http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–7 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2014.884117
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RESEARCH ARTICLE
Impact of surfactant selection on the formulation and characterization of microparticles for pulmonary drug delivery Elizabeth Cocks1, Oya Alpar2, Satyanarayana Somavarapu2, and David Greenleaf3 1
School of Pharmacy, DeMontfort University, Leicester, UK, 2UCL School of Pharmacy, London, UK, and 33M Healthcare Ltd, Loughborough, UK
Abstract
Keywords
The effect of suspension stabilizers, internal aqueous phase volume and polymer amount were investigated for the production of protein loaded poly(D,L lactide-co-glycolide) (PLGA) microparticles suitable for pulmonary drug delivery. PLGA microparticles were produced adopting water-in-oil-in-water (W/O/W) solvent evaporation technique and were investigated for surface morphology, particle size, encapsulation efficiency (EE%) and in-vitro release profile. Porous surface morphologies with a narrow size distribution were observed when employing 0.5 ml internal aqueous phase; 23.04 mm (±0.98), 15.05 mm (±0.27) and 22.89 mm (±0.41) for PVA, Tween 80 and oleic acid. Porous microparticles exhibited increased size and reduction in EE% with increasing internal aqueous phase, with non-porous microparticles produced when adopting 2.0 ml internal aqueous phase. The selection of stabilizer influences the size of the pores formed thus offers potential for the aerodynamic properties of the microparticles to be manipulated to achieve suitable aerosolization characteristics for pulmonary delivery of proteins.
Inhalation, poly(D,L lactide-co-glycolide), porous microspheres, solvent evaporation, water-in-oil-in-water
Introduction In recent years there has been continued interest in the use of the pulmonary route for the delivery of systemic drugs and in particular proteins and peptides1. However, the structure of the lungs, rapid clearance via mucociliary clearance and macrophage uptake represent major barriers to the successful delivery of drugs to the lungs2,3. Large porous particles are thought to be particularly suited to pulmonary drug delivery due to their low mass density relative to their size, in addition to their potential to target the lower regions of the lung. Increased geometric size results in a decreased tendency to aggregate hence, in conjunction with mass density, this leads to a more efficient aerosolization in a given air field. In addition, large porous particles deposited in the pulmonary region may escape clearance by alveolar macrophages due to their size, therefore allowing prolonged and more efficient drug release4–7. Porous microparticles may be prepared using a variety of carriers but one of the most versatile strategies is the bioengineering of inhalable particles using biocompatible polymers8–12. Synthetic polymers such as poly(D,L lactideco-glycolide) (PLGA) and poly-lactic acid (PLA) have been used extensively for the preparation of microparticles5,13–15. The synthetic nature of PLGA ensures reproducibility and it has Food and Drug Administration (FDA) approval for human use16 in addition it is also is listed as an additive for dry powder inhaler formulations2.
Address for correspondence: Elizabeth Cocks, School of Pharmacy, DeMontfort University, Leicester LE1 9BH, UK. Tel: +44 1162078871. E-mail: [email protected]
History Received 10 August 2013 Revised 7 January 2014 Accepted 8 January 2014 Published online 20 March 2014
In order to prepare porous PLGA microparticles various formulation parameters including the choice of excipients, method of manufacture and the process of manufacture, have been found to influence porosity17,18. The double emulsion water-in– oil-in-water (W/O/W) method is commonly used for the encapsulation of a variety of water soluble drugs. Manufacture variables including method and duration of mixing as well as the dimensions of equipment have all been proven to influence the properties of the resultant microparticles8,9,19–25. In addition, formulation parameters also have an impact on the formation of porous microparticles and their characteristics. Key formulation factors which influence the physicochemical properties of porous microparticles include choice and volume of solvent as well as the concentration of polymer8,10,20,24,26,27. Total volume of the internal aqueous phase and the concentration of surfactants (or suspensions stabilizers) has also been shown to be influential over porous microparticle properties8,28–30. This paper focuses on the impact of the formulation and in particular the choice of suspension stabilizer in the internal aqueous phase on the production of porous microparticles. Suspension stabilizers are employed in the primary emulsion for the short-term stabilization of the suspended droplets, prior to the process of solvent evaporation at which point the polymer droplets harden to prevent coalescence and aggregation. However, the concentration and type of suspension stabilizer have also been shown to affect the key characteristics of microparticles30–32. Typically poly vinyl alcohol (PVA) has been used extensively for the production of microparticles; however, it has yet to obtain approval for pulmonary drug delivery, it is therefore critical that suspension stabilizers which are biocompatible with the inhaled route of administration are adopted.
