Effect of Formulation and Process Parameters on Chitosan Microparticles Prepared by an Emulsion Crosslinking Technique

There are many studies about the synthesis of chitosan microparticles; however, most of them have very low production rate, have wide size distribution, are difficult to reproduce, and use harsh crosslinking agents. Uniform microparticles are necessary to obtain repeatable drug release behavior. The main focus of this investigation was to study the effect of the process and formulation parameters during the preparation of chitosan microparticles in order to produce particles with narrow size distribution. The technique evaluated during this study was emulsion crosslinking technique. Chitosan is a biocompatible and biodegradable material but lacks good mechanical properties; for that reason, chitosan was ionically crosslinked with sodium tripolyphosphate (TPP) at three different ratios (32, 64, and 100%). The model drug used was acetylsalicylic acid (ASA). During the preparation of the microparticles, chitosan was first mixed with ASA and then dispersed in oil containing an emulsifier. The evaporation of the solvents hardened the hydrophilic droplets forming microparticles with spherical shape. The process and formulation parameters were varied, and the microparticles were characterized by their morphology, particle size, drug loading efficiency, and drug release behavior. The higher drug loading efficiency was achieved by using 32% mass ratio of TPP to chitosan. The average microparticle size was 18.7 μm. The optimum formulation conditions to prepare uniform spherical microparticles were determined and represented by a region in a triangular phase diagram. The drug release analyses were evaluated in phosphate buffer solution at pH 7.4 and were mainly completed at 24 h.     

The Effect of Chitosan as Internal or External Coating on the 5-ASA Release from Calcium Alginate Microparticles

The effect of chitosan as internal or external coating on the mesalamine (5-ASA) release from calcium alginate microparticles (CaAl) was studied, and a delayed release of 5-ASA system intended for colonic drug delivery was developed. The external chitosan coating was developed by immersion of wetted CaAl in chitosan solution and the internal coating by mixing 5-ASA with chitosan solution and drying before the preparation of CaAl. Both systems were coated with Acryl-EZE^® using combined fluid bed coating and immersion procedure. The results showed that in phosphate medium (pH 7.5), chitosan as 5-ASA coating promotes a quick erosion process accelerating drug release, but chitosan as external coating (CaAlCS) does not increase the T ₅₀ value compared with the microparticles without chitosan (CaAl). Chitosan as internal or external coating was not effective to avoid the quick 5-ASA release in acidic medium (pH 1.2). The presence of β-glucosidase enzymes increases significantly the 5-ASA release for CaAl, while no effect was observed with chitosan as internal or external coating. Fourier transform infrared spectroscopy, thermogravimetric analysis, and X-ray data revealed that 5-ASA did not form a solid solution but was dispersed in the microparticles. The Acryl-EZE^® coating of microparticles was effective because all the formulations showed a low release, less than 15%, of 5-ASA in acid medium at pH 1.2. Significant differences in the percentage of 5-ASA released between formulations were observed in phosphate buffer at pH 6.0. In phosphate buffer at pH 7.2, all the formulations released 100% of 5-ASA.     

Polyethylene Glycol on Stability of Chitosan Microparticulate Carrier for Protein

Stability enhancement of protein-loaded chitosan microparticles under storage was investigated. Chitosan glutamate at 35 kDa and bovine serum albumin as model protein drug were used in this study. The chitosan microparticles were prepared by ionotropic gelation, and polyethylene glycol 200 (PEG 200) was applied after the formation of the particles. All chitosan microparticles were kept at 25°C for 28 days. A comparison was made between those preparations with PEG 200 and without PEG 200. The changes in the physicochemical properties of the microparticles such as size, zeta potential, pH, and percent loading capacity were investigated after 0, 3, 7, 14, and 28 days of storage. It was found that the stability decreased upon storage and the aggregation of microparticles could be observed for both preparations. The reduction in the zeta potential and the increase in the pH, size, and loading capacity were observed when they were kept at a longer period. The significant change of those preparations without PEG 200 was evident after 7 days of storage whereas those with PEG 200 underwent smaller changes with enhanced stability after 28 days of storage. Therefore, this investigation gave valuable information on the stability enhancement of the microparticles. Hence, enhanced stability of chitosan glutamate microparticles for the delivery of protein could be achieved by the application of PEG 200.     

