Diclofenac sodium-loaded solid lipid nanoparticles

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Jun 23, 2010 - (Sanyo Ultra-Low Temperature Freezer MDF-192,. Japan) and then the samples were moved to the freeze-drier (Free Zone 12 Liter, Labconco ...

J Nanopart Res (2011) 13:2375–2386 DOI 10.1007/s11051-010-9998-y

RESEARCH PAPER

Diclofenac sodium-loaded solid lipid nanoparticles prepared by emulsion/solvent evaporation method Dongfei Liu • Sunmin Jiang • Hong Shen Shan Qin • Juanjuan Liu • Qing Zhang • Rui Li • Qunwei Xu



Received: 30 December 2009 / Accepted: 11 June 2010 / Published online: 23 June 2010 Ó Springer Science+Business Media B.V. 2010

Abstract The preparation of solid lipid nanoparticles (SLNs) suffers from the drawback of poor incorporation of water-soluble drugs. The aim of this study was therefore to assess various formulation and process parameters to enhance the incorporation of a water-soluble drug (diclofenac sodium, DS) into SLNs prepared by the emulsion/solvent evaporation method. Results showed that the entrapment efficiency (EE) of DS was increased to approximately 100% by lowering the pH of dispersed phase. The EE of DS-loaded SLNs (DS-SLNs) had been improved by the existence of cosurfactants and increment of PVA concentration. Stabilizers and their combination with PEG 400 in the dispersed phase also resulted in higher EE and drug loading (DL). EE increased and DL decreased as the phospholipid/DS ratio became greater, while the amount of DS had an opposite effect. Ethanol turned out to be the ideal solvent making DS-SLNs. EE and DL of DS-SLNs were not

D. Liu  S. Jiang  S. Qin  J. Liu  Q. Zhang  R. Li (&)  Q. Xu (&) School of Pharmacy, Nanjing Medical University, 140 Hanzhong Road, Nanjing, People’s Republic of China e-mail: [email protected] Q. Xu e-mail: [email protected] H. Shen Neuro-Psychiatric Institute, Nanjing Brain Hospital Affiliated to Nanjing Medical University, 264 Guangzhou Road, Nanjing, People’s Republic of China

affected by either the stirring speed or the viscosity of aqueous and dispersed phase. According to the investigations, drug solubility in dispersion medium played the most important role in improving EE. Keywords Solid lipid nanoparticles  Entrapment efficiency  Drug loading  Emulsion/solvent evaporation  Diclofenac sodium  Water-soluble  Drug delivery  Nanomedicine

Introduction Solid lipid nanoparticles (SLNs), introduced in 1991, have attracted more and more attention during the last few years. It is regarded as an alternative carrier system (Muller and Runge 1998) to traditional colloidal ones such as emulsions, liposomes and biodegradable polymer nanoparticles. SLNs are produced by replacing the liquid lipid (oil) of an o/w emulsion with a solid lipid or a blend of solid lipids, i.e., the lipid particle matrix being solid at both room and body temperature (Lucks and Muller 1991). SLNs are composed of 0.1% (w/w) to 30% (w/w) solid lipid dispersed in an aqueous medium and stabilized with preferably 0.5% (w/w) to 5% (w/w) surfactant if necessary (Pardeike et al. 2009). The average particle size of SLNs is in the submicron rage, ranging from about 40 to 1000 nm (Lucks and Muller 1991). The potential of site-specific drug delivery in optimizing drug therapy has given impetus to significant

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advancements in the pharmaceutical engineering of novel dosage forms such as SLNs (Muller et al. 2000). Several articles have highlighted the capability of SLNs to improve the bioavailability and targeting ability of various drugs (Kaur et al. 2008). Methods universally used in preparing SLNs include highpressure homogenization, high-shear homogenization and ultrasound, emulsion solvent/evaporation, solvent injection and microemulsion, etc. (Mehnert and Mader 2001). Among the present encapsulation techniques, double emulsion (w/o/w) method is a relatively simple and efficient way to prepare SLNs loaded with hydrophilic drugs (Almeida and Souto 2007). However, the average size is often in the micrometer range without restrict control of formulation parameters (Liu et al. 2007), and the drug loading (DL) capacity of the carriers is reduced remarkably due to rapid migration which results in the loss of drug into the external aqueous phase. The emulsion/solvent evaporation method represents an easy and reproducible technique, which has been widely used to prepare SLNs (Mehnert and Mader 2001). In the modified emulsion/solvent evaporation method, lipophilic material is dissolved in an organic solvent emulsified in an aqueous phase to give an oil/water (o/w) emulsion. After evaporation of the organic solvent, the emulsion is poured into cold water, and SLNs are formed. A nanoparticle system with maximal DL and high entrapment efficiency (EE) will reduce the quantity of carriers required for the administration of sufficient amount of active compound (drug) to the target site as well as drug wastage during manufacturing (Govender et al. 1999). Mainly water insoluble drugs have been incorporated into SLNs using the emulsion/solvent evaporation method. However, loading with watersoluble molecules can only be achieved to a very low extent by solubilization in the lipid melt. This limits the use of SLNs to low-dose lipophilic drugs (Muchow et al. 2008). From this point, there is a definite need to create a lipid nano-delivery system for high-dose hydrophilic drugs having a substantial loading capacity. Furthermore, while literature is replete with studies investigating drug incorporation into particles by the double emulsion method (Almeida and Souto 2007), there is a lack of published data on approaches to promote the incorporation of water-soluble drugs by the emulsion/solvent evaporation method.

