflammation and endothelial dysfunction. Considering that antioxidants' polyphenol compounds are easily degraded in the gastrointestinal tract, recent strategies to reduce the degradation and improve the bioavailability of CE are also presented and discussed. In particular, we report on results obtained with nanoparticles (NP) based on chitosan derivatives (Ch-der), which improved the mucoadhesive properties of the chitosan polymers, as well as their positive charge, to favor high cellular interaction and polyphenols intestinal absorption, compared with a non-mucoadhesive negative surface charged poly(lactic-co-glycolic) acid NP. The advantages and safety of different nanosystems loaded with natural CE or other nutraceuticals are also discussed.

Impact of Different Mucoadhesive Polymeric Nanoparticles Loaded in Thermosensitive Hydrogels on Transcorneal Administration of 5-Fluorouracil.

In a previous paper a thermosensitive hydrogel formulation based on chitosan or its derivatives (TSOH), containing medicated chitosan nanoparticles (Ch NP) for transcorneal administration of 5-fluorouracil (5-FU) was described. The Ch NP-containing TSOH allowed a time-constant 5-FU concentration in the aqueous for 7 h from instillation. The aim of the present work was to study the impact of the surface characteristics of new NP contained in TSOH on ocular 5-FU bioavailability. The Ch derivatives used to prepare NP were quaternary ammonium-Ch conjugate (QA-Ch), S-protected derivative thereof (QA-Ch-S-pro), and a sulphobutyl chitosan derivative (SB-Ch). All NP types had 300-400 nm size, 16-18% encapsulation efficiency, and retained the entrapped drug for at least 15 h. Drug release from TSOH containing NP based on QA-Ch or QA-Ch-S-pro was virtually equal, whereas with TSOH containing NP based on SB-Ch was significantly slower. Instillation, in rabbit eyes, of NP-containing TSOH based on QA-Ch or SB-Ch led to a plateau in the aqueous concentration vs. time plot in the 1-10 h range with significantly enhanced area under curve (AUC). Negative charges on the NP surface slowed down 5-FU release from TSOH while positive charges increased NP contact with the negatively charged ocular surface. Either results in enhanced ocular bioavailability.

Anti-Inflammatory Effect of Cherry Extract Loaded in Polymeric Nanoparticles: Relevance of Particle Internalization in Endothelial Cells.

This study aimed at evaluating the anti-inflammatory effect of natural cherry extract (CE), either free or encapsulated in nanoparticles (NPs) based on chitosan derivatives (Ch-der) or poly(lactic-co-glycolic acid) (PLGA), on human umbilical vein endothelial cells (HUVEC). CE from Prunus avium L. was characterized for total polyphenols, flavonoids, and anthocyanins content. CE and CE-loaded NP cytotoxicity and protective effect on lipopolysaccharide (LPS)-stressed HUVEC were tested by water-soluble tetrazolium salt (WST-1) assay. Pro- and anti-inflammatory cytokines (TNF-α, IL-6, IL-10, and PGE2) released by HUVEC were quantified by enzyme-linked immunosorbent assay (ELISA). All NP types were internalized into HUVEC after 2 h incubation and promoted the anti-inflammatory effect of free CE at the concentration of 2 µg gallic acid equivalents (GAE)/mL. CE-loaded Ch-der NPs showed the highest in vitro uptake and anti-inflammatory activity, blunting the secretion of IL-6, TNF-α, and PGE2 cytokines. Moreover, all NPs reduced the production of nitric oxide and NLRP3 inflammasome, and had a stronger anti-inflammatory effect than the major corticosteroid dexamethasone. In particular, the results demonstrate that natural CE protects endothelial cells from inflammatory stress when encapsulated in NPs based on quaternary ammonium chitosan. The CE beneficial effects were directly related with in vitro internalization of CE-loaded NPs.

Cherry Extract from Prunus avium L. to Improve the Resistance of Endothelial Cells to Oxidative Stress: Mucoadhesive Chitosan vs. Poly(lactic-co-glycolic acid) Nanoparticles.

