Preparation of Polycaprolactone Nanoparticles via Supercritical Carbon-dioxide Extraction Of Emulsions.

Preparation of Polycaprolactone Nanoparticles via Supercritical Carbon-dioxide Extraction Of Emulsions. Adejumoke Lara Ajiboye* a, Vivek Trivedi a, *, and John Mitchell a a University of Greenwich, Central Avenue, Chatham Maritime. Kent. ME4 4TB * A.O.Ajiboye@greenwich.ac.uk, V.Trivedi@greenwich.ac.uk ABSTRACT Polycaprolactone (PCL) nanoparticles were produced through supercritical fluid extraction of emulsions () of an oil-in-water emulsion via supercritical fluid (SCF) processing. The efficiency of the supercritical carbon dioxide (scco 2 ) extraction was investigated and compared to that of solvent extraction at atmospheric pressure. The effects of process parameters including polymer concentration (.6 1 % w/w in acetone) and polymer: surfactant concentration (.7 and.14 % w/w) on the particle size and surface morphology were also investigated. Spherical PCL nanoparticles with mean particle sizes between 19 35 nm were obtained depending on the polymer concentration. The polymer concentration was the most important factor where increase in the particle size was directly related to total polymer content in the formulation. Nanoparticles produced were analysed using dynamic light scattering and scanning electron microscopy. The results indicated that can be applied for the preparation of PCL nanoparticles without agglomeration and in a comparatively short duration of only 1 hour. Keywords: Solvent extraction; Polycaprolactone; Nanoparticles; Supercritical carbon dioxide; Supercritical fluid extraction of emulsions 1. INTRODUCTION The use of drug delivery systems, particularly the micro- and nano- scale intelligent systems for therapeutic molecules has rapidly increased over the years because they can successfully maximise the efficacy of the drug molecules [1]. Nanoparticles are particles with a diameter range from 1 to 1 nm, even though the dynamic range can cover the whole nanometre scale [2]. As a drug delivery system, nanoparticles can entrap drugs or biomolecules into their internal structures, and/or adsorb these drugs or biomolecules onto their external surfaces [3]. Due to their particle size, nanoparticles can move freely through the body via the smallest capillary vessels, cell and tissue gaps in order to reach their target organs [4]. These properties of nanoparticles help to modify the bio-distribution and pharmacokinetic properties of the adsorbed/ entrapped drug molecules leading to an improvement in the efficacy of the drug, decrease in unwanted side-effects, and increase in patient compliance [5]. Nanoparticles can be made from inorganic or polymeric materials. Polymeric

nanoparticles are more common and appropriate as they can be chemically modified to be biodegradable and biocompatible [6]. Polycaprolactone (PCL) is a semi- crystalline polyester that is hydrophobic, biodegradable and biocompatible. When compared to other polymers the biodegradation of PCL is slow, hence it can be highly suitable for the design of controlled-release delivery systems [7-9]. The glass transition temperature (T g ) of -6 C and low melting point (59-64 C) of PCL allows for its easy formability at reasonably low temperatures [8]. Furthermore, PCL has an excellent blend compatibility with other polymers which facilitates for the tailoring of desired properties like degradation kinetics, hydrophilicity, and muco-adhesion [1, 11]. Various techniques can be employed to prepare nanoparticles, most of which will involve a type of emulsion evaporation. Emulsification of polymer mixtures is known to be an efficient method to produce nanoparticles but it can be associated with limitations concerning process efficiency and control of the particle size and distribution when particles are being recovered from the emulsion through conventional processes [12]. Supercritical fluid extraction of emulsion () is a novel particle formation technique where it employs a SCF such as scco 2 to rapidly extract the solvent or oil phase of an emulsion. The removal of the solvent leads to the precipitation of the solute resulting into an aqueous suspension containing nanoparticles. Particles can thereafter be recovered from the aqueous suspension by centrifugation and/or by evaporation at room temperature [13]. Particles generated via have an ordered size and morphology because emulsification process provides a template and the fast kinetics of the sc CO 2 extraction prevents particle agglomeration [14]. The objective of this work was to study the effect of various parameters influencing the preparation of PCL nanoparticles during. The effect of parameters including polymer and surfactant concentration on the particle s hydrodynamic diameter, polydispersity index (PDI) and zeta potential was investigated. The time and the optimum CO 2 flow rate were also determined for the complete extraction of oil phase/solvent at both high pressures in scco 2 and atmospheric conditions. 2. EXPERIMENTAL SECTION 2.1. MATERIALS Polycaprolactone (PCL) (Mw= 45 Da) was obtained from ShenZhen ESUN, China and tween 8 and acetone were obtained from Sigma Aldrich, UK. Liquid CO 2 at 99.9% was delivered by BOC ltd, UK. All chemicals were of analytical grade and used without any further purification. Distilled water was used throughout the study. 2.2. METHODS 2.2.1. Oil-in-water emulsion preparation Method used for the preparation of PCL nanoparticles was adapted from the work reported by Prieto and Calvo [14]. In summary, appropriate quantity of PCL was first dissolved in acetone (.6-1 % w/w) in a thermostatic water bath at 4 C before the addition of surfactant to obtain required concentrations (.7 and.14 %). The mixture was transferred to a centrifuge tube thereafter and the contents in the tube were mixed together via vortexing for 1 minute. After vortexing, 35.8 g of warm distilled water (4 C) was added to the tube and the tube was placed back on the vortex mixer for 5 minutes in order to form an emulsion. The temperature of the emulsion was maintained at 4 C until all the acetone was evaporated. 2.2.2. Solvent extraction via

