Preparation, characterization, and drug release in vitro of chitosan-glycyrrhetic acid nanoparticles

Preparation, Characterization, and Drug Release In Vitro of Chitosan-Glycyrrhetic Acid Nanoparticles YONGLI ZHENG,1 YAN WU,1 WULI YANG,1 CHANGCHUN WANG,1 SHOUKUAN FU,1 XIZHONG SHEN2 1

Department of Macromolecular Science, Key Laboratory of Molecular Engineering of Polymers of Educational Ministry, Fudan University, Shanghai 200433, People’s Republic of China 2

Zhongshan Hospital, Fudan University, Shanghai 200032, People’s Republic of China

Received 23 January 2005; revised 26 April 2005; accepted 28 April 2005 Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.20399

ABSTRACT: A suitable carrier chitosan (CS) was used to prepare CS-Glycyrrhetic acid (GLA) nanoparticles under very mild conditions by polyelectrolyte complexation. These nanoparticles were well dispersed and stable in aqueous solution, and the physicochemical properties of which were investigated by FT-IR, dynamic light scattering, transmission electron microscope, fluorescence spectra and zeta potential. It was found that only when the weight ratio of CS to GLA was lower than 16.7, could the nanoparticles be formed. The prepared nanoparticles carried a positive charge and had the dried TEMassessed size in the range from 20 to 30 nm. The mean hydrated diameter, size distribution and zeta potential of the nanoparticles could be controlled by some factors including the weight ratio of CS to GLA, the average molecular weight of CS and the pH value of the medium. It was also found that GLA encapsulation efficiency into the nanoparticles increased with the increase of the weight ratio of CS to GLA. The experiment of GLA release in vitro showed that the effect of CS encapsulation on GLA release was obvious and the CS-GLA nanoparticles system might be used to provide a continuous release. ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 95:181–191, 2006

Keywords: chitosan; glycyrrhetic acid; nanoparticles; encapsulation efficiency; in vitro release; light-scattering; microscopy; fluorescence spectroscopy; biocompatibility; FTIR

INTRODUCTION Polysaccharides have been frequently studied for drug delivery and medical applications. Among these polymers, chitosan (CS) has received increasing attention in the pharmaceutical field for a wide range of drug-delivery applications.1,2 Chitosan is a deacetylation derivative of chitin, the second abundant polysaccharide present in nature. Because of its biocompatibility and mucoadhesivity, CS has been formulated as films, beads, intragastric floating tablets, microspheres, and nanoparticles in the pharmaceutical field.3–7 Correspondence to: Shoukuan Fu (Telephone: 86 21 65642385; Fax: 86 21 65640293; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 95, 181–191 (2006) ß 2005 Wiley-Liss, Inc. and the American Pharmacists Association

Recently the use of complexation of oppositely charged macromolecules to prepare CS complexes and nanoparticulate structures as controlled drug-release formulations has attracted much attention,8–10 because this process is simple, feasible, and can usually be performed under mild conditions. Another advantage of this system is that preparation of the complex entails physical cross-linking by electrostatic interactions instead of chemical cross-linking, and the possibility of toxicity associated with the cross-linking reagent involved in chemical cross-linking processes can be eliminated. Glycyrrhetic acid (GLA) is an aglycone and active metabolite of glycyrrhizin (GLZ), showing various therapeutic effects, such as antiinflammatory, antihepatotoxic, and interferon-inducing actions.11 Some reports indicated that GLZ could

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inhibit Con A-induced hepatitis without affecting cytokine expression and anti-Fas antibodyinduced hepatitis by acting upstream of CPP32like protease activation.12,13 Since only GLA appears in the blood circulation after oral administration of GLZ, GLA is considered to play an important role in the biological action of oral administration of GLZ.14 Once absorbed, GLA is transported, and mainly is taken up into the liver by capacity-limited carriers, where it is metabolized into glucuronide and sulfate conjugates. These conjugates are transported efficiently into the bile. After outflow of the bile into the duodenum, the conjugates are hydrolyzed to GLA by commensal bacteria. GLA is subsequently reabsorbed, causing a pronounced delay in the terminal plasma clearance. When considerable doses of GLA are consumed habitually, mineral cortiocoid excesslike side effects may occur due to the inhibition of the enzyme 11-b-hydroxysteroid dehydrogenase. Many pharmacokinetic studies on GLZ and GLA have analyzed their behavior in the body. Prolonged supply of GLA can make the effect higher and longer, but it may cause side-effects such as hypertension, edema, and electrolyte disturbances.15 In order to decrease these sideeffects, GLA may be entrapped by carrier forming nanosize controlled release system.16 However, so far very few studies have been reported on this subject. In this study, we reported the formation of novel nanosize particles driven by polyelectrolyte complexation between the hydrophilic polyelectrolyte carbohydrates CS and GLA. The nanoparticles were characterized in terms of their size, surface charge, morphology, and infrared spectra, and the drug release of CS-GLA nanoparticles was also evaluated in vitro.