Polyphenolic compounds contained in cherry extract (CE) are well known for their antioxidant and anti-inflammatory properties. Unfortunately, most of these natural compounds have low oral bioavailability, reducing their widespread use. Here, different concentrations of polyphenol-rich CE from Tuscany (Italy), encapsulated in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), were compared with those encapsulated in two NP types, different from each other in terms of mucoadhesivity, obtained with chitosan derivatives (Ch-der), regarding CE gastrointestinal (GI) permeability and protective effect on oxidative stress. Different NP systems were physico-chemically characterized, and the antioxidant GI permeability was evaluated in a triple-cell co-culture model (Caco-2/HT29-MTX/Raji B), resembling the intestine. PLGA NPs efficiently entrapped CE (up to 840 µg gallic acid equivalent (GAE)/mL) without altering size (210 nm), polydispersity index (0.05), or zeta potential (-10.7 mV). Such NPs promoted permeation of encapsulated CE at a CE polyphenolic concentration of at least 2 µg GAE/mL. More mucoadhesive NPs from Ch-der, coded quaternary ammonium S-protected thiolated chitosan (QA-Ch-S-pro) NP, promoted CE GI permeation of 0.5 µg GAE/mL. At higher concentrations of Ch-der polymers, the resulting NPs containing CE were toxic toward Caco-2 and HT29-MTX cells. CE protected human umbilical vein endothelial cells (HUVECs) from oxidative stress and maintained its activity when entrapped in PLGA NPs. CE encapsulated in QA-Ch-S-pro NP protected HUVECs from oxidative stress, even more effectively than non-encapsulated CE. Furthermore, mucoadhesive NPs from Ch-der were more effective antioxidant protectors than PLGA NPs, but less cytotoxic PLGA NPs could be more useful when comparatively high therapeutic antioxidant doses are needed.

Chitosan-Based Nanoparticles Containing Cherry Extract from Prunus avium L. to Improve the Resistance of Endothelial Cells to Oxidative Stress.

Cherries are known for their nutraceutical properties, in particular for their antioxidant ability due to their polyphenol content, which causes a reduction of cardiovascular disease (CVD) risk factors. However, once ingested these molecules are degraded in the Gastrointestinal (GI) tract before reaching the blood, which is the action site. The object of the present work is to evaluate the ability of cherry extract (CE), encapsulated in nanoparticles (NPs) based on different chitosan (Ch) derivatives, to promote a protective effect of human umbilical vein endothelial cells (HUVECs) involved in vascular dysfunction against oxidative stress. CE-loaded NPs based on quaternary ammonium chitosan (NP1) and an S-protected thiolated derivative thereof (NP2) were prepared. The mean particle size (NP1 344.9 ± 17.8, NP2 339.9 ± 68.2 nm), the polydispersity index, the encapsulation efficiency (NP1 78.4 ± 4.5, NP2 79.8 ± 0.6%), and the zeta potential (NP1 14.8 ± 0.3, NP2 15.8 ± 0.5 mV) did not appear to be significantly different. Both NP types improved the CE apparent permeation parameters with respect to the control. Conversely, CE-loaded NP2 protected HUVECs from oxidative stress and reduced reactive oxygen species (ROS) production more than CE-loaded NP1 and free CE. In addition to promoting HUVEC resistance, NP2 could be a useful tool to overcome the problem of cherry seasonality.

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.

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

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.

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.

Red grape skin and seeds polyphenols: Evidence of their protective effects on endothelial progenitor cells and improvement of their intestinal absorption.

SCOPE: To evaluate the ability of grape skin and seeds to protect endothelial progenitor cells (EPC) from oxidative stress induced by hyperglycemia (HG) compared to red wine (RW) and prepare innovative pharmaceutical systems for the oral administration of red grape extract allowing the overcoming of its poor intestinal absorption. METHODS AND RESULTS: Human EPC were characterized by expression of cell surface markers. Cells were incubated with different concentrations of total polyphenols from grape components or RW in the presence or absence of HG. Cell viability, migration, adhesion, and reactive oxygen species (ROS) production were assayed. Intestinal permeation of polyphenols was studied in the absence or presence of a quaternary ammonium-chitosan conjugate (N⁺(60)-Ch). Grape components and RW increased EPC viability, adhesion and migration, and prevented the HG effect (P < 0.01). ROS production induced by HG was significantly reduced only by grape seed extract and RW (P < 0.01). N⁺(60)-Ch acted as an effective enhancer of polyphenol permeability across the excised rat intestine. CONCLUSIONS: Red grape components are a source of antioxidant compounds that ameliorate EPC viability and function, while preventing endothelial dysfunction. The use of polycationic chitosan derivatives can promote the absorption of polyphenols across intestinal epithelium, thus increasing their bioavailability and potential therapeutic value in atherosclerosis.

New chitosan derivatives for the preparation of rokitamycin loaded microspheres designed for ocular or nasal administration.

