W emulsion stabilized by Labrafil®

Colloids and Surfaces B: Biointerfaces 52 (2006) 47–51

Preparation of biodegradable polymeric hollow microspheres using O/O/W emulsion stabilized by Labrafil® Hyuk Sang Yoo ∗ School of Bioscience and Bioengineering, Kangwon National University, Hyoja2-dong, Chuncheon 200-701, Republic of Korea Received 22 June 2006; received in revised form 29 June 2006; accepted 5 July 2006 Available online 14 July 2006

Abstract Biodegradable hollow microspheres were prepared by double oil and water emulsion using a lipophilic surfactant, Labrafil® M 1944 CS. Olive oil was emulsified in biodegradable polymer-dissolved dichloromethane mixed with Labrafil by vigorous sonication. This oil-in-oil emulsion was directly re-emulsified in 0.1% poly(vinyl alcohol) solution, subsequently solidified by evaporating dichloromethane. Olive oil and Labrafil were extracted from the microspheres by using hexane. After vigorous washing with n-hexane, the hollow microsphere was freeze-dried and examined under scanning electron microscopy, confirming the morphology of hollow microspheres with thin walls and huge blank cores inside. The concentration of poly(l-lactide) in dichloromethane affected the size of hollow microspheres while the volume of olive oil or dichloromethane did not. This hollow microsphere is expected to be employed as an imaging contrast agent and a novel drug delivery vehicle. © 2006 Elsevier B.V. All rights reserved. Keywords: Biodegradable; Hollow; Microsphere; Labrafil

1. Introduction Microspheres have been prepared by various fabrication methods including phase separation, solvent evaporation, and cold precipitation [1–3]. Among them, solvent evaporation technique has been widely employed, where a volatile organic phase containing dissolved polymer is emulsified in an aqueous phase under constant stirring [3,4]. In this method, drugs are usually incorporated into the organic phase in dissolved or dispersed states. However, water-soluble compounds including proteins and many hydrophilic drugs were not suitable for oil-in-water (O/W) emulsion, therefore, should be made to a primary emulsion (W1 /O1 ) before preparing a secondary emulsion (O1 /W2 ) [5–7]. Many surfactants including poly(vinyl alcohol) were extensively employed in aim to stabilize primary and secondary emulsion because each emulsion should be well-stabilized in order to efficiently encapsulate pharmaceuticals drugs [8]. Polymeric hollow microspheres have been extensively prepared for their unique structures including low density and usefulness of inner spaces [9]. These properties enabled them

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to be applied to cosmetics, coatings, inks, pharmaceutics, and other biological fields [10,11]. Recently, many efforts have been made to develop hollow microspheres for drug delivery systems and clinical imaging contrast agents [12,13]. Therefore, many researchers concentrated on effective formulation of biocompatible hollow microspheres. However, most hollow microspheres have been made of non-biodegradable or non-biocompatible polymers, therefore, were not suitable for clinical purposes. Labrafil® M 1944 CS was extensively used as a pharmaceutical emulsifying agent for the purpose of preparing microemulsions [14,15]. Biodegradable ibuprofen-loaded PLGA microspheres for intraarticular administration were prepared to measure the effects of Labrafil [14]. The addition of Labrafil lowered initial burst of ibuprofen, prolonging the drug release rate. They concluded that 10% addition of Labrafil to biodegradable microsphere formulation was the most suitable for the drug release. In addition, biodegradable gel formulation was prepared using Labrafil and vegetable oil for the delivery of contraceptive steroids. They confirmed that drug release characteristics were affected by surfactants including Labrafil [15]. In this study, biodegradable hollow microspheres were fabricated by using double oil emulsion and oil-in-water emulsion. First, olive oil and dichloromethane was made to oil-in-oil emulsion using a non-ionic and lipophilic surfactant, Labrafil and

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H.S. Yoo / Colloids and Surfaces B: Biointerfaces 52 (2006) 47–51

Fig. 1. Schematic presentation of preparing hollow microspheres using O/O/W emulsion.