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Hence, the main purpose of this study was to assess formulation and process parameters to enhance the incorporation of a water-soluble drug into SLNs by the emulsion/solvent evaporation technique. Glycerol monostearate (GMS) was selected since it has emerged as the widely used solid lipids for pharmaceutical use due to its biocompatibility, low toxicity and high physico-chemical stability (Mehnert and Mader 2001). In all investigations, diclofenac sodium (DS) has been used as a model drug because it is water soluble, ease to analyze, readily available, and of low cost (Araujo et al. 2009; Attama et al. 2008).

Experimental section Materials Diclofenac sodium (DS) was purchased from Dongtai Pharm Co., Ltd. ([99.0%, Henan, China). Phospholipid (Epikuron 200) was obtained from Lukas Meyer (Hamburg, Germany). Polyvinyl alcohol (PVA, Av.Mw 30–70 kDa, 88% hydrolysis) was supplied by Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Polyethylene glycol 400 (PEG 400), PluronicÒ F68 (F68) and Cremophor EL were all generous gifts from the BASF SE (Rhineland-Palatinate, Germany). Glycerol monostearate (GMS), Tween-80, Tween-60, Tween-20, glycerol, mannitol, sorbitol and any other chemicals were all bought from Shanghai chemicals and agency factory (Shanghai, China). HPLC grade methanol was obtained from Jiangsu Hanbon Sci. & Tech. Co., Ltd. Distilled water was purified by Hitech-K flow Water Purification System (Hitech Instruments Co. Ltd., Shanghai, China). All other chemicals were of analytical grade. Preparation of SLNs The DS-loaded SLNs (DS-SLNs) were prepared according to a modified emulsion/solvent evaporation method. In brief, 17.5 mg DS, 42.5 mg phospholipid, and 100 mg GMS were dissolved in ethanol (organic phase) at 60 °C. The aqueous phase (1% PVA containing 1% PEG 400, w/v) was heated to the same temperature of the organic phase. Then the organic phase was dropped into the hot aqueous phase under rapid stirring at 1200 rpm (RET Controlvisc C, IKA, Germany) for dispersion. After that the

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homogeneous suspension was poured into the dispersed phase (1% Tween-60 containing 1% PEG 400, w/v) under stirring at 1000 rpm for 4 h at 2 °C in an ice bath to allow for the hardening of the SLNs. The resulting suspension was filtered through a membrane with 0.45 lm pore size (Phenomenex, 25 mm filter, CA, USA).

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(Sanyo Ultra-Low Temperature Freezer MDF-192, Japan) and then the samples were moved to the freeze-drier (Free Zone 12 Liter, Labconco Corp., USA). The drying time was controlled in 72 h and then to get the SLNs powder. The recovery rate of SLNs was calculated from Eq. 1: Recovery (% ) ¼

HPLC methodology HPLC determination of DS concentration was done using a Shimadzu LC-10AT, SPD-10A HPLC system (Shimadzu, Japan) at 275 nm. The column used was Shim-pack VP-ODS, 150 mm 9 4 mm column (Shimadzu, Japan) with this solvent system: methanol/ distilled water/acetic acid (80:20:0.5, v/v/v). The standard HPLC method used for DS was as described previously (Baydoun and Muller-Goymann 2003). An injection volume of 20 ll and a flow rate of 1.0 mL/min were used, and DS could be detected at a retention time of 4.9 min. Linear correlation between peak area and DS concentration was obtained within the concentration range of 2–40 lg/mL, with a limit of quantification of 1.15 lg/mL. The equation describing the calibration curves for DS was y = 47551x ? 1701.7 (R2 = 1.0000), where x is the concentration of DS and y is the peak area. Particle size analysis The particle size and polydispersity index (PDI) were determined using Zetasizer 3000 HS (Malvern Instruments Ltd, Malvern, UK) based on quasi-elastic light scattering. Size measurements were performed in triplicate following a proper dilution of the nanoparticles suspension in distilled water at 25 °C. The PDI range was comprised between 0 and 1. Lyophilization and the recovery rate of SLNs The SLNs dispersions were fast frozen under -75 °C in a deep-freeze for 5 h in ultra low refrigeratory,

DL (%) ¼

analyzed weight of SLNs theoretical weight of SLNs  100%

ð1Þ

Encapsulation efficiency and drug loading For the drug EE and DL tests, nanoparticle suspensions were divided into 2 different samples. The percentage of drug entrapped by the SLNs was determined indirectly (Attama et al. 2008) after centrifugation in a membrane concentrator (Amicon Ultra 15, MWCO 100 K, Millipore, Ireland) for 10 min at 21,0009g at 4 °C, in a Sigma-3k30 Centrifuges (Sigma-Aldrich, Germany). The drug concentration in the aqueous continuous phase was determined by the HPLC method. Total amount of the drug in the suspension was analyzed by dissolving the sample in methanol, which was also determined by the HPLC method. The amount of drug inside the particles was calculated by subtracting the amount of drug in the aqueous continuous phase from the total amount of the drug in the suspension. The EE of DS in SLNs was determined using the following Eq. 2: EE (%) drug in suspension  drug in continuous phase ¼ drug in suspension  100% ð2Þ DL was calculated as drug analyzed in the nanoparticles versus the total amount of drug inside the particles and the lipid matrix added (GMS and phospholipid) during the preparation according to Eq. 3:

