Development and characterization of anti-inflammatory activity of curcumin-loaded biodegradable microspheres with potential use in intestinal inflammatory disorders.

This research addresses the development and in vitro evaluation of a microparticulate system intended for intestine-targeted delivery of curcumin (CRM), a natural polyphenol with anti-inflammatory properties. Microspheres (Ms) based on zein (ZN) and Gantrez® AN119 (PVMMA) were prepared by spray-drying and coated with a pH-sensitive polymer (Eudragit® FS30D). An experimental design was performed to optimize the microparticulate formulation. A detailed characterization of systems was carried out by SEM, DSC, FTIR, particle size, ζ potential measurements and in vitro CRM release. The optimized formulation was evaluated in LPS-stimulated RAW 264.7 macrophages to investigate its anti-inflammatory activity. FTIR and DSC studies suggest a predominant presence of α-helix structure for ZN when formulated and also, a strong interaction between components. The stabilization of α-helix by PVMMA or CRM would take place by hydrogen bonds. Although the encapsulation efficiency was high (89%) for ZN/PVMMA Ms, the coating process with Eudragit® led to an EE decrease of 62%. Coating of Ms was found to retain a 20% of drug within 6h of release, although a strong initial burst release was observed. Cells viability and apoptosis were not affected when cells were co-incubated with coated Ms with CRM. The exposure of unstimulated cells to Ms did not show any effect on NO and PGE2 production. However, a reduction in NO and PGE2 production was obtained when CRM-loaded Ms were co-incubated with stimulated macrophages. Further, this inhibition was significantly higher compared to the decrease obtained when Ms with pure CRM were used in culture, which suggested a synergistic effect of CRM and Ms. Finally, CRM-loaded Ms caused a significant inhibition of analysed pro-inflammatory cytokines (TNFα, IL-1ß, NOS2, COX-2) in macrophages stimulated with LPS. All these results confirm the advantageous features of ZN/PVMMA microspheres as a serious alternative for delivering CRM to reduce the inflammatory activity at intestinal regions affected by inflammatory bowel diseases.

Pharmacokinetic model of florfenicol in turbot (Scophthalmus maximus): establishment of optimal dosage and administration in medicated feed.

The pharmacokinetics of florfenicol (FF) in turbot (Scophthalmus maximus) was studied after single intravenous (10 mg kg-1 ) and oral (100 mg kg-1 ) administration. The plasma concentration-time data of florfenicol were described by an open one-compartment model. The elimination half-life (t1/2 ) was estimated to be 21.0 h, and the total body clearance, Cl, was determined as 0.028 L kg h-1 . The apparent volume distribution (Vd ) was calculated to be 0.86 L kg-1 and the mean residence time (MRTiv ) was 30.2 h. Following oral administration, the maximum plasma concentration (Cmax ) of 55.4 µg mL-1 was reached at 12 h (Tmax ). The absorption constant (ka ) was 0.158 h-1 . The bioavailability was estimated to be 57.1%. The low bioavailability observed at higher doses was explained by the saturation of the mechanisms of absorption. The drug absorption process was limited by its inherent low solubility, which limited the amount of available FF absorbed in the gastrointestinal tract. Based on the pharmacokinetic data, an optimal dosing schedule for FF administration is hereby provided. Based on the minimum inhibitory concentration found for susceptible strains of Aeromonas salmonicida, oral FF administration of first, an initial dose of 30 mg FF kg-1 , followed by 6 maintenance doses at 18 mg kg-1 /daily could be effective against furunculosis in turbot.

Inflammatory responses and side effects generated by several adjuvant-containing vaccines in turbot.

Several of the adjuvants used in fish vaccines cause adhesions in internal organs when they are injected intraperitoneally. We describe the damage caused by vaccines containing different adjuvants in the turbot Scophthalmus maximus and show that internal adhesions can be greatly reduced by injecting the fish in a specific way. Injection of fish with the needle directed towards the anterior part of the peritoneal cavity induced formation of a single cell-vaccine mass (CVM) that became attached to the parietal peritoneum. However, injection of the fish with the needle pointing in the opposite direction generated many small CVM that became attached to the visceral and parietal peritoneum and in some cases caused internal adhesions. We describe the structural and cellular changes in the adjuvant-induced CVMs. The CVMs mainly comprised neutrophils and macrophages, although most of the former underwent apoptosis, which was particularly evident from day 3 post-injection. The apoptotic cells were phagocytosed by macrophages, which were the dominant cell type from the first days onwards. All of the vaccines induced angiogenesis in the area of contact between the CVM and the mesothelium. Vaccines containing oil-based adjuvants or microspheres induced the formation of granulomas in the CVM; however, no granulomas were observed in the CVM induced by vaccines containing aluminium hydroxide or Matrix-Q(®) as adjuvants. All of the vaccines induced strong migration of cells to the peritoneal cavity. Although some of these cells remained unattached in the peritoneal cavity, most of them formed part of the CVM. We also observed migration of the cells from the peritoneal cavity to lymphoid organs, indicating bidirectional traffic of cells between the inflamed areas and these organs.

