Synthesis of L-methionine-loaded chitosan nanoparticles for controlled release and their in vitro and in vivo evaluation

Abstract Therapeutically popular controlled release-enabling technology has forayed into the nutrition sector. Polymer coated forms of L-methionine used in soy protein diets, and its intermediate metabolite, S-adenosyl-L-methionine, used in myriad of medical conditions have proved more efficacious over (highly catabolized) free forms. In this premier study, L-methionine-loaded chitosan nanoparticles (M-NPs) were synthesized using ionic gelation method and their efficacy was evaluated. Biophysical characterization of the NPs was done using a Nanopartica SZ 100 analyser, transmission electron microscopy, and Fourier transform infrared spectroscopy. The M-NPs were spherical and smooth and 218.9 ± 7.4 nm in size and in vitro testing confirmed the controlled release of methionine. A 60-days feeding trial in L. rohita fish fingerlings was conducted. A basal diet suboptimal (0.85%) in methionine was provided with one of the supplements as under: none (control), 0.8% chitosan NPs (0.8% NPs), 1.2% L-methionine (1.2% M) (crystalline free form), 0.6% M-NPs and 1.2% M-NPs. While the addition of 0.6% M-NPs to the basal diet complemented towards meeting the established dietary requirement and resulted in significantly highest (P < 0.05) growth and protein efficiency and sero-immunological test scores (serum total protein, serum globulin, serum albumin: globulin ratio, phagocytic respiratory burst/NBT reduction and lysozyme activity), 1.2% supplementation in either form (free or nano), for being 0.85% excess, was counterproductive. Liver transaminases and dehydrogenases corroborated enhanced growth. It was inferred that part of the methionine requirement in nano form (M-NPs) can confer intended performance and health benefits in animals relying on plant proteins-based diets limiting in this essential amino acid. The study also paves the way for exploring chitosan NPs-based sustained delivery of amino acids in human medical conditions

Chitosan nanoencapsulated exogenous trypsin biomimics zymogen-like enzyme in fish gastrointestinal tract.

Exogenous proteolytic enzyme supplementation is required in certain disease conditions in humans and animals and due to compelling reasons on use of more plant protein ingredients and profitability in animal feed industry. However, limitations on their utility in diet are imposed by their pH specificity, thermolabile nature, inhibition due to a variety of factors and the possibility of intestinal damage. For enhancing the efficacy and safety of exogenous trypsin, an efficient chitosan (0.04%) nanoencapsulation-based controlled delivery system was developed. An experiment was conducted for 45 days to evaluate nanoencapsulated trypsin (0.01% and 0.02%) along with 0.02% bare trypsin and 0.4% chitosan nanoparticles against a control diet on productive efficiency (growth rate, feed conversion and protein efficiency ratio), organo-somatic indices, nutrient digestibility, tissue enzyme activities, hematic parameters and intestinal histology of the fish Labeo rohita. All the synthesized nanoparticles were of desired characteristics. Enhanced fish productive efficiency using nanoencapsulated trypsin over its bare form was noticed, which corresponded with enhanced (P<0.01) nutrient digestibility, activity of intestinal protease, liver and muscle tissue transaminases (alanine and aspartate) and dehydrogenases (lactate and malate), serum blood urea nitrogen and serum protein profile. Intestinal tissues of fish fed with 0.02% bare trypsin showed broadened, marked foamy cells with lipid vacuoles. However, villi were healthier in appearance with improved morphological features in fish fed with nanoencapsulated trypsin than with bare trypsin, and the villi were longer in fish fed with 0.01% nanoencapsulated trypsin than with 0.02% nanoencapsulated trypsin. The result of this premier experiment shows that nanoencapsulated trypsin mimics zymogen-like proteolytic activity via controlled release, and hence the use of 0.01% nanoencapsulated trypsin (in chitosan nanoparticles) over bare trypsin can be favored as a dietary supplement in animals and humans

Lipotropes Protect against Pathogen-Aggravated Stress and Mortality in Low Dose Pesticide-Exposed Fish

<div><p>The decline of freshwater fish biodiversity corroborates the trends of unsustainable pesticide usage and increase of disease incidence in the last few decades. Little is known about the role of nonlethal exposure to pesticide, which is not uncommon, and concurrent infection of opportunistic pathogens in species decline. Moreover, preventative measures based on current knowledge of stress biology and an emerging role for epigenetic (especially methylation) dysregulation in toxicity in fish are lacking. We herein report the protective role of lipotropes/methyl donors (like choline, betaine and lecithin) in eliciting primary (endocrine), secondary (cellular and hemato-immunological and histoarchitectural changes) and tertiary (whole animal) stress responses including mortality (50%) in pesticide-exposed (nonlethal dose) and pathogen-challenged fish. The relative survival with betaine and lecithin was 10 and 20 percent higher. This proof of cause-and-effect relation and physiological basis under simulated controlled conditions indicate that sustained stress even due to nonlethal exposure to single pollutant enhances pathogenic infectivity in already nutritionally-stressed fish, which may be a driver for freshwater aquatic species decline in nature. Dietary lipotropes can be used as one of the tools in resurrecting the aquatic species decline.</p></div

Fish injected with pathogenic bacteria <i>Aeromonas hydrophila</i> showing one or more typical signs of infection according to the stage of disease.