then oil-in-water emulsion was prepared in poly(vinyl alcohol) solution. The formation of emulsion was monitored by light microscopy and the morphology of hollow microspheres was examined by field emission scanning electron microscopy. 2. Materials and methods 2.1. Materials Poly(l-lactide) [PLA] (MW = 2 kDa) was purchased from Aldrich. Labrafil® M 1944 CS, a mixture of mono-, di- and triglycerides and mono- and di-fatty esters of polyethylene glycol 300 (PEG), oleic acid was supplied by Gattefoss´e (France). Polyvinyl alcohol (PVA) (MW = 49 kDa) and Sudan III were supplied by Sigma–Aldrich. Dichloromethane was purchased from Merck. 2.2. Methods 2.2.1. Preparation of hollow microspheres Biodegradable hollow microsphere was fabricated by a solvent evaporation method using O/O/W emulsion (Fig. 1). PLA (10–50 mg) was completely dissolved in dichloromethane (5 ml) and Labrafil® M 1944 CS was added to the organic phase (10%). Olive oil (0.2–1 ml) (O1 ) was emulsified into the dichloromethane phase (O2 ) and the mixture was sonicated with a probe-type sonicator (S-250A, Branson) for 30 s. The O1 /O2 emulsion was re-emulsified into distilled deionized water (DDW) containing 0.1% PVA with vigorous homogenization for 3 min. After completely evaporating dichloromethane with a gentle stirring, microspheres were collected by centrifugation at 1000 rpm for 5 min and then washed with DDW twice. The microsphere was directly examined under a light microscope for visualizing the inner oil phase. For this visualization, Sudan III was previously mixed with olive oil (O1 phase). The microsphere was freeze-dried for the following step. 2.2.2. Removing inner oil phase of microspheres The freeze-dried microsphere was sticky because of olive oil and Labrafil sticking to outer and inner spaces of microsphere. Therefore, those oil phases were completely extracted

by n-hexane. One gram of freeze-dried microsphere was mixed with 10 ml of n-hexane and vigorously vortexed for 1 min. After centrifugating at 3000 rpm for 1 min, the supernatant was completely removed. This step was repeated three times until no olive oil was visibly detected. The hollow microsphere was subsequently freeze-dried for further use. 2.2.3. Field emission scanning electron microscopy (FE-SEM) The morphology of freeze-dried microsphere was examined by a field-emission scanning electron microscope (XL30SFEG, Philips). Completely dried hollow microspheres were coated by gold-sputtering and subjected to FE-SEM. The hollow microspheres were cut by a surgical blade for examining microsphere cross-sections. 2.2.4. Image analysis In order to measure average diameter of hollow microspheres, FE-SEM images were image-analyzed by a computer software, SigmaScan® (Systat, CA). Ten microspheres per an image were counted and each value was statistically analyzed by SigmaPlot® (Systat) for statistical significance. A Student’s t-test was performed and p < 0.05 was considered statistically significant. 3. Results and discussion Table 1 summarizes hollow microspheres prepared in this study and volume of each phase and concentration of

Table 1 Preparation of biodegradable polymeric hollow microspheres Microspheres

O1 volume (ml)

Concentration of PLA in O2 (mg/ml)

O2 volume (ml)

W volume (ml)

A B C D

0.2 0.2 1 1

10 50 10 50

5 5 5 5

100 100 100 100

O1 , O2 and W are olive oil, dichloromethane and water, respectively. Details are described in Section 2.2.

H.S. Yoo / Colloids and Surfaces B: Biointerfaces 52 (2006) 47–51

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Fig. 2. Light microscopy of O/O/W emulsion before hardening of microspheres. Sudan III dissolved in O1 phase (olive oil) is shown in red. A–D are hollow microspheres prepared according to Table 1. Magnification was 60× (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article).

