Cranberry proanthocyanidins composite electrospun nanofibers as a potential alternative for bacterial entrapment applications.

The interaction between A-type interflavan bonds from cranberry proanthocyanidins (PAC) and surface virulence factors of extra-intestinal pathogenic Escherichia coli (ExPEC) was studied. Electrospun nanofibers (ESNF) were fabricated using PAC and polycaprolactone (PCL) solutions and their physical and chemical properties were characterized. The ability of PAC:PCL composite ESNF to interact with and entrap ExPEC strain 5011 (ExPEC-5011) was evaluated in vitro by plate culturing and when formulated as a biofilter and nanocoating. As a biofilter, the PAC:PCL ESNF exhibited a dose-dependent ability to entrap ExPEC-5011. Images from scanning electron and fluorescent microscopies revealed that ESNF sections with higher amounts of PAC led to higher bacterial entrapment. The effectiveness PAC:PCL ESNF to bind ExPEC when applied as a nanocoating was studied using ESNF-coated polyvinyl chloride intermittent catheter. Results indicate that ExPEC-5011 was entrapped well into the PAC:PCL ESNF coating on the catheter. Overall, our results suggest that incorporating the biomolecule PAC in ESNF is a potential means for applications requiring bacterial entrapment, such as biofunctionalization, biofiltration, and surface coating, among others.

Cranberry Proanthocyanidins-PANI Nanocomposite for the Detection of Bacteria Associated with Urinary Tract Infections.

Consumption of cranberries is associated with the putative effects of preventing urinary tract infections (UTIs). Cranberry proanthocyanidins (PAC) contain unusual double A-type linkages, which are associated with strong interactions with surface virulence factors found on UTI-causing bacteria such as extra-intestinal pathogenic Escherichia coli (ExPEC), depicting in bacterial agglutination processes. In this work, we demonstrated the efficacy of cranberry PAC (200 µg/mL) to agglutinate ExPEC (5.0 × 108 CFU/mL) in vitro as a selective interaction for the design of functionalized biosensors for potential detection of UTIs. We fabricated functionalized screen-printed electrodes (SPEs) by modifying with PAC-polyaniline (PANI) nanocomposites and tested the effectiveness of the PAC-PANI/SPE biosensor for detecting the presence of ExPEC in aqueous suspensions. Results indicated that the PAC-PANI/SPE was highly sensitive (limit of quantification of 1 CFU/mL of ExPEC), and its response was linear over the concentration range of 1-70,000 CFU/mL, suggesting cranberry PAC-functionalized biosensors are an innovative alternative for the detection and diagnosis of ExPEC-associated UTIs. The biosensor was also highly selective, reproducible, and stable.

Minicell-based fungal RNAi delivery for sustainable crop protection.

Spray-induced gene silencing (SIGS) using topical dsRNA applications has risen as a promising, target-specific, and environmentally friendly disease management strategy against phytopathogenic fungi. However, dsRNA stability, efficacy, and scalability are still the main constraints facing SIGS broader application. Here we show that Escherichia coli-derived anucleated minicells can be utilized as a cost-effective, scalable platform for dsRNA production and encapsulation. We demonstrated that minicell-encapsulated dsRNA (ME-dsRNA) was shielded from RNase degradation and stabilized on strawberry surfaces, allowing dsRNA persistence in field-like conditions. ME-dsRNAs targeting chitin synthase class III (Chs3a, Chs3b) and DICER-like proteins (DCL1 and DCL2) genes of Botryotinia fuckeliana selectively knocked-down the target genes and led to significant fungal growth inhibition in vitro. We also observed a compensatory relationship between DCL1 and DCL2 gene transcripts, where the silencing of one gene upregulated the expression of the other. Contrary to naked-dsRNAs, ME-dsRNAs halted disease progression in strawberries for 12 days under greenhouse conditions. These results elucidate the potential of ME-dsRNAs to enable the commercial application of RNAi-based, species-specific biocontrols comparable in efficacy to conventional synthetics. ME-dsRNAs offer a platform that can readily be translated to large-scale production and deployed in open-field applications to control grey mould in strawberries.

Antimicrobial proanthocyanidin-chitosan composite nanoparticles loaded with gentamicin.