MATERIALS AND METHODS Materials Chitosan (CS) was purchased from Shanghai Kako Trading Co., Ltd (Shanghai, China), the degree of deacetylation (DD) was 95.3%, and the weight average molecular weights (Mws) of CS was about 420 kDa. CS of DD 95.3% with different Mws (9.0, 17.9, 24.0, and 210 kDa) was prepared according to the reference [17]. The Mws were determined by viscometric methods.18 Glycyrrhetic acid (GLA) was purchased from Tianshan pharmaceutical Co., Ltd. (Neimenggu, China). All other chemicals were of analytical grade and used without further purification. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

Preparation of CS-GLA Nanoparticles CS-GLA nanoparticles were prepared by mixing a positively charged CS acetic acid solution and a negatively charged GLA ammonia solution at room temperature. Briefly, 0.1 g of CS was dissolved in dilute acetic acid solution (0.1 g CH3COOH þ 100 g deionized water) and the solution was stirred for 24 h, then filtered by paper filter for use. GLA 0.01 g was dissolved in a dilute ammonia solution (0.1 g 25% NH3
H2O þ 100 g deionized water). Afterwards, the GLA solution was used as a base material and the CS solution was dropped into it in different ratio at a dropping rate of one drop per second with a syringe under magnetic stirring. The opalescent suspension was formed. The obtained suspension was then filtered by paper filter for characterization. Naoparticles were prepared with different processing parameters to study the effect of a number of variables on their physicochemical properties. Some process parameters were varied. The Mws of CS were 9.0, 17.9, 24.0, 210, and 420 kDa respectively. CS-GLA nanoparticles were prepared at pH 5.0 and dilute ammonia or dilute acetic acid was added to obtain different pH values (pH ¼ 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0). The weight ratio of CS to GLA was varied from 6.5 to 25, and the range of these variable values were selected based on preliminary experiments. FT-IR Spectrum Analysis FT-IR spectrum was recorded on a Nicolet Magna 550 FT-IR spectrometer to determine the interaction between CS and GLA. CS-GLA nanoparticles were separated from the aqueous suspension medium by ultra centrifugation with 154000g at 258C for 30 min. Washed by dilute acetic acid (pH ¼ 5.0) solution and separated by ultracentrifugation for three times, the precipitate was obtained by discarding the supernatant. The precipitate was frozen by liquid nitrogen and lyophilized by free dryer system to obtain dried CS-GLA nanoparticles. These gained CS-GLA nanoparticles were mixed with KBr and pressed to a plate for measurement at room temperature.

Stead-State Fluorescence Spectroscopy The fluorescence measurement was performed on a spectrofluorometer FLS-920 (Edinburg Instruments). The spectral resolution of both excitation