amount of drug loaded  100% amount of drug loaded þ amount of lipid matrix added

ð3Þ

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Evaluation of process variables In order to optimize the EE and DL of DS-SLNs prepared by the emulsion/solvent evaporation method, different process variables were evaluated. Unless otherwise stated the standard preparation described above was used. Influence of the cosurfactant In order to find out if cosurfactants had any observable influence on the EE and DL of DS-SLNs, the nanoparticles were prepared with 1,2-propanediol, PEG 400, n-butanol, isopropanol, tert-butyl alcohol, and glycerol presented in the aqueous phase. The concentration of cosurfactant was kept at 1% (w/v) and PVA was selected as the emulsifier. Influence of the emulsifier concentration Batches with different concentrations of PVA were prepared in order to determine the effects of PVA on the EE and DL. The concentrations of PVA were varied from 0.5 to 2.0% (w/v) while the concentration of PEG 400 remained constant and equal to 1% (w/v). Influence of the organic solvent The SLNs were prepared using various solvents and their combinations and the effect of solvent on the EE and DL was studied. The selected organic solvents included ethanol, chloroform, acetone, acetic ether, and tetrahydrofuran and the volume ratio of combinations was set at 1:1. Influence of different stabilizers and the presence of PEG 400 Different stabilizers in the dispersion medium were tested in order to examine their influence on the EE and DL. The concentrations of stabilizers (1%, w/v) and PEG 400 (1%, w/v) were kept constant. The selected stabilizers included F68, PVA, mannitol, sorbitol, Tween-20, Tween-60, Tween-80, and Cremophor EL. Influence of the phospholipid/DS ratio Phospholipids/DS ratio varied from 1:4, 1:2, 1:1, 2:1, and 4:1 were applied in this study to examine its

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influence on EE and DL. The amount of DS was kept at 17.5 mg. Influence of the amount of DS Batches with rising amount of DS were prepared in order to evaluate its influence on the EE and DL of DS-SLNs. The amount of DS varied from 10 to 50 mg, while the amount of phospholipid (42.5 mg) was kept constant. Influence of viscosity of the aqueous and dispersed phase The effect of viscosity of the aqueous and dispersed phase on the EE and DL was studied through glycerol addition. Different amounts of glycerol (2, 4, 6, 8, and 10%, w/v) were dissolved in the aqueous and dispersed phase before the preparation of DS-SLNs. Influence of the pH conditions of the aqueous and dispersed phase The influence of acidity of the aqueous and dispersed phase on the EE and DL was studied. DS-SLNs were prepared following the standard methodology, varying the pH of the aqueous and dispersed phase from 1 to 13 with HCl and NaOH. Influence of stirring rate The effect of stirring speed on the EE and DL was studied. The stirring speed values were set at 800, 1000, 1200, and 1400 rpm, respectively.

Results and discussion Influence of different cosurfactants As shown in Table 1, PEG 400 was found out to be the ideal cosurfactant for making the DS-SLNs with a little higher EE and relative smaller particle size. This result was in good agreement with those reported by Castro et al. (2009), which indicated that cosurfactants influenced the EE of retinoic acid-loaded SLNs. In this case, the cosurfactants incorporated into lipid matrix, in association with the surfactant, increased the hydrophilicity of SLNs and favored the entrapment

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of DS (Wan et al. 2008), thus explaining the enhancement of EE. An alternative explanation of the improved EE would be that the increased amount of lattice defects triggered by the introduction of cosurfactants in the lipid matrix, which was related to easier drug incorporation into SLNs (Castro et al. 2009). It was also found in Table 1 that the recoveries of the SLNs were all more than 90% and the cosurfactants had no significant effect on the DL. Influence of the emulsifier concentration Table 2 showed that both the EE and particle size kept rising until it reached a plateau with the increasing concentration of PVA. PVA contained a large number of hydroxyl groups which could form hydrogen bonds between or inside molecules. The stronger intramolecular interaction via hydrogen bonds resulted in an increase of aqueous phase viscosity (Song et al. 2008) and a larger particle size. Meanwhile, the strong intermolecular interaction of hydroxyl groups adhering to the SLNs’ surface might result in coalescence of nanoparticles, thereby the particle size of SLNs increased. Larger nanoparticles had higher drug content, because fewer drug molecules had sufficient time to diffuse into the aqueous phase. That might be the reason why the EE followed the same trend as the particle size, such that larger particles contained more drugs. At high Table 1 Effect of different cosufactants on the DS-SLNs (n = 3)

Table 2 Effect of the PVA concentration on the DS-SLNs (n = 3)