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This study investigates the potential application of alternative suspensions stabilizers namely oleic acid and Tween 80 for the production of porous microparticles. Oleic acid has been selected as it is currently used in commercial pMDIs as a valve lubricant2,33 and Tween 80 as it is approved for use in the Pulmicort nebulizer product2. In addition to their approval for pulmonary use the aforementioned stabilizers are commonly adopted in water-in-oil (W/O) emulsions. This study was designed to assess the effects of formulation parameters on porous PLGA microparticles characteristics such as surface morphology, particle size, encapsulation efficiency (EE) and in-vitro release characteristics. Emphasis has been placed on key characteristics critical to pulmonary drug delivery namely high encapsulation efficiencies and narrow size distribution. The aforementioned factors are prerequisites to ensure the successful delivery of drugs to the lungs.
Materials and methods Materials PLGA 50:50 (molecular weight, Mw 45–75 000 kDa), BSA, oleic acid, Tween 80 and PVA (Mw 13–23 000 kDa, 98% hydrolyzed) were purchased by Sigma Aldrich, UK. Dichloromethane (DCM) was obtained from Fisher Scientific (Loughborough, UK). Methods Microparticle method of preparation Microparticles were prepared by the standard W/O/W double emulsion, solvent evaporation technique34. Formulations were prepared are described in Table 1. Briefly, an oil phase comprising of 100 mg or 250 mg of PLGA was dissolved in 2 ml of DCM. This was homogenized (Silverson, UK) at 10 000 rpm for 4 min with an internal aqueous phase comprising of 6 mg BSA in appropriate volume (0.5 ml, 1.5 ml, 2.0 ml) of 5%w/w suspension stabilizer to produce a primary emulsion. Oleic acid was added to the oil phase at a concentration of 5%w/w due to lack of solubility in water. The primary emulsion was added dropwise under constant stirring to a bulk external phase (75 ml 1.5%w/w PVA) and was homogenized for 6 min at 10 000 rpm to produce a W/O/W emulsion (Table 1). DCM was removed from the formulation by constant magnetic stirring (100 rpm) for 24 h. The microparticles were washed with double distilled water and recovered by centrifugation (Beckman-Coulter Instruments Inc., High Wycombe, UK) at 16 000 rpm for 40 min following triplicate washes. The resultant microspheres were then freezedried for 48 h adopting the following parameters; additional of microspheres to an initial shelf temperature 15 C, drying at a shelf temperature of 40 C for 48 h prior to an increase in shelf
temperature to 15 C prior to analysis. All formulations were prepared in triplicate. Particle size determination Particle size analysis was determined using a Malvern Mastersizer X (Malvern Instruments, Malvern, UK) fitted with a 100 mm receiver lens, and 15 ml magnetically stirred cell. A representative sample of microspheres was dispersed in 0.01% w/v Tween 20. The data generated are presented as 10th (D0.1), 50th (D0.5, volume mean diameter; VMD) and 90th (D0.9) percentile of the cumulative particle under size frequency distribution. Scanning electron microscopy Surface morphology of microparticles was assessed by scanning electron microscopy (SEM) using a Cambridge Instrument Stereoscan 90B. Samples were prepared by placing a representative sample onto an aluminum specimen stub, and were sputter coated with gold prior to imaging. Determination of protein loading in microparticles Five milligram samples of microparticles were dissolved in 500 ml DCM at 37 C. Once dissolved 500 ml of 5 mmol sodium dodecyl sulfate (SDS) in phosphate buffered saline (PBS) was added and centrifuged at 15 000 rpm for 15 min. The upper aqueous layer was recovered and the process repeated twice. The harvested solution was assayed using a microBCA assayÔ (Pierce, Rockford, IL) for the presence of protein. EE is expressed as the ratio of the actual and theoretical loading. In-vitro BSA release study In-vitro release rates of the various microparticle formulations were analyzed in triplicate by dispersing in release buffer comprising of 5 mmol SDS in PBS (pH 7.4) containing 0.01% w/v sodium azide as a preservative. Five milligrams of microparticles were placed in a glass vial and 1000 ml of release buffer was added. The samples were stored at 37 C under constant agitation (20 rpm) (Gallenkamp INR 201, flask shaker, Gallenkamp, UK). At appropriate intervals, after centrifugation (15 000 rpm) 100 ml of supernatant was taken and 100 ml fresh buffer added. Protein content of the samples was assessed using the microBCA assayÔ.