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and emission was 1 nm. Recrystallized pyrene was used as fluorescence probe, and the final concentration was 6.0  107 mol/L. Before the fluorescence measurement, the aqueous solutions containing a known mass of nanoparticles with different weight ratio of CS to GLA were stirred for at least 12 h after the addition of pyrene at 408C. The emission and excitation spectra were recorded with an excitation and emission wavelength of 335 and 390 nm, respectively. The slit opening for excitation were at 0.4 nm. The spectra was accumulated with an integration time of 5 s/ nm. Each sample was repeated measured three times and the values reported were the mean value for two replicate samples. pH Value Measurement CS-GLA nanoparticles were prepared at pH 5.0 and dilute ammonia or dilute acetic acid was added to obtain different pH values. The pH value was measured at 25  18C in a PHS-3TC digital pH-meter with an error of 0.01 pH units. A combined glass electrode E-201 was employed and the pH-meter was calibrated with two buffer solutions supplied by Shanghai Hongbei Reagent Factory. Physicochemical Characterization of the Nanoparticles Hydrodynamic diameter (Dh) and polydispersity index (PI, m2/hGi2)19 of the CS-GLA nanoparticles were measured by dynamic light scattering (DLS) in buffer solution with different pH values. A commercial laser light-scattering spectrometer (Malvern Autosizer4700) equipped with a multi-i digital time correlation (Malvern PCS7132) and Compass 315M-100 Diode-Pumped Laser (output power  100 mW, CW at l0 ¼ 532 nm) as a light source was used. All DLS measurements were done at 25  0.18C and at a scattering angle of 908. The measured time correlation functions were analyzed by automatic progress equipped with the correlator. The zeta potential of the CS-GLA nanoparticles were measured at 258C with a Zeta Plus zeta potential Analyzer (Brookhaven Instruments Corp., USA). The samples were diluted with 0.1 mM NaCl solution at a series of pH values in order to maintain a constant ionic strength and measured in automatic mode. Each sample was repeated measured three times and the values reported were the mean value for two replicate samples.

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The morphology and dried TEM-assessed size measurement of the CS-GLA nanoparticles were obtained using a Hitachi HU-11B transmission electron microscopy (TEM) operating at 250 kV. The sample was prepared as follows: A drop of CSGLA nanoparticles opalescent suspension was mounted onto carbon-coated copper grill. They were dried at room temperature, and then were examined using a TEM without being negative stained.

Determination of Encapsulation Efficiency and Loading Capacity To determinate the encapsulation efficiency (EE) and loading capacity (LC), CS-GLA nanoparticles were separated from the aqueous suspension medium by ultra centrifugation with 154000g at 258C for 30 min. Washed by dilute acetic acid (pH ¼ 5.0) solution and separated by ultracentrifugation for three times, the precipitate was frozen by liquid nitrogen and lyophilized by free dryer system to obtain dried CS-GLA nanoparticles. A known mass of dried nanoparticles was dissolved in dilute acetic acid solution (pH ¼ 2.5) and the amount of GLA in this solution was measured by HPLC. The amount of GLA subtracted from the amount dried nanoparticles gave the amount of CS. HPLC was carried out using a LC-4A HPLC system equipped with a LC-4A pump, and a SPD-10A UV detector (Shimadzu, Kyoto, Japan). The detective wavelength was set at 254 nm. HPLC analysis of samples was performed using a Science C18 column (4.6  250 mm, 5 mm, Japan) preceded by a C18 guard column (GL Sciences, Japan). The column temperature was maintained at room temperature. The mobile phase was a mixture of methanol/3.6% acetic acid (80:20, v/v). The flow rate was maintained at 1.0 mL/min. Each sample was repeated measured three times and the values reported were the mean value for two replicate samples. The GLA concentration range used to construct calibration curve was from 0.005 to 0.1 mg/mL. The GLA encapsulation efficient of the process and the GLA loading capacity of the nanoparticles were calculated with the following equation: EE ¼

The amount of GLA in the nanoparticles  100% Total amount of GLA

LC ¼

The amount of GLA in the nanoparticles  100% Total amount of nanoparticles weight

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In Vitro Drug Release from the Nanoparticles Dried CS-GLA nanoparticles were prepared described as discussed in experiment section (Determination of encapsulation efficiency and loading capacity), then redispersed in 4.0 mL of phosphate buffer saline (PBS) (pH ¼ 7.4, I ¼ 0.3) and placed in a dialysis membrane bag with a molecular weight cut-off of 10 kDa, tied and placed into 40.0 mL of PBS medium. The entire system was kept at 37  0.58C in a beaker (220  2 r/min). After a predetermined period, 3.0 mL of the medium was removed and the amount of GLA was analyzed by HPLC measurement. The released GLA was determined by a calibration curve.20 In order to maintain the original volume, each time, 3.0 mL fresh PBS solution was added to the system. The GLA release experiments were repeated three times. Bring into comparison, the release of GLA from the dialysis bag under the same conditions was also evaluated.

