Factors Affecting Particle Composition

Numerous variables are known to affect the particle formation and AI encapsulation and release when using a single oil-in-water emulsion technique, both within emulsification and particle hardening. During emulsification, process parameters, such as emulsification time and intensity are well known to affect the resultant microparticle size.75 The type of material used for particle synthesis is a key factor in predicting the AI release rate. A diverse range of materials have been applied as the

Figure 1.6: Schematic representation of microparticle synthesis via single oil-in-water solvent evaporation

1) Emulsification 2) Particle Hardening

Aqueous Phase

Organic Phase

15 particle matrix. However, as a consequence of the good biocompatibility and biodegradability observed with biodegradable polymers, polyesters, such as PLA, PCL and poly(lactic-co-glycolic acid) (PLGA) continue to be the pivotal materials selected for the design and synthesis of innovative microparticle delivery vehicles.76- 78 _{Several reports have distinguished a clear influence of the polymer molecular weight} on the AI release rate, for instance, Makino et al., investigated the release of the

steroid, estradiol, from PLGA particles at three different PLGA molecular weights.79 Characterisation of the drug release by fluorescence spectroscopy revealed that as the molecular weight increased, the release rate decreased as a consequence of the decreased surface area: volume ratio (Figure 1.7).79 Furthermore, the release of estradiol was considered to be a consequence of diffusion between the device interior and the bulk solution, polymer degradation and change in water content within the particle. Further investigations into emulsification parameters have shown that the polymer concentration, microparticle size and the encapsulated AI can all have a significant effect on the particle release rate.80-82 Therefore, overall, it can be surmised that the rate of AI release from particles synthesised via a single oil-in-water technique is foremostly governed by the properties of the polymer and the encased AI.

16

Figure 1.7: Graph displaying the difference in release rate obtained from PLGA microparticles with varying PLGA molecular weight79

To achieve the optimum conditions for successful particle hardening, the organic droplets are required to remain dispersed and stable to coalescence, within the continuous aqueous phase. Moreover, the particles must remain stable long enough for evaporation of the organic solvent to occur, thus permitting the formation of stable, solidified microparticles. Therefore, to prevent droplet coalescence, the aqueous phase typically contains a surfactant that can stabilise the dispersed phase droplets.75 Indeed, the surfactant aligns at the droplet surface, thus acting to lower the free energy at the water-oil droplet interface and subsequently increasing the particle stability.83 Multiple surfactants have been applied, however, as a consequence of its good biocompatibility, the non-ionic poly(vinyl alcohol) (PVA) has been the most widely investigated.84, 85 Furthermore, investigations by Sansdrap et al., have reported that an increase in surfactant concentration can decrease the particle size.86 _{The decreased} particle size can result in an increased rate of AI release as a consequence of the

17 decreased total volume of the particle. Hence, the applied stabiliser and stabiliser concentration can significantly influence the AI release profile.

The organic solvent has an integral role in both emulsification and particle hardening.87 In general, the organic solvent should be able to easily solubilise the polymer and drug, be immiscible with the continuous phase and have a relatively low boiling point. Several solvents have been applied for the synthesis of microparticles via solvent evaporation emulsion technique, however, as a consequence of its high

volatility, low boiling point and high immiscibility in water, most reports have focussed on dichloromethane.84 The rate of solvent removal is a key factor in controlling the particle morphology and AI encapsulation and release rate. Many factors are known to affect the time required for solvent extraction, for example, the ratio of the continuous phase to the organic phase, temperature, organic solvent volatility, etc.72, 88 Indeed, Jeyanthi et al. investigated the effect of increasing the

organic solvent extraction rate by transferring the prepared emulsion into a larger continuous phase.89 However, they discovered the decreased extraction time (less than 30 min) resulted in increased porosity within the particle. Additional reports have noted the increased extraction of the solvent resulted in increased loss of encapsulated AI during particle synthesis and consequently a decreased encapsulation efficiency.90 In a different approach to enhance the solvent extraction rate, several studies have investigated the effect of the addition of a co-solvent (e.g., methanol) into the aqueous phase.91 However, even though an initial increase in solvent removal rate was observed, the overall time required for particle solidification did not significantly change.