<p>Signs included hemorrhagia (large and irregular hemorrahages), shallow to deep necrotizing ulcers, and abdominal distension with sero-hemorrhagic fluids exuded from the inflamed vent. White arrows indicate edges of the ulcers. A yellow arrow indicates vent. Abdominal distension is clear in the right side fish. All fish shown are infected.</p

Secondary stress response to low dose endosulfan-exposure in <i>L. rohita</i> fish fingerlings unfed or fed with lipotropes for 37 days: Pre-challange^┼ and post-challenge^┼┼ hematological profile.

┼<p>Pre-challange blood samples were taken after 37 days of experiment. Subsequently, fish were challenged with the infectious bacteria, <i>A. hydrophila</i>, injected intraperitoneally and ^┼┼post-challange samples were taken at the end of 7 days in surviving fish or just before sacrificing severely morbid fish.</p><p>Units: RBC count (x 10⁶ cells/mm³), WBC count (x 10³ cells/mm³) and Hemoglobin (Hb) g/dL.</p><p>**Indicates significant difference from pre-challenge values (P<0.01) with in a group by student's t-test.</p>a, b, c, d<p>Means bearing different superscript letters in a row differ significantly against the P value indicated in the last column. Data expressed as Mean ± SE (n = 6).</p

Secondary stress response to low dose endosulfan exposure in fish unfed or fed with lipotropes for 37 days: Histoarchitectural response.

<p>Histoarchitecture of the liver revealed a protective role for lipotropes in fish exposed to a nonlethal dose of endosulfan and intraperitoneally injected with the infectious bacteria, <i>A. hydrophila</i>. Pre-challenge samples were collected after 37 days of experiment. Post-challenge samples were collected at the end of 7 days in surviving fish or just before sacrificing severely morbid fish. Section from control fish (A), <i>A. hydrophila</i> injected fish (B), fish exposed to nonlethal dose of endosulfan (C) and also injected with <i>A. hydrophila</i> but either fed with no supplements (D), or fed with choline (E), betaine (F) or lecithin (G). Blue arrowhead: sinusoids, Red arrowhead: hepatocyte with nucleus, Black arrowhead: vacuole in hepatocyte, White arrow: central vein, White arrowhead: ghost cell without nucleus (due to karyolysis), Yellow arrowhead: focal inflammatory infiltrate. Histological changes are described in detail in the text.</p

Secondary stress response to low dose endosulfan-exposure in <i>L. rohita</i> fish fingerlings unfed or fed with lipotropes for 37 days: Effect on body composition and some metabolites.

<p>OM¹, Organic Matter, CP², Crude Protein and ³Ash expressed as % DM.</p><p>Glycogen expressed as mg glycogen/g tissue. Data expressed as Mean ± SE (n = 6).</p

Composition of the basal diet.

a, b, c, d<p>Sources: ^aprocured from local market, ^bHiMedia (JTJ Enterprises, Mumbai, India), ^cPrepared manually and all components from HiMedia Ltd and ^dSD Fine-Chemicals Ltd (Mumbai, India).</p>c<p>Composition of vitamin mineral mix (quantity/250 g starch powder): Vitamin A 550,000 IU; Vitamin D₃ 110,000 IU; Vitamin B₁ 20 mg; Vitamin B₂ 200 mg; Vitamin E 75 mg; Vitamin K 100 mg; Vitamin B₁₂ 0.6 μg; Calcium Pantothenate 250 mg; Nicotinamide 1000 mg; Pyridoxine 100 mg; Mn 2,700 mg; I 100 mg; Fe 750 mg; Zn 500 mg; Cu 200 mg; Co 45 mg; Ca 50 g; P 30 g; Selenium 5 ppm.</p

Secondary stress response to low dose endosulfan exposure in fish unfed or fed with lipotropes for 37 days: Cellular responses.

<p>Secondary cellular stress responses included activities/levels of: antioxidant enzymes superoxide dismutase (SOD) and catalase; phase II metabolism enzymes glutathione-s-transferase (GST) and SAM-dependent methyl transferase (MT); heat shock protein (HSP70); and caspase. While MT was measured in serum, all other attributes were quantified in the liver and gills. Abbreviations for exposure/diet treatments of fish are the same as used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093499#pone-0093499-g001" target="_blank">Figure 1:</a> Ctr, control; EE, endosulfan-exposed; Cho, choline; Bet, betaine and Lec, lecithin. The values reported in bar charts represent the mean±SE. Bars bearing different letters (a, b, c) indicate significant differences between treatment means for the level/activity of a marker in respective tissue or serum. Probability (P) values: SOD liver (P = 0.002), SOD gill (P = 0.005), catalase gill (P = 0.003), catalase liver (P = 0.005), GST gill (P = 0.02), GST liver (P = 0.03), serum MT (P<i> = </i>0.001), HSP70 and caspase (P = 0.001). Number of observations (n): n = 6 for SOD, GST, catalase, HSP70 and caspase, and n = 7 for MT.</p

Secondary stress response to low dose endosulfan-exposure in <i>L. rohita</i> fish fingerlings unfed or fed with lipotropes for 37 days: Pre-challange^┼ and post-challenge^┼┼ serum protein profile.

┼<p>Pre-challange blood samples were taken after 37 days of experiment. Subsequently, fish were challenged with the infectious bacteria, <i>A. hydrophila</i>, injected intraperitoneally and ^┼┼post-challange samples were taken at the end of 7 days in surviving fish or just before sacrificing severely morbid fish.</p><p>TP indicates Total Protein. Serum proteins expressed as g/dL.</p><p>*Indicates significant difference from pre-challenge value (P<0.01) with in a group by student's t-test.</p>a, b, c<p>Means bearing different superscript letters in a row differ significantly against P value indicated in the last column. Data expressed as Mean ± SE (n = 6).</p