RESULTS The molecular structures of CS and GLA are shown in Figure 1. The protonable NH2 groups in CS molecular were expected to interact with the deprotonable COOH groups of GLA.

Figure 1. Chemical structure of CS (a) and GLA (b). *protonable groups, **deprotonable group.

FTIR Analysis The interaction of CS and GLA was first investigated spectrophotometrically. As seen in Figure 2, for the IR spectrum of CS, characteristic absorption bands appear at 1630 and 1380/cm.21 In the spectrum of GLA, the characteristic absorption bands appear at 1719/cm (COOH), 1458/cm (COO), and 1647/cm (C – O). These results indicate that some carboxylic groups of GLA have been dissociated into COO groups, which will be able to complex with the protonated amino groups of CS through electrostatic interactions to form the polyelectrolyte complex. Hence, in the IR spectrum of GLA-CS nanoparticles the 1719/cm peak in GLA becomes weak and only shows a shoulder peak, 1458/cm is shifted to 1460/cm and the peak of 1630/cm in CS is shifted to 1637/cm. Fluorescence Measurenent The aggregation of GLA-CS complexes in aqueous solution was investigated by fluorescence probe technique, with pyrene as the probe. It has been JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

Figure 2. FI-IR spectra of CS, GLA, and CS-GLA nanoparticles (WCS:WGLA ¼ 11:1).

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reported that the intensity ratio between the first and third highest energy (frequency) emission peaks, known as the I1/I3 ratio, has been shown to correlate well with solvent polarity.22 The I1 peak, which arises from the (0, 0) transition from the lowest excited electronic state, is a ‘‘symmetryforbidden’’ transition that can be enhanced by the distortion of the P-electron cloud. On the other hand, the I3 peak is not forbidden and thus is relatively solvent-insensitive.23,24 Thus the ratio (I3/I1) serves as a measure of the polarity of the environment. These values range from 0.526 for water, to 1.05 for a polystyrene film, and to about 2.0 for nonpolar solvents such as hexane and are thus very helpful for determination the location of the pyrene probe in the micelles. The I337/I334 (I3/ I1) fluorescence intensity ratio was measured for different weight ratio of GLA to CS in aqueous solution (Fig. 3), and it was found to increase substantially above the weight ratio of 0.04. The result shows the transfer of pyrene molecules from a hydrophilic environment to a relative hydrophobic environment, and indicates that the CS and GLA molecules can form aggregates in aqueous solution depending on their weight ratio. Chitosan, which has an apparent pKa of 6.3, assumes a stiff extended conformation in the dilute acetic acid solution due to the charge

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repulsion of highly protonated amino groups. With the addition of the CS solution into the GLA solution, interaction of GLA and CS lead to the formation of aggregation resulting in the transfer of pyrene molecules from a hydrophilic environment to a hydrophobic environment. When the weight ratio of GLA to CS was lower than 0.04, the pyrene fluorescence spectrum corresponded to that in water, with an I3/I1 ratio of about 0.524, indicating that there was no aggregation present in the system because of the weak interaction of CS and GLA. As the weight ratio increased to 0.06, stronger interaction between GLA and CS caused the formation of aggregation and pyrene was progressively solubilized into the hydrophobic interior, as illustrated by the drastically increased I3/I1 ratio in the intermediate region of the weight ratio of GLA to CS. The I3/I1 values for CS-GLA complexion (0.575) were found to be below those for hydrophobically modified chitosan.25 These results indicated that the hydrophobic interior is formed by complexion of CS and GLA, which showed weak hydrophilic environment. Mean Particle Size The mean particle hydrated size, size distribution (PI) of CS-GLA nanoparticles were determined by

Figure 3. Plot of the fluorescence intensity ratio, I337/I334, versus weight ratio of WGLA:WCS, at room temperature. The pyrene concentration was 6.0  107 M, CS Mw 17.9 kDa. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