Results and discussion Particle size of microparticles A low internal phase volume did result in the formation of porous microparticles in some instances. This effect is independent of the
Table 1. VMD and surface morphology of prepared microparticles. 100 mg PLGA Stabilizer
Int. phase vol. (ml)
PVA PVA PVA Tween 80 Tween 80 Tween 80 Oleic acid Oleic acid Oleic acid
0.5 1.5 2.0 0.5 1.5 2.0 0.5 1.5 2.0
VMD (mm)
250 mg PLGA
Morphology
20.36 28.95 5.28 14.30 26.18 8.07
(±0.56)* (±3.42) (±0.34) (±0.48)* (±1.22) (±0.20) – 21.21 (±0.31) –
Morphology: NP: non porous, P: porous, NF: not formed. *Optimum surface morphology and size distribution observed.
P P NP P P NP NF P NF
VMD (mm) 23.04 35.33 7.86 15.05 26.17
(±0.98)* (±2.88) (±0.47) (±0.27)* (±2.35) – 22.89 (±0.41)* 35.77 (±1.86) –
Morphology P P NP P P NF P P NF
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DOI: 10.3109/03639045.2014.884117
internal phase surfactant employed and the polymer concentration. Porous microparticles exhibited a larger physical particle size than non-porous microparticles, with an observed increase in size with corresponding increase in internal aqueous phase. For example, microparticles prepared using 100 mg PLGA with Tween 80 as the suspension stabilizer porous microparticles exhibiting a VMD of 14.30 (±0.48) mm and 26.18 (±1.22) mm were observed with an internal aqueous phase of 0.5 ml and 1.5 ml, respectively. In contrast a VMD of 8.07 (±0.20) mm was observed when a non-porous morphology was observed with an internal aqueous phase volume of 2.0 ml (Table 1). This effect was independent of the suspension stabilizer and may be attributed to the aqueous phase providing a greater resistance to mechanical breakdown during the second emulsification step following the inclusion of suspension stabilizer19,20,35. A wide size distribution was observed following the use of 1.5 ml, 5% w/v PVA and was independent of the PLGA loading. A VMD of 28.95 mm (±3.42), was observed with D(0.1) of 9.14 mm (±2.12) and D(0.9) of 67.79 mm (±1.68) when employing 100 mg PLGA, with VMD 35.33 mm (±2.88), D(0.1) of 10.16 mm (±2.68) and D(0.9) of 59.06 mm (±4.05) following inclusion of 250 mg PLGA. The increase in size with corresponding increase in polymer concentration correlates with the findings of Yang36, Yan37, Mao et al.8, and Gaignaux et al.27. The wide size distribution observed may suggestive of poor primary emulsion stability hence the formation of a broad size range of microparticles38 or poor selection of suspension stabilizer10. Microparticle morphology Porous microparticles were produced with an internal aqueous phase volume of 0.5 ml or 1.5 ml containing PVA or Tween 80 (Table 1). Superior surface morphologies supported by a narrow
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size distribution were seen for several formulation and these are indicated with an asterix (*) in Table 1. A similar effect was observed following the inclusion of Tween 80, although poor quality microparticles were produced adopting 100 mg PLGA with respect to size distribution and morphology. In contrast, the use of oleic acid did not produce microparticles in all instances. Although porous microparticles were produced, there was variation in surface morphology (Figure 1). The surface morphology of microparticles produced following the inclusion of Tween 80 exhibited larger pores compared to those produced following the inclusion of oleic acid. The variation in surface morphology following the inclusion of different suspension stabilizers is supported by the findings of Mohamed and Van der Walle39, and Blanco and Alonso31, when investigating poloxamers. The data suggest the composition of the internal aqueous phase has a greater impact on the surface morphology and formation of porous microparticles than the viscosity of the oil phase and the bulk continuous phase. The formation of porous microparticles is attributed to the hardening of the microsphere as the solvent evaporates40. High oil phase viscosity is a prerequisite to preparing porous microparticles due to the viscosity of the polymer phase providing a fast rate of polymer precipitation32,36. The data also illustrate the detrimental effect of rapid polymer solidification rate with high polymer concentrations10,24,28. An internal aqueous phase to oil phase ratio of 1:1 resulted in the formation of non-porous microparticles or unsuccessful production. With 1:4 or 3:4 ratios, porous microparticles were produced. The differences observed between the successful formation of porous microparticles may be attributed to the stability of the primary emulsion. Suspension stabilizers form a mechanical barrier around the water droplets therefore stabilizing against coalescence38,41. This barrier prevents the migration of internal water droplets to the air–water interface, and thus primary
Figure 1. SEM of porous microparticles produced containing 250 mg PLGA, with a 0.5 ml aqueous phase comprising 5% w/v (a) oleic acid, (b) PVA and (c) Tween 80.
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50
Figure 2. Graph illustrating EE% and burst release (BR) of microparticles prepared using 250 mg PLGA.