Cosurfactants (1%, w/v)

concentrations, more PVA could be oriented at organic solvent/water interface to reduce the interfacial tension (Galindo-Rodriguez et al. 2004), which resulted in significant increase in the net shear stress (Tesch and Schubert 2002; Budhian et al. 2007) during emulsification and promoted the formation of smaller emulsion droplets. Thus, the EE of SLNs should decrease as the PVA concentration went up. Consider all of the above factors together, the change of particle size and EE with PVA concentration in this study was predominantly a result of the increasing viscosity dominated over the reduction of interfacial tension. Table 2 also showed the effect of PVA concentration on the DL of DS-SLNs. As the concentration of PVA increased, drug content increased slightly and then reached a plateau. The change trend of DL was similar to that of EE, and this also might be the result of the dynamic equilibrium of viscosity and interfacial tension. On the other hand, the recovery rate of the SLNs decreased with the increasing concentration of PVA, which might be the result of larger particle size and increasing aqueous phase viscosity. Influence of different organic solvents As outlined in Table 3, the influence of various organic solvents and their combinations on the particle size, EE and DL was studied. Ethanol was

Mean particle size (nm)

PDI

EE (%)

DL (%)

Recovery (%)

Without cosurfactant

251.9 ± 3.2

0.174 ± 0.025

63.6 ± 1.2

7.3 ± 0.6

95.7 ± 1.3

PEG 400

226.5 ± 2.8

0.219 ± 0.017

77.0 ± 1.4

8.0 ± 0.4

95.4 ± 1.6

1,2-Propanediol n-Butanol

217.3 ± 2.1 229.7 ± 4.5

0.186 ± 0.021 0.197 ± 0.024

70.4 ± 1.6 73.4 ± 0.7

7.9 ± 0.6 7.3 ± 0.2

92.1 ± 0.7 94.3 ± 1.2

Isopropanol

234.0 ± 3.6

0.173 ± 0.019

74.0 ± 2.9

7.4 ± 0.8

93.5 ± 1.4

tert-Butyl alcohol

298.4 ± 2.3

0.243 ± 0.025

71.0 ± 2.3

7.9 ± 0.5

91.9 ± 1.2

Glycerol

241.6 ± 2.7

0.192 ± 0.022

70.1 ± 0.4

7.7 ± 0.1

94.6 ± 1.3

Concentration (%, w/v)

Mean particle size (nm)

PDI

EE (%)

DL (%)

Recovery (%)

0.5

164.8 ± 2.5

0.184 ±0.016

63.9 ± 3.9

6.5 ± 0.5

98.7 ± 1.3

1.0

234.3 ± 3.9

0.197 ± 0.023

74.7 ± 3.0

7.3 ± 0.9

96.9 ± 0.8

1.5 2.0

288.2 ± 2.4 290.1 ± 5.6

0.210 ± 0.025 0.219 ± 0.027

81.6 ± 2.2 81.9 ± 0.1

7.8 ± 0.1 7.9 ± 0.2

94.3 ± 1.6 91.1 ± 1.4

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Table 3 Effect of different organic solvents on the DS-SLNs (n = 3) Solvent

Mean particle size (nm)

PDI

EE (%)

DL (%)

Recovery (%)

Ethanol (EtOH)

226.5 ± 3.7

0.169 ± 0.025

78.3 ± 2.7

7.9 ± 0.9

95.6 ± 1.8

Chloroform (Chl)

380.9 ± 4.5

0.367 ± 0.021

76.1 ± 1.9

7.4 ± 0.5

81.3 ± 2.2

Acetone (Ace) Acetic ether (AE)

273.4 ± 4.1 253.8 ± 3.3

0.234 ± 0.018 0.158 ± 0.026

72.8 ± 2.2 77.9 ± 1.4

6.7 ± 0.8 7.7 ± 0.4

93.5 ± 1.2 93.4 ± 1.6

Tetrahydrofuran (THF)

236.5 ± 3.2

0.213 ± 0.027

72.9 ± 3.5

6.6 ± 1.1

94.8 ± 1.5

EtOH/Ch = 1:1 (v/v)

241.0 ± 3.8

0.285 ± 0.021

77.7 ± 0.9

7.8 ± 0.3

93.0 ± 0.7

EtOH/Ace = 1:1 (v/v)

234.9 ± 3.6

0.206 ± 0.019

71.8 ± 1.8

7.1 ± 0.5

95.2 ± 1.3

EtOH/AE = 1:1 (v/v)

331.1 ± 4.3

0.291 ± 0.023

73.4 ± 2.6

7.5 ± 0.9

85.6 ± 0.9

EtOH/THF = 1:1 (v/v)

235.6 ± 2.9

0.253 ± 0.024

72.0 ± 1.9

7.5 ± 0.5

94.3 ± 1.8

Chloroform/Ace = 1:1 (v/v)

240.7 ± 3.2

0.280 ± 0.029

74.1 ± 3.2

7.2 ± 1.1

95.4 ± 1.3

Chloroform/AE = 1:1 (v/v)

252.7 ± 2.6

0.274 ± 0.025

71.0 ± 1.6

7.1 ± 0.4

96.1 ± 1.7

Chloroform/THF = 1:1 (v/v)

238.2 ± 3.0

0.264 ± 0.018

72.0 ± 2.0

6.8 ± 0.8

95.9 ± 1.1

Acetone/AE = 1:1 (v/v)

269.3 ± 2.7

0.283 ± 0.022

73.6 ± 1.3

7.5 ± 0.4

96.7 ± 1.5

Acetone/THF = 1:1 (v/v)