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DLS. The mean size was affected by various processing parameters. Light scattering results showed that the Mw of CS was a critical parameter in controlling the size of the nanoparticles (Tab. 1). As the Mw of CS increased, the mean particle sizes and polydispersity index increased rapidly. When the Mw of CS was 17.9 kDa, the Dh and PI have the lowest value, 298 nm and 0.04, respectively. Mw 9.0 kDa CS was also used, however, no nanoparticle was formed at any specified pH value and the weight ratio of CS to GLA. This result might be due to the weak ionic interaction between CS and GLA. The particles hydrated size and size distributions of CS-GLA nanoparticles, prepared by dropwise addition of CS solution into a GLA solution at varying weight ratio of CS to GLA, were determined by DLS at pH 6.0. The results in Table 2 indicated that the mean size and size distributions of the samples decreased firstly and then increased with an increase in the weight ratio of CS to GLA, arriving at the least value 298 nm and 0.04, respectively, when the weight ratio of CS to GLA was approximately 11. In order to investigate the effect of pH on the mean hydrated size and distributions of CS-GLA nanoparticles, a series of experiments were undertake (Tab. 3). Results of these experiments showed that these nanoparticles were stable in a range of pH values from 4.0 to 6.5, but dissolved in a few minutes at pH value lower than 3.5 and aggregated quickly at pH value higher than 7.5. Table 3 showed the result of the mean size and zetapotential of the CS-GLA nanoparticles under different pH values. When the pH value was in the range from 4.0 to 7.0, the nanoparticles had positive zeta-potential. Increase of pH value decreased the mean size and size distributions of the CS-GLA nanoparticles until pH 6.5, and then increase of pH value only increased the mean size and size distributions slightly. However, when Table 1. Effect of the Mw of CS on the Mean Particle Size and Polydispersity Index of the CS-GLA Nanoparticlesa Mw/kDa 17.9 24.0 210 420

Dh (nm)

PI

298 407 503 716

0.04 0.18 0.26 0.47

a

WCS:WGLA ¼ 11:1, media pH 6.0.

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Table 2. Effect of the Weight Ratio of CS to GLA on the Mean Size and Polydispersity Index of the CS-GLA Nanoparticlesa WCS:WGLA 6.5 7 8.0 9.0 10.0 11.0 12.0 17.0 25.0

Dh/nm

PI

396 372 366 362 350 298 340 345 385

0.33 0.24 0.20 0.17 0.16 0.04 0.10 0.15 0.18

a

CS Mw 17.9 kDa, media pH 6.0.

the pH value was up to 7.5, the mean size and size distributions increased distinctly and the nanoparticles had negatively zeta-potential. When the pH was arriving at 8.0, some deposition formed. Morphology The TEM image provided a detailed morphology of the samples with different weight ratio of CS to GLA (Fig. 4). TEM showed that ionic interaction between CS and GLA lead to a matrix structure which could not form nanoparticles when the weight ratio of CS and GLA was higher than 16.7 (Fig. 4a). Based on the image, the nanoparticles had the smallest size 20 nm probably when the weight ratio of CS to GLA was 16.7 (Fig. 4b). Decrease the weight ratio to 12.5, the size of the nanoparticles increased slightly to 40 nm (Fig. 4c). However when the weight ratio decreased further to 11, the size of the nanoparticles decreased to 25 nm (Fig. 4d). The results in Table 3. Effect of pH Values on the Mean Size and Zeta-Potentiala pH Value 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 a

Dh (nm)

PI

Zeta Potential (mV)

— 377 388 366 332 298 288 292 2022 —

— 0.13 0.13 0.15 0.07 0.04 0.03 0.05 1.00 —

— 32 29 27 25 23 10 4 12 —

CS, Mw 17.9 kDa. The weight ratio of CS to GLA (WCS: 11:1.

WGLA)is

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Figure 4. TEM photograph of the CS–GLA nanparticles with different weight ratio of CS to GLA. a ¼ 25, b ¼ 16.7, c ¼ 12.5, d ¼ 11 (CS, Mw 17.9 kDa, DD 95.34%; media pH 6.5.)

Figure 4 also indicated that the shape of the particles becomed more regular as the weight ratio of CS and GLA decreased. When the weight ratio of CS to GLA was 11 (Fig. 4d), the particles had almost a spherical shape. Encapsulation Efficiency and Loading Capacity When the weight ratio of CS to GLA was less than 11, a linear relationship between EE and the weight ratio was observed and the maximum EE occurred at the weight ratio of 11 (Fig. 5). However, increase in the weight ratio of CS to GLA did not enhanced the EE any more when the weight ratio of CS to GLA was higher than 11.