Tactile Detection Thresholds for a Single Asperity on an Otherwise Smooth Surface

An investigation was made of the capacities of humans to detect, by actively touching with the fingertip, the presence of a single, small asperity on a very smooth background. The asperity consisted of either a raised dot having a diameter of 602, 231, or 40 μum, or an edge, each etched into a silicon wafer using the methods of contact photolithography. The height of each dot or edge was varied and the subject was asked to make a forced choice on each test trial as to which of two wafers, one of which was blank, contained the asperity. The mean detection threshold, or minimal height of asperity corresponding to a d' of 1.35, was lowest for edges (0.85 ± 0.22 μm, SD) and increased with decreases in the diameter of dot from 1.09 ± 0.19 μm for a diameter of 602 μm to 2.94 ± 1.19 μm and 5.97 ± 2.02 μm for diameters of 231 μm and 40 μm, respectively. The type of skin displacement required for the detection of these small asperities was believed to be a local lateral deformation of the papillary ridges.     

Fine structure and motility of spermatozoa and composition of the seminal plasma in the perch

The fine structure and motility of spermatozoa and the composition of the seminal plasma of the perch Perca fluviatilis are investigated by electron microscopy, computer assisted cell motility analysis (CMA) and biochemical methods. The spermatozoon is asymmetrical as the flagellum inserts mediolateral on the nucleus. It lacks an acrosome, has an ovoid head and a small midpiece with one mitochondrion. Sperm motility–initiated in distilled water (10° C)–is characterized as follows: 85·0 ± 2·7% of the spermatozoa are motile, the main swimming type (10 ± 1 s after motility initiation) is the linear motion (61·4 ± 24·4%) and the average swimming velocity is 122·4 ± 21·9 μm s^–1. When motility is initiated with NaCl, glucose or sucrose solutions of 100 mosmol kg^–1 the percentage of motile spermatozoa and the swimming types are similar as in water, but the swimming velocity (174·0 ± 22·3 μm s^–1) is significantly higher. Motility is inhibited by high osmolality of the diluent: when increasing the osmolality of the saline solutions to 350 mosmol kg^–1 sperm motility is totally suppressed while potassium (10–40 mmol 1^–1) does not affect motility parameters. pH optimum for sperm motility is between pH 7·0 and 8·5. The seminal fluid contains 124·01 ± 21·68 mmol 1^–1 sodium, 10·22 ± 1·11 mmol 1^–1 potassium and 0·72 ± 0·26 mmol 1^–1 calcium. pH is 8·25 ± 0·09, and osmolality 283·90 ± 37·19 mosmol kg^–1. The following organic components were determined: monosaccharides (glucose 63 ± 19 μmol 1^–1, fructose 54 ± 28 μmol 1^–1, galactose 59 ± 25 μmol 1^–1), lipids (cholesterol 5·51 ± 6·42 μmol 1^–1, triglycerides 72 ± l00 μmol l^–1, cholesteryloleate 15–150 μmol 1^–1, phosphatidylcholine 26 · 31 μmol 1^–1, glycolipids 1–10 mg 100 m1^–1), lactate 108 ± 99 μmol 1^–1, hydroxybutyrate 102 ± 99 nmol 1^–1, choline 59 ± 159 μmol 1^–1, protein 344·75 ± 59·06 mg 100m1^–1, enzymes (β-d -glucuronidase l.4 ± 0.7 μmol h^–1 100 ml^–1, protease (caseolytic activity) 1·0 ± 0·6 μmol h^–1 100 ml^–1, alkaline phosphatase 2520·0 ± 861·0 μmol h^–1 100 ml^–1, acid phosphatase 44.0 ± 16.0 μmol h^–1 100 ml^–1, glucose-6-phosphate dehydrogenase 38·9 ± 86·9 μmol h^–1 100 ml^–1, lactate dehydrogenase 134·4 ± 69·6 μmol h^–1 100 ml^–1, butyrylcholine esterase 0·014 ± 0·010 μmol h^–1 100 ml^–1, adenosine triphosphatase 562·8 ± 665·4 μmol h ^–1 100 ml^–1).     

