Preparation and properties of ionically cross-linked chitosan ...

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H. LIU AND C. GAOdrug toxicity as a result of long term application, for examplearthritis damage for children, various sustained release carrierssuch as microcapsules, gels and liposomes have been developed.Yet so far less attention has been given to the nanoparticlecarriers, in particular the chitosan nanoparticles.EXPERIMENTALMaterialsChitosan (CS, M h ¼ 620 kDa, degree of deacetylation ¼ 90%,viscosity ¼ 115 cps) is a commercial product of Haidebei HalobiosCo., Ltd. (Ji’nan, China). Sodium tripolyphosphate (TPP) waspurchased from Dongsheng Chemical Reagent Factory (Wenzhou,China). Ciprofloxacin hydrochloride (CH, C 17 H 18 FN 3 O 3 HClH 2 O)was purchased from Zhejiang Medicine Co. Ltd., XinchangPharmaceutical Factory (Shaoxing, China). Phosphotungstic acidwas purchased from Sinopharm Group Chemical Reagent Co., Ltd.(Shanghai, China). All other chemicals were of analytical grade andused as received. The water used in all experiments was tripledistilled.Preparation of the chitosan nanoparticlesThe nanoparticles were obtained based on ionic gelation of TPPwith chitosan. [10] Briefly, chitosan and TPP were dissolved inacetic acid and triple distilled water to obtain solutions of 1.0–5.0and 0.25–2.0 mg/ml, respectively. The chitosan nanoparticleswere obtained upon addition of 14 ml TPP solution into 35 mlchitosan solution under mild mechanical stirring (550 rpm) atroom temperature. The drug-loaded nanoparticles were similarlyprepared by using a chitosan solution containing the drug.CharacterizationMorphological examination of the chitosan nanoparticles wasperformed by transmission electron microscopy (TEM) (JEOLJEM-200CX, Japan). The sample was stained with 2% (w/v)phosphotungstic acid and a drop of the sample was applied ontoa carbon-coated copper meshwork, and dried at roomtemperature. The acceleration voltage was 100 kV. Zeta potentialof the particles was recorded on a Zetasizer 2000. Each value wasaveraged from three parallel measurements.The particle size was measured by dynamic light scattering(DLS) (90 Plus/BI-MAS). The wavelength of laser is 658 nm.Brookhaven’s unique dust filter algorithm can remove theinfluence of a contaminating fraction of large particles (dust).Processing the fluctuating signal with a state-of-the-art digitalauto-correlator yields the particle’s diffusion coefficient, fromwhich the equivalent spherical particle size is calculated using theStokes–Einstein equation. When a broad distribution of spheres ispresent in the samples, the following auto-correlation function isused:ZgðtÞ ¼ GðGÞe Gt dGan average diameter and a distribution width (polydispersity) areobtained. It has to be noted that the most important pieces ofinformation obtained in photon correlation spectroscopy usingany multimodal distribution analysis are the positions of thepeaks and the ratio of the peak areas. The widths of the peaks,however, are not particularly reliable, since they will narrow,typically, with increasing duration of experiment until a limit isreached. For more details, the readers can refer to the machinemanual. Each measurement was taken for 1 min with a 908 fixedangle detector. The size was averaged from five parallelmeasurements.Stability of the chitosan nanoparticlesThe stability of the chitosan nanoparticles obtained at differentfabricating conditions was examined in terms of the particle size.The nanoparticle suspensions were stored at 48C and the sizes ofthe nanoparticles were monitored by DLS.Biocompatibility of the chitosan nanoparticlesIn vitro cell culture was performed to determine the biocompatibilityof the as-prepared chitosan nanoparticles. The chitosannanoparticles were sterilized by UV irritation before cell culture.Human dermal fibroblasts were isolated from foreskins androutinely expanded. [20] The cells were cultured at 378C, in 5% CO 2 ,and 95% humidity in DMEM supplemented with penicillin (100 U/ml), streptomycin (100 mg/ml), and 10% fetal bovine serum (FBS)(complete medium), which was changed every 3 days. Cells werepassaged at confluence and the 4–8th passage of fibroblasts witha density of 2 10 5 /ml were seeded in a 96-well polystyreneplate. After 24 hr, 60 ml of nanoparticle suspension was addedinto each well. The final nanoparticle concentration was highenough for close contact between the cells and the nanoparticles.The culture medium was changed every 2 days. Meanwhile,normal culture medium was used in parallel as a negative control.The cell proliferation was measured by methylthiazoletetrazolium(MTT) assay. [21] The absorbance was recorded at a wavelength of570 nm by a microplate reader (Bio-Rad model 550). Threeparallel samples were analysed and data were expressed asmean standard deviation.Quantification of the relative CH amount in the chitosannanoparticlesThe prepared nanoparticle suspension containing the CH wascentrifugated at 