Development and optimization of pH-responsive PLGA-chitosan nanoparticles for triggered release of antimicrobials.

The aim of this work was to develop and optimize a pH-responsive nanoparticle based on poly(D,L-lactide-co-glycolide) (PLGA) and chitosan (CHIT) for delivery of natural antimicrobial using trans-cinnamaldehyde (TCIN) as a model compound. The optimization was performed using a central composite design and the desirability function approach. The optimized levels of variables considering all significant responses were 4% (w/w) of TCIN and 6.75% (w/w) of CHIT. After, optimized nanoparticles were produced and characterized according to their physicochemical properties and their antimicrobial activity against Salmonella Typhimurium and Staphylococcus aureus. Optimized nanoparticles characterization indicated a satisfactory TCIN encapsulation (33.20 ±â€¯0.85%), spherical shape, pH-responsive controlled release, with faster release in the presence of CHIT at low pH, and enhanced antimicrobial activity against both pathogens. TCIN encapsulation using PLGA coated with CHIT enhanced its antimicrobial activity and generated a delivery system with pH-sensitivity for controlled release with promising properties for food safety applications.

Self-assembled amphiphilic chitosan nanoparticles for quercetin delivery to breast cancer cells.

Novel drug delivery strategies are needed to meet the complex challenges associated to cancer therapy. Biocompatible pH-sensitive drug delivery nanocarriers based on amphiphilic co-polymers seem to be promising for cancer treatment. In the present study, a drug delivery system was produced by encapsulating quercetin into novel pH-sensitive self-assembled amphiphilic chitosan nanoparticles. Up to 83% of quercetin was entrapped by the nanoparticles. The particle diameter, as measured by dynamic light scattering (DLS), ranged from ∼235 to ∼312 nm for the blank and ∼490 to ∼502 nm for the loaded carriers. The results showed that the payload release is larger at acidic pH (5.0) than at physiological pH (7.4). Fitting the data to the Korsmeyer-Peppas model indicated that anomalous diffusion is the predominant release mechanism at pH 5.0, while Fickian diffusion operates at pH 7.4. The MTT assay revealed that blank nanoparticles were non-antiproliferative for the cell tested. The results further revealed that quercetin maintains its metabolism inhibition against MCF-7 cells after encapsulation. Cellular uptake experiments showed that nanoparticles accumulated on the cell surface, whereas few were internalized. Haemocompatibility test results suggest that the nanoparticles exhibit suitable blood compatibility for biological applications. Results suggest that nanoparticles might be a promising pH-sensitive drug delivery system for applications in anticancer treatment.

Synthesis and Characterization of pH-Sensitive Genipin Cross-Linked Chitosan/Eudragit® L100 Hydrogel for Metformin Release Study Using Response Surface Methodology.

BACKGROUND: In this study, central composite design was utilized for the optimization of genipin cross-linked chitosan/Eudragit®-L 100 interpenetrating hydrogel network films fabricated through solvent evaporation technique. METHODS: Hydrogel formulations were studied using response surface methodology; regression analysis and the surface plots were used to evaluate the effect of variables on T50% (the time for 50% of drug release) and dynamic swelling with optimum formulation selection. Initial burst release of drug was observed from the formulated hydrogels during the first 2 hours of dissolution at simulated gastric pH 1.2 and then slow release during the next 10 hours in the simulated intestinal fluid at pH 7.4. Different polymer ratios in formulation showed significant influence on T50% and dynamic swelling of hydrogel. The highest T50% was observed at 9.89 hour and dynamic swelling at 7.86 h. RESULT: It was observed that by changing the polymer ratio with cross-linker, release rate of metformin could be modified. Cross-linker also affects drug release rate, i.e. the release rate is decreased with the increase in its concentration. The physical state of hydrogel was investigated by scanning electron microscope. CONCLUSION: It indicated the uniform distribution of drug in hydrogel matrix system. Moreover, the presence of hydrogen and ionic bonds between polymers and crosslinking agent formed interpenetrating hydrogel network, likely responsible for increased value of T50%, as confirmed by FTIR. Acute oral toxicity study was performed to investigate the toxic effect of crosslinking agent and polymer used in formulations.