271.6 ± 2.5

0.296 ± 0.025

77.4 ± 2.5

8.0 ± 0.9

95.9 ± 1.7

Acetic Ether/THF = 1:1 (v/v)

254.8 ± 3.1

0.272 ± 0.028

75.1 ± 1.4

7.8 ± 0.6

97.4 ± 1.8

found out to be the ideal solvent making DS-SLNs with lower toxicity, smaller particle size, and higher EE. SLNs were also made with chloroform, acetone, acetic ether, tetrahydrofuran, and their combinations. The physicochemical properties of different solvents such as viscosity, vapor pressure, and surface tension could play a crucial role in the overall particle properties, such as particle size and PDI (Italia et al. 2007). However, none of the solvents and their combinations tested influenced the DL, which suggested the less possible role of organic solvents on DS entrapment under the studied conditions. It was also found that recovery rate of the SLNs was below 90% when the particle size was bigger than 300 nm. It is known that DS could not be directly dissolved in chloroform and other organic solvents, except ethanol and tetrahydrofuran. However, it is possible to change DS’s lipophilicity and solubility by using specific ampholytic surfactants. When the concentration of ampholytic surfactants in organic solvents beyond their critical micelle concentrations (CMC), reverse micelles formed with the lipophilic part of the surfactant molecule facing the organic phase and the hydrophilic part representing the inner core. Thus, solubilization of water-soluble drugs was possible in the reverse micelles. Phospholipid was a widely used excipient in drug development, and was able to form reverse micelles in different organic phases (Muller-Goymann 2004). DS

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could be solubilized into the reverse micelles since the phospholipid was contained in the organic phase (Cui et al. 2006). In our preliminary study, it was found that the presence of phospholipid had tended to reduce the gap of DS’ solubility in different organic solvents. It was considered that DS had been complexed with phospholipid in all the solvents (Liu et al. 2007). That might be the reason why various types of solvents made no difference in the DL, and the EE was all about 70%. Influence of the stabilizers and the presence of PEG 400 The choice of stabilizer was specific to each drug candidate and formulation procedure. The stabilizer (or mixture of stabilizers) should exhibit sufficient affinity for the particle surface in order to stabilize the SLNs (Kocbek et al. 2006). As shown in Table 4, the type of compounds employed for stabilization had a pronounced effect on the particle size, EE, DL, and recovery rate. The EE, DL, and recovery rate of DSSLNs without the stabilizers was lower than those with the presence of stabilizers. However, the particle size of DS-SLNs without the stabilizers was the biggest one, which might respond to the lowest recovery rate. The stabilizer was an important factor, as they had a tendency to increase the stability of phospholipid complex and/or to reduce the solubility of phospholipid complex in the dispersed phase

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(Patist et al. 1997), which therefore explained the enhancement of EE and DL. The EE and DL of SLNs stabilized with Tween20, Tween-60, Tween-80, or cremophor EL were significantly higher than those stabilized with PVA, sorbitol, or F68. The surfactant nature was accountable for the differences in EE and DL of the SLNs. An important function of stabilizers was to form a mechanical and thermodynamic barrier at the interface that retarded the coalescence of individual emulsion droplets (Myers 1999). It seemed that interactions between DS and Tween-20, Tween-60, or Tween-80 were substantial for high EE and consequently led to the high DL of DS-SLNs. The EE of the yielded SLNs with the combination of stabilizers (Tween-20 and Tween-80) and PEG 400 significantly greater than those obtained with stabilizers alone (Table 4). PEG 400 associated with the stabilizers in the dispersed phase might participate in the redistribution of water inside the reverse micelles (Talukder et al. 2003), therefore increased Table 4 Effect of different stabilizers and the presence of PEG 400 in the dispersed phase on the DS-SLNs (n = 3)

Table 5 Effect of the phospholipid:DS ratio on the DS-SLNs (n = 3)

Stabilizers (1%, w/v)

the solubility of DS in them, which accounted for the increase of EE. However, the existence of PEG 400 made no significant difference in the DL. Influence of the phospholipid/DS ratio As shown in Table 5, both the particle size and EE increased as the phospholipid/DS ratio became greater. However, the recovery rate had an opposite trend, which might be the result of increasing particle size. The increase of phospholipid content was expected to improve the EE by providing more space to incorporate the drug. Increment of the phospholipid content also enlarged the particle size and reduced the possibility of drugs to enter the external phase, which accounted for the enhancement of EE (Shah et al. 2007). The additional explanation of the increased particle size and improved EE would be an excess of lecithin which might possibly form multilayers around the particles and/or leak into the aqueous phase leading to the formation of liposomes,

Mean particle size (nm)

PDI

EE (%)

DL (%)

Recovery (%) 86.2 ± 1.7

Without stabilizer

324.7 ± 8.3

0.281 ± 0.027

59.4 ± 2.1

6.0 ± 0.6

F68

253.4 ± 4.7

0.162 ± 0.015

55.4 ± 1.5

5.7 ± 0.5

92.3 ± 1.1

PVA Mannitol

229.3 ± 3.1 276.9 ± 2.6

0.259 ± 0.020 0.236 ± 0.026

61.6 ± 0.4 69.4 ± 1.3

5.9 ± 0.2 7.4 ± 0.4

93.5 ± 1.4 90.9 ± 1.6

Sorbitol

231.6 ± 3.4

0.224 ± 0.021

58.1 ± 2.7

6.3 ± 0.8

91.3 ± 1.8

Tween-20

235.8 ± 3.1

0.260 ± 0.029

73.0 ± 3.8

7.5 ± 1.1

94.7 ± 1.2

Tween-20 ? PEG 400

225.2 ± 3.3

0.254 ± 0.018

77.6 ± 0.6

8.2 ± 0.2

92.1 ± 0.9

Tween-60

242.1 ± 2.5

0.263 ± 0.014

75.9 ± 1.9

8.6 ± 0.6

91.0 ± 1.3

Tween-60 ? PEG 400

230.6 ± 3.7

0.205 ± 0.022

78.3 ± 0.5

7.9 ± 0.2

93.6 ± 0.7

Tween-80

238.1 ± 3.0

0.227 ± 0.023

72.6 ± 1.2

7.6 ± 0.4

90.4 ± 0.9

Tween-80 ? PEG 400

214.8 ± 2.2

0.238 ± 0.028

78.2 ± 1.6

8.1 ± 0.5

92.7 ± 1.2

Cremophor EL

183.4 ± 1.0

0.241 ± 0.027

71.0 ± 3.3

7.3 ± 0.9

96.2 ± 1.5

Cremophor EL ? PEG 400

194.3 ± 0.9

0.252 ± 0.024

74.0 ± 2.6

7.3 ± 0.8

95.8 ± 1.3

Phospholipid:DS

Mean particle size (nm)

PDI

EE (%)

DL (%)

Recovery (%)

1:4

208.4 ± 3.5

0.237 ± 0.025

73.3 ± 0.6

9.2 ± 0.1

94.8 ± 1.3

1:2

212.9 ± 2.6

0.183 ± 0.011

76.1 ± 2.5

8.7 ± 0.8

93.6 ± 0.9

1:1 2:1

225.2 ± 3.1 244.3 ± 2.8

0.225 ± 0.028 0.208 ± 0.023

76.1 ± 1.0 79.4 ± 1.4

8.0 ± 0.1 6.4 ± 0.2

92.3 ± 1.4 92.2 ± 1.3

4:1

271.7 ± 3.3

0.199 ± 0.017

80.5 ± 2.1

4.4 ± 0.6

87.0 ± 1.8

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Table 6 Effect of the amount of DS on the DS-SLNs (n = 3)

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Amount of DS (mg)

Mean particle size (nm)

EE (%)

DL (%)

Recovery (%)

10.0

221.3 ± 2.2

0.124 ± 0.017

79.8 ± 2.1

4.6 ± 1.1

93.8 ± 1.6

20.0

238.9 ± 3.5

0.140 ± 0.008

75.7 ± 1.5

8.7 ± 0.6

91.5 ± 1.2

30.0 40.0

233.4 ± 2.6 239.7 ± 2.8

0.157 ± 0.015 0.148 ± 0.026

72.6 ± 1.8 71.1 ± 0.9

12.5 ± 0.9 15.2 ± 0.2

93.1 ± 0.9 92.7 ± 0.8

50.0

232.3 ± 3.1

0.161 ± 0.023

68.7 ± 1.3

19.0 ± 0.5

93.2 ± 1.2

mixed micelles or other aggregates (Schubert et al. 2006; Heiati et al. 1996). A decrease in DL was also observed as the phospholipid/DS ratio increased from 1:4 to 4:1. As the amount of phospholipid increased, the mass of lipid matrix got larger. The change of the DL with the amount of phospholipid was mainly a result of the increasing of lipid matrix dominated over the enhancement of DS entrapped. Influence of the amount of DS The effect of the initial amount of drug on the particle size, EE, and DL was studied. As the amount of DS varied from 10 to 50 mg, the DL increased, while the EE decreased slightly (Table 6). However, both the particle size and recovery rate were not influenced by the initial DS weight in the formulation. The enhancement of DL was probably caused by the linear increase (correlation coefficient was 0.9986) of DS entrapped into SLNs. The DS concentration in the organic phase increased and more drug molecules could incorporate into the lipid matrix, responsible for the rise of the entrapment amount of DS. Nevertheless, the EE of DS-SLNs decreased as the relative amount of the drug substance went up. This phenomenon could be explained by the fact that the osmotic pressure difference between the outer and inner phase changed with the increasing amount of model drug inside the nanoparticles, which might cause some damages to the formation of emulsion droplets and made it easier for the drug to escape from the inner phase (Peltonen et al. 2004). Influence of viscosity of the aqueous and dispersed phase Changes in the viscosity of the aqueous phase might affect the kinetics of the outward diffusion of the drug and consequently the drug entrapment of the

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SLNs. To verify this hypothesis, DS-SLNs were prepared with aqueous and dispersed phase of up to 10% glycerol, i.e., with viscosity ranges of 0.89 cP for pure water to 1.15 cP in the latter case (Shahgaldian et al. 2003). As shown in Table 7, there was no significant variation in the EE and DL when the concentration of glycerol increased from 2 to 10% in both the aqueous and dispersed phase. This phenomenon was in contradiction with the result found in the influence of the emulsifier concentration, in which the aqueous viscosity enhancement due to PVA increased EE. According to the work of Alexy et al. (2004) in which the interaction of PVA and glycerol was investigated, it was shown that the role of glycerol was to accelerated the degradation of PVA. In this case, during the preparation of SLNs, PVA might be degraded in the presence of glycerol, which might offset the effect of viscosity increment. This should be the reason why the addition of glycerol had no influence on the EE and DL. Table 7 also showed the effect of both the aqueous and dispersed phase viscosity on the particle size and recovery rate. Particle size enlarged slightly with the increasing concentration of glycerol in dispersed phase. The increment of particle size when glycerol added into the aqueous phase was greater than that added into the dispersed phase. The recovery rate of the DS-SLNs decreased with the increasing concentration of glycerol in both the aqueous and dispersed phase, which might also be the result of larger particle size and increasing viscosity. However, the decreased degree of recovery rate as glycerol added into the aqueous phase was smaller than that added into the dispersed phase. Influence of pH conditions of the aqueous and dispersed phase This factor was evaluated since it might play an important role in the EE and DL of compounds (Song

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Table 7 Effect of the viscosity of aqueous and dispersed phase on the DS-SLNs (n = 3) Concentration of glycerol (w/v) Aqueous phase

Dispersed phase

0

Mean particle size (nm)

PDI

EE (%)

DL (%)

Recovery (%)

201.3 ± 2.2

0.139 ± 0.016

78.5 ± 2.4

8.1 ± 0.2

95.1 ± 0.7

2.0

218.6 ± 3.7

0.182 ± 0.004

72.3 ± 3.3

7.4 ± 0.3

94.6 ± 0.5

4.0

231.1 ± 4.4

0.198 ± 0.011

73.7 ± 1.6

7.4 ± 0.2

94.4 ± 2.3

6.0

240.5 ± 1.2

0.125 ± 0.014

74.0 ± 2.8

7.5 ± 0.6

94.0 ± 0.9

8.0

269.4 ± 3.1

0.117 ± 0.019

74.1 ± 1.4

7.4 ± 0.1

93.5 ± 1.3

10.0

284.2 ± 1.9

0.101 ± 0.018

77.7 ± 3.8

7.7 ± 0.4

93.5 ± 1.6

0

220.3 ± 1.5

0.137 ± 0.012

77.6 ± 3.5

8.0 ± 0.7

95.1 ± 1.4

2.0

218.7 ± 4.3

0.145 ± 0.017

74.6 ± 0.4

7.3 ± 0.1

94.8 ± 1.0

4.0

229.8 ± 3.6

0.158 ± 0.016

74.8 ± 0.1

7.4 ± 0.4

94.2 ± 1.1

6.0

233.4 ± 2.7

0.166 ± 0.020

75.8 ± 3.3

7.5 ± 0.6

94.7 ± 1.3

8.0

245.0 ± 2.8

0.148 ± 0.023

76.9 ± 1.6

7.5 ± 0.1

92.9 ± 1.5

10.0

241.9 ± 1.1

0.134 ± 0.023

77.5 ± 3.1

7.7 ± 0.6

91.7 ± 0.9

Table 8 Effect of pH conditions of the aqueous and dispersed phase on the DS-SLNs (n = 3)

pH value

Aqueous phase

Dispersed phase

6.7

Mean particle size (nm)

PDI

EE (%)

DL (%)

Recovery (%)

221.3 ± 2.2

0.139 ± 0.016

78.5 ± 2.4

8.1 ± 0.2

93.1 ± 0.7

9.0

265.2 ± 1.6

0.260 ± 0.023

83.8 ± 0.5

9.1 ± 0.6

92.6 ± 0.2

11.0 13.0

255.4 ± 1.2 275.7 ± 2.8

0.296 ± 0.028 0.408 ± 0.016

79.0 ± 1.3 83.2 ± 2.2

8.2 ± 0.2 8.9 ± 0.5

93.5 ± 1.3 90.8 ± 1.1

1.0

398.8 ± 9.5

0.567 ± 0.026

99.4 ± 0.4

2.4 ± 0.1

71.9 ± 0.8

3.0

367.6 ± 8.1

0.432 ± 0.019

96.1 ± 1.2

8.7 ± 0.2

83.2 ± 0.6

4.0

301.1 ± 5.4

0.385 ± 0.017

76.0 ± 3.0

7.9 ± 0.5

87.4 ± 1.2

5.0

249.8 ± 4.0

0.239 ± 0.018

78.2 ± 2.7

8.9 ± 0.5

90.4 ± 0.9

6.7

210.3 ± 1.5

0.137 ± 0.012

77.6 ± 3.5

8.0 ± 0.7

93.1 ± 1.4

9.0

283.1 ± 1.9

0.244 ± 0.025

77.8 ± 2.1

8.3 ± 0.4

93.7 ± 1.1

11.0

270.9 ± 2.3

0.303 ± 0.014

73.1 ± 1.3

7.8 ± 0.3

93.4 ± 1.3

13.0

289.2 ± 3.1

0.341 ± 0.013

77.6 ± 1.6

8.3 ± 0.2

92.5 ± 1.7

et al. 1997), whose solubility is pH-dependent. As seen in Table 8, EE was strongly increased by lowering the pH of dispersed phase (from 77.6% at pH = 13 to 99.9% at pH = 1). Particle size of DSSLNs first decreased and then increased as the pH value of dispersed phase increased form 1 to 13. While the recovery rate of DS-SLNs had an opposite trend compared with the particle size. DS has a dissociation constant (pKa) of 4.0 ± 0.2 at 25 °C in water. When pH value is more than 1 unit below pKa, DS is mostly in its free acid form, which is even less soluble than the salt (Bertocchi et al. 2005). Therefore, the amount of DS in the dispersed phase at pH 1 was very little. This might be the reason why the EE