45 40 35
BR 59.73% BR 53.05% (± 1.75) (± 7.32) BR 45.41% (± 3.69)
BR 47.86% (± 8.93)
BR 41.87% (± 2.42)
25
BR 50.27% (± 0.48) BR 77.89% (± 6.51)
20 15 10 5 0 0.5
1.5
2
Internal Aqueous Phase Volume (mL)
60
Figure 3. Graph illustrating EE% and burst release (BR) of microparticles prepared using 100 mg PLGA.
Tween 80
BR 76.48% ( ± 6.32)
PVA 50
Oleic Acid
40 EE %
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EE %
30
BR 54.56% ( ± 5.58)
BR 60.54% ( ± 4.81)
30
20
BR 47.53% (± 3.98)
BR 41.90% (± 3.95)
BR 44.53% (± 9.71)
BR 97.76% (± 1.34)
10
0 Porous 0.5
Porous 1.5
Non Porous 2
Internal Aqueous Phase Volume (mL)
emulsion stability is achieved. However, the formation of porous particles is achieved through the presence of water droplets on the surface of the microparticle as it solidifies. Based on the size of the pores achieved following the inclusion of Tween 80 it may be inferred that primary emulsion stability is less than with inclusion of oleic acid and PVA due to larger water droplets formed and captured at the polymer air interface (Figure 1). This is supported by the findings of Mao et al.8, who suggest increased coalescence leads to the formation of larger pores and a less tortuous network. EE of microparticles An increased EE% was observed following the inclusion of 250 mg PLGA in the oil phase, and was independent of the suspension stabilizer employed and may be attributed to the
increased viscosity in the oil phase preventing the BSA from partitioning out8,37,42,43 (Figures 2 and 3). The data suggest there is an optimum volume of internal aqueous phase to achieve higher EE%. However, the change in particle morphology should be considered in conjunction with the EE% (Figure 2). Lower EE%s are expected for porous microparticles as the internal aqueous phase has migrated to the polymer/external phase interface to produce pores increasing the likelihood of BSA migration into the external phase prior to polymer solidification. Microparticles exhibiting the same morphology show a reduction in EE% following an increase in internal aqueous phase (Figure 2). A reduction in EE% from 29.61% (±0.48) to 17.79% (±2.33) was observed with an increase in internal aqueous phase volume from 0.5 ml to 1.5 ml when employing PVA as a
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DOI: 10.3109/03639045.2014.884117
0.5 ml Tween 80, 100 mg PLGA 1.5mL Tween 80, 100 mg PLGA 2.0mL Tween 80, 100 mg PLGA
100 80 60 40 20
100 80 60 40 20 0
0 0
2
24
168
336
504
672
0
2
24
Time in Hours
168
336
504
672
Time in Hours
Figure 4. Cumulative release profile from microparticles prepared using (a) PVA and (b) Tween 80.
suspension stabilizer (Figure 2). The EE% of microparticles produced using Tween 80 exhibited the same trend, however very low EE%s were observed. The low EE% observed by microparticles prepared with Tween 80 combined with large pores sizes support the findings of Schlicher et al.38. The increased EE% observed with PVA suggest improved emulsion stability due to an interaction between PVA and BSA at the interface preventing diffusion to the external phase35. This may have contributed to the higher EE% observed compared to Tween 80. Based on the data generated oleic acid appears to achieve higher EE% than PVA providing the formulation is optimized (Table 1). It is worth noting that BSA is an efficient surfactant31 improving protein and antigen stability when preparing microparticles30,44. This suggests that BSA and PVA compete at the interfaces however, with oleic acid there is no such competition. Based on the data generated oleic acid appears that to achieve higher EE% than PVA suggesting oleic acid is able to prevent the migration of BSA towards the external interface. However, the formulation requires further optimization. In-vitro release from microparticles All porous microsphere formulations assessed for their in-vitro release behavior show a continuous pattern of release as the BSA escapes through the porous network. Based on the surface morphology illustrating a reduced porous morphology with increasing internal aqueous phase volume, there is a corresponding reduction in burst effect observed. A burst effect of 60.54% (±4.82) compared to 44.53% (±9.70) was observed after 2 h when comparing porous versus nonporous microparticles obtained using PVA and 100 mg PLGA (Figure 4a). Data obtained on the highly porous microparticles adopting a primary emulsion comprising of 1.5 ml Tween 80 and 100 mg PLGA suggest the BSA encapsulated was surface bound. In this instance a burst effect of 97.76% (±1.34) after 2 h was observed (Figure 4b). The high burst effect correlates with the low EE observed suggesting that Tween 80 is ineffective at preventing the migration of BSA towards the external aqueous phase. A similar effect was observed for formulations prepared using oleic acid. Although a porous morphology and narrow size distribution was obtained, the burst effect is indicative of surface bound material, a burst effect of 76.47% (±6.32) with a corresponding EE% of 32.61% (±3.56). There is a direct correlation between the surface morphology of the microparticles and the release profiles exhibited when comparing formulations which had a narrow size distribution. The surface morphology increased in porosity in the following
120
% cummulative Release
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0.5 mL PVA, 100 mg PLGA 1.5 mL PVA, 100 mg PLGA 2.0 mL PV, 100 PLGA
(b) 120 % cumulative Release
% cumulative Release
(a) 120
5
100 80 60
0.5 ml oleic acid
40
1.5 mL oleic acid
20
1.5 mL oleic acid (100mg PLGA)
0 0
2
24
168
336
504
672
Time in Hours
Figure 5. Cumulative release profile from microparticles prepared using oleic acid.