Effect of the Presence of Surfactants and Immobilization Conditions on Catalysts' Properties of Rhizomucor miehei Lipase onto Chitosan.

Lipase from Rhizomucor miehei (RML) was immobilized onto chitosan support in the presence of some surfactants added at low levels using two different strategies. In the first approach, the enzyme was immobilized in the presence of surfactants on chitosan supports previously functionalized with glutaraldehyde. In the second one, after prior enzyme adsorption on chitosan beads in the presence of surfactants, the complex chitosan beads-enzyme was then cross-linked with glutaraldehyde. The effects of surfactant concentrations on the activities of free and immobilized RML were evaluated. Hexadecyltrimethylammonium bromide (CTAB) promoted an inhibition of enzyme activity while the nonionic surfactant Triton X-100 caused a slight increase in the catalytic activity of the free enzyme and the derivatives produced in both methods of immobilization. The best derivatives were achieved when the lipase was firstly adsorbed on chitosan beads at 4 °C for 1 h, 220 rpm followed by cross-link the complex chitosan beads-enzyme with glutaraldehyde 0.6% v.v-1 at pH 7. The derivatives obtained under these conditions showed high catalytic activity and excellent thermal stability at 60° and 37 °C. The best derivative was also evaluated in the synthesis of two flavor esters namely methyl and ethyl butyrate. At non-optimized conditions, the maximum conversion yield for methyl butyrate was 89%, and for ethyl butyrate, the esterification yield was 92%. The results for both esterifications were similar to those obtained when the commercial enzyme Lipozyme® and free enzyme were used in the same reaction conditions and higher than the one achieved in the absence of the selected surfactant.

Transient aggregation of chitosan-modified poly(d,l-lactic-co-glycolic) acid nanoparticles in the blood stream and improved lung targeting efficiency.

Chitosan (CS)-modified poly(d,l-lactic-co-glycolic) acid (PLGA) nanoparticles (NPs) were prepared and their lung targetability after intravenous administration was elucidated. PLGA NPs (mean diameter: 225nm; polydispersity index: 0.11; zeta potential: -15mV), 0.2% (w/v) CS-coated PLGA NPs (CS02-PLGA NPs, mean diameter: 264nm; polydispersity index: 0.17; zeta potential: -7mV), and 0.5% (w/v) CS-coated PLGA NPs (CS05-PLGA NPs, mean diameter: 338nm; polydispersity index: 0.23; zeta potential: 12mV) were fabricated by a modified solvent evaporation method. PLGA NPs maintained their initial particle size in different media, such as human serum albumin (HSA) solution, rat plasma, and distilled water (DW), while CS05-PLGA NPs exhibited the formation of aggregates in early incubation time and disassembly of those into the NPs in late incubation time (at 24h). According to the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, the binding affinity of CS05-PLGA NPs with HSA and rat plasma was higher than that of PLGA NPs. By a near-infrared fluorescence (NIRF) imaging test in the mouse, the selective accumulation of CS05-PLGA NPs, rather than PLGA NPs, in lung tissue was demonstrated. These findings suggest that CS05-PLGA NPs can form transient aggregates in the blood stream after intravenous administration and markedly improve lung targeting efficiency, compared with PLGA NPs.

A comparative study of chitosan and poloxamer based thermosensitive hydrogel for the delivery of PEGylated melphalan conjugates.