12,000 rpm for 70 min to obtain the supernatant,which was diluted over hundreds of times and the absorbance at276 nm was recorded by a UV–vis spectrophotometer (ShimadzuUV-2550). The supernatant of blank nanoparticles was obtainedunder the same conditions and used as a control. Theconcentration of CH in the supernatant was quantified byreferring to a calibration curve recorded from a known amount ofCH at the same condition. A lower concentration found in thesupernatant implies a higher loading efficiency in the chitosannanoparticles.614where G is related to the relaxation of the fluctuations. Forobtaining information from this equation, a method of Cumulantanalysis is applied. To get the solution, an approximation is usedby the non-negative constrained least squares (NNLS) algorithm.Moreover, Mie theory is used to calculate the weight and numberfractions from the intensity fractions. For routine determinations,Release of CHCH release was performed using a dialysis technique. The CHloaded chitosan nanoparticles were centrifugated at 12,000 rpmfor 70 min to obtain the nanoparticle precipitate, which waswashed with water twice. The nanoparticles were transferred intowww.interscience.wiley.com/journal/pat Copyright ß 2008 John Wiley & Sons, Ltd. Polym. Adv. Technol. 2009, 20 613–619
IONICALLY CROSS-LINKED CHITOSAN NANOPARTICLESa dialytic-bag with a cut-off molecular weight of 3.5 kDa. Thedialytic-bag was then immersed into 100 ml phosphate bufferedsaline (PBS, pH 7.4) or H 2 Oat378C under continuous shaking. Atpredetermined time, 1 ml dialytic medium was taken out foranalysis and an equal volume of freshly prepared medium wassupplemented. Absorbance of the dialytic medium at 270 nm wasrecorded by an UV–vis spectroscopy. The released amount of CHwas quantified by referring to a calibration curve recorded fromknown amounts of CH at the same condition.RESULTS AND DISCUSSIONFabrication and properties of chitosan nanoparticleThe chitosan molecules are gelled when they encounter TPPmolecules via electrostatic interaction. [10] Firstly, the zone ofparticle formation is investigated. Chitosan was dissolved in 1.5%acetic acid solution to form a 10 mg/ml solution, which wasdiluted with water to various concentrations: 1.0, 2.0, 3.0, 4.0, and5.0 mg/ml. The pH value of these solutions was adjusted to 4.8.TPP was dissolved in water at various concentrations: 0.50, 0.75,1.0, 1.25, 1.50, 1.75, 2.0, and 2.5 mg/ml. Chitosan nanoparticleswere prepared by the addition of 14 ml TPP solution into 35 mlchitosan solution at room temperature under mechanicalagitation. By visual observation, the systems were classified asclear solution (), opalescent suspension (H), and aggregates (#).The results are summarized in Table 1. A clear solution wasobserved when both the chitosan and TPP concentrations weretoo small, whereas aggregates were formed spontaneously whenthey were too large. The zone of the opalescent suspension,which should represent a suspension of colloidal particles, wasfound when the chitosan and the TPP concentrations wereappropriate. Illuminating that formation of the nanoparticles isonly possible for some specific concentrations of chitosan andTable 1. Conditions for formation of the chitosan nanoparticlesCS (mg/ml)TPP (mg/ml)0.25 0.5 0.75 1.0 1.25 1.5 2.0 2.51.0 H H H H # # #2.0 H H H H H H #3.0 H H H H H H4.0 H H H H H H, clear solution; H, opalescent suspension; #, aggregates.TPP. Confirmed by DLS measurement and microscopy observation,the chitosan nanoparticles with a size of 300 nm wereobtained and well dispersed in the solution when theconcentration of chitosan and TPP was 2.0 and 1 mg/ml,respectively.Figure 1a shows that the number average size of the chitosannanoparticles was measured as 321 nm by DLS, which isconsistent with the TEM observation (Fig. 1c, d). No severeagglomeration of the particles was observed. A magnified TEMimage (Fig. 1d) illustrates that the chitosan nanoparticles had acore–shell structure, i.e. with denser skirts and looser interiors.After loading with CH, the particle size increased slightly to337 nm (Fig. 1b) with a homogeneous structure (Fig. 1e, f),suggesting that the drug was surely loaded.For the next study, the chitosan concentration was fixed at2 mg/ml with a pH value of 4.8. Fig. 2 shows that both the particlesize (Fig. 2a) and zeta potential (Fig. 2b) decreased along with theincrease of TPP concentration. This alteration tendency isconsistent with the results of Gan et al., [18] but is the oppositeFigure 1. (a,b) Size distribution and (c–f) TEM images of (a,c,d) chitosan nanoparticles and (b,e,f) CH loaded chitosan nanoparticles, respectively. Thechitosan and TPP concentrations were 2 and 1 mg/ml, respectively. This figure is available in color online at www.interscience.wiley.com/journal/pat.Polym. Adv. Technol. 2009, 20 613–619 Copyright ß 2008 John Wiley & Sons, Ltd. www.interscience.wiley.com/journal/pat615
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