25 g emulsion was taken in a 5 ml beaker and introduced in the high pressure vessel preheated to 4 C. The vessel was then closed and liquid CO 2 was pumped until a pressure of 1 bar was achieved. The system was left to equilibrate under stirring for 15 minutes and then continuously flushed with fresh CO 2 for the duration of the experiment. The experiments were carried out at the scco 2 flow rate of 1, 4 or 1 g/min. The system was then depressurized at a rate of 6 bar/ minute, and the beaker was removed from the high pressure vessel. The amount of acetone extracted was calculated using the weight difference before and after the procedure. 2.2.3. Solvent extraction at atmospheric pressure 25 g emulsion in a 5 ml beaker was left to stir at atmospheric pressure at a rate of 3 rpm in a thermostatic water bath set to 4 C. The amount of acetone lost was measured at time, 1, 2, 3, 4 and 24 hours using the weight difference before and after solvent extraction. 2.3. Physicochemical characterization of nanoparticle 2.3.1. Particle size and zeta potential measurement The average particle sizes were measured by dynamic light scattering using zetasizer from Malvern Instrument at room temperature (25 C). The mean particle size was the average of three independent measurements. The zeta potential of the aqueous nanoparticle dispersions was determined by using Nano-ZS Malvern at 25 C. 2.3.2. Particle morphology Scanning Electron Microscopy (SEM) was performed in order to determine the shape and surface morphology of the nanoparticles. SEM micrographs were obtained from freeze-dried samples using Hitachi SU83 scanning electron microscope. 3. RESULTS AND DISCUSSION 3.1. Solvent extraction The temperature (4 C) and pressure (1 bar) selected for was based on the low melting point (59-64 C) of the PCL polymer and the complete miscibility of acetone at these conditions [15]. Figure 1 shows that at a constant exposure time of 1 hour, the % weight of acetone extracted from the emulsion increases with an increasing flow rate of scco 2. Extraction of the organic phase can occur either by diffusion mechanism in the aqueous phase or by direct extraction upon contact between the organic phase and scco 2 in the emulsion droplet. Thus, acetone extraction at higher sc CO 2 flow rates improves as it allows for a larger quantity of CO 2 to be in contact with both the emulsion and organic phase [16].

Figure 1 : 12 Acetone extraction via at different CO₂ flow rate for 1 hour. % Weight acetone loss 1 8 6 4 2 1 4 1 CO₂ flow rate (g/min) When compared to acetone extraction by scco 2, complete acetone extraction at atmospheric pressure (Figure 2) occurs after 24 hours. The extraction at atmospheric pressure also leads to the agglomeration of particles as seen in Figure 3 consequently decreasing the total product yield. Like a gas the scco 2 displays lesser viscosity and greater diffusivity relative to the liquid thus facilitating a rapid solvent extraction that is able to avoid particle agglomeration [17]. Figure 2: %Weight acetone loss 12 1 8 6 4 2 Acetone extraction at atmospheric pressure 1 2 3 4 24 Time (hours) Figure 3: An image of the typical agglomerate seen after extraction of acetone at atmospheric pressure for 24 hours

3.2 Effect of polymer concentration The effect of increasing the polymer concentration in the dispersed phase at a constant surfactant concentration and oil-water ratio, on the emulsion droplet size, particle size and zeta-potential, and particle size distribution was investigated. As seen in Figure 5, an increase in particle size and size distribution occurs as the polymer concentration increases. These results are in line with findings of Santos et al. [18] and Kwon et al. [19]. An increase in the polymer content increases both the hydrophobic content and viscosity of the oil phase, which can lead to a negative effect on the nanoparticle formation process [19]. The results also show a marked difference between the size before and after acetone extraction (Table 1), which could be as a result of the presence of empty spaces in the polymer matrix as acetone extracted out of the matrix by sc CO 2. Free spaces within the polymer matrix may cause the particles to shrink and reduce in size [2]. Table 1: Size, size distribution and z-potential before and after extraction of a 25 g emulsion made up of 14.15 g acetone, 35.8 g of distilled water,.7 % w/w surfactant concentration, and varying polymer concentrations PCL concentratio n (% w/w in acetone) Mean emulsion droplet size (nm) ± SD Mean particle size (nm) ± SD after PDI ± SD before.6 266 ± 2 19 ± 39.133 ±.7 1 271 ± 5 182 ± 8.194 ±.6 2 341 ± 65 24 ± 27.92 ±.7 4 446 ± 19 286 ± 2.168 ± 6 567 ± 79 354 ± 52.23 ±.7 8 577 ± 63 338 ± 15.29 ±.5 1 549 ± 68 356 ± 4.26 ± PDI ± SD after.114 ±.1.92 ±.7.158 ±.8.219 ±.266 ±.265 ±.2.251 ±.2 Z- Potential (mv) ± SD before Z- Potential (mv) ± SD after -11.7 ± 2.1-1.4 ± 1. -16. ± 3.4-11.8 ± 1.3-13.6 ± 1.1-15.4 ± 1.9-16.2 ± 2.1-26.8 ± 5.7-22.2 ± 5.4-17.8 ± 4.8-17.6 ± 2.3-25.8 ± 2.4-17.8 ± 1.4-24. ± 3.5 Figure 4 and 5 show that a minimal change in size occurs between 6 to 1 % PCL concentration, and particles made at these concentrations have a high standard error. This probably suggests that at a constant surfactant concentration, an increase in PCL concentration can have an effect on the particle size up to an extent. Z-potential measurements provide information about the particle surface charge and in a way, characterizes the index for particle stability [21, 22]. The z-potential recorded increases with an increasing polymer concentration and also indicate that the nanoparticles produced have a negative surface charge. The negative surface charge could be as a result of the orientation of the nanoparticle structure where the -COO group of PCL is at the surface.