Physicochemical and Preclinical Evaluation of a Novel Buccal Measles Vaccine

The aim of this study is to develop an orally disintegrating film (ODF) containing a microparticulate measles vaccine formulation for buccal delivery. The measles vaccine microparticles were made with biocompatible and biodegradable bovine serum albumin (BSA) and processed by spray drying. These vaccine microparticles were incorporated in the ODF, consisting of Lycoat RS720®, Neosorb P60W® and Tween 80. The yield of the microparticles was approximately 85–95%, w/w. The mean size of the vaccine microparticles was 3.65?±?1.89 μm and had a slightly negative surface charge of 32.65?±?2.4 mV. The vaccine particles were nontoxic to normal cells at high concentrations (500 μg/2.5?×?10⁵ cells) of vaccine particles. There was a significant induction of innate immune response by vaccine microparticles which was observed in vitro when compared to blank microparticles (P?<?0.05). The vaccine microparticles also significantly increased the antigen presentation and co-stimulatory molecules expression on antigen presenting cells, which is a prerequisite for Th1 and Th2 immune responses. When the ODF vaccine formulation was dosed in juvenile pigs, significantly higher antibody titers were observed after week 2, with a significant increase at week 4 and plateauing through week 6 comparative to naïve predose titers. The results suggest that the ODF measles vaccine formulation is a viable dosage form alternative to noninvasive immunization that may increase patient compliance and commercial distribution.     

Alginate/chitosan hydrogels as perspective transport systems for cefotaxime

This work reports synthesis of pH-responsive alginate/chitosan hydrogel spheres with the average diameter of 2.0 ± 0.05 mm, which contain cefotaxime that is an antibiotic of the cefalosporine group. The spheres provided the cefotaxime encapsulation efficiency of 95 ± 1%. An in vitro release of cefotaxime from the spheres in the media that simulate human biological fluids in peroral delivery conditions was found to be a pH-dependent process. The analysis of cefotaxime release kinetics by the Korsmeyer–Peppas model revealed a non-Fickian mechanism of its diffusion, which may be related to intermolecular interactions occurring between the antibiotic and chitosan. Conductometry, UV spectroscopy, and IR spectroscopy were used to study complexation of chitosan with cefotaxime in aqueous media with varied pH, characterize the composition of the complexes, and calculate their stability constants. The composition of the cefotaxime–chitosan complexes was found to correspond to the 1.0:4.0 and 1.0:2.0 molar ratios of the components at pH 2.0 and 5.6, respectively. Quantum chemical modeling was used to evaluate energy characteristics of chitosan–cefotaxime complexation considering the influence of a solvent.     

Evidence for a calcium-binding site on the eggshell of Globodera rostochiensis with a role in hatching