order; PVA, oleic acid and Tween 80 (Figures 4 and 5) with a corresponding increase in burst effect 60.54% (±4.82), 76.47% (±6.32) and 97.76% (±1.34), respectively. The increased porosity of the microparticles has resulted in increased surface area exposed to the release media, and thus a higher burst effect45. The data may also suggest that although Tween 80 and oleic acid are suitable suspension stabilizers to achieve porous microsphere morphology they do not appear to prevent the migration of BSA towards the external aqueous phase and thus achieve the desired release characteristics. This is supported by the low EE% observed, particularly following the inclusion of Tween 80.
Conclusions Microparticles exhibiting a porous morphology may be produced by adopting various suspension stabilizers providing a low internal aqueous phase is used. The variation in surface morphology may be attributed to the size of the water droplets in the internal aqueous phase. Although porous microparticles with large pores and thus small aerodynamic diameters may be desirable for pulmonary delivery they may not possess suitable encapsulation or release characteristics. However, the research shows the potential for successfully employing Tween 80 in the production of porous microparticles. The use of Tween 80 is beneficial when developing pulmonary based formulations as it one of a limited number of excipients that have FDA approval for pulmonary use. Therefore, formulation of microparticles employing Tween as a stabilizer may offer a potential for pulmonary drug
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delivery of vaccines. Further optimization of the formulation parameters is required with the alternative suspension stabilizers to ensure suitable EE% and particle sizes can be achieved.
Declaration of interest
Drug Dev Ind Pharm, Early Online: 1–7
20. 21.
This work completed as part of a PhD studentship funded by 3M Healthcare Ltd, Morley Street, Loughborough, Leicestershire. 22.
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RESEARCH ARTICLE
Impact of surfactant selection on the formulation and characterization of microparticles for pulmonary drug delivery Elizabeth Cocks1, Oya Alpar2, Satyanarayana Somavarapu2, and David Greenleaf3 1
School of Pharmacy, DeMontfort University, Leicester, UK, 2UCL School of Pharmacy, London, UK, and 33M Healthcare Ltd, Loughborough, UK
Abstract
Keywords
The effect of suspension stabilizers, internal aqueous phase volume and polymer amount were investigated for the production of protein loaded poly(D,L lactide-co-glycolide) (PLGA) microparticles suitable for pulmonary drug delivery. PLGA microparticles were produced adopting water-in-oil-in-water (W/O/W) solvent evaporation technique and were investigated for surface morphology, particle size, encapsulation efficiency (EE%) and in-vitro release profile. Porous surface morphologies with a narrow size distribution were observed when employing 0.5 ml internal aqueous phase; 23.04 mm (±0.98), 15.05 mm (±0.27) and 22.89 mm (±0.41) for PVA, Tween 80 and oleic acid. Porous microparticles exhibited increased size and reduction in EE% with increasing internal aqueous phase, with non-porous microparticles produced when adopting 2.0 ml internal aqueous phase. The selection of stabilizer influences the size of the pores formed thus offers potential for the aerodynamic properties of the microparticles to be manipulated to achieve suitable aerosolization characteristics for pulmonary delivery of proteins.
Inhalation, poly(D,L lactide-co-glycolide), porous microspheres, solvent evaporation, water-in-oil-in-water
Introduction In recent years there has been continued interest in the use of the pulmonary route for the delivery of systemic drugs and in particular proteins and peptides1. However, the structure of the lungs, rapid clearance via mucociliary clearance and macrophage uptake represent major barriers to the successful delivery of drugs to the lungs2,3. Large porous particles are thought to be particularly suited to pulmonary drug delivery due to their low mass density relative to their size, in addition to their potential to target the lower regions of the lung. Increased geometric size results in a decreased tendency to aggregate hence, in conjunction with mass density, this leads to a more efficient aerosolization in a given air field. In addition, large porous particles deposited in the pulmonary region may escape clearance by alveolar macrophages due to their size, therefore allowing prolonged and more efficient drug release4–7. Porous microparticles may be prepared using a variety of carriers but one of the most versatile strategies is the bioengineering of inhalable particles using biocompatible polymers8–12. Synthetic polymers such as poly(D,L lactideco-glycolide) (PLGA) and poly-lactic acid (PLA) have been used extensively for the preparation of microparticles5,13–15. The synthetic nature of PLGA ensures reproducibility and it has Food and Drug Administration (FDA) approval for human use16 in addition it is also is listed as an additive for dry powder inhaler formulations2.