OBJECTIVE: Although the melphalan (ML) used extensively for the management of breast cancer, its clinical application is limited due to significant hemolytic activity. In the present work, a comparative analysis of two distinct in situ-based thermogelling polymers of PEGylated ML was performed. METHODS: Briefly, the PEGylated conjugate of the melphalan (MLPEG 5000) for local and sustained drug release action is loaded into two different thermogelling polymeric systems, namely chitosan- and poloxamer-based systems. The synthesized conjugate was loaded to a chitosan (MLP 5000) and poloxamer-based (MPX-CG) thermogelling injectable hydrogels. These thermogelling hydrogels were evaluated for in vitro hydrolysis, in vitro hemolytic activity. and in vitro anticancer activity. RESULTS: The lower percent cumulative hydrolysis was witness for both the hydrogels. MPX-CG and MLP 5000 hydrogels as predicted had shown lower percent cumulative hydrolysis of 3.31 ± 0.1 and 1.67 ± 0.1 after 6 h. The percentage hemolysis of MPX-CG and MLP 5000 even at a concentration of 32 µg/ml was found to be 39.23 ± 1.24% and 34.23 ± 2.24%, observed at 1 h, respectively. Both the hydrogels showed similar anticancer pattern, the MPX-CG hydrogel showed low cell viability of 8.4 ± 1.1% at a concentration of 150 µM and the MLP-5000 hydrogel showed slight higher cell viability (13.12 ± 5.4%) as compared with MPX-CG hydrogel. CONCLUSION: Hence, from the present study it can be well understood that both the chitosan- and the poloxamer-based thermogelling hydrogel proves to be an effective drug delivery systems for the delivery of the PEGylated conjugates.

Effectiveness of submicronized chitosan-coated liposomes in oral absorption of indomethacin.

The plasma profile of indomethacin (IMC) after oral administration of IMC-loaded submicronized chitosan-coated liposomes (ssCS-Lip) was evaluated to reveal the effectiveness of the mucoadhesive function for improving the absorption of this poorly absorbable drug. The stomach and small intestine were removed from rats after 1, 2, and 4 hours of oral administration of submicron-sized liposomes (ssLip) or ssCS-Lip containing fluorescent dye, and the retentive properties were confirmed by measuring the amount of dye in each part of the gastrointestinal (GI) tract. Results showed that ssCS-Lip tended to be better retained in the upper part of the GI tract, compared with ssLip, at 1, 2, and 4 hours after administration, and was significantly better retained in the small intestine at 4 hours. The plasma profile and bioavailability of IMC after oral administration of both types of liposomes were improved, compared with oral administration of IMC solution. The maximum residence time of ssCS-Lip was significantly longer than those of ssLip. The extended plasma profile of ssCS-Lip was attributed to its prolonged retention in the upper region of the GI tract, and its delayed migration to the lower part of the intestine, the neutral pH of which is more soluble for IMC, an acidic drug. Therefore, the chitosan-coated ssLip, with its higher retention in the GI tract, is a promising drug carrier for the oral administration of poorly absorbed compounds.

Long circulating chitosan/PEG blended PLGA nanoparticle for tumor drug delivery.

Polymeric nanoparticles have long been sought after as carriers for systemic and targeted drug delivery. The ability of these particles to circulate in the bloodstream for a prolonged period of time is often a prerequisite for successful targeted delivery. To achieve this, paclitaxel loaded chitosan and polyethylene glycol coated PLGA (PLGA-CS-PEG) nanoparticles were formulated and characterized that could efficiently encapsulate hydrophobic drugs, and also evade the phagocytic uptake by reducing opsonization by blood proteins, hence increasing the bioavailability of the drug. In our study, we primarily assessed a rational approach for designing and formulating ideal long-circulating nanoparticles by optimizing the concentration of chitosan (CS) and polyethylene glycol (PEG). Uptake efficiency and in vitro cytotoxicity of the formulated nanoparticles was also evaluated in different cancer cell lines (retinoblastoma, breast cancer and pancreatic cancer). PLGA-CS-PEG nanoparticles showed dramatic prolongation in blood circulation, as well as reduced macrophage uptake, with only a small amount of the nanoparticles sequestered in the liver, when compared to PLGA-CS and PLGA nanoparticles. Superior anti-proliferative effect and cell cycle inhibition was observed in case of PLGA-CS nanoparticles and PLGA-CS-PEG nanoparticles over PLGA nanoparticles and native paclitaxel, which may be due to higher cellular uptake resulting in greater antiproliferative activity of nanoparticles. The present results thus suggest that, a combinational coating of PEG and chitosan may represent a significant step in the development of long-circulating drug delivery carriers for tumor drug delivery.