Two inhibitors of hatching in Globodera rostochiensis, ruthenium red and lanthanum, have been shown to bind to the eggshell using the techniques of microdensitometry for ruthenium red and X-ray microanalysis for lanthanum. Neither inhibitor penetrated or adhered to unhatched or hatched viable juveniles. Scatchard analysis for binding of lanthanum and ruthenium red to eggshells gave dissociation constants (K) of K_La 32.5 ± 14.0 μM and K_Rured 33.5 ± 5.0 μM respectively. Both values are within the 95% fiducial limits of those shown to cause 50% inhibition of hatch in previous work. Pretreatment with sodium hypochlorite separated an outer part of the eggshell from an inner region which exclusively bound ruthenium red. It is the inner lipoprotein layer that is believed to include the membranes controlling the permeability of the tylenchid eggshell. The rate of binding of ruthenium red was similar for intact and isolated eggshells with 50% binding occurring after 6.11 ± 0.91 min and 4.95 ± 2.38 min but the latter gave a significantly higher maximum binding suggesting that rupture of the eggshells made available additional binding sites on their inner surface. The binding of ruthenium red to the eggshells was pH dependant over most of the range pH 2.8–8.5 with 50% binding, given with its standard deviation, occurring at pH 5.75 ± 0.85. Competitive binding of lanthanum influenced the binding of ruthenium red to the eggshells from which Scatchard analysis gave K_la of 176 ± 79 μM. Similarly, calcium influenced the binding but this caused a biphasic plot with high and low affinity binding sites of K“_ca of 0.423 ± 1.16 μm and K‘_ca of 1078 ± 462 μM. The existence of a high affinity site for calcium that also binds ruthenium red, suggests that the eggshell membrane includes a calcium binding glycoprotein as found in some other receptor mechanisms.     

Drying Using Supercritical Fluid Technology as a Potential Method for Preparation of Chitosan Aerogel Microparticles

Supercritical fluid technology offers several advantages in preparation of microparticles. These include uniformity in particle size, morphology, and drug distribution without degradation of the product. One of the recent advantages is preparation of porous aerogel carrier with proper aerodynamic properties. In this study, we aimed to prepare chitosan aerogel microparticles using supercritical fluid (SCF) technology and compare that with microparticles produced by freeze drying (FD). Loading the prepared carriers with a model drug (salbutamol) was also performed. Comparisons of the particle properties and physicochemical characterizations were undertaken by evaluating particle size, density, specific surface area, and porosity. In vitro drug release studies were also investigated. The effect of many variables, such as molecular weight of chitosan oligomers, concentrations of chitosan, and concentrations of tripolyphosphate on the release, were also investigated. Chitosan aerogels were efficiently produced by SCF technology with an average particle size of 10 μm with a tapped density values around 0.12 g/mL, specific surface area (73–103) m²/g, and porosity (0.20–0.29) cc/g. Whereas, microparticles produced by FD method were characterized as cryogels with larger particle size (64 microns) with clear cracking at the surface. Sustained release profile was achieved for all prepared microparticles of salbutamol produced by the aforementioned methods as compared with pure drug. The results also demonstrates that chitosan molecular weight, polymer concentration, and tripolyphosphate concentration affected the release profile of salbutamol from the prepared microparticles. In conclusion, SCF technology was able to produce chitosan aerogel microparticles loaded with salbutamol that could be suitable for pulmonary drug delivery system.KEY WORDS: aerodynamic, aerogels, chitosan, salbutamol, supercritical fluid technology     

Taxonomy,phylogeny and host-parasite interaction of two novel Myxobolus species infecting Brycon orthotaenia from the São Francisco River,Brazil