Cranberry proanthocyanidin-chitosan nanoparticles (PAC-CHT NPs) loaded with antibiotic gentamicin (GEN) (PAC-CHT-GEN NPs) were formulated and characterized according to size, polydispersity (PDI), surface charge, morphology, and encapsulation efficiency (EE). PAC-CHT-GEN NPs were evaluated for their ability to agglutinate E. coli, S. aureus, and P. aeruginosa and their bacteriostatic and bactericidal activity. Results indicate that the PAC-CHT-GEN NPs at 0.5:1.0, 1.0:1.0, and 2.0:1.0 weight ratios formed stable nanoparticles with sizes from 242.9 to 277.4 nm, a PDI from 0.344 to 0.391, and a zeta potential from 34.5 to 38.5 mV, and up to 94% EE. Results indicate that PAC-CHT-GEN NPs have the ability to agglutinate E. coli, S. aureus, and P. aeruginosa. Furthermore, PAC-CHT-GEN NPs exhibited greater bactericidal activity than GEN alone. Results suggested PAC-CHT-GEN NPs form stable, round-shaped, and bioactive nanoparticles with the potential to be use in the treatment of bacterial infections.

Composition of Anthocyanins and Proanthocyanidins in Three Tropical Vaccinium Species from Costa Rica.

Total polyphenol content (TPC), total flavonoid content (TFC), total anthocyanin content (TAC), and proanthocyanidin (PAC) content were determined in fruit from three tropical Vaccinium species (Vaccinium consanguineum, Vaccinium floribundum, and Vaccinium poasanum) from Costa Rica sampled at three stages of fruit development. Results show that TAC increased as the fruit developed, while TPC, TFC, and PAC content decreased. Anthocyanin profiles were evaluated using electrospray ionization tandem mass spectrometry. Cyanidin and delphinidin glycosides were the predominant anthocyanins for the three tropical Vaccinium species. Proanthocyanidins were characterized using attenuated total reflection Fourier transform infrared spectroscopy, nuclear magnetic resonance, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The presence of procyanidin structures with B-type interflavan bonds were observed, but deconvolution of mass spectrometry isotope patterns indicated that PACs with one or more A-type interflavan bonds accounted for more than 74% of the oligomers at each degree of polymerization.

Characterization of proanthocyanidin-chitosan interactions in the formulation of composite nanoparticles using surface plasmon resonance.

Chitosan (CHT) interacts with proanthocyanidins (PAC) by a mechanism involving hydrogen bonding and ion-dipole interactions, allowing the spontaneous formation of PAC-CHT composite nanoparticles (PAC-CHT NPs). The interaction between PAC and CHT was characterized by ellipsometry, infrared spectroscopy, and surface plasmon resonance (SPR) to determine the effect of CHT molecular weight (MW), PAC to CHT ratios, and pH on the formulation of PAC-CHT NPs. These parameters also affect the size and morphology of PAC-CHT NPs. Results indicate that CHT MW and pH of the solution impact the interactions of PAC-CHT in two ways: (1) greater CHT MW increases the amount of PAC molecules that attach to the CHT chain, and (2) lower pH of the CHT solutions increases the amount PAC molecules that attach to the CHT chain. Results also show that higher CHT MW, CHT concentration, and pH of the CHT solutions increase the size of PAC-CHT NPs. In contrast, greater PAC concentrations decreases the size of PAC-CHT NPs. This study demonstrates that SPR is a useful technique for measuring the effect of changes in the interaction between PAC and CHT, which in turn affects the size and morphology of PAC-CHT NPs.

Proanthocyanidin-chitosan composite nanoparticles prevent bacterial invasion and colonization of gut epithelial cells by extra-intestinal pathogenic Escherichia coli.

Cranberry proanthocyanidin-chitosan composite nanoparticles (PAC-CHT NPs) were formulated using 2:1, 5:1, 10:1, 15:1 20:1, 25:1, and 30:1 PAC to CHT weight ratio to form round shaped particles. The PAC-CHT NPs were characterized by size, polydispersity, surface charge, morphology, and PAC content. PAC-CHT NPs bioactivity was measured by agglutination of extra-intestinal pathogenic Escherichia coli (ExPEC) and inhibition of gut epithelial cell invasion by ExPEC. Results indicate that by increasing the PAC to CHT ratio 10:1 to 30:1 formed stable nanoparticles with diameters of 122.8 to 618.7 nm, a polydispersity index of approximated 0.4 to 0.5, and a zeta potential of 34.5 to 54.4 mV. PAC-CHT NPs ratio 30:1 agglutinated ExPEC and decreased the ability of ExPEC to invade epithelial cells in a dose-dependent manner. PAC-CHT NPs ratio 10:1 to 30:1 form stable, round-shaped, and bioactive nanoparticles for potential applications in the treatment of ExPEC bacterial infections.

