Chitosan microparticle preparation for controlled drug release by response surface methodology

The objectives were to investigate the effects of formulation variables on the release of drug and to optimize the formulation of chitosan microparticles loaded with drug for controlled release using response surface methodology. Chitosan microparticles were prepared by dropping a chitosan solution into sodium tripolyphosphate (TPP) through ionic cross-linking. The release behaviour of felodipine as a model drug was affected by preparation variables. A central composite design was used to evaluate and optimize the effect of preparation variables, chitosan concentration (X1), the pH of the TPP solution (X2) and cross-linking time (X3) on the cumulative per cent drug release (Y) in 24 h. Chitosan concentration and cross-linking time affected negatively the release of felodipine, while the pH of the TPP did so positively and was the highest influential factor. The optimum rate of drug release, 100% in 24 h, was achieved at 1.8% chitosan concentration, a pH 8.7 for the TPP solution and 9.7 min cross-linking time.     

Chitosan microparticle preparation for controlled drug release by response surface methodology

The objectives were to investigate the effects of formulation variables on the release of drug and to optimize the formulation of chitosan microparticles loaded with drug for controlled release using response surface methodology. Chitosan microparticles were prepared by dropping a chitosan solution into sodium tripolyphosphate (TPP) through ionic cross-linking. The release behaviour of felodipine as a model drug was affected by preparation variables. A central composite design was used to evaluate and optimize the effect of preparation variables, chitosan concentration (X₁), the pH of the TPP solution (X₂) and cross-linking time (X₃) on the cumulative per cent drug release (Y) in 24 h. Chitosan concentration and cross-linking time affected negatively the release of felodipine, while the pH of the TPP did so positively and was the highest influential factor. The optimum rate of drug release, 100% in 24 h, was achieved at 1.8% chitosan concentration, a pH 8.7 for the TPP solution and 9.7 min cross-linking time.     

Preparation and characterization of chitosan microparticles intended for controlled drug delivery

Chitosan microparticles were prepared with tripolyphosphate (TPP) by ionic crosslinking. The particle sizes of TPP-chitosan microparticles were in range from 500 to 710 microm and encapsulation efficiencies of drug were more than 90%. The morphologies of TPP-chitosan microparticles were examined with scanning electron microscopy. As pH of TPP solution decreased and molecular weight (MW) of chitosan increased, microparticles had more spherical shape and smooth surface. Release behaviors of felodipine as a model drug were affected by various preparation processes. Chitosan microparticles prepared with lower pH or higher concentration of TPP solution resulted in slower felodipine release from microparticles. With decreasing MW and concentration of chitosan solution, release behavior was increased. The release of drug from TPP-chitosan microparticles decreased when cross-linking time increased. These results indicate that TPP-chitosan microparticles may become a potential delivery system to control the release of drug.     

Design and development of gliclazide-loaded chitosan for oral sustained drug delivery: in vitro/in vivo evaluation

Gliclazide (GLZ)/Chitosan microparticles were prepared with tripolyphosphate (TPP) by ionic cross-linking. The particle sizes of TPP-chitosan microparticles were in the range 675-887 μm and the loading efficiencies of drug was more than 94.0%. Chitosan concentration, TPP solution pH and glutaraldehyde volume added to the TPP cross-linking solution had an effect on the drug release characteristics. The microparticles were examined with scanning electron microscopy and infrared spectroscopy. Furthermore, pectin can interact with cationic chitosan on the surface of these TPP/chitosan microparticles to form a polyelectrolyte complex film for the improvement of the drug sustained-release performances. In vivo testing of the GLZ-chitosan microparticles in diabetic albino rabbits demonstrated significant antidiabetic effect of GLZ/chitosan microparticles after 8 h which lasts for 18 h, compared with GLZ powder which produced maximum hypoglycaemic effect after 4 h, suggesting that GLZ/chitosan microparticles are a valuable system for the long-term delivery of GLZ.     