Acanthamoeba spp. are the causative agents of granulomatous amoebic encephalitis (GAE) and amoebic keratitis. Recent studies performed by Rassu et al. showed that, compared with the free drug, the loading of rokitamycin in chitosan microspheres improves and prolongs the in vitro antiamoebic activity of rokitamycin. This could be useful in transporting the drug for either ocular application to treat amoebic keratitis or nasal administration as an alternative route for the administration of the drug to the brain in GAE therapy. Starting from the previous study, our goal was to optimize the technological parameters in order to obtain chitosan microparticles loaded with rokitamycin and to evaluate the use of new quaternary ammonium chitosan derivatives in the preparation of spray dried microspheres containing the macrolide; these derivatives showed better characteristics (solubility, penetration enhancement) compared with chitosan itself. Toxicity studies on new polymers were performed. Spray dried loaded microspheres based on chitosan or chitosan derivatives were obtained by using appropriate preparative parameters. Microparticles containing chitosan derivatives showed similar or often better properties than formulations made of chitosan with respect to size, in vitro release behaviour and mucoadhesiveness thus making them more suitable for ocular or nasal administration. New polymers did not demonstrate cytotoxicity.

Improved synthesis of quaternary ammonium-chitosan conjugates (N+ -Ch) for enhanced intestinal drug permeation.

The pH-induced structural modifications of the reaction product between chitosan and 2-diethylaminoethyl chloride are studied with the purpose of testing and comparing the resulting chitosan derivatives on the basis of their intestinal drug permeability-enhancing properties. The reaction reproducibly yielded conjugates comprising short pendant chains of n adjacent diethyl-dimethylene-ammonium groups substituted onto the primary amino group of the chitosan repeating unit. The more significant derivatives, N(+)-Ch-7 (degree of substitution, DS=41%; n=3) and N(+)-Ch-8 (DS=59%; n=1.7) were prepared at pH 7 and 8, respectively. The apparent permeability (P(app)) of excised rat intestine was determined by means of Ussing type chambers. The hydrophilic fluorescein sodium (NaFlu) and fluorescein isothiocyanate dextran (MW 4400 Da) (FD-4), and the lipophilic rhodamine 123 (Rh-123), were applied in Ringer buffer to the apical side. Apical to basolateral transport was measured in the absence or presence of 0.5% (w/v) of the polymer under test. N(+)-Ch-7 and N(+)-Ch-8 were more effective P(app) enhancers than N-trimethyl-chitosan. Both N(+)-Ch-7 and N(+)-Ch-8 enhanced the P(app) of NaFlu (enhancement ratio, ER=1.84 and 1.33, respectively), while N(+)-Ch-8 was the more effective enhancer for FD-4 (ER=2.14). The P(app) of Rh-123 was significantly enhanced only by N(+)-Ch-7 (ER=1.37). Permeant-polymer binding counteracted the enhancement effect of polymer on transmembrane permeant flux. Contact with the chitosan conjugates did not impair the mucosal epithelium integrity.

Polymeric enhancers of mucosal epithelia permeability: synthesis, transepithelial penetration-enhancing properties, mechanism of action, safety issues.

Transmucosal drug administration across nasal, buccal, and ocular mucosae is noninvasive, eliminates hepatic first-pass metabolism and harsh environmental conditions, allows rapid onset, and further, mucosal surfaces are readily accessible. Generally, however, hydrophilic drugs, such as peptides and proteins, are poorly permeable across the epithelium, which results in insufficient bioavailability. Therefore, reversible modifications of epithelial barrier structure by permeation enhancers are required. Low molecular weight enhancers generally have physicochemical characteristics favoring their own absorption, whereas polymeric enhancers are not absorbed, and this minimizes the risk of systemic toxicity. The above considerations have warranted the present survey of the studies on polymeric transmucosal penetration-enhancers that have appeared in the literature during the last decade. Studies on intestinal permeation enhancers are also reviewed as they give information on the mechanism of action and safety of polymers. The synthesis and characterization of polymers, their effectiveness in enhancing the absorption of different drugs across different epithelium types, their mechanism of action and structure-efficacy relationship, and the relevant safety issues are reviewed. The active polymers are classified into: polycations (chitosan and its quaternary ammonium derivatives, poly-L-arginine (poly-L-Arg), aminated gelatin), polyanions (N-carboxymethyl chitosan, poly(acrylic acid)), and thiolated polymers (carboxymethyl cellulose-cysteine, polycarbophil (PCP)-cysteine, chitosan-thiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugates).