Strategic approaches for improving entrapment of hydrophilic peptide drugs by lipid nanoparticles

Colloids and Surfaces B: Biointerfaces 70 (2009) 248–253

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Strategic approaches for improving entrapment of hydrophilic peptide drugs by lipid nanoparticles Hong Yuan a , Sai-Ping Jiang b , Yong-Zhong Du a , Jing Miao b , Xing-Guo Zhang a , Fu-Qiang Hu a,∗ a b

College of Pharmaceutical Science, Zhejiang University, 388 Yuhangtang Road, Hangzhou 310058, PR China The First Affiliated Hospital of College of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, PR China

a r t i c l e

i n f o

Article history: Received 16 October 2008 Received in revised form 18 December 2008 Accepted 20 December 2008 Available online 31 December 2008 Keywords: Solid lipid nanoparticles Leuprolide Hydrophobic ion pairing O/O emulsion-evaporation method Solvent diffusion method in an aqueous system

a b s t r a c t In order to introduce hydrophilic peptide drugs into solid lipid nanoparticles (SLN), a technique of combining hydrophobic ion pairing (HIP) and non-aqueous oil-in-oil (O/O) emulsion-evaporation was developed. Leuprolide (LR) was selected as the model drug, while sodium stearate (SA-Na) was used as the negative charged ion pairing material. The formation of leuprolide-sodium stearate (LR-SA-Na) complex was confirmed by differential scanning calorimetry (DSC). It was observed that when the molar ratio of SA-Na/LR reached 2/1, ca 88.5% LR was incorporated into the hydrophobic ion complexes with SA-Na. Compared with the conventional method of solvent diffusion in an aqueous system, the efficiency of LR drug entrapment with SLN increased from 28.0% to 74.6% by the combined technique of HIP and O/O emulsion-evaporation. In vitro drug release tests revealed that employing technique of HIP obviously reduced the burst release and slowed down the rate of drug release. At meanwhile, applying the method of non-aqueous O/O emulsion-evaporation, the longer time of drug release but relatively higher drug burst release ratio was observed in comparison with those by the solvent diffusion method in an aqueous system. The drug entrapment and release behaviors of LR-SA-Na SLN prepared by the O/O emulsion-evaporation method suggested that it could potentially be exploited as an oral delivery system for leuprolide. © 2008 Elsevier B.V. All rights reserved.

1. Introduction Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) composed of lipid materials are novel colloidal carriers for controlled drug delivery system [1,2]. Compared with the other nano- or microparticulate carriers, SLN and NLC combine the advantages of polymeric nanoparticles and o/w fat emulsions for drug delivery administration, such as a good tolerability [3,4], protection of incorporated active compounds against chemical degradation [5], a high bioavailability by oral administration [6], large-scale production by high pressure homogenization [7]. SLN and NLC are usually applicable for entrapping lipophilic drugs [2,8]. However, for hydrophilic drugs, they were disadvantageous to the lower drug entrapment efficiency due to their limited solubility in lipid matrix. To solve this problem, basically three approaches were considered to be adoptable: (I) Increasing the lipophilicity of hydrophilic drugs, such as by means of hydrophobic ion pairing (HIP), namely pairs of hydrophilic drugs and the amphipathic molecules with opposite charges [9], or lipophilic prodrug formed by chemical conjugation with hydrophobic component [10]; (II) Non-aqueous techniques during encapsulation so as to

∗ Corresponding author. Tel.: +86 571 88208441; fax: +86 571 88208441. E-mail address: [email protected] (F.-Q. Hu). 0927-7765/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.colsurfb.2008.12.031

reduce drug leakage, for example, compressed gas anti-solvent precipitation techniques [11], oil-in-oil (O/O) emulsion-evaporation method [12], etc.; (III) Improving the hydrophilicity of nanoparticles by using hydrophilic materials or introducing functional groups that can be easily substituted or conjugated to the particle matrix. For example, substituting acyl groups on poly(glycerol adipate) backbone (PGA) with various amounts of pendant C18 chain by which the dexamethasone phosphate loaded nanoparticles were yielded [13]. Leuprolide (LR) is a synthetic analogue of luteinizing hormonereleasing hormone which acts as a potent agonist towards anterior pituitary receptor [14]. It is a water-soluble peptide drug with molecular weight 1209.41. To enhance its lipophilic partition coefficient, hydrophobic ion pairing (HIP) of leuprolide with amphipathic molecules such as sodium oleate [14] and sodium docusate [9] were reported. In this study, because stearic acid was used as solid lipid material of SLN, sodium stearate (SA-Na) was chosen as amphipathic molecules to prepare hydrophobic ion pairing (HIP) of leuprolide, the characterization of hydrophobically ion-paired complex was then assessed. On the other hand, the double-emulsion solvent evaporation methods are basically applied for encapsulation of most watersoluble peptides, e.g., water-in-oil-in-water (w/o/w) technique [12]. However, the main problems were reported that, by this method, the instability of water-in-oil pre-emulsion induced the

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lower drug encapsulation efficiency and the peptide or protein drug was inactivated during the preparation process [15]. In our previous study, a solvent diffusion method in an aqueous system was successfully applied for the loading of gonadorelin into SLN [16]. This method was also trialed for preparing leuprolide loaded SLN. However, due to the leakage of hydrophilic drugs was severe in a mass of aqueous phase during encapsulation, the amount of drug incorporated into the particle matrix was much smaller than that we expected. Additionally, the non-aqueous oil-in-oil (O/O) emulsionevaporation method was reported to succeed in encapsulating the bovine serum albumin (BSA) into poly (d,l lactide-co-glycolide) (PLG) microspheres [12]. It was said, by this method, the leakage of drug was restricted, and the stability of biomacromolecule drugs during encapsulation was maintained [17]. Therefore, in this paper, we tried to combine the merits of above two methods into the preparation of SLN loading leuprolide. Physical–chemical properties of resultant SLN, such as mean diameter, drug encapsulation efficiency and in vitro drug release profiles were investigated in comparison with those by solvent diffusion method. 2. Materials and methods 2.1. Materials Stearic acid (Shanghai Chemical Reagent Co. Ltd., China) was used as solid lipid material for the preparation of SLN. Sodium stearate (Wenzhou Chemical Reagent Co. Ltd., China) was chosen as amphipathic molecules for the preparation of ion complex. Leuprorelide was kindly donated by Shenzhen Hanyu bioengineering Co. Ltd., China. Liquid paraffin was purchased from Guangzhou Siyuan Chemical Reagent Co. Ltd., China. The surfactants, sodium dodecyl sulfate and poloxamer 188 were provided by Guangzhou Chemical Reagent Co. Ltd., China, Jiqi Pharmaceutical Industry of Shen Yang Pharmaceutical University Co. Ltd., China, respectively. Tween-80 and Span-80, chromatogram reagent grade methanol and acetonitrile, analytical reagent grade ethanol, acetone and other chemicals were purchased from Wenzhou Qingming Chemical Reagent Co. Ltd., China. All reagents were used without any further treatment. 2.2. Hydrophobic ion pairing of leuprolide The so-called hydrophobic ion pairing technique was performed as following. Concentrations of sodium stearate (SA-Na), 0.625, 1.25, 2.5, 3.75, 5 mg/mL were formulated in 10 mL distilled water at 80 ◦ C. The resultant solutions, 0.2 mL, were slowly dropwise added into leuprolide (LR, 5 mg/mL) with equivalent volume, and then surged vertically (XW-80A, Instruments factory of Shanghai Medical University) for 3 min. As a result, the dispersions of leuprolide-sodium stearate (LR-SA-Na) ion complex corresponding to the molar ratios of SA-Na:LR, 0.5:1, 1:1, 2:1, 3:1, 4:1, respectively, were obtained. The above dispersions were frozen at −75 ◦ C in a deep-freezer (Sanyo Ultra Low Temperature Freezer MDF-192, Japan) for 5 h, and then moved to the freeze-drier (Freezone 2.5 L, LABCONCO, USA) for drying 72 h. The powders of LR-SA-Na complex were collected for further characterization and preparation of SLN. 2.3. Characterization of LR-SA-Na complex 2.3.1. Formation efficiency of leuprolide in ion complex The dispersions of LR-SA-Na complex were centrifuged for 10 min at 20,000 rpm (3K30, Sigma, Germany). The LR content in the supernatant was measured by HPLC equipped with an Agilent

249

G1310A pump (1100 Series) unit control, an Agilent G1314A Variable Wavelength Detector (1100 Series) set at 220 nm, and Hypersil C18 column (150 mm × 3.9 mm). The eluting phase was consisted of 0.1 M phosphate buffered saline (PBS) solution (pH adjusted to 3.0 by triethylamine), methanol and acetonitrile (67:12:26, v/v). The calibration curve of peak area against concentration of leuprolide was y = 37.507x + 16.869 in the range of concentration of LR, 0.2–40 ␮g/mL (R2 = 0.9985, where y = peak area and x = LR concentration). The error of detection was 0.06 ␮g/mL. Formation efficiency of LR (RL ) was calculated by Eq. (1). RL =

W − W  a s Wa

× 100%

(1)

where Wa and Ws were initial weight and weight of LR in supernatant, respectively. 2.3.2. DSC analysis for LR-SA-Na complex Differential scanning calorimetry (DSC) analysis was carried out by Delta Series DSC7 (PE, USA). 10 mg of powdered LR, SA-Na, physically mixed LR and SA-Na (1:1, w/w) and LR-SA-Na complex were measured, respectively, at 10 ◦ C/min in the range of 40–250 ◦ C. 2.3.3. Dissociation property of LR-SA-Na complex The free leuprolide dissociated from LR-SA-Na complex was measured in vitro. Briefly, 1.2 mg LR-SA-Na complex with SANa:LR = 2:1 or 0.8 mg of free leuprolide (stability test) were dispersed in 25 mL phosphate buffered saline (PBS) solution at pH 6.8 containing 0.2% sodium dodecyl sulfate, and then shaken horizontally (SHELLAB1227-2E, SHELLAB, USA) at 37 ◦ C and 60 strokes per min. The dispersion, 1 mL, was extracted from the system in a definitive time interval and filtrated with a 100 nm filter. The filtrate was determined by HPLC as described above. 2.4. Preparation of SLN loading LR SLN loading LR was prepared by two methods: solvent diffusion method in an aqueous system and O/O emulsion-evaporation method. The former was reported in our previous study [18], while the latter was described as followings. 200 mg stearic acid and 2 mg LR or 3 mg LR HIP complex (equivalent gram of free LR was 2 mg) were completely dissolved in 2 mL ethanol at 70 ◦ C. The resultant organic solution was dispersed in 10 mL liquid paraffin containing 2.5% (w/v) Span-80 by a ultrasonic probe (JY92-II, Scientz Biotechnology Co., Ltd., China) performing for 40 times at 400 W. The O/O emulsion was obtained. Subsequently, in order to evaporate ethanol, the emulsion was vacuumed for 3 h at room temperature, while stirred magnetically at 200 rpm. SLN precipitate was then centrifuge separated from the dispersion (20,000 rpm for 10 min). The liquid paraffin was cleaned up by washing the precipitates twice with petroleum ether. 2.5. Measurement of particles size The volume average diameter of SLN was determined by Zetasizer (3000HS, Malvern Instruments, UK). 2.6. Determination of drug entrapment efficiency The amount of LR in SLN was determined by HPLC. For SLN prepared by the solvent diffusion method in an aqueous system, ethanol/water (4:1, v/v) was used as the solvent to dissolve SLN precipitate at 70 ◦ C. Thereafter, it was filtrated with 100 nm filter at room temperature. As for SLN produced by the O/O emulsion-evaporation method, SLN firstly was washed with water (10 mL) to remove the unencapsulated drug, and then

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centrifuged with 20,000 rpm for 10 min, the precipitate exactly treated with the same way. The drug concentration in filtrate was determined. The drug entrapment efficiency (Ee ) and drug loading (L) in the nanoparticles was calculated by Eqs. (2) and (3). Ee = L=

W 



d

Wa

× 100%

Wd Wa + WL



× 100%

(2) (3)

where Wd , Wa and WL were the weight of drug in dissolved solution, the weight of drug and lipid added in system, respectively. 2.7. In vitro drug release studies 2.7.1. Selection of release medium The media of LR release were selected in this study. Briefly, LR, 0.5 mg, and free SLN, 40 mg, were added in phosphate buffered saline (PBS) solution at pH 6.8 or PBS (pH 6.8) containing 0.2% sodium dodecyl sulfate, 0.1% poloxamer 188, or 0.06% Tween-80, respectively, and then shaken horizontally at 37 ◦ C and 60 strokes per min. The dispersion sampled in given time intervals was filtrated with a 100 nm filter and LR in filtrate was determined by HPLC. These results were used to evaluate the stability of free LR in PBS (pH 6.8). 2.7.2. Measurement of in vitro drug release The profiles of LR release were measured in vitro by recycling the following operations. SLN was re-dispersed in 5 mL PBS of pH6.8 containing 0.2% sodium dodecyl sulfate, and then incubated in a glass test-tube at 37 ◦ C. In given time intervals, the incubated SLN were recovered by centrifugation (20,000 rpm for 10 min). The supernatant was used for measurements of HPLC, but the precipitate was dispersed again with the equivalent fresh media.