Two new Myxobolus species were described infecting Brycon orthotaenia from the São Francisco River, in the state of Minas Gerais, Brazil. From a total of 39 B. orthotaenia collected, two specimens (5.1%) exhibited infection of the ovary and 12 specimens (30.8%) displayed infection of the liver. The plasmodia of both Myxobolus species were white and spherical measuring around 1 mm in length. The plasmodium found in the ovary showed mature myxospores, which were oval shaped from the frontal view and measured 9.2–11.0 (9.8 ± 0.4) μm in length, 5.9–6.9 (6.5 ± 0.3) μm in width and 4.6–5 (4.9 ± 0.1) μm in diameter. The two polar capsules were the same size and measured 3.9–6.2 (4.7 ± 0.5) μm in length and 1.8–2.4 (2.1 ± 0.2) μm in width. The polar tubules had 9 coils. The plasmodium found in the liver showed mature myxospores which were ellipsoidal in shape from the frontal view and measured 10.0–11.4 (10.7 ± 0.5) μm in length, 7.3–8.6 (8.1 ± 0.4) μm in width and 5.3–7.0 (6.8 ± 0.4) μm in diameter. The two polar capsules were the same size and measured 4.2–5.4 (4.9 ± 0.3) μm in length and 1.9–2.9 (2.7 ± 0.3) μm in width. The polar tubules had 8 coils. Ultrastructural analysis revealed an asynchronous sporogenesis process, with young developmental myxospore stages more often found in the periphery of the plasmodium and mature myxospores in the centre of the plasmodium. The plasmodial wall was formed by a single membrane which was not surrounded by a layer of host tissue. A thick layer of fibrous material was found in the peripheral ectoplasm close to the plasmodial wall of the plasmodium found in the ovary. Phylogenetic analysis based on the small-subunit ribosomal DNA – ssrDNA sequences and using the closest myxozoan sequences to each one of the species studied here based on previous GenBank data and Henneguya/Myxobolus/Thelohanellus species parasitizing fish from South American, revealed that the new species are grouped in a subclade together with other Myxobolus species parasitizing bryconid hosts.     

Two New Species of Eimeria from Peacocks (Pavo cristatus) in Saudi Arabia1

Fifteen fecal samples from peacocks (Pavo cristatus) in Saudi Arabia contained oocysts of Eimeria riyadhae n. sp. in two peacocks and oocysts of E. arabica n. sp. in one peacock. Sporulated oocysts of Eimeria riyadhae are ellipsoidal, 27–30.5 times 20.5–25 (28.8 ± 1.3 × 22.4 ± 1.6) μm, with a two-layered wall and bilobed polar body, but without a micropyle or residuum. The sporocysts are ovoid, 11–14.5 × 6.5–8 (13.2 ± 1.2 × 7.2 ± 0.6) μm with a thick, knob-like Stieda body and a residuum. Sporulated oocysts of Eimena arabica are spheroidal, 17.5–21.5 × 17.5–21.5 (19.2 ± 1.6 × 19.2 ± 1.6) μm, with a two-layered wall and two refractile polar bodies, but without a micropyle or residuum. The sporocysts are elongate ovoid, 9.5–12 × 4–6.5 (11.2 ± 0.9 × 5.5 ± 0.88), with a small crescent-shaped Stieda body. The host bird belongs to the order Galliformis.     

Synthesis,characterization, and anticancer evaluation of novel 4-hydrazinothiazole analogs

Single-step synthesis of novel 4-hydrazinothiazole derivatives 6a–e was achieved under mild conditions using the sequential four-components method involving isothiocyanate, aminoguanidine, carbonyl adduct, and α-haloketone derivatives. Deprotection of these hydrazinothiazoles was influenced by acylation, providing a novel group of diacylated molecular structures with a broader scope for the design of thiazolyl-containing drugs 7a and 7b . FTIR, ¹H/¹³C NMR, LC–MS spectroscopy, and CHN elemental analyses were used to study the compound chemical structures. Using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay on human periodontal ligament fibroblast (HPDLF) cells, the 4-hydrazinothiazole derivatives were screened for cytotoxicity in an in vitro cytotoxicity investigation. The 4-hydrazinothiazole compound 6b bearing an isopropylidene-hydrazino group demonstrated strongly potent cytotoxicity against CAKI1 (IC₅₀ = 1.65 ± 0.24 μM) and A498 (IC₅₀ of 0.85 ± 0.24 μM). Furthermore, the chloroacetyl-containing thiazole compound 7a displayed efficient inhibition of growth against the test cell lines CAKI1 and A498 at low micromolar concentrations, IC₅₀ 0.78 and 0.74 μM, respectively.     