The LC, which measured the mount of GLA associated with unit weight of nanoparticles, was less sensitive to the weight ratio of CS to GLA when the weight ratio of CS to GLA was in the range of 7–11 (Fig. 5). Increasing the weight ratio of CS to GLA from 11 to 14, LC value decreased from 7.4 to 5.9% sharply. According to the data of the weight ratio of CS to GLA and EE in Figure 5, the weight ratio of CS to GLA within the nanoparticles was calculated. The results show that the weight ratio of CS to GLA within the nanoparticles was 12.5 invariably when the weight ratio of CS to GLA used in the precusor solution was lower than 11. When the weight ratio of CS to GLA used in the precusor solution was JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

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Figure 5. Encapsulation efficiency (EE) (a) and the loading capacity (LC) (b) obtained as a function of the weight ratio of CS to GLA. CS, Mw 17.9 kDa.

higher than 11, increase the weight ratio CS to GLA lead to the decrease the weight ratio of CS to GLA in the nanoparticles. These results indicated that CS was gelled with GLA at a stoichiometrical ratio about 12.5.26 In Vitro Release In vitro release of GLA into PBS solution was evaluated of pure GLA and CS-GLA nanoparticles formulated with GLA encapsulation efficiency 88% and the weight ratio of CS to GLA was 11. The release profile is shown in Figure 6. Pure GLA escaped rapidly from the dialysis bag 91.3% within the first 3 h and the release of GLA was almost complete 99.8% by 5 h (Fig. 6a). The profiles of the CS-GLA nanoparticles showed a rapid release of GLA 30% (w/w) within the first 3 h, JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

Figure 6. Typical in vitro release profile of pure GLA (a) and GLA loaded CS-GLA nanoparticles (b) in PBS at 378C (CS, Mw 17.9 kDa).

followed by a release of GLA gradually up to 86% (w/w) in 48 h after the start of the release test, and then the release of GLA became very slow (Fig. 6b). Comparing the drug release of pure GLA with GLA loaded CS-GLA nanoparticles, it could be seen that the effect of CS encapsulation on GLA release was obvious and the CS-GLA nanoparticles system might be used to provide a continuous release.

DISCUSSION The CS-GLA nanoparticles were produced by ionotropic gelation between the protonated amino of CS chain and the deprotonated carboxyl group of GLA chain. They are formed rapidly when the two polymers are mixed, as recognized by solution

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turbidity. This type of phase separation was usually described as complex coacervation. FTIR spectra of CS-GLA nanoparticles demonstrated spectral shifts in the carboxyl group and amine regions, confirming the existence of an electrostatic interaction between the protonated amino and the deprotonated carboxyl group. According to the results in the stead-state fluorescence measurement (Fig. 3), one could consider that the particle forming process relied on the formation of hydrophobic segments on the polymer chains by neutralization and segregation of these segments into particles stabilized by free unpaired charges. In the dilute acetic acid solution (pH ¼ 4.0), chitosan, which has an apparent pKa of 6.3, assumes a stiff extended conformation due to the charge repulsion of highly protonated amino groups. Mixing CS acetic acid solution with GLA ammonia solution, CS molecule tended to globule because of the ionotropic interaction. When the weight ratio of CS to GLA in the precusor solution was 11, GLA was gelled stochiometrically. The weight ratio of CS to GLA in the nanoparticles was about 12.5 and the molar ratio between the protonated amino group of CS chain and the deprotonated carboxyl group of GLA was about 22. Thus, excessive CS chain constituted the corona of the particles, and CS-GLA nanoparticles consisted of hydrophobic interior formed by the complexion of CS and GLA and positively charged corona of unpaired CS chain27 (Fig. 7). With a high weight ratio of CS to GLA in the precusor solution, weak ionic interactions between the protonated amino of CS chain and the deprotonated carboxyl group of GLA chain lead to a small hydrophobic interior and a thick corona of uncomplexed CS chain. Addition of more GLA consumed the excessive CS chain enlarging the hydrophobic interior, meanwhile leading the corona be thinner. When the weight ratio of CS to GLA was reduced to 11, GLA was gelled stociochemically and the stronger ionic interactions tended to form a more compass hydrophobic interior. According to this process, as the weight ratio of CS to GLA decreased, the mean size of the nanoparticles decreased based on the DLS measurement (hydrodynamic diameter), but increased firstly and then decreases based on the TEM image (only the hydrophobic interior formed by the complexion of CS and GLA could be seen in TEM, and the corona of the segments of CS was invisible). In additon, due to the drying step of the sample preparation for TEM measurement inducing a collapse of the large and mobile outer