Fig. 1. Formation efficiency of LR-SA-Na complex for leuprolide against the molar ratios of Na-SA to LR (n = 3).

3. Results and discussion 3.1. Characteristics of LR-SA-Na complex LR-SA-Na complex was prepared by hydrophobic ion pairing (HIP) and its properties were assessed subsequently. Fig. 1 shows the formation efficiency of IR in LR-SA-Na complex with different molar ratios of SA-Na and LR. As shown in Fig. 1, the percentage of LR hydrophobically ion-paired with SA-Na was firstly increased as the SA-Na:LR molar ratios increased, and when the ratio reached to 2:1, the highest amount of LR-SA-Na complex (88.5% LR) was obtained, then the complex efficiency of LR decreased even further increasing the molar ratio. It was consistent to the turbidity observation of dispersion. The turbidity of dispersion gradually increased with the increase of SA-Na: LR molar ratio until it reached 2:1, and then decreased as further increasing the ratio. It was normal during the preparation of ion complex of LR [14]. Leuprolide has two basic amino acid groups in the peptide chain, thus stoichiometrically may

Fig. 2. Differential scanning calorimetry scans of sodium stearate (SA-Na, A), leuprolide (LR, B), physical mixture of leuprolide and sodium stearate (LR and SA-Na mixture, C), leuprolide-sodium stearate complex (LR-SA-Na complex, D).

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combine two negatively charged SA-Na molecules. When SA-Na: LR ratio is lower than 2:1, the insoluble LR-SA-Na complex gradually aggregated. However, as the ratio surmounted 2:1, the complex tended to dissociate and form isolated micelles, which resulted in the increase of solubility of leuprolide. These results indicated that the efficiency of LR attained the highest one at the ratio of 2:1. Therefore, LR-SA-Na complex of the molar ratio 2:1 was selected to be freeze-dried for DSC characterization and SLN preparation. Fig. 2 shows DSC curves of pure compounds of SA-Na and LR, the physically mixed LR and SA-Na, and LR-SA-Na complexes. As shown in Fig. 2, three endothermic peaks were observed on SA-Na curve, i.e., at 114.89, 132.83, 204.52 ◦ C, respectively (Fig. 2A), while one main endothermic peak at 70 ◦ C on LR curve (Fig. 2B). Hence, it was clear that there was no new observable interaction in the physically mixed sample because, as shown in Fig. 2C, peaks on the curve of physically mixed sample were attributable, to a great extent, to those of pure compounds. However, as for LR-SA-Na complex (Fig. 2D), it was noticed that the endothermic peaks belonging to SA-Na almost disappeared. It implied that most of SA-Na was ion-paired, thus no isolated domain formed in complexes aggregates. The endothermic peak of LR still retained. It indicated that the domains of LR long-chain aggregates in LR-SA-Na complex dominated the thermo-behavior of complex. Furthermore, a new peak at 233.25 ◦ C was discovered in Fig. 2D (arrow marked). It should be attributed to the ionic domains consisted of amino cations and carboxylic anions. This result further confirmed that the complex of LR-SA-Na was prepared. As well known, the ion-paired biomacromolecule drugs such as LR in complex did not have any their pharmaceutical effect in vivo. Therefore, the dissociation of obtained LR-SA-Na complex was investigated. As shown in Fig. 3, by using the sample containing ca 88.5% of ion-paired LR, the quick dissociation of LR was observed from 0 to 4 h. After 4 h, however, the dissociated ratio decreased. As LR was stable at pH 6.8 in the 24 h test (Fig. 3), the decreased dissociation ratio was probably because some dissociated LR was ion-paired with SA-Na once again. These results are helpful for the selection of in vitro drug release method.