Figure 4: 7 Emulsion droplet size Mean droplet size (nm) 6 5 4 3 2 1.6 1 2 4 6 8 1 PCL concentration (%w/w in acetone) Figure 5: Mean particle size (nm) 45 4 35 3 25 2 15 1 5 Particle size after extraction.6 1 2 4 6 8 1 PCL concentration (%w/w in acetone) 3.3. Effect of surfactant concentration The surfactant concentration was doubled to.14 % w/w in order to be able to overcome the effects of the varying polymer concentration on the particle size. An increase in surfactant concentration also allowed for a reduction in the w/w PCL- Tween 8 weight ratio. Table 2: Size, size distribution and z-potential before and after extraction of a 25 g emulsion made up of 14.15 g acetone, 35.8 g of distilled water,.14 % w/w surfactant concentration, and varying polymer concentrations PCL concentration (% w/w in acetone) Mean emulsion droplet size (nm) ± SD Mean particle size (nm) ± SD after PDI ± SD before.6 3 ± 38 165 ± 7.89 ±.3 PDI ± SD after.114 ±.5 Z- Potential (mv) ± SD before Z- Potential (mv) ± SD after -16.9 ± 4.2-14.1 ±.6

1 261 ± 34 219 ± 37.137 ±.5 2 361 ± 59 231 ± 12.9 ± 4 42 ± 8 249 ± 29.14 ±.8 6 454 ± 145 32 ± 25.163 ±.6 8 484 ± 39 272 ± 22.189 ± 1 456 ± 29 331 ± 15.187 ±.6.193 ±.143 ±.7.188 ±.8.224 ±.12.263 ±.9.238 ±.5-15.2 ± 1.4-17. ± 3.4-18.5 ± 2. -18.6 ± 3. -27.6 ± 3.1-21.6 ± 2.4-29.1 ± 2.4-25. ± 3.2-36. ± 9. -28.5 ± 3.4-3.6 ± 3.4-29.1 ± 3. Figure 6: Graph comparing the mean emulsion droplet size (nm) of.7 % and.14 % w/w surfactant concentrations at the different PCL concentration (% w/w in acetone) 7 Emulsion droplet size Mean droplet size (nm) 6 5 4 3 2 1.7% conc..14% conc..6 1 2 4 6 8 1 PCL concentration (% w/w in acetone) Figure 7: Graph comparing the mean particle size (nm) of.7 % and.14 % w/w surfactant concentrations at the different PCL concentration (% w/w in acetone) Mean particle size (nm) 45 4 35 3 25 2 15 1 5 Particle size after.63 1 2 4 6 8 1 PCL concentration (%w/w in acteone).7%.14%

At a doubled surfactant concentration, the particles have a smaller size. This can be anticipated from the role of surfactant as an emulsion stabilizer to form nanoparticles. At a lower surfactant concentration, some nanoparticles tend to aggregate and form larger particles due to insufficient quantity of surfactant required to stabilize all the nanoparticles [23]. A similar change in particle size between PCL concentrations 6-1 % is seen at both surfactant concentrations. This could suggest that at increasing polymer concentrations there is a limited effect of increasing surfactant concentration on particle size. 3.3 Particle morphology SEM micrographs were obtained using freeze-dried PCL nanoparticle samples in order to investigate the shape and surface morphology of produced particles. Figure 8: SEM micrographs of particles processed by : (a) 1% w/v PCL conc. (b) 4% w/v PCL conc. (c) 6% w/v PCL conc. (d) 8% w/v PCL conc. (e) 1% w/v PCL conc. (a) (b) (c) (d) (e) From the SEM micrographs, it is clearly seen that the particles made by exhibited a spherical structure. SEM micrographs also confirm DLS results that there is an increase in particle size as the polymer concentration increases.

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