Use of 1H NMR STD, waterLOGSY, and Langmuir monolayer techniques for characterization of drug-zein protein complexes.

Zein is a protein based natural biopolymer containing a large amount of nonpolar amino acids, which has shown the ability to form aggregates and entrap solutes, such as drugs and amino acids to form stable protein-drug complexes. In this work, π-A isotherm, NMR, and Dynamic light scattering were used to detect the formation of protein aggregates and the affinity between zein and two different drugs: tetracycline and indomethacin. An effective interaction of zein and the two drugs was evidenced by means of liquid NMR reinforced by means of changes in the surface pressure by π-A isotherm. The effective interactions zein/drugs under air/water interface were evidenced as a change in the surface pressure of the π-A isotherm of zein in the presence of drug solutions. The presence of tetracycline in the subphase decreased the area occupied by the monolayer at the expanded region until pressures of 12 mN/m were the areas became similar, but indomethacin produces an increment of the area in both expanded and collapsed region. The feasible methodology employed, focused in the functionality of the protein-drug interaction, can be very promising in the drug delivery field.

Biodegradable microparticles covalently linked to surface antigens of the scuticociliate parasite P. dicentrarchi promote innate immune responses in vitro.

The histiophagous scuticociliate endoparasite Philasterides dicentrarchi is an emerging pathogen that infects the turbot Scophthalmus maximus and thus causes important economic losses in turbot aquaculture. This in vitro study investigated the adjuvant capacity of biodegradable microspheres (MS) composed of two polymers (chitosan and Gantrez(®)) covalently coupled to surface antigens (Ag) of P. dicentrarchi. The coupled MS-Ag significantly stimulated the phagocytic response of both murine macrophages (RAW 264.7 cells) and leukocytes from the anterior kidney of turbot (HLK), at a level similar to that induced by zymosan A. The MS-Ag also significantly increased production of reactive oxygen and nitrogen species, as shown by the increased O(2) consumption and stimulation of the respiratory burst and nitric oxide production by murine and in particular by turbot HLK. The MS-Ag stimulated the production of the proinflammatory cytokine tumour necrosis factor alpha (TNFα) by murine and turbot HLK. The results confirm the high adjuvant capacity of biodegradable MS covalently bound to Ag as regards stimulating the innate immune response, and they justify the use of MS in the production of safe and effective vaccines against fish pathogens.

A vaccine based on biodegradable microspheres induces protective immunity against scuticociliatosis without producing side effects in turbot.

The histiophagous scuticociliate parasite Philasterides dicentrarchi is an emergent pathogen in aquaculture and causes significant economic losses on turbot (Scophthalmus maximus) farms. In this study, the surface antigens (Ag) of the parasite were encapsulated and covalently linked to a polymeric microparticle formulation composed of two biodegradable polymers (chitosan and Gantrez). The antigenicity of the formulation and the protection provided were compared in mice and turbot. This formulation induced a higher antibody (Ab) response in mice at doses of 5mg of microspheres (MS) conjugated with approximately 230 µg of Ag (MS-Ag(c)). However, Ab levels were significantly lower than in mice vaccinated with the same concentration of Ag in complete Freund's adjuvant (FCA). In turbot, the MS-Ag(c) formulation induced a higher level of Abs than that induced by the same vaccine emulsified in FCA. The challenge experiments performed with P. dicentrarchi and vaccinated turbot also showed a clear correlation between Ab levels and survival levels. Growth was significantly affected in fish vaccinated with FCA, but not in fish vaccinated with MS. The high adjuvant capacity of MS, together with its biodegradability and low toxicity to fish, makes this new vaccine an economical, effective and safe alternative to oil-based adjuvants for the immunoprophylaxis of scuticociliatosis in turbot.

Amoxicillin-Loaded Sponges Made of Collagen and Poly[(methyl vinyl ether)-co-(maleic anhydride)] for Root Canal Treatment: Preparation, Characterization and In Vitro Cell Compatibility.