Preparation and characterization of chitosan microparticles for oral sustained delivery of gliclazide: in vitro/in vivo evaluation

Chitosan microparticles were prepared with tripolyphosphate (TPP) by ionic cross‐linking with gliclazide (GLZ) as a model drug. The particle sizes of TPP‐chitosan microparticles ranged from 675–887 µm with loading efficiencies of greater than 94%. Chitosan concentration, TPP solution pH, and glutaraldehyde volume solution added to the TPP cross‐linking solution affected drug release characteristics. Pectin interactions with cationic chitosan on the surface of TPP/chitosan microparticles led to the formation of polyelectrolyte complex films that improved drug sustained release performance. In vivo testing of the GLZ‐chitosan microparticles in diabetic albino rabbits demonstrated a significant antidiabetic effect of GLZ/chitosan microparticles after 8 h that lasts for 18 h compared with GLZ powder that produced a maximal hypoglycemic effect at 4 h, suggesting that GLZ/chitosan microparticles represent an improved system for the long‐term delivery of GLZ. Drug Dev Res 72: 235–246, 2011. © 2010 Wiley‐Liss, Inc.     

Design and characterisation of doxorubicin-releasing chitosan microspheres for anti-cancer chemoembolisation

The aims of this study were to design and characterise doxorubicin-loaded chitosan microspheres for anti-cancer chemoembolisation. Doxorubicin-loaded chitosan microspheres were prepared by emulsification and cross-linking methods. Doxorubicin–chitosan solution was initially complexed with tripolyphosphate (TPP) to improve drug loading capabilities. Doxorubicin-loaded chitosan microspheres were highly spherical and had approximately diameters of 130–160?µm in size. Drug loading amount and loading efficiency were in the range 3.7–4.0% and 68.5–85.8%, respectively, and affected by TPP concentration, drug levels and cross-linking time. Doxorubicin release was affected by TPP complexation, cross-linking time and release medium. Especially, lysozyme in release media considerably increased drug release. Synergistic anti-cancer activities of doxorubicin-releasing chitosan microspheres were confirmed to VX2 cells in the rabbit auricle model compared with blank microspheres. Doxorubicin-loaded chitosan microspheres can efficiently be prepared by TPP gelation and cross-linking method and developed as multifunctional anti-cancer embolic material.     

The use of chitosan-coated flexible liposomes as a remarkable carrier to enhance the antitumor efficacy of 5-fluorouracil against colorectal cancer

Surface-coated nanocarriers have been extensively used to enhance the delivery of anticancer drugs and improve their therapeutic index. In this study, chitosan (CS)-coated flexible liposomes (chitosomes) containing 5-fluorouracil (5-FU) were designed and characterized for use as a novel approach to target colon cancer cells. 5-FU-loaded flexible liposomes (F1, F2, and F3) and 5-FU-loaded chitosomes (F4, F5, and F6) were prepared using film hydration and electrostatic deposition techniques, respectively. The particle size, polydispersity index (PDI), zeta potential, entrapment efficiency (EE%), morphology, and in vitro drug release ability, and cytotoxicity of the formulations were determined. The results revealed that the size of chitosomes ranged from 212 to 271 nm with a positive surface charge of 6.1 to 14.7 mV, whereas the particle size of liposomes ranged from 108 to 234 nm with negative surface charges of ?2.3 to ?16.3. F3 and F6 had a spherical shape with a rough surface structure. The in vitro drug release study revealed that chitosomes retard 5-FU release as opposed to the 5-FU solution and liposomes. The cytotoxicity study using a colon cancer cell line (HT-29) showed that 5-FU-loaded chitosomes were more effective in killing cancer cells in a sustained manner than liposomes and the 5-FU solution. Chitosomes were therefore successfully developed as nanocarriers of 5-FU, with potential cytotoxicity for colorectal cancer cells.     

Evaluation of ultrasonic atomization as a new approach to prepare ionically cross-linked chitosan microparticles

Ultrasonic atomization was evaluated as a new approach for the preparation of ionically cross-linked controlled-release chitosan microparticles loaded with theophylline as the model drug, using tripolyphosphate (TPP) as counter-ion. It was possible to nebulize both 2% and 3% (w/v) chitosan solutions as a function of their viscosity, usually not processed by employing the conventional nebulizer. The results of the chitosan molecular characterization using the SEC-MALS analysis revealed that ultrasonic atomization caused a certain depolymerization, probably due to the main chain scission of the 1,4-glycosidic bond; however, Fourier transform-infrared spectroscopy revealed the absence of other chemical modifications. The ultrasonic atomization allowed preparation of TPP cross-linked chitosan microparticles mostly ranging between 50 and 200 mum. As regards manufacturing parameters, the linking time and washing medium were found to affect the properties of the microparticles, while the stirring rate of the TPP solution did not show any influence. The evaluation of the formulation variables revealed that chitosan concentration strongly affected both the feasibility of the ultrasonic atomization and the drug release. All the microparticles showed an encapsulation efficiency of > 50 % and, after an initial burst effect, a controlled release of drug for 48 h. In conclusion, the ultrasonic atomization could be proposed as a robust and innovative single-step procedure with scale-up potential to successfully prepare ionically cross-linked chitosan microparticles.     