Solvents used in microencapsulation may be retained in the microsphere as a residual volatile organic impurity.72 Bitz et al. determined the total amount of residual

18 dichloromethane in a microsphere via multiple headspace gas chromatography (GC).92 A rotary evaporator was used to remove the dichloromethane from the microsphere before storing the particles under vacuum for three days. The researchers found that the level of residual dichloromethane was below the recommended 600 ppm (as outlined by the European Medicines Agency).93 However, dichloromethane is known to be non-biocompatible, therefore, several reports have focussed on finding and utilising ‘greener’ solvents.94-96 As a consequence of its decreased toxicity, in comparison to dichloromethane, and its good volatility, ethyl acetate appeared to be a promising alternative. However, the partial miscibility of ethyl acetate with water (a factor of 4.5 times higher than dichloromethane), resulted in the formation of fibre- like agglomerates as a consequence of the fast precipitation of the polymer which is associated with rapid extraction of the dispersed phase.95 Several modifications were considered, including pre-saturation of the continuous phase with ethyl acetate and decreasing the polymer concentration. However, the resultant particles appeared to be partly collapsed and displayed low E of the respective encapsulated AIs.96

Typically, hydrophilic AIs diffuse out of the particle matrix as the organic solvent evaporates, this can result in a very low E and a large initial burst release. With the aim to increase the E of a hydrophilic AI, several investigations into the formation of a single oil-in-oil emulsion have been reported.97, 98 To achieve a single oil-in-oil emulsion, the AI and polymer are dissolved in a water miscible oil phase (such as acetonitrile), before being suspended and emulsified with a second immiscible oil continuous phase. The water miscible oil-phase can then be either evaporated or extracted into water, resulting in particle hardening. Investigations into the development of water-in-oil emulsions and double oil-in-water emulsions have also reported increased E of encapsulated hydrophilic AI’s.99, 100

19 1.2.1.3 Double Oil-in-Water Solvent Evaporation

The double oil-in-water emulsion technique has attracted particular attention as a consequence of its simple methodology and ability to successfully encapsulate hydrophilic AI’s with increased E compared to the single oil-in-water emulsion. To create a double emulsion, initially a water-in-oil emulsion is prepared (Figure 1.8); the drug is dissolved within an aqueous phase before emulsifying with an excess of organic solvent. The emulsion is then added to an excess of a second aqueous phase, thus resulting in the formation of a water-in-oil-in-water emulsion. The second aqueous phase typically contains an oil soluble surfactant, which is usually a fatty acid ester e.g., polyoxyethylene or sorbitan to stabilise the particles.

Figure 1.8: Schematic representation of microparticle synthesis via double oil-in-water solvent evaporation technique

Similar to a single oil-in-water technique, a wide array of parameters are known to influence the particle formation and subsequent AI release when using a double emulsion technique.100 _{Specifically, Siepmann et al., investigated the effect of the size}

20 of biodegradable microparticles on the release rate of the encapsulated drug.81 To achieve this, Siepmann et al. prepared microparticles of PLGA loaded with the drug 5-Fluorouracil (5-FU) via the double emulsion technique. Analysis of the degradation behaviour of the polymer and morphological changes of the microparticles upon exposure to the release medium via DSC, SEM and SEC revealed that the release rate of the drug increased as the microparticle size increased.81

The double emulsion technique has proven to be a great alternative for the encapsulation of drugs that are insoluble in organic solvents. However, similarly to a single oil-in-water emulsion system, the particle size and morphology obtained using a double emulsion is highly dependent on the emulsification parameters.101, 102 Furthermore, as a consequence of the uncontrolled particle synthesis attained with emulsification of an organic and aqueous phase, both the single and double oil-in- water emulsion procedures display broad particle size distributions, with standard deviations of the distribution equal to 25%-50% of the average size.67 Even though these characteristic broad particle size distributions are usually reproducible, the lack of control observed with a conventional emulsion technique prevents the controlled synthesis of advanced, hierarchical particle morphologies (e.g., Janus particles), which have been shown to be highly desirable for enhanced drug delivery.103