Address for correspondence: Elizabeth Cocks, School of Pharmacy, DeMontfort University, Leicester LE1 9BH, UK. Tel: +44 1162078871. E-mail: [email protected]
History Received 10 August 2013 Revised 7 January 2014 Accepted 8 January 2014 Published online 20 March 2014
In order to prepare porous PLGA microparticles various formulation parameters including the choice of excipients, method of manufacture and the process of manufacture, have been found to influence porosity17,18. The double emulsion water-in– oil-in-water (W/O/W) method is commonly used for the encapsulation of a variety of water soluble drugs. Manufacture variables including method and duration of mixing as well as the dimensions of equipment have all been proven to influence the properties of the resultant microparticles8,9,19–25. In addition, formulation parameters also have an impact on the formation of porous microparticles and their characteristics. Key formulation factors which influence the physicochemical properties of porous microparticles include choice and volume of solvent as well as the concentration of polymer8,10,20,24,26,27. Total volume of the internal aqueous phase and the concentration of surfactants (or suspensions stabilizers) has also been shown to be influential over porous microparticle properties8,28–30. This paper focuses on the impact of the formulation and in particular the choice of suspension stabilizer in the internal aqueous phase on the production of porous microparticles. Suspension stabilizers are employed in the primary emulsion for the short-term stabilization of the suspended droplets, prior to the process of solvent evaporation at which point the polymer droplets harden to prevent coalescence and aggregation. However, the concentration and type of suspension stabilizer have also been shown to affect the key characteristics of microparticles30–32. Typically poly vinyl alcohol (PVA) has been used extensively for the production of microparticles; however, it has yet to obtain approval for pulmonary drug delivery, it is therefore critical that suspension stabilizers which are biocompatible with the inhaled route of administration are adopted.
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This study investigates the potential application of alternative suspensions stabilizers namely oleic acid and Tween 80 for the production of porous microparticles. Oleic acid has been selected as it is currently used in commercial pMDIs as a valve lubricant2,33 and Tween 80 as it is approved for use in the Pulmicort nebulizer product2. In addition to their approval for pulmonary use the aforementioned stabilizers are commonly adopted in water-in-oil (W/O) emulsions. This study was designed to assess the effects of formulation parameters on porous PLGA microparticles characteristics such as surface morphology, particle size, encapsulation efficiency (EE) and in-vitro release characteristics. Emphasis has been placed on key characteristics critical to pulmonary drug delivery namely high encapsulation efficiencies and narrow size distribution. The aforementioned factors are prerequisites to ensure the successful delivery of drugs to the lungs.
Materials and methods Materials PLGA 50:50 (molecular weight, Mw 45–75 000 kDa), BSA, oleic acid, Tween 80 and PVA (Mw 13–23 000 kDa, 98% hydrolyzed) were purchased by Sigma Aldrich, UK. Dichloromethane (DCM) was obtained from Fisher Scientific (Loughborough, UK). Methods Microparticle method of preparation Microparticles were prepared by the standard W/O/W double emulsion, solvent evaporation technique34. Formulations were prepared are described in Table 1. Briefly, an oil phase comprising of 100 mg or 250 mg of PLGA was dissolved in 2 ml of DCM. This was homogenized (Silverson, UK) at 10 000 rpm for 4 min with an internal aqueous phase comprising of 6 mg BSA in appropriate volume (0.5 ml, 1.5 ml, 2.0 ml) of 5%w/w suspension stabilizer to produce a primary emulsion. Oleic acid was added to the oil phase at a concentration of 5%w/w due to lack of solubility in water. The primary emulsion was added dropwise under constant stirring to a bulk external phase (75 ml 1.5%w/w PVA) and was homogenized for 6 min at 10 000 rpm to produce a W/O/W emulsion (Table 1). DCM was removed from the formulation by constant magnetic stirring (100 rpm) for 24 h. The microparticles were washed with double distilled water and recovered by centrifugation (Beckman-Coulter Instruments Inc., High Wycombe, UK) at 16 000 rpm for 40 min following triplicate washes. The resultant microspheres were then freezedried for 48 h adopting the following parameters; additional of microspheres to an initial shelf temperature 15 C, drying at a shelf temperature of 40 C for 48 h prior to an increase in shelf