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Fig. 3. Dissociation profile of leuprolide from LR-SA-Na complex and the stability of free leuprolide in pH 6.8 PBS containing 0.2% SDS (n = 3).

emulsion-evaporation method, was ca 400 nm, and no significant difference was found in use of LR or LR-SA-Na. On the other hand, the polydispersity of SLN prepared by O/O emulsion-evaporation method was higher than that prepared by solvent diffusion method. The effects of preparation method on the drug entrapment efficiency and loading capacity of stearic acid SLN were investigated. The results are shown in Table 2. It is clear that the drug entrapment efficiency of SLN increased from 28.0% to 46.1% and from 53.1% to 74.6%, respectively, when LR was hydrophobic ion-paired. Obviously, this was related to the improved lipophilicity of LR, which prevented the water-soluble LR from diffusing out of lipophilic SLN during the preparation [14]. In addition, comparing with solvent diffusion method, the higher drug entrapment efficiency and loading capacity in SLN were obtained by O/O emulsion-evaporation method. It was attributed to the non-aqueous liquid paraffin used as dispersed phase in O/O emulsion-evaporation method. The limited solubility of LR in liquid paraffin was advantageous to restrict drug leakage of water-soluble LR in comparison to an aqueous solvent diffusion method.

3.2. Preparation of SLN of LR

3.3. In vitro drug release profiles from SLN

SLN of LR was prepared by solvent diffusion method in an aqueous system or O/O emulsion-evaporation method, respectively. Table 1 shows particle sizes of obtained SLN. It was observed that the particles size either prepared by solvent diffusion or O/O

3.3.1. Drug release medium During the prolonged in vitro drug release, the biomacromolecule drug might be degraded and absorbed on the particles surface, and thus affect in vitro drug release studies [19]. Therefore,

Table 1 Particle size and polydispersity index (PI) of leuprolide (LR) and leuprolide-sodium stearate (LR-SA-Na) complex loaded SLN obtained by solvent diffusion method or emulsionevaporation method. Preparation method

Solvent diffusion method Emulsion-evaporation method

LR SLN

LR-SA-Na SLN

Size (nm)

PI

Size (nm)

418.6 ± 16.4a 397.5 ± 10.5b

0.192 ± 0.004c 0.398 ± 0.011c

403.8 ± 14.2a 411.3 ± 9.6b

PI 0.213 ± 0.007 0.316 ± 0.09

Data are represented with mean ± S.D. (n = 3). a,b Indicate no significant difference (P > 0.05). c Indicates significant difference (P < 0.05). Table 2 Drug encapsulation efficiencies of leuprolide (LR) and leuprolide-sodium stearate (LR-SA-Na) complex loaded SLN obtained by solvent diffusion method or O/O emulsionevaporation method (n = 3). Preparation method

Drug encapsulation efficiency (%) LR SLN

Solvent diffusion method Emulsion-evaporation method a,b,c,d

Indicate significant difference (P < 0.05).

28.0 ± 2.5 53.1 ± 4.4b,c

a,c

Drug loading capacity (%)

LR–SA-Na SLN

LR SLN

LR–SA-Na SLN

46.1 ± 4.0 74.6 ± 5.8b,d

0.28 ± 0.03 0.53 ± 0.04

0.46 ± 0.04 0.76 ± 0.06

a,d

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Fig. 4. The release medium selection results for leuprolide (n = 3).

it is essential to select a suitable drug release medium to protect the biomacromolecule drug from degradation and absorption on the nanoparticles during in vitro drug release period. The results are shown in Fig. 4. It was observed that the free LR was in-stable in pH 6.8 PBS in the 48 h test. Comparing with that of LR in PBS alone, at the same time point, the determined amount was reduced when a certain number of drug free SLN existed in the medium. As described in our previous study [20], the stearic acid SLN prepared by solvent diffusion method in an aqueous system had high zeta potential, −46.4 mV. It probably resulted in the drug absorption by ionic interact due to the positively charged leuprolide. On the other hand, only a part of LR was determined when LR and drug free SLN were added in PBS with 0.1% poloxamer 188 or 0.06% Tween-80. On the contrary, almost all of LR was determined in PBS with 0.2% SDS and blank SLN in 72 h. It was confirmed that LR was stable in PBS with 0.2% SDS (Fig. 3). These results indicated that SDS excellently prevented LR from degradation and absorption by SLN. Anyway, the anionic SDS could form ionic pair with cationic LR to enhance the stability of LR, and also could displace biomacromolecule adsorbed on the particles surface [19]. Therefore, pH 6.8 PBS with 0.2% SDS was selected as the release medium of LR. 3.3.2. Drug in vitro release As discussed above, the dissociated LR could form ion complex again with SA-Na in pH 6.8 PBS containing 0.2% SDS. Therefore, SANa should be removed timely during the drug release tests. Herein, the release medium was completely withdrawn at predetermined time and replaced with fresh buffer. Fig. 5 gives the release curves of SLN obtained by solvent diffusion method in an aqueous system. Obviously, by using LR-SA-Na, the release rate was slower than that by using free LR. For those by using free LR, ca 43.9% LR was released at the first 2 h, which was considered as the burst release. After 24 h, the accumulated drug release reached to 98.1%. However, as for those by using LR-SANa, the burst release reduced to 8.3%, and 85.3% LR was released in the first 24 h. The release went on for 48 h. It was prolonged compared to 24 h of complete release of SLN by using free LR. These results indicated that hydrophobic ion pairing (HIP) not only favored to improve the encapsulation efficiency, but also prolong the release time. Two reasons were possibly responsible for the prolonged release. Firstly, the improved lipophilicity was advantageous to entrap LR in the core of SLN, thus release of LR was slower by either the molecular diffusion or the matrix erosion. Secondly, the process of free leuprolide dissociated from negatively charged sodium stearate was possibly another important factor. The drug release profiles of SLN prepared by the O/O emulsionevaporation method were shown in Fig. 6. The same results were