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Figure 7. Formation of colloidal polyelectrolytes complexes based on chitosan (blue) and GLA (red) through the hydrophobic sesgregation of complexed segments.

shell of the CS chain, the results were smaller than those from the DLS measurement. These results were consistent with the experiments. For positively charged surface of CS-GLA nanoparticles, being more hydrophilic, uncomplexed segments of CS constituted the corona of the particles. Thus, the higher the CS chain length, the thicker the corona and the higher the hydrodynamic volume of particles (Tab. 1). As mentioned above, CS-GLA nanoparticles were stabilized by the positively charged corona of unpaired CS chain. Thus, the particles hydrated size and size distribution were affected by the pH value of the medium. In this work, CS-GLA nanoparticles were prepared at pH 5.0. Adding dilute acetic acid to the CS-GLA nanoparticles suspension, the complexion between CS and GLA was destroyed partly resulting in more CS chain unpaired, which entwined onto the surface of the nanoparticles leading to the increase in nanoparticle size. When the pH value decreased to 3.5, the interaction between CS and GLA was destroyed completely resulting in the dissolve of CS molecule and GLA. Adding dilute ammonia to the media, the protonated glucosamine units were neutralization leading to a collapse of chitosan chains in the particle corona, resulting in the decrease in particle size. The progressive neutralization of chitosan was confirmed by the concomitant decrease of electrophoretic mobilities. As pH value was up to JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

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6.5, the mean size and distribution of the nanoparticles were arriving at the least value. Increasing pH to 7.5, the effective over-charge of the particles was reduced to such an extent that precipitation was formed. When the weight ratio of CS to GLA in the precusor solution was 11, GLA was gelled stochiometrically. Because of the high water solubility, GLA molecules were associated to the CS chains partly and form the nanoparticles with the highest loading capacity 7.4%. Upon lyophilization with dextrose, yellowish powder was obtained that was easy to redisperse. Under optimal conditions, particles of very similar size and drug loading capacity could be reproducibly manufactured. Moreover, after storage in a refrigeratory at 2–88C for 2 months, they manifested the same particle size and drug content as the initial preparation. In vitro release studies revealed that those GLA molecules associated to the nanoparticles surface diffused out easily and caused the rapid release of GLA in the incubation time.28

CONCLUSION Antihepatotoxic drug CS-GLA nanoparticles were prepared using a complex coaceration process. The interaction involved in the complex coaceration was mainly electrostatic interaction. The remarkable advantage of this system is that all CS-GLA nanoparticles are obtained under mild conditions without any organic solvents and surfactants. Only when the weight ratio of GLA and CS in the precusor solution was higher than 0.06, could the nanoparticles were formed. These nanoparticles are stable at pH value ranging from 4.0 to 7.0. Encapsulation efficient of GLA within the nanoparticles increased from 55.5 to 88.1% when the weight ratio of CS to GLA in the precusor solution increased from 7 to 14, but the GLA loading capacity of the nanoparticles decreased from 7.3 to 6.4%. The optimal conditions for formation CS-GLA nanoparticles were the Mw of CS 17.9 kDa, pH 6.5, and a weight ratio of CS to GLA of 11. These conditions produced the smallest mean hydrated size 298 nm. The polydispersity index of 0.04 was consistent with a relatively narrow size distribution. Electron microscopy suggested that the particles are spherical nanoparticles with a smooth surface. The experiment of GLA release in vitro showed that the effect of CS encapsulation on GLA release was obvious and the CS-GLA nanoparticles JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006

system might be used to provide a continuous release.

ACKNOWLEDGMENTS This study is supported by STCSM (No. 034319242) and the Special Funds for Nanoparticles Research Projects of Shanghai, China (0352102).

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JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 95, NO. 1, JANUARY 2006