could increase to approximately 100% (diclofenac was not taken into account). These findings were consistent with the recent report which pointed that drug entrapment of procaine hydrochloride had been increased from 11.0 to 58.2% with the pH of aqueous phase raised from 6.2 to 9.3 (Govender et al. 1999). The explanation of the larger particle size in acid conditions would be the present of diclofenac which might form multilayers around the lipid particle pore and/or leak into the aqueous phase leading to the formation of diclofenac particles. The effect of the dispersed phase pH conditions on the DL was also investigated. As in general, an increase in EE increases DL. It was also shown in

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Table 9 Effect of the stirring rate on the DS-SLNs (n = 3)

J Nanopart Res (2011) 13:2375–2386

Stirring rate (rpm)

Mean particle size (nm)

EE (%)

DL (%)

Recovery (%)

800

219.5 ± 4.1

0.146 ± 0.023

80.7 ± 0.8

9.4 ± 1.3

93.4 ± 1.7

1000

222.6 ± 3.7

0.135 ± 0.018

79.2 ± 4.3

8.4 ± 0.2

94.9 ± 1.2

1200 1400

225.9 ± 2.2 224.8 ± 3.4

0.142 ± 0.019 0.133 ± 0.020

78.2 ± 4.9 74.2 ± 1.3

8.0 ± 0.9 7.6 ± 0.1

94.1 ± 2.0 93.2 ± 1.1

Table 8 that the EE increased when the dispersed phase pH conditions decreased from pH 3 to 1, however, the DL decreased dramatically. This observation might be explained by the fact that although the percentage of DS entrapped increased (diclofenac was not taken into account) by lowering the pH conditions, the absolute amount of DS entrapped decreased. That might be the reason why the change trend of DL was different from the EE’s. The effect of aqueous phase pH conditions on the EE and DL was also tested (Table 8). When pH value of aqueous phase was below 6.7, the phospholipid rapidly precipitated and the homogeneous suspension was not obtained. In this case, the pH of aqueous phase only varied from 6.7 to 13. DL was not affected by the aqueous phase pH conditions, and EE at pH 6.7 was a little different from the groups of pH 9 and 13. It was also found that particle size enlarged while the recovery rate decreased with the increasing pH condition of aqueous phase. Influence of stirring rate The effect of stirring speed on particle size, EE, DL and recovery rate was also investigated. At a stirring speed below 600 rpm, the uniformity of the mixing force throughout the emulsion mixture decreased, and the homogeneous suspension was not obtained. In contrast, stirring speeds higher than 800 rpm were vigorous and uniform enough to form small and homogeneous size distribution nanoparticles. The EE and DL of the DS-SLNs prepared at stirring speeds between 800 and 1400 rpm was shown in Table 9. Neither EE nor DL was significantly affected by the stirring speed of the system, which indicated that the EE and DL were not sensitive to the stirring speed. The stirring speed had a little effect on the EE of microspheres was also reported by Haznedar and Dortunc (2004). The plateau of EE and DL at high stirring rates might also suggest that PVA at the concentration of 1.0% was efficient for the droplet

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PDI

cover and the coalescence suppression (Valot et al. 2009). The complete cover of the emulsion droplets at these stirring speeds guarantied the stability and the size remains of O/W emulsion. It was also found in Table 9 that stirring speed had no obvious effect on the particle size and recovery rate of DS-SLNs.

Conclusions Emulsion/solvent evaporation technique has its limits on efficiently loading water-soluble drug substances inside the SLNs. However, by lowering the pH of the dispersed phase, the drug EE of hydrophilic DS was increased from 75 to 80% to a level high as approximately 100%, which may be a result of the decreasing ionization and the consequent lower drug solubility in the dispersed phase. According to the investigations, drug solubility in dispersion medium has an important influence on the drug EE. Most of the process variables have less influence on the DL than the EE. It is because the amount of DS is relatively low compared with the lipid matrix added, and DL is not sensitive to the change of process variables. It was also found that the recovery rate of DS-SLNs had an opposite trend compared with the particle size, which means when the particle size enlarges, the recovery rate decreases. In conclusion, this study has revealed that formulation variables can be exploited in order to enhance the incorporation of a water-soluble drug into SLNs by the emulsion/solvent evaporation technique. Water-soluble drugs’ EE and DL depend not only on the formulation and process parameters, but mostly on the drug molecule. Improving the liposolubility of water-soluble drugs is an attractive way to enhance their loading efficiency as far as hydrophobic lipid carriers are concerned. Some efforts will be made to increase their affinity for hydrophobic carrier materials in subsequent studies.

J Nanopart Res (2011) 13:2375–2386

Acknowledgment This work was financially supported by the Jiangsu Natural Science Funds (Project No. BK2009420).

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