biodegradable polymers in O2 phase. O1 volume was 0.2 ml or 1 ml, which was 4% and 20% of O2 volume, respectively. PLA was dissolved in O2 phase at 10 mg/ml or 50 mg/ml, which was 1% and 5% of O2 volume (w/v), respectively. O2 phase and W phase were fixed at 5 m and 100 ml, respectively. In order to stabilize O1 /O2 emulsion, Labrafil® M 1944 CS was dissolved in O2 phase at 10% (w/v). Because a hydrophile/lipophile balance (HLB) of Labrafil® M 1944 CS was 4, it was expected that hydrophobic tail of Labrafil® M 1944 CS interacted with olive oil better than O2 phase or W phase, stabilizing O1 /O2 emulsion when O1 /O2 phase was emulsified into W phase. In fact, Labrafil has been employed as a co-surfactant in pharmaceutical systems such as microemulsions, contributing to stabilizing microemulsions in water. Core oil phase was visualized by light microscopy with staining inner oil phase with a hydrophobic dye, Sudan III. As shown in Fig. 2, emulsion was characterized by a red oil phase, confirming O1 phase was successfully stabilized by a surfactant, Labrafil. Therefore, a lipophilic and non-ionic surfactant including Labrafil was confirmed to be employed as a core oilstabilizing agent in oil in oil emulsion. Several other surfactants having this HLB can be possibly employed, this substance, however, can be widely employed as an imaging contrast agent or a drug delivery vehicle because of its biocompatibility and biodegradability. In addition, the size of emulsion was shown to be 5–20 ␮m in diameter when dichloromethane was not completely evaporated. There was no difference in size among four samples prepared when light microscopic examination was performed. In this state, the size of microsphere cannot be exactly measured because the dichloromethane in O2 phase was not evaporated enough to solidify hollow microspheres.

Field emission scanning electron microscopy confirmed the morphologies of hollow microspheres as shown in Fig. 3. Some cross-sections of hollow microspheres clearly showed that hollow microspheres were successfully prepared by this O/O/W emulsion technique. PLA shells were very thin compared to the entire size of microspheres, therefore, it could be expected that this hollow microsphere showed very low density compared to conventional biodegradable microspheres. In fact, these hollow microspheres were floated over water when the dried hollow microspheres were added to water (data not shown). It is interesting that distorted morphologies are confirmed throughout the all microspheres (A–D) because the distorted morphology of microsphere was not usually observed when a conventional O/W emulsion technique was employed. This can be attributed that olive oil and Labrafil were not completely removed from the hollow microspheres although the microsphere was washed with n-hexane several times. This sticky oil still adhered to the surface of hollow microspheres, contributing roughness of hollow microspheres. In addition, low molecular weight of PLA also contributed to distortion of hollow microspheres. Because low molecular weight PLA showed a low viscosity in O2 phase, many smaller and distorted microspheres could be formed without forming proper morphology of round-shaped microspheres. Therefore, more refined methods should be devised to prepare mono-dispersed microspheres and to completely remove unwanted oil phases. The diameter of hollow microsphere was image-analyzed by a image-analyzing software as shown in Fig. 4. The size of hollow microsphere ranged from 10 to 30 ␮m in diameter. It is of interest that A and C were smaller than B and D with a statistical significance (p < 0.05). This can be attributed to

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Fig. 3. Field emission scanning electron microscopy (FE-SEM) of hollow microspheres. (A–D) are hollow microspheres prepared according to Table 1.

PLA concentration in O2 phase. As the concentration of PLA increased, the viscosity of O2 phase increased, inhibiting emulsifying process when O2 phase was emulsified into W phase with homogenization. Therefore, increased density of PLA in O2 phase increased the size of hollow microspheres (B and D) compared to A and D. In addition, it should be mentioned that O1 volume did not alter both size of microsphere or inner diameter of hollow microspheres. This can be explained that O1 phase was clearly separated from W phase by stabilized emulsion in O2 phase. Only O2 emulsion could contact with W phase because

Labrafil effectively stabilized O1 /O2 emulsion. Therefore, only a viscosity of O2 phase affected the size of hollow microspheres. 4. Conclusion Biodegradable hollow microsphere was successfully prepared because Labrafil effectively stabilized O1 /O2 emulsion when PLA was solidified in water phase. SEM and light microscopy characterized he morphology of the hollow microspheres. The concentration of PLA in dichloromethane affected particle size of hollow microspheres. Acknowledgement This research was supported by the Ministry of Commerce, Industry and Energy in Republic of Korea. (0001487-2005-22). References

Fig. 4. Average diameters of hollow microspheres. The average diameter was calculated from image-analyzing FE-SEM images by an image-analysis software. Ten microspheres per an image was counted. ‘*’ and ‘**’ in the figure are statistically different (p < 0.05).

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