Physicochemical and structural properties of starch from five Andean crops grown in Bolivia.

Three Andean grains - amaranth (Amaranthus caudatus), quinoa (Chenopodium quinoa), canihua (Chenopodium pallidicaulle) - and two Andean roots starches - achira (Canna indica), maca (Lepidium meyenii) - were studied. Physicochemical properties such as granule size, crystallinity, pasting properties among other as well as structural properties such as root-mean-square radius (rrms), weight-average molar mass (Mw) and apparent density (ρapp) were analyzed in order to evaluate the relation between them. Grains were similar in most of their characteristics as roots in their i.e. granule size, shape, type of crystallinity, Mw and rrms varied according to botanical source. The starch granules from grains were in a narrow diameter range (0.5 to 2 µm) and displayed A-type X-ray diffraction pattern (XRD). Roots starch had a wide granule diameter range (1 to 100 µm) and displayed a B-type XRD. The amylose content varied between 0 and 48% where amaranth had the lowest value and achira had the highest. Furthermore, quinoa and canihua starches had very low breakdown in pasting properties, indicating high stability during cooking. A model is proposed that relates pasting properties i.e. peak viscosity and final viscosity with ρapp, gelatinization enthalpy, granule size and amylose content.

Electrospun plant mucilage nanofibers as biocompatible scaffolds for cell proliferation.

Electrospun nanofibers (ESNFs) were prepared from mucilage isolated from chan and linaza beans and mozote stem commercially available in Costa Rica. Poly(vinyl alcohol) (PVA) was used as an aiding agent. Mucilage/PVA mixed solutions of different volume ratios (100:0, 80:20, 60:40, 40:60, 20:80 and 0:100) were prepared and adjusted to be similar in viscosity and electrical conductivity suitable for electrospinning. Morphology of the ESNFs was examined using scanning electron microscopy (SEM). Fourier transform infrared spectrometer (FTIR) and differential scanning calorimetry (DSC) studies were used to characterize chemical composition and thermal characteristics of the nanofibers (NFs). The ability of the NFs to support fibroblast cell proliferation was investigated in vitro using the optimized mucilage/PVA solutions. Results show plant mucilage-based ESNFs are well-suited for fibroblast cell growth, significantly better than ESNFs of PVA; and the mucilage of chan beans is better than those of mozote and linaza for supporting cell proliferation.

Cranberry proanthocyanidin-chitosan hybrid nanoparticles as a potential inhibitor of extra-intestinal pathogenic Escherichia coli invasion of gut epithelial cells.

Chitosan interacts with proanthocyanidins through hydrogen-bonding, which allows encapsulation and development of stable nanoparticles via ionotropic gelation. Cranberry proanthocyanidins (PAC) are associated with the prevention of urinary tract infections and PAC inhibit invasion of gut epithelial cells by extra-intestinal pathogenic Escherichia coli (ExPEC). We determined the effect of cranberry proanthocyanidin-chitosan hybrid nanoparticles (PAC-CHTNp) on the ExPEC invasion of gut epithelial cells in vitro. PAC-CHTNp were characterized according to size, morphology, and bioactivity. Results showed a decrease in the size of the nanoparticles as the concentration of PAC was increased, indicating that PAC increases cross-linking by hydrogen-bonding on the surface of the chitosan nanoparticles. Nanoparticles were produced with diameters ranging from 367.3 nm to 293.2 nm. Additionally, PAC-CHTNp significantly inhibited the ability of ExPEC to invade the enterocytes by ~80% at 66 µg GAE/mL and by ~92% at 100 µg GAE/mL. Results also indicate that chitosan nanoparticles alone were not significantly different from controls in preventing ExPEC invasion of enterocytes (data not shown) and also there were not significant differences between PAC alone and PAC-CHTNp, suggesting that the new PAC-CHTNp could lead to an increase in the stability of encapsulated PAC, maintain the molecular adhesion of PAC to ExPEC.