Serum selenium levels in Slovak population

Blood serum selenium levels were measured in 576 healthy middle aged adults (40–60 yr, 255 men and 321 women) residing in both urban and rural areas in four districts of Slovakia. Serum selenium was determined by electrothermal AAS. The mean (±SD) serum selenium concentration was 0.852±0.335 μmol/L, ranging from 0.219–2.30 μmol/L. A large proportion of the individuals (19.62%) exhibited serum selenium levels under 0.57 μmol/L (45 μmol/L). There was no significant correlation between serum, selenium concentration and age, sex, and smoking status. There were significant differences between districts. The lowest mean (±SD) serum selenium was 0.664±0.269 μmol/L, the highest mean serum selenium (±SD) was 0.975±0.361 μmol/L. This differences could probably be attributed to the selenium, content in the soil of the different areas, which would contribute to the average daily selenium intake. In comparison with serum selenium levels in other European countries, the concentrations of selenium in the Slovak population are relatively low.     

Ubiquinone-10 plasma concentrations in healthy European children

The reduced form of ubiquinone-10 (coenzyme Q) has been shown to represent an important physiologic antioxidant principle in human blood. In order to establish a reference range for infants, we measured plasma levels of ubiquinone in 50 healthy European children aged 2 months to 15 years. A mean ±SD) value of 0.75±0.27 μg/ml plasma (0.87±0.31 μM) was determined; ubiquinone concentrations were not found to be sex-dependent (0.7±0.24μg/ml for girls, n=17, and 0.7±0.28μg/ml for boys, n=33) but correlated negatively with age (r = -0.37, P=0.0075). This negative correlation was mainly due to relatively high levels in infants approximately 1 year old.

The mean value determined does not significantly differ from the average ubiquinone plasma concentrations determined in healthy Nigerian children (0.85±0.40 μg/ml, n= 18) in a previous study (Becker K, Boetticher D, Leichsenring M. Internat J Vitam Nutr Res 1995, in press).     

Preparation of alginate coated chitosan microparticles for vaccine delivery

Background  

Absorption of antigens onto chitosan microparticles via electrostatic interaction is a common and relatively mild process suitable for mucosal vaccine. In order to increase the stability of antigens and prevent an immediate desorption of antigens from chitosan carriers in gastrointestinal tract, coating onto BSA loaded chitosan microparticles with sodium alginate was performed by layer-by-layer technology to meet the requirement of mucosal vaccine.     

The effect of protein kinase C activator and nitric oxide donor on oocyte activation and cortical granule exocytosis in porcine eggs