Electrosprayed bovine serum albumin-loaded tripolyphosphate cross-linked chitosan capsules: synthesis and characterization

Bovine serum albumin (BSA)-loaded tripolyphosphate (TPP) cross-linked chitosan capsules were prepared using an electrospraying technique, in which a sufficiently strong electric field was applied to overcome the surface tension of a droplet. A comprehensive investigation was conducted on the effects of concentrations of initial chitosan and TPP solutions, flow rate and BSA/chitosan weight ratio on the physical properties of the mixtures; the morphology, size and yield of the capsules; BSA encapsulation efficiency (EE) and loading capacity (LC); and in vitro release. A high voltage was required to obtain a continuous and stable spray for the mixtures with a high viscosity at high chitosan concentration. The capsules were spherical in shape. Capsule size increased with increasing flow rate, but did not change significantly (p < 0.05) with increases in concentrations of chitosan and TPP. Increasing concentrations of chitosan and TPP solutions increased the yields, while yields decreased with increases in the flow rate. EE and LC increased with increasing chitosan concentration, BSA/chitosan weight ratio and TPP concentration and decreased with increasing flow rate. High EE enhanced the BSA release rate, while a high degree of cross-linking slowed its release.     

Electrosprayed bovine serum albumin-loaded tripolyphosphate cross-linked chitosan capsules: Synthesis and characterization

Bovine serum albumin (BSA)-loaded tripolyphosphate (TPP) cross-linked chitosan capsules were prepared using an electrospraying technique, in which a sufficiently strong electric field was applied to overcome the surface tension of a droplet. A comprehensive investigation was conducted on the effects of concentrations of initial chitosan and TPP solutions, flow rate and BSA/chitosan weight ratio on the physical properties of the mixtures; the morphology, size and yield of the capsules; BSA encapsulation efficiency (EE) and loading capacity (LC); and in vitro release. A high voltage was required to obtain a continuous and stable spray for the mixtures with a high viscosity at high chitosan concentration. The capsules were spherical in shape. Capsule size increased with increasing flow rate, but did not change significantly (p?<?0.05) with increases in concentrations of chitosan and TPP. Increasing concentrations of chitosan and TPP solutions increased the yields, while yields decreased with increases in the flow rate. EE and LC increased with increasing chitosan concentration, BSA/chitosan weight ratio and TPP concentration and decreased with increasing flow rate. High EE enhanced the BSA release rate, while a high degree of cross-linking slowed its release.     

Preparation and characterization of drug‐loaded chitosan–tripolyphosphate microspheres by spray drying

The present study reports on the preparation of chitosan–tripolyphosphate (TPP) microspheres by the spray‐drying method using acetaminophen as a model drug substance. Chitosan–TPP microspheres were spherical and had a smooth surface. Perfectly spherical chitosan–TPP microparticles loaded with acetaminophen were obtained in the size range of 3.1–10.1 µm. Spray‐dried chitosan–TPP microspheres were positively charged (zeta potential ranged from +18.4 to +31.8). The encapsulation efficiency of these microspheres was in the range of 48.9–99.5%. The swelling capacity of chitosan–TPP microspheres increased with increases in the molecular weight of chitosan and decreases with increasing volume of 1% wt/vol TPP solution used for the cross‐linking reaction. The effect of chitosan concentration, drug loading, volume of TPP solution used for cross‐linking, and chitosan molecular weight on surface morphology and drug release rate was extensively investigated. Microparticles with spherical shape and slower release rates were obtained from chitosan–TPP microspheres prepared using a higher concentration of chitosan, higher volume of TPP solution, a higher molecular weight chitosan and/or a higher drug loading. Most importantly, the drug release rate was mainly controlled by the chitosan–TPP matrix density and, thus, by the degree of swelling of the hydrogel matrix. Drug release from chitosan–TPP microspheres occurred via diffusion as the best fit for drug release was obtained using the Higuchi equation. Drug Dev. Res. 64:114–128, 2005. © 2005 Wiley‐Liss, Inc.     