temperature to 15 C prior to analysis. All formulations were prepared in triplicate. Particle size determination Particle size analysis was determined using a Malvern Mastersizer X (Malvern Instruments, Malvern, UK) fitted with a 100 mm receiver lens, and 15 ml magnetically stirred cell. A representative sample of microspheres was dispersed in 0.01% w/v Tween 20. The data generated are presented as 10th (D0.1), 50th (D0.5, volume mean diameter; VMD) and 90th (D0.9) percentile of the cumulative particle under size frequency distribution. Scanning electron microscopy Surface morphology of microparticles was assessed by scanning electron microscopy (SEM) using a Cambridge Instrument Stereoscan 90B. Samples were prepared by placing a representative sample onto an aluminum specimen stub, and were sputter coated with gold prior to imaging. Determination of protein loading in microparticles Five milligram samples of microparticles were dissolved in 500 ml DCM at 37 C. Once dissolved 500 ml of 5 mmol sodium dodecyl sulfate (SDS) in phosphate buffered saline (PBS) was added and centrifuged at 15 000 rpm for 15 min. The upper aqueous layer was recovered and the process repeated twice. The harvested solution was assayed using a microBCA assayÔ (Pierce, Rockford, IL) for the presence of protein. EE is expressed as the ratio of the actual and theoretical loading. In-vitro BSA release study In-vitro release rates of the various microparticle formulations were analyzed in triplicate by dispersing in release buffer comprising of 5 mmol SDS in PBS (pH 7.4) containing 0.01% w/v sodium azide as a preservative. Five milligrams of microparticles were placed in a glass vial and 1000 ml of release buffer was added. The samples were stored at 37 C under constant agitation (20 rpm) (Gallenkamp INR 201, flask shaker, Gallenkamp, UK). At appropriate intervals, after centrifugation (15 000 rpm) 100 ml of supernatant was taken and 100 ml fresh buffer added. Protein content of the samples was assessed using the microBCA assayÔ.
Results and discussion Particle size of microparticles A low internal phase volume did result in the formation of porous microparticles in some instances. This effect is independent of the
Table 1. VMD and surface morphology of prepared microparticles. 100 mg PLGA Stabilizer
Int. phase vol. (ml)
PVA PVA PVA Tween 80 Tween 80 Tween 80 Oleic acid Oleic acid Oleic acid
0.5 1.5 2.0 0.5 1.5 2.0 0.5 1.5 2.0
VMD (mm)
250 mg PLGA
Morphology
20.36 28.95 5.28 14.30 26.18 8.07
(±0.56)* (±3.42) (±0.34) (±0.48)* (±1.22) (±0.20) – 21.21 (±0.31) –
Morphology: NP: non porous, P: porous, NF: not formed. *Optimum surface morphology and size distribution observed.
P P NP P P NP NF P NF
VMD (mm) 23.04 35.33 7.86 15.05 26.17
(±0.98)* (±2.88) (±0.47) (±0.27)* (±2.35) – 22.89 (±0.41)* 35.77 (±1.86) –
Morphology P P NP P P NF P P NF
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DOI: 10.3109/03639045.2014.884117
internal phase surfactant employed and the polymer concentration. Porous microparticles exhibited a larger physical particle size than non-porous microparticles, with an observed increase in size with corresponding increase in internal aqueous phase. For example, microparticles prepared using 100 mg PLGA with Tween 80 as the suspension stabilizer porous microparticles exhibiting a VMD of 14.30 (±0.48) mm and 26.18 (±1.22) mm were observed with an internal aqueous phase of 0.5 ml and 1.5 ml, respectively. In contrast a VMD of 8.07 (±0.20) mm was observed when a non-porous morphology was observed with an internal aqueous phase volume of 2.0 ml (Table 1). This effect was independent of the suspension stabilizer and may be attributed to the aqueous phase providing a greater resistance to mechanical breakdown during the second emulsification step following the inclusion of suspension stabilizer19,20,35. A wide size distribution was observed following the use of 1.5 ml, 5% w/v PVA and was independent of the PLGA loading. A VMD of 28.95 mm (±3.42), was observed with D(0.1) of 9.14 mm (±2.12) and D(0.9) of 67.79 mm (±1.68) when employing 100 mg PLGA, with VMD 35.33 mm (±2.88), D(0.1) of 10.16 mm (±2.68) and D(0.9) of 59.06 mm (±4.05) following inclusion of 250 mg PLGA. The increase in size with corresponding increase in polymer concentration correlates with the findings of Yang36, Yan37, Mao et al.8, and Gaignaux et al.27. The wide size distribution observed may suggestive of poor primary emulsion stability hence the formation of a broad size range of microparticles38 or poor selection of suspension stabilizer10. Microparticle morphology Porous microparticles were produced with an internal aqueous phase volume of 0.5 ml or 1.5 ml containing PVA or Tween 80 (Table 1). Superior surface morphologies supported by a narrow