Fig. 5. In vitro drug release profile of leuprolide (LR) and leuprolide-sodium stearate (LR-SA-Na) complex loaded SLN which was obtained by solvent diffusion method. (A) Drug release in 24 h; (B) drug release in 48 h (n = 3).

acquired by using LR-SA-Na, i.e., the slower rate of release. From Fig. 6A, it can be seen that 40.3% LR was released during the first 2 h, and this amount increased to 92.3% after 24 h for LR SLN. While for LR-SA-Na SLN, the drug burst release significantly decreased to 23.7% and totally 77.3% LR was released in the first 24 h. Combined with the relatively higher drug entrapment efficiency result obtained in Section 3.2, the LR-SA-Na SLN prepared by the O/O emulsion-evaporation method could be administered orally for further in vivo research. Comparing with solvent diffusion method in an aqueous system (Fig. 5), the relatively higher ratio of burst release was observed by using LR-SA-Na in O/O emulsion-evaporation method. In the first 4 h, about 45% LR was released from LR-SA-Na SLN prepared by O/O emulsion-evaporation method, however, only about 18% LR was released from LR-SA-Na SLN prepared by solvent diffusion method. This was probably related to the use of aqueous phase for solvent diffusion method, which reduced the amount of leuprolide locating on the SLN surface due to the LR existed as a hydrophobic LR-SA-Na form in SLN. After 4 h, the LR release rate from LR-SA-Na SLN prepared by O/O emulsion-evaporation method became slow than that from LR-SA-Na SLN prepared by solvent diffusion method. About 15% LR was released from LR-SA-Na SLN prepared by O/O emulsion-evaporation method between 4 and 8 h, while about 50% LR was released from LR-SA-Na SLN prepared by solvent diffusion method. The fast drug release from LR-SA-Na SLN prepared by solvent diffusion method after 4 h might be due to the erosion of the SLN surface by SDS. It could be consider the LR uniformly distributed in the lipid matrix of SLN prepared by O/O emulsion-evaporation

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in an aqueous system and O/O emulsion-evaporation method was applied to prepare SLN encapsulating LR and its complex, respectively. The size of SLN was about 400 nm. HIP obviously increased the entrapment efficiency of LR and prolonged the release time of SLN due to the improved lipophilicity of LR. Comparing with solvent diffusion method in an aqueous system, SLN produced by O/O emulsion-evaporation method exhibited higher entrapment efficiency and longer release time as well, which could be explained by the non-aqueous dispersed phase used in the preparation procedure. According to the drug entrapment and release behaviors of LR-SA-Na SLN prepared by the O/O emulsion-evaporation method, it could potentially be exploited as an oral delivery system for leuprolide. Acknowledgment We appreciate the financial support of National Basic Research Program of China (973 Program) under contract 2009CB930300. References

Fig. 6. In vitro drug release profiles leuprolide (LR) and leuprolide-sodium stearate (LR-SA-Na) complex loaded SLN which was obtained by O/O emulsion-evaporation method. (A) Drug release in 24 h; (B) drug release in 96 h (n = 3).

method, while the LR mainly existed in the core of SLN prepared by solvent diffusion method. 4. Conclusion Leuprolide-sodium stearate complex was successfully produced by hydrophobic ion pairing technique. Solvent diffusion method

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