Preparation and characterization of spray dried inhalable powders containing chitosan nanoparticles for pulmonary delivery of isoniazid

The aim of this study was to prepare spray dried inhalable powders containing isoniazid-loaded chitosan/tripolyphosphate (TPP) nanoparticles for sustained delivery of the drug to the lung. Nanoparticles were prepared by ionic gelation method. In-vitro drug release study indicated that the rate of drug release from nanoparticles was decreased by increasing the amount of chitosan. Entrapment of isoniazid into chitosan/TPP nanoparticles decreased minimum inhibitory concentrations (MIC) of the drug against mycobacterium avium intracellulare. Nanoparticles were spray dried using excipients such as lactose, mannitol and maltodextrin alone or with leucine. Results showed that the obtained powders had different aerosolization property. It was observed that by adding leucine, the particle size of microparticles deceased and the process yield and fine particle fraction (FPF) increased significantly. The in-vitro deposition data indicated that spray drying of isoniazid-loaded nanoparticles with lactose in the presence of leucine resulted in the production of inhalable powders with the highest FPF (45%).     

Development and in vitro/in vivo evaluation of Zn-pectinate microparticles reinforced with chitosan for the colonic delivery of progesterone

The colon is a promising target for drug delivery owing to its long transit time of up to 78?h, which is likely to increase the time available for drug absorption. Progesterone has a short elimination half-life and undergoes extensive first-pass metabolism, which results in very low oral bioavailability (～25%). To overcome these shortcomings, we developed an oral multiparticulate system for the colonic delivery of progesterone. Zn-pectinate/chitosan microparticles were prepared by ionotropic gelation and characterized for their size, shape, weight, drug entrapment efficiency, mucoadhesion and swelling behavior. The effect of cross-linking pH, cross-linking time and chitosan concentration on progesterone release were also studied. Spherical microparticles having a diameter of 580–720?µm were obtained. Drug entrapment efficiency of ～75–100% was obtained depending on the microparticle composition. Microparticle mucoadhesive properties were dependent on the pectin concentration, as well as the cross-linking pH. Progesterone release in simulated gastric fluids was minimal (3–9%), followed by burst release at pH 6.8 and a sustained phase at pH 7.4. The in vivo study revealed that the microparticles significantly increased progesterone residence time in the plasma and increased its relative bioavailability to ～168%, compared to the drug alone. This study confirms the potential of Zn-pectinate/chitosan microparticles as a colon-specific drug delivery system able to enhance the oral bioavailability of progesterone or similar drugs.     

Effect of manufacturing parameters on the characteristics of vitamin C encapsulated tripolyphosphate-chitosan microspheres prepared by spray-drying

To prepare the sustained release vitamin C carriers, vitamin C was successfully encapsulated in tripolyphosphate (TPP) cross-linked chitosan (TPP-chitosan) microspheres by the spray-drying method at different manufacturing conditions. Manufacturing parameters (inlet temperature, liquid flow rate, chitosan concentration and volume of 1% w/v TPP solution) had a significant influence on the characteristics of thus prepared microspheres. The optimum spray-drying conditions such as inlet temperature, liquid flow rate and compressed air flow rate for the encapsulation of vitamin C in TPP-chitosan microspheres was found to be 170 degrees C, 2 ml min(-1) and 101 min(-1), respectively. The size and yield of the TPP-chitosan microspheres ranged from 3.9-7.3 microm and 54.5-67.5%, respectively. The encapsulation efficiency of TPP-chitosan microspheres ranged from 45.72-68.7% and it decreased with the increasing volume of 1% w/v TPP solution. At the same cross-linking extent, the encapsulation efficiency of TPP-chitosan microspheres increased when the concentration of chitosan was increased from 0.5-1% w/v. Effect of volume of 1% w/v TPP solution on the surface morphology of chitosan microspheres was examined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). These studies revealed that chitosan solution (250 ml) cross-linked with 15 ml 1% w/v TPP solution produced more porous microspheres than that cross-linked with 5 and 10 ml TPP solution. The release rate of vitamin C from TPP-chitosan microspheres decreased when the concentration of chitosan was increased from 0.5-1.0% w/v. Vitamin C release rate was also modulated by varying the volume of 1% w/v TPP solution. The release rate of vitamin C from TPP-chitosan microspheres decreased with increasing volume (5-15 ml) of 1% w/v TPP solution.     