Pharmacokinetics and biodegradation mechanisms of a versatile carboxymethyl derivative of chitosan in rats: in vivo and in vitro evaluation.

Carboxymethyl chitosan (CM-chitosan), which is a water-soluble derivative of chitosan, has attracted much attention as a new biomedical material. The safety study of this material was persuasive for its potential application. The present study was conducted to assess the tissue distribution, pharmacokinetics, biodegradation mechanism, and excretion of CM-chitosan in rats. After the rats were intraperitoneally injected at the dose of 50 mg/kg, the fluorescein isothiocyanate (FITC)-labeled CM-chitosan was absorbed rapidly and distributed to different organs, including liver, spleen, and kidney. The highest level of CM-chitosan was found in liver. It was at the level of 1.6 +/- 0.6 mg/liver and made up approximately 10-22% of the total injected FTC-CM-chitosan. Urinary excretion was the predominant way of excretion of FITC-labeled CM-chitosan, and 85% of the dose was excreted in urine over the period of 11 days. The molecular weights of body distributed FTC-CM-chitosan and urinary excreted FTC-CM-chitosan were analyzed by gel chromatography. The results indicated that the FTC-CM-chitosan was degraded in abdominal dropsy. The absorbed CM-chitosan forms were found with a relatively high molecular weight (approximately 300 kDa), whereas the molecular weight of the urinary excreted FTC-CM-chitosan was less than 45 kDa. In vitro research revealed that the CM-Chi was also degradable in plasma and homogenate of liver. The CM-chitosan with a molecular weight of approximately 800k was thoroughly degraded to a small molecule after it was incubated in homogenate of liver at 37 degrees C for 24 h. The results suggested that the liver plays a central role in biodegradation of CM-chitosan. The excellent biodegradability of CM-chitosan could potentially contribute to the clinical applications. The results also provide important clues for further modification of CM-chitosan as the postsurgical and other biomedical materials.

[An experimental study of plasma-perfusion with a novel aminated chitosan on liver failure in a canine model].

OBJECTIVE: To study the efficacy and safety of plasma-perfusion with a novel aminated chitosan on liver failure in a canine model. METHODS: A canine model of liver failure was established. Plasma-perfusion with a novel aminated chitosan was performed on those dogs. Blood pressure and body temperature during plasma-perfusion were monitored. Total plasma bilirubin, direct bilirubin and indirect bilirubin at the entrance and exit of a column before and after plasma-perfusion and at the time of 15, 30, 60, and 120 min during plasma-perfusion were examined. Blood alanine aminotransferase, aspartate aminotransferase, ammonia, plasma prothrombin time, electrolytes and other blood parameters were examined before and after the plasma-perfusion. RESULTS: After the plasma-perfusion, total bilirubin decreased from 177.4+/-18.1 to 46.1+/-3.7 (P less than 0.05), direct bilirubin decreased from 124.2+/-10.3 to 30.5+/-1.7 (P less than 0.05), indirect bilirubin decreased from 53.2+/-2.8 to 15.6+/-2.0 (P less than 0.05). Compared at the entrance of the column, there were significant decreases in the levels of total bilirubin, direct bilirubin and indirect bilirubin of plasma at the exit of the column at the times of 15 and 30 min during plasma-perfusion (P less than 0.05); there were no further significant decreases at 60 and 120 min (P more than 0.05). Compared with pre-plasma-perfusion, there were significant decreases in the levels of blood alanine aminotransferase, aspartate aminotransferase, and ammonia (P less than 0.05); the plasma prothrombin time was significantly increased (P less than 0.05), the electrolytes, hematocrit level, platelet count, and white cell count were not affected significantly by the perfusion (P more than 0.05); blood pressure and body temperature were not affected significantly during the plasma-perfusion. CONCLUSION: Plasma-perfusion with a novel aminated chitosan resin is an effective and safe method for treating liver failure in this canine model.

