The encapsulation of an amphiphile into polystyrene microspheres of narrow size distribution
© Pellach et al 2010
Received: 31 August 2011
Accepted: 6 December 2011
Published: 6 December 2011
Encapsulation of compounds into nano- or microsized organic particles of narrow size distribution is of increasing importance in fields of advanced imaging and diagnostic techniques and drug delivery systems. The main technology currently used for encapsulation of molecules within uniform template particles while retaining their size distribution is based on particle swelling methodology, involving penetration of emulsion droplets into the particles. The swelling method, however, is efficient for encapsulation only of hydrophobic compounds within hydrophobic template particles. In order to be encapsulated, the molecules must favor the hydrophobic phase of an organic/aqueous biphasic system, which is not easily achieved for molecules of amphiphilic character.
The following work overcomes this difficulty by presenting a new method for encapsulation of amphiphilic molecules within uniform hydrophobic particles. We use hydrogen bonding of acid and base, combined with a pseudo salting out effect, for the entrapment of the amphiphile in the organic phase of a biphasic system. Following the entrapment in the organic phase, we demonstrated, using fluorescein and (antibiotic) tetracycline as model molecules, that the swelling method usually used only for hydrophobes can be expanded and applied to amphiphilic molecules.
Encapsulation of hydrophobic molecules into hydrophobic particles, on both the nano- and microscale has been applied in drug-delivery systems of lipophilic drugs and in imaging or diagnostic systems [1–6]. Encapsulation allows for protection of the encapsulated material from harsh physical or chemical environments, as well as retention of micro- or nanoparticle surface properties, which may be consequently modified as desired.
Several methods of nano- and microencapsulation have been described in the literature, and the choice of method used depends on hydrophilicity or hydrophobicity of the compound . For hydrophobic compounds, an oil-in-water method is often used, in which the polymer is dissolved together with the compound to be encapsulated in an organic phase. The organic solution is then emulsified in an aqueous continuous phase, and solvent evaporation results in solid particles dispersed in water . Another possibility for entrapment of hydrophobic compounds is by emulsion polymerisation of a hydrophobic vinylic monomer, e.g., styrene, in the presence of the hydrophobic compound. However, these methods present difficulties in obtaining particles of narrow size distribution. The oil-in-water approach as well as a variety of other methods for microencapsulation into biodegradable polylactic acid microparticles has been discussed by Wischke and Schwenderman . Modern methods for obtaining monodispersed polymeric microparticles, for example using microporous membrane emulsification , have been discussed by V-T Tran et al. .
The swelling method of encapsulation may be applied using hydrophobic polymeric microspheres, and is generally limited to encapsulation of hydrophobic substances, e.g. hydrophobic UV absorbing agents  or imaging agents [4, 10] into polystyrene particles. Amphiphilic substances exhibit solubility problems in organic solvents, and the surfactant in the aqueous phase, used for forming an emulsion, acts as a solubilising agent, increasing the solubility in water and thus further preventing possibility of encapsulation into a hydrophobic matrix. Properties of fluorescein and alkyl derivatives in surfactant micelles have been investigated by Song et al. .
The following work demostrates that fluorescein, an amphiphilic molecule with limited solubility in both water and most common organic solvents, as well as an amphiphilic antibiotic, tetracycline, may be solvated and trapped in the organic phase of a biphasic system, and thus encapsulated into hydrophobic polymeric microspheres. We show that their encapsulation may be performed using methods typically used for hydrophobic molecules and that absolute lipophilicity is not necessarily a prerequisite for encapsulation into a hydrophobic carrier such as polystyrene.
Our findings were that the solubility of fluorescein in dichloromethane (DCM) is increased with addition of triethylamine (TEA). This is probably due to interaction between the nitrogen atom of TEA and the fluorescein hydroxyl groups. In aqueous solutions, fluorescein can exist in a number of pH-dependent forms: cationic, neutral, monoanionic and dianionic (as in Figure 2) [16, 17]. Presence of TEA thus also increases its solubility in water, with the formation of one or more of the anionic forms of fluorescein, and solute-solvent interactions and H-bonds between the water molecules and hydrophilic groups of the solute molecule [16, 18]. Solubility in both DCM and water is also increased, to a lesser extent, in acidic conditions, with addition of an organic acid such as acetic acid (AcOH). H-bonds may form to sterically protect the hydroxyl groups in an organic solvent, and the formation of the cationic form of fluorescein in the aqueous phase can also occur (Figure 2).
Overall, our results show the entrapment of amphiphilic molecules into a hydrophobic environment, achieved by solvation, and by controlling the molecular surroundings. Until now, encapsulation using the method of swelling, which allows for retention of uniform particle size distribution, has been efficient only for encapsulation of hydrophobic substances into hydrophobic particles. The present work suggests a solution to problems with reagent solubility, and expands the swelling method to also include amphiphilic substances.
In future, we plan to extend this work for additional amphiphiles and hydrophobic polymeric particles, e.g., biodegradable polymers such as polymethylmethacrylate and polylactic acid. Future plans also include coating of the hydrophobic polymeric particles with functional groups, so that they may be conjugated to a targeting agent, and used for purposes of diagnostics or targeted drug delivery.
Synthesis of polystyrene microspheres
Polystyrene microspheres of 1.9 ± 0.3 μm were synthesised in our lab by dispersion polymerisation, as previously described [4, 21]. Briefly, polyvinylpyrrolidone (MW 360 000, 1.5% w/v of total solution), dissolved in a mixture of ethanol (150 mL) and 2-methoxyethanol (62.5 mL), was introduced into a 1 L flask. The solution was deaerated using nitrogen, heated to 73°C, and a deaerated solution of benzoyl peroxide (1.5 g) in styrene (37.5 mL) was then added. The polymerisation continued under nitrogen for 24 h, and was terminated by cooling. The microspheres formed were washed by centrifugation with ethanol followed by water, and then dried by lyophilisation. The diameter of the microspheres is variable, and was controlled by varying parameters such as monomer, initiator or stabiliser concentrations.
Swelling and deswelling method of encapsulation
TEA (70 μL, 0.5 mmol) was added to fluorescein (0.4 mg, 1.2 μmol) or tetracycline (2 mg, 4.5 μmol) in DCM (2 mL), followed by addition of AcOH (30 μL, 0.5 mmol). An (immiscible) aqueous solution of 1% sodium dodecyl sulphate (SDS, 10 mL) was added to the organic solution. The mixture was then sonicated for one minute to give emulsion droplets containing the fluorescein or tetracycline. An aqueous 7% suspension of template polystyrene microspheres (3.5 mL, 245 mg microspheres) was then added to the emulsion, and the mixture was allowed to stir. The swelling process is complete when all the droplets have diffused into the microspheresas verified by light microscopy. The disappearance of the emulsion droplets containing the amphiphile from the aqueous phase is also indication for the complete entrapment of the fluorescein molecules within the polystyrene template microspheres. The swelling solvent was then slowly evaporated, at room temperature. The microspheres were then washed with water by repeated centrifugation cycles.
Particle Size and size distribution
Particle size and size distribution was determined by DLS with PCCS (Nanophox particle analyser, Sympatec GmbH, Germany).
Fluorescent micrographs were obtained with an Olympus microscope, model BX51, or Zeiss microscope, model LSM510. Confocal microscopy was employed for verification of encapsulation of fluorescent material, and was performed using an Olympus FV1000 confocal microscope.
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