Nitric oxide (NO) and protein kinase C (PKC) are involved in the activation of mammalian oocytes, although their role in the exit from the metaphase II stage and cortical granule (CG) exocytosis is still not fully understood. The aim of this study was to verify whether the NO-donor together with specific PKC-activators induce the complete activation of porcine oocytes assessed as meiosis resumption and a cortical reaction. Pig maturated oocytes were treated with the NO-donor S-nitroso-N-acetylpenicillamine (SNAP, 2 mM) or PKC-activators such as phorbol-12-myristate-13-acetate (PMA, 100 nM), 1-oleoyl-2-acetyl-sn-glycerol (OAG, 400 μM) and l-α-phosphatidylinositol-3,4,5-trisphosphate dipalmitoyl heptaammonium salt (DPAM, 2 μM). To study the combined effect of NO-donor and PKC-activators, aliquots of oocytes were also incubated with SNAP (0.5 mM) together with PKC-activators at the same concentration as above (SNAP–DPAM, SNAP–OAG and SNAP–PMA groups). After in vitro maturation, an aliquot of oocytes was placed in a fresh medium without NO-donor or PKC-activators (Control group). Another aliquot of oocytes was activated by calcium ionophore A23187 (25 μM, 5 min). The results showed that 0% of the control oocytes reassumed meiosis. However, both the PKC-activators (DPAM 44.0 ± 10.0%, OAG 63.3 ± 1.0% and PMA 45.0 ± 16.5%) as well as the NO-donor alone (48.7 ± 21.0%) significantly induced exit from MII. Interestingly, the combination of PKC-activators and SNAP mainly restrained to the meiosis resumption (SNAP–OAG 0, SNAP–DPAM 17.4 ± 2.5% and SNAP–PMA 38.4 ± 8.5%). Control oocytes did not show a cortical reaction and the area occupied by CG reached 25.9 ± 1.7%, whereas CGs were partially released after Ca²⁺ ionophore treatment (13.0 ± 3.2%). Treatment with PKC-activators induced a cortical reaction compared with the control group (8.6 ± 2.5, 6.7 ± 1.9 and 0.7 ± 0.4%, respectively, for DPAM, OAG and PMA groups). However, treatment with the NO-donor alone (SNAP group 17.2 ± 2.2%) or combined with any PKC-activator prevented cortical reaction (SNAP–DPAM 20.7 ± 2.6%, SNAP–OAG 16.7 ± 2.9% or SNAP–PMA 20.0 ± 2.4%). Besides, meiosis resumption was not always accompanied by a cortical reaction, indicating that these two activation events are independent. In conclusion, PKC-activators alone induce CG exocytosis to the same degree as calcium ionophore. However, an NO-donor alone or combined with PKC-activators is not able to induce a cortical reaction in pig oocytes.     

Chitosan and silver nanoparticles are attractive auxin carriers: A comparative study on the adventitious rooting of microcuttings in apple rootstocks

Nanocarriers for encapsulation and sustained release of agrochemicals such as auxins have emerged as an attractive strategy to provide enhanced bioavailability and efficacy for improved crop yields and nutrition quality. Here, a comparative study was conducted on the effectiveness of chitosan-as a biopolymeric nanocarrier- and silver-as a metallic nanocarrier- on in vitro adventitious rooting potential of microcuttings in apple rootstocks, for the first time. Auxins indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA) loaded silver (nAg) or chitosan nanoparticles (nChi) were synthesized. Scanning electron microscopy and transmission electron microscopy studies showed the spherical shape of the nanoparticles. The average particle size of IAA-nChi was 167.5 ± 0.1 nm while that of IBA-nChi was 123.2 ± 2.6 nm. The hydrodynamic diameter of the nAg-IAA and nAg-IBA particles were measured as 93.66 ± 5 nm and 71.41 ± 3 nm, respectively. Fourier transform infrared spectroscopy analyses confirmed the encapsulation of IAA or IBA in the chitosan nanoparticles. Meanwhile, the characteristic peaks of IAA or IBA were detected on silver nanoparticles. In-vitro adventitious rooting of microcuttings of Malling Merton 106 (MM 106) was significantly higher both in chitosan and silver nanoparticles loaded with IAA or IBA (91.7%–62.5%) compared to free IAA or IBA applications (50.0%–33.3%), except for 2.0 mg L^–1 IBA (66.7%). However, the application of 2 mg L^–1 IBA and IBA-nChi at all concentrations caused an undesirable large callus development.     

Formulation and <Emphasis Type="Italic">In Vitro</Emphasis> Characterization of Thiolated Buccoadhesive Film of Fluconazole

The present work is focused on the development of thiolated film for fluconazole buccal delivery. To this end, unmodified polymers chitosan and sodium carboxymethylcellulose (NaCMC) backbone was covalently modified by thioglycolic acid (TGA) and cysteine, respectively. The thiolated buccoadhesive film was evaluated in terms of thickness, weight uniformity, water-uptake capacity, drug content, and release patterns. Moreover, mucoadhesion profile was investigated on buccal mucosa. The resulting chitosan–TGA and NaCMC–cysteine conjugates displayed 171?±?13 and 380?±?19 μmol thiol groups per gram of polymer (mean?±?SD; n?=?3), respectively. The water binding capacity of the thiolated film was significantly ～2-fold higher (p?<?0.05) as compared to unmodified film. The obtained thiolated film displayed 5.8-fold higher mucoadhesive properties compared with corresponding film. Controlled release of drugs from film was observed over 8 h. The transport of fluconazole across excised buccal mucosa was enhanced up to 17-fold in comparison with fluconazole applied in buffer. Based on these findings, thiolated film seems to be promising for fluconazole buccal delivery.     