The effect of conjugation to gold nanoparticles on the ability of low molecular weight chitosan to transfer DNA vaccine.

Nonviral gene delivery systems based on conventional high molecular weight chitosans are efficient as DNA vaccine delivery system, but have poor physical properties such as aggregated shapes, low solubility at neutral pH, high viscosity at concentrations used for in vivo delivery and a slow onset of action. Furthermore, Chitosan oligomers shorter than 14 monomers units were recently found to form only weak complexes with DNA, resulting in physically unstable polyplexes in vitro and in vivo. Here, low molecular weight chitosans with an average molecular mass of 6kDa (Chito6) have been covalently attached to gold nanoparticles (GNPs), and the potency of the resulting Chito6-GNPs conjugates as vectors for the delivery of plasmid DNA has been investigated in vitro and in vivo. After delivery by intramuscular immunization in BALB/c mice, the Chito6-GNPs conjugates induced an enhanced serum antibody response 10 times more potent than naked DNA vaccine. Additionally, in contrast to naked DNA, the Chito6-GNPs conjugates induced potent cytotoxic T lymphocyte responses at a low dose.

Bioanalysis and pharmacokinetics of chitosan ester in rabbit serum by HPLC with postcolumn fluorescence derivatization.

Interest in antiatherosclerotic activity of chitosan ester (PS916) with a new form of sulfate amino polysaccharide derived from marine chitin has necessitated the development of a sensitive and specific method to study its pharmacokinetics. A sensitive and reproducible high-performance liquid chromatography (HPLC) with postcolumn fluorescence derivatization method was developed and validated for the determination of PS916 in rabbit serum. Chromatography was carried out using a C8 reversed-phase column with an isocratic mobile phase consisting of methanol-water (20:80, v/v) at a flow rate of 0.2 ml/min. The derivatization procedure involved postcolumn reaction with guanidine hydrochloride in an alkaline medium at 110 degrees C. The fluorometric detector was operated at 250 nm (excitation) and 435 nm (emission). The assay was linear over the concentration range of 5-100 microg/ml. The lower limit of detection (LLOD) was found to be 1.0 microg/ml. The proposed method was successfully applied for a pharmacokinetic study of PS916 in rabbits.

Labeling efficiency and biodistribution of Technetium-99m labeled nanoparticles: interference by colloidal tin oxide particles.

The interference of colloidal tin oxides on the biodistribution of (99m)Technetium radiolabeled chitosan nanoparticles has been overcome by using sodium borohydride instead of commonly used stannous salts as reducing agent for the reduction of (99m)Tc (VII) to lower valency states. Biodistribution of radiolabeled chitosan nanoparticles prepared by using stannous chloride method revealed localization of the radioactivity mainly in the liver and spleen while that of radiolabeled chitosan nanoparticles prepared by using sodium borohydride method manifested the presence of radioactivity in blood up to an extent of 10% even after 2 h. Interestingly, the reduction of radioactivity in the latter case with the progress of time was not manifested through an increase in activity in the liver. Rather, a time dependent increased accumulation of radioactive materials was observed in the stomach. From the results it has been concluded that the biodistribution is strongly influenced by the presence of colloidal particles of tin oxides and (99m)Tc labeled chitosan nanoparticles are RES evading and long circulating in blood when Tc (VII) is reduced by sodium borohydride and not by stannous chloride during radiolabeling process.