Cranberry Proanthocyanidins - Protein complexes for macrophage activation.

In this work we characterize the interaction of cranberry (Vaccinium macrocarpon) proanthocyanidins (PAC) with bovine serum albumin (BSA) and hen egg-white lysozyme (HEL) and determine the effects of these complexes on macrophage activation and antigen presentation. We isolated PAC from cranberry and complexed the isolated PAC with BSA and HEL. The properties of the PAC-protein complexes were studied by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), gel electrophoresis and zeta-potential. The effects of PAC-BSA complexes on macrophage activation were studied in RAW 264.7 macrophage like cells after treatment with lipopolysaccharide (LPS). Fluorescence microscopy was used to study the endocytosis of PAC-BSA complexes. The effects of the PAC complexes on macrophage antigen presentation were studied in an in vitro model of HEL antigen presentation by mouse peritoneal mononuclear cells to a T-cell hybridoma. The mass spectra of the PAC complexes with BSA and HEL differed from the spectra of the proteins alone by the presence of broad shoulders on the singly and doubly charged protein peaks. Complexation with PAC altered the electrophoretic mobility shift assay in native agarose gel and the electrophoretic mobility (ζ-potential) values. These results indicate that the PAC-protein complexes are stable and alter the protein structure without precipitating the protein. Fluorescence microscopy showed that the RAW 264.7 macrophages endocytosed BSA and PAC-BSA complexes in discrete vesicles that surrounded the nucleus. Macrophages treated with increasing amounts of PAC-BSA complexes had significantly reduced COX-2 and iNOS expression in response to treatment with lipopolysaccharide (LPS) in comparison to the controls. The PAC-HEL complexes modulated antigen uptake, processing and presentation in murine peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas the PAC-HEL complex had already reached the maximum IL-2 expression. Cranberry PAC may increase the rate of endocytosis of HEL and subsequent expression of IL-2 by the T-cell hybridomas. These results suggest that PAC-protein complexes modulate aspects of innate and acquired immune responses in macrophages.

Estudio preliminar de la obtención de compuestos híbridos de quitosano y polifenoles derivados de lignina a partir de subproductos agropecuarios y pesquería de camarón / Preliminary study of the production of hybrid compounds of chitosan and polyphenols derived from lignin from agroindustry and shrimp fishery

En este estudio se prepararon y caracterizaron microcápsulas híbridas del conjugado de polifenoles derivados de la lignina proveniente de la cáscara de piña, y el quitosano obtenido a partir de la quitina de la cáscara del camarón; ambos materiales fueron obtenidos como residuos de la industria agropecuaria y pesquería de camarón de Costa Rica. Con el objetivo de preparar compuestos fenólicos derivados de la lignina, y utilizarlos en la síntesis de las microcápsulas, se realizó la hidrólisis enzimática de la misma en un reactor a presión atmosférica a un pH de 6.8, en buffer de citrato 1 M, durante 6 h a 37ºC. Las enzimas utilizadas fueron extraídas de los cultivos de hongos de Gloeophyllum trabeum (Pers.) Murrill y Phanerochae chrysosporiumin Burdsall. Para la obtención del quitosano se realizó la desacetilación alcalina a partir de exoesqueletos del camarón Heterocarpus vicarius Fazon. Para la preparación de las microcápsulas se empleó una disolución de quitosano en ácido acético, el cual fue mezclado con una disolución acuosa del producto obtenido de la hidrólisis de la lignina y luego añadido a una disolución de vaselina para microemulsionar. Posteriormente, se agregó el glutaraldehído como agente entrecruzante. Se obtuvieron microcápsulas con tamaños entre 5 y 10 µm. Estas microcápsulas son un material promisorio ya que, mediante la formación del complejo, se puede aumentar la solubilidad del quitosano y estabilizar los polifenoles, manteniendo así sus propiedades antioxidantes. Los resultados preliminares obtenidos en esta investigación, muestran el potencial de este material para el encapsulamiento de fármacos y pesticidas.