Enhanced protection against pulmonary mycobacterial challenge by chitosan‐formulated polyepitope gene vaccine is associated with increased pulmonary secretory IgA and gamma‐interferon+ T cell responses

Induction of local (pulmonary) immunity plays a critical role in preventing dissemination of Mycobacterium tuberculosis (M. tb) during the early infection stage. To induce specific mucosal immunity, chitosan, a natural cationic polysaccharide, was employed as a mucosal gene carrier and complexed with pHSP65pep, our previously constructed multi‐epitope gene vaccine, which induces splenic gamma‐interferon (IFN‐γ)⁺ T helper cell 1 responses. The resultant chitosan–pHSP65pep was administered intranasally to BALB/c mice with four doses of 50 μg DNA followed by mycobacterial challenge 4 weeks after the final immunization. It was found that the chitosan formulation significantly induced production of secretory immunoglobulin A (P < 0.05) as determined by measuring its concentrations in lung lavage fluid and enhanced pulmonary CD4⁺ and CD8⁺IFN‐γ⁺ T cell responses (P < 0.001) compared with naked gene vaccine. Improved protection against Mycobacterium bovis bacillus Calmette–Guérin (BCG) challenge was consistently achieved by the chitosan–DNA formulation both as the vaccine alone or in a BCG prime‐vaccine boost immunization scenario. Our study shows that mucosal delivery of gene vaccine in a chitosan formulation remarkably enhances specific SIgA concentrations and mucosal IFN‐γ⁺ T cell response, which correlated positively with immunological protection.     

Sodium,potassium, and protein concentrations and 2D-SDS-page of epididymal luminal and ejaculated seminal fluids of the adult chimpanzee (Pan troglodytes)

The concentration of soluble protein and of sodium and potassium ions was estimated in chimpanzee caput epididymal luminal fluid, cauda epididymal luminal fluid, and ejaculated seminal fluid. Protein concentration was 48.5 ± 1.5 μg/μl in caput fluid, 26.8 ± 2.0 μg/μl in cauda fluid, and 53.0 ± 7.9 μg/μl in seminal fluid. Sodium concentration was 127.0 ± 7.0 mM in caput fluid, 34.5 ± 1.8 mM in cauda fluid, and 18.8 ± 1.8 mM in seminal fluid. Potassium concentration was 58.0 ± 0.0 mM in caput fluid, 56.8 ± 5.2 mM in cauda fluid, and 77.0 ± 1.7 mM in seminal fluid. Proteins in caput epididymal, cauda epididymal, and ejaculated seminal fluids, with approximate molecular weights (kDa) between <14.4 and 45.0 kDa and apparent isoelectric points (pIs) between 4.5 and 7.5, were resolved by two-dimensional SDS-polyacrylamide gel electrophoresis (2D-SDS-PAGE) and silver stained. In caput fluid, the most intensely stained polypeptides resolved between 14.4 and 21.5 kDa (pI 5.4–7.4). In cauda fluid, the number and intensity of stained components increased markedly, and the most intensely stained polypeptides resolved between 21.5 and 31.0 kDa (pI 5.7–7.3). In seminal fluid, polypeptides between <14.4 and 45.0 kDa (pI 5.7–7.4) appeared characteristically diffuse and distributed. These results demonstrate that the ions and the polypeptides in the luminal microenvironment change significantly along the epididymal duct of the male chimpanzee. © 1994 Wiley-Liss, Inc.