Controlled drug release properties of ionically cross-linked chitosan beads: the influence of anion structure

By adopting a novel chitosan cross-linked method, i.e. chitosan/gelatin droplet coagulated at low temperature and then cross-linked by anions (sulfate, citrate and tripolyphosphate (TPP)), the chitosan beads were prepared. Scanning electron microscopy (SEM) observation showed that sulfate/chitosan and citrate/chitosan beads usually had a spherical shape, smooth surface morphology and integral inside structure. Cross-sectional analysis indicated that the cross-linking process of sulfate and citrate to chitosan was much faster than that of TPP due to their smaller molecular size. But, once completely cross-linked, TPP/chitosan beads possessed much better mechanical strength and the force to break the beads was approximately ten times higher than that of sulfate/chitosan or citrate/chitosan beads. Release media pH and ionic strength seriously influenced the controlled drug release properties of the beads, which related to the strength of electrostatic interaction between anions and chitosan. Sulfate and citrate cross-linked chitosan beads swelled and even dissociated in simulated gastric fluid (SGF) and hence, model drug (riboflavin) released completely in 5 h; while in simulated intestinal fluid (SIF), beads remained in a shrinkage state and drug released slowly (release % usually <70% in 24 h). However, swelling and drug release of TPP/chitosan bead was usually insensitive to media pH. Chitosan beads, cross-linked by a combination of TPP and citrate (or sulfate) together, not only had a good shape, but also improved pH-responsive drug release properties. Salt weakened the interaction of citrate, especially sulfate with chitosan and accelerated beads swelling and hence drug release rate, but it was insensitive to that of TPP/chitosan. These results indicate that ionically cross-linked chitosan beads may be useful in stomach specific drug delivery.     

Sustained release of antineoplastic drugs from chitosan-reinforced alginate microparticle drug delivery systems

Alginate based microparticle drug delivery systems were prepared for the sustained release of antineoplastic drugs. Two drugs, 5-fluorouracil (5-FU) and tegafur, were encapsulated into the microparticles. The drug loaded microparticles were fabricated using a very convenient method under very mild conditions, i.e., directly shredding the drug loaded beads into microparticles in a commercial food processor. The mean sizes of the obtained microparticles were between 100 and 200 μm. To effectively sustain the drug release, alginate microparticles were reinforced by chitosan during gelation. The drug release from the chitosan-reinforced alginate microparticles was obviously slower than that from the unreinforced microparticles. The effect of the reinforcement conditions on the drug release property of the microparticles was studied, and the optimized concentration of chitosan solution for reinforcement was identified. The effects of drug feeding concentration and pH value of the release medium on the drug release were investigated.     

Preparation and characterization of spray dried inhalable powders containing chitosan nanoparticles for pulmonary delivery of isoniazid

The aim of this study was to prepare spray dried inhalable powders containing isoniazid-loaded chitosan/tripolyphosphate (TPP) nanoparticles for sustained delivery of the drug to the lung. Nanoparticles were prepared by ionic gelation method. In-vitro drug release study indicated that the rate of drug release from nanoparticles was decreased by increasing the amount of chitosan. Entrapment of isoniazid into chitosan/TPP nanoparticles decreased minimum inhibitory concentrations (MIC) of the drug against mycobacterium avium intracellulare. Nanoparticles were spray dried using excipients such as lactose, mannitol and maltodextrin alone or with leucine. Results showed that the obtained powders had different aerosolization property. It was observed that by adding leucine, the particle size of microparticles deceased and the process yield and fine particle fraction (FPF) increased significantly. The in-vitro deposition data indicated that spray drying of isoniazid-loaded nanoparticles with lactose in the presence of leucine resulted in the production of inhalable powders with the highest FPF (45%).     