Hybrid microcapsules of the conjugate of polyphenols derived from lignin were prepared and characterized. They were obtained from pineapple peel and chitin from shrimp shell from agroindustry or shrimp fishery of Costa Rica. The phenolic compounds were obtained by hydrolysis of lignin, using the enzyme derivatives from the fungus culture of Gloeophyllum trabeum (Pers.) Murrill and Phanerochaete chrysosporium Burdsall. The reaction was carried out in a reactor with atmospheric pressure, pH 6.8, with a citrate buffer of 1M, for 6 hours at 37°C. The chitosan was obtained by alkaline deacetylation of the Heterocarpus vicarious Fazon, shrimp exoskeletons. Microcapsules were prepared mixing a solution of chitosan dissolved in acetic acid and a solution of polyphenol derivatives from lignin. Afterwards, they were added to a vaseline aqueous solution for the microemulsion formation and glutaraldehyde was added as a crosslinking agent. Microcapsules with sizes between 5 to 10 µm were obtained. These microcapsules are a promising material to increase chitosan solubility and for preventing the oxidation of polyphenols. The preliminary results obtained in this research show the potential of this material for the encapsulation of drugs and pesticides.

Non-covalent pomegranate (Punica granatum) hydrolyzable tannin-protein complexes modulate antigen uptake, processing and presentation by a T-cell hybridoma line co-cultured with murine peritoneal macrophages.

In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas.

Production of poly(3-hydroxybutyrate) by Halomonas boliviensis in an air-lift reactor.

BACKGROUND: Microbial polyesters, also known as polyhydroxyalkanoates (PHAs), closely resemble physical and mechanical features of petroleum derived plastics. Recombinant Escherichia coli strains are being used in industrial production of PHAs in Stirred Tank Bioreactors (STRs). However, use of Air-Lift Reactors (ALRs) has been known to offer numerous technical operating options over STRs, and as such has been successfully implemented in many bioprocesses. Halomonas boliviensis is a halophilic bacterium that is known to assimilate various carbohydrates and convert them into a particular type of PHA known as poly(3-hydroxybutyrate) (PHB). Owing to this capability, it has been used to synthesize the polyester using hydrolysates of starch or wheat bran in stirred tank bioreactors. RESULTS: This research article firstly describes the production of PHB in shake flasks by H. boliviensis using different combinations of carbohydrates and partially hydrolyzed starch as carbon sources. The highest PHB yields, between 56 and 61 % (wt.), were achieved when either starch hydrolysate or a mixture of glucose and xylose were used as carbon sources. The starch hydrolysate obtained in this study was then used as carbon source in an ALR. The largest amount of PHB, 41 % (wt.), was attained after 24 hrs of cultivation during which maltose in the hydrolysate was assimilated more rapidly than glucose during active cell growth; however, the rate of assimilation of both the carbohydrates was found to be similar during synthesis of PHB. An incomplete pentose phosphate pathway, which lacks 6-phosphogluconate dehydrogenase, was deduced from the genome sequence of this bacterium and may result in the characteristic assimilation of glucose and maltose by the cells. CONCLUSIONS: This study showed that the production of PHB by H. boliviensis using cheap substrates such as starch hydrolysate in a simple production system involving an ALR is feasible. Both maltose and glucose in the hydrolysate induce cell growth and PHB synthesis; most likely the cells balance adequately CoA and NAD(P)H during the assimilation of these carbohydrates. The combination of cheap substrates, simple production systems and the use of non-strict sterile conditions by the halophile H. boliviensis are desirable traits for large scale production of PHB, and should lead to a competitive bioprocess.

Scattering of light by colloidal aluminosilicate particles produces the unusual sky-blue color of Río Celeste (Tenorio volcano complex, Costa Rica).