The difficulty of eliminating Enterococcus faecalis and other bacteria infecting dental root canals makes it desirable to develop formulations capable of sustained release of antibiotics within the canal. With this function in view, in this work we compared the mechanical, drug release and biocompatibility properties of amoxicillin-loaded collagen (CL) and CL complexed with poly[(methyl vinyl ether)-co-(maleic anhydride)] (PVMMA), with or without glutaraldehyde (GTA) or the natural product genipin (GN) as cross-linker. Collagen was not denatured by complexation with PVMMA. Only CL-PVMMA-GN sponges did not disintegrate during 7 days exposure to cell culture medium (un-cross-linked CL disintegrated within 24 h and un-cross-linked CL-PVMMA within 4 days), and CL-PVMMA-GN sponges also exhibited the most appropriate combination of mechanical properties (hardness, modulus of deformability and plasticity). CL-PVMMA-GN sponges absorbed aqueous medium faster than other cross-linked formulations, but their maximum uptake was less; and drug release from CL-PVMMA-GN sponges tended to be faster than from any other, except un-cross-linked CL-PVMMA, maximum release taking about 4 days. No formulation significantly altered the viability of L929 fibroblast-like mouse connective tissue cells, but cells growing on sponges showed signs of non-adherence. It is concluded that genipin-cross-linked CL-PVMMA sponges merit further investigation as antibiotics vehicles and aids to tissue regeneration in the dental root canal.

Development of a novel AMX-loaded PLGA/zein microsphere for root canal disinfection.

The aim of this study was to develop polymeric biodegradable microspheres (MSs) of poly(D-L lactide-co-glycolide) (PLGA) and zein capable of delivering amoxicillin (AMX) at significant levels for root canal disinfection. PLGA/zein MSs were prepared using a spray-drying technique. The systems were characterized in terms of particle size, morphology, drug loading and in vitro release. Drug levels were reached to be effective during the intracanal dressing in between visits during the endodontic treatment. In vitro release studies were carried out to understand the release profile of the MSs. Antimicrobial activity of AMX was performed by antibiograms. Enterococcus faecalis was the bacteria selected due to its prevalence in endodontic failure. Drug microencapsulation yielded MSs with spherical morphology and an average particle size of between 5 and 38 µm. Different drug-release patterns were obtained among the formulations. Release features related to the MSs were strongly dependent on drug nature as it was demonstrated by using a hydrophobic drug (indomethacin). Finally, AMX-loaded MSs were efficient against E faecalis as demonstrated by the antibiogram results. In conclusion, PLGA/zein MSs prepared by spray drying may be a useful drug delivery system for root canal disinfection.

Cyclodextrins: more than pharmaceutical excipients.

Cyclodextrins are pharmaceutical excipients used to enhance the solubility, stability, safety and bioavailability of drugs. Recent findings have shown them to display adjuvant activity in vaccine therapy and prophylactic and therapeutic activity in the treatment of several host-pathogen infections. This article focuses on their activity and mechanism of action.

Iontophoretic delivery of ropinirole hydrochloride: effect of current density and vehicle formulation.

PURPOSE: The objectives of this work were 1) to establish the feasibility of the transdermal iontophoretic delivery of ropinirole hydrochloride; 2) to investigate the possibility of delivering therapeutic doses of this drug; and 3) to determine the key factors that control ropinirole electrotransport. METHODS: A series of in vitro transdermal iontophoretic experiments were instituted to study the effects of drug concentration, co-ion concentration, intensity of current, and application time on ropinirole flux. The convective contribution to ropinirole electrotransport was evaluated by following the transport of the electroosmotic marker mannitol. RESULTS: Ropinirole flux decreased dramatically in the presence of competing ions. This effect was observed even when the molar fraction of the two competing cations was kept constant. Anodal flux of mannitol decreased with drug concentration, indicating a possible alteration of the skin permselectivity. In the absence of competing co-ions, ropinirole transport number reached a maximum value (8-13%). In these conditions, the main factor controlling drug delivery was the intensity of current applied. CONCLUSIONS: Transdermal iontophoresis allowed the delivery of therapeutic doses of ropinirole. The dose administered and the input rate were controlled by the judicious choice of the key delivery factors here described.

Iontophoretic permselectivity of mammalian skin: characterization of hairless mouse and porcine membrane models.

PURPOSE: To evaluate the transport number of Na+, and the isoelectric point, of two skin membranes frequently used for iontophoretic in vitro research. METHODS: Na+ transport numbers were determined by the Hittorf method or by the measurement of membrane potential. The skin isoelectric point was deduced from the electroosmosis of mannitol (a polar non-electrolyte) as a function of pH. RESULTS: The Na+ transport number across porcine skin, like that for hairless mouse, indicated a modest cation permselectivity. Consistent with this observation, the isoelectric points of porcine and hairless mouse skin were determined to be in the ranges of 3.5-3.75 and 4.5-4.6, respectively. That is, at physiological pH, both of these model membranes supports a net negative charge. CONCLUSIONS: The permselective properties of porcine and hairless mouse skin are similar (but with the porcine membrane having apparently fewer basic or more weakly-acidic groups than that of the mouse) and consistent with the characteristics, which have been deduced elsewhere, of human skin.