Layer-by-layer assembly of poly(l-glutamic acid)/chitosan microcapsules for high loading and sustained release of 5-fluorouracil

Hollow polyelectrolyte microcapsules based on poly(l-glutamic acid) (PLGA) and chitosan (CS) with opposite charges were fabricated by layer-by-layer (LbL) assembly technique using melamine formaldehyde (MF) microparticles as sacrificial templates. The LbL assembly of polyelectrolytes and the resultant PLGA/CS microcapsules were characterized. A hydrophilic anticancer drug, 5-fluorouracil (5-FU), was chosen to investigate the loading and release properties of the microcapsules. The PLGA/CS microcapsules show high loading capacity of 5-FU under conditions of high drug concentration and salt adding. The high loading can be ascribed to spontaneous deposition of 5-FU induced by hydrogen bonding between 5-FU and PLGA/CS microcapsules. The PLGA/CS microcapsules show sustained release behavior. The release rate of 5-FU drastically slows down after loading in PLGA/CS microcapsules. The 5-FU release from PLGA/CS microcapsules can be best described using Ritger-Peppas or Baker-Londale models, indicating the diffusion mechanism of 5-FU release from the PLGA/CS microcapsules. In vitro cytotoxicity evaluation by the MTT assay shows good cell viability over the entire concentration range of PLGA/CS microcapsules. Therefore, the novel PLGA/CS microcapsules are expected to find application in drug delivery systems because of the properties of biodegradability, high loading, sustained release and cell compatibility.     

Preparation,In Vitro Characterization,and In Vivo Pharmacokinetic Evaluation of Respirable Porous Microparticles Containing Rifampicin

This study aimed to prepare and evaluate rifampicin microparticles for the lung delivery of rifampicin as respirable powder. The microparticles were prepared using chitosan by the spray-drying method and evaluated for aerodynamic properties and pulmonary drug absorption. To control the drug release, tripoly-phosphate in different concentrations 0.6, 0.9, 1.2, and 1.5 was employed to get a sustained drug release profile. The microparticles were evaluated for drug loading, % entrapment efficiency, tapped density, morphological characteristics, and in vitro drug release studies. Aerosol properties were determined using the Andersen cascade impactor. Porous microparticles with particle sizes (d_0.5) less than 10 μm were obtained. The entrapment of rifampicin in microparticles was up to 72%. In vitro drug release suggested that the crosslinked microparticles showed sustained release for more than 12 hrs. The drug release rate was found to be decreased as the TPP concentration was increased. The microparticles showed a fine particle fraction in the range of 55–63% with mass median aerodynamic diameter (MMAD) values below 3 μm. The in vivo pulmonary absorption of the chitosan microparticles suggested a sustained drug release profile up to 72 hrs with an elimination rate of 0.010 per hr. The studies revealed that the spray-dried porous microparticles have suitable properties to be used as respirable powder in rifampicin delivery to the lungs.     

Preparation of cross-linked chitosan microspheres by spray drying: effect of cross-linking agent on the properties of spray dried microspheres

Chitosan microspheres cross-linked with three different cross-linking agents viz, tripolyphosphate (TPP), formaldehyde (FA) and gluteraldehyde (GA) have been prepared by spray drying technique. The influence of these cross-linking agents on the properties of spray dried chitosan microspheres was extensively investigated. The particle size and encapsulation efficiencies of thus prepared chitosan microspheres ranged mainly between 4.1-4.7 microm and 95.12-99.17%, respectively. Surface morphology, % erosion, % water uptake and drug release properties of the spray dried chitosan microspheres was remarkably influenced by the type (chemical or ionic) and extent (1 or 2% w/w) of cross-linking agents. Spray dried chitosan microspheres cross-linked with TPP exhibited higher swelling capacity, % water uptake, % erosion and drug release rate at both the cross-linking extent (1 and 2% w/w) when compared to those cross-linked with FA and GA. The sphericity and surface smoothness of the spray dried chitosan microspheres was lost when the cross-linking extent was increased from 1 to 2% w/w. Release rate of the drug from spray dried chitosan microspheres decreased when the cross-linking extent was increased from 1 to 2% w/w. The physical state of the drug in chitosan-TPP, chitosan-FA and chitosan-GA matrices was confirmed by the X-ray diffraction (XRD) study and found that the drug remains in a crystalline state even after its encapsulation. Release of the drug from chitosan-TPP, chitosan-FA and chitosan-GA matrices followed Fick's law of diffusion.