Río Celeste (Sky-Blue River) in Tenorio National Park (Costa Rica), a river that derives from the confluence and mixing of two colorless streams--Río Buenavista (Buenavista River) and Quebrada Agria (Sour Creek)--is renowned in Costa Rica because it presents an atypical intense sky-blue color. Although various explanations have been proposed for this unusual hue of Río Celeste, no exhaustive tests have been undertaken; the reasons hence remain unclear. To understand this color phenomenon, we examined the physico-chemical properties of Río Celeste and of the two streams from which it is derived. Chemical analysis of those streams with ion-exchange chromatography (IC) and inductively coupled plasma atomic emission spectroscopy (ICP-OES) made us discard the hypothesis that the origin of the hue is due to colored chemical species. Our tests revealed that the origin of this coloration phenomenon is physical, due to suspended aluminosilicate particles (with diameters distributed around 566 nm according to a lognormal distribution) that produce Mie scattering. The color originates after mixing of two colorless streams because of the enlargement (by aggregation) of suspended aluminosilicate particles in the Río Buenavista stream due to a decrease of pH on mixing with the acidic Quebrada Agria. We postulate a chemical mechanism for this process, supported by experimental evidence of dynamic light scattering (DLS), zeta potential measurements, X-ray diffraction and scanning electron microscopy (SEM) with energy-dispersive spectra (EDS). Theoretical modeling of the Mie scattering yielded a strong coincidence between the observed color and the simulated one.

In vitro uptake of lysozyme-loaded liposomes coated with chitosan biopolymer as model immunoadjuvants.

Chitosan binds to negatively charged soy lecithin liposomes by an electrostatic interaction driven by its cationic amino group. This interaction allows developing stable coated vesicles suitable as a targeted carrier and controlled release system for drugs and vaccines. In this work, we studied the effect of chitosan-coated liposomes on the uptake and antigen presentation of hen egg-white lysozyme (HEL) in Peyer's patches peritoneal macrophages isolated from mice. Chitosan-coated liposomes were characterized according to size, zeta potential, and antigen-loading and release properties. Results showed an increase in the positive net charge and size of the liposomes as the concentration of chitosan was increased, suggesting an electrostatic interaction and an effective coating, followed by fluorescence microscopy. About 85% of the antigen loaded remained in the chitosan-coated liposomes after release studies for 4 hours in phosphate-buffered saline. After 4 hours of preincubation with a T-cell hybridoma line cocultured with murine peritoneal macrophages, only trace amounts of interleukin-2 (IL-2) were detected in the cocultures treated with HEL alone, whereas cocultures treated with HEL-liposomes had an important production of IL-2, and the HEL chitosan-coated liposomes had already reached maximum IL-2 expression. Confocal microscopy studies showed that chitosan-coated liposomes had a higher uptake rate of the fluorescently labeled HEL than uncoated liposomal vesicles after 30 minutes of incubation with the peritoneal macrophages. Since uptake by macrophage cells is the first step in vaccination, our results suggest that the chitosan-coated liposomal system is a potential candidate as an immunoadjuvant for vaccine delivery systems.

Chitosomes loaded with cranberry proanthocyanidins attenuate the bacterial lipopolysaccharide-induced expression of iNOS and COX-2 in raw 264.7 macrophages.

Chitosan binds to negatively charged soy lecithin liposomes by an electrostatic interaction driven by its positively charged amino group. This interaction allows stable covered vesicles (chitosomes) to be developed as a suitable targeted carrier and controlled release system. This study investigated the effect of chitosomes on the activation of cranberry proanthocyanidins (PAC) in Raw 264.7 macrophages. Chitosomes were characterized according to size, zeta potential, PAC-loading, and release properties. Results showed an increase in the net positive charge and size of the liposomes as the concentration of chitosan was increased, suggesting an effective covering of the vesicles by means of electrostatic interactions, as shown by transmission electron microscopy and fluorescence microscopy. About 85% of the PAC that was loaded remained in the chitosomes after release studies for 4 hours in phosphate-buffered saline. Cyclo-oxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) are associated with inflammation. Activated RAW 264.7 macrophages increase the expression of COX-2 and iNOS in response to bacterial infection and inflammation; we, therefore, tested the ability of the PAC-loaded chitosomes to attenuate COX-2 and iNOS expression in LPS (lipopolysaccharide)-stimulated macrophages. Increasing the amount of PAC loaded into the chitosomes caused a dose-dependent attenuation of iNOS and COX-2 expression in LPS-stimulated macrophages. A 2% v/v PAC-loaded chitosomes formulation almost completely attenuated the LPS-induced expression of iNOS and COX-2. PAC-loaded chitosomes were more active than PAC alone, suggesting that the macrophage response to LPS occurs after endocytosis of the PAC-loaded chitosomes.