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size distribution were seen for several formulation and these are indicated with an asterix (*) in Table 1. A similar effect was observed following the inclusion of Tween 80, although poor quality microparticles were produced adopting 100 mg PLGA with respect to size distribution and morphology. In contrast, the use of oleic acid did not produce microparticles in all instances. Although porous microparticles were produced, there was variation in surface morphology (Figure 1). The surface morphology of microparticles produced following the inclusion of Tween 80 exhibited larger pores compared to those produced following the inclusion of oleic acid. The variation in surface morphology following the inclusion of different suspension stabilizers is supported by the findings of Mohamed and Van der Walle39, and Blanco and Alonso31, when investigating poloxamers. The data suggest the composition of the internal aqueous phase has a greater impact on the surface morphology and formation of porous microparticles than the viscosity of the oil phase and the bulk continuous phase. The formation of porous microparticles is attributed to the hardening of the microsphere as the solvent evaporates40. High oil phase viscosity is a prerequisite to preparing porous microparticles due to the viscosity of the polymer phase providing a fast rate of polymer precipitation32,36. The data also illustrate the detrimental effect of rapid polymer solidification rate with high polymer concentrations10,24,28. An internal aqueous phase to oil phase ratio of 1:1 resulted in the formation of non-porous microparticles or unsuccessful production. With 1:4 or 3:4 ratios, porous microparticles were produced. The differences observed between the successful formation of porous microparticles may be attributed to the stability of the primary emulsion. Suspension stabilizers form a mechanical barrier around the water droplets therefore stabilizing against coalescence38,41. This barrier prevents the migration of internal water droplets to the air–water interface, and thus primary
Figure 1. SEM of porous microparticles produced containing 250 mg PLGA, with a 0.5 ml aqueous phase comprising 5% w/v (a) oleic acid, (b) PVA and (c) Tween 80.
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50
Figure 2. Graph illustrating EE% and burst release (BR) of microparticles prepared using 250 mg PLGA.
45 40 35
BR 59.73% BR 53.05% (± 1.75) (± 7.32) BR 45.41% (± 3.69)
BR 47.86% (± 8.93)
BR 41.87% (± 2.42)
25
BR 50.27% (± 0.48) BR 77.89% (± 6.51)
20 15 10 5 0 0.5
1.5
2
Internal Aqueous Phase Volume (mL)
60
Figure 3. Graph illustrating EE% and burst release (BR) of microparticles prepared using 100 mg PLGA.
Tween 80
BR 76.48% ( ± 6.32)
PVA 50
Oleic Acid
40 EE %
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EE %
30
BR 54.56% ( ± 5.58)
BR 60.54% ( ± 4.81)
30
20
BR 47.53% (± 3.98)
BR 41.90% (± 3.95)
BR 44.53% (± 9.71)
BR 97.76% (± 1.34)
10
0 Porous 0.5
Porous 1.5
Non Porous 2
Internal Aqueous Phase Volume (mL)
emulsion stability is achieved. However, the formation of porous particles is achieved through the presence of water droplets on the surface of the microparticle as it solidifies. Based on the size of the pores achieved following the inclusion of Tween 80 it may be inferred that primary emulsion stability is less than with inclusion of oleic acid and PVA due to larger water droplets formed and captured at the polymer air interface (Figure 1). This is supported by the findings of Mao et al.8, who suggest increased coalescence leads to the formation of larger pores and a less tortuous network. EE of microparticles An increased EE% was observed following the inclusion of 250 mg PLGA in the oil phase, and was independent of the suspension stabilizer employed and may be attributed to the
increased viscosity in the oil phase preventing the BSA from partitioning out8,37,42,43 (Figures 2 and 3). The data suggest there is an optimum volume of internal aqueous phase to achieve higher EE%. However, the change in particle morphology should be considered in conjunction with the EE% (Figure 2). Lower EE%s are expected for porous microparticles as the internal aqueous phase has migrated to the polymer/external phase interface to produce pores increasing the likelihood of BSA migration into the external phase prior to polymer solidification. Microparticles exhibiting the same morphology show a reduction in EE% following an increase in internal aqueous phase (Figure 2). A reduction in EE% from 29.61% (±0.48) to 17.79% (±2.33) was observed with an increase in internal aqueous phase volume from 0.5 ml to 1.5 ml when employing PVA as a
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DOI: 10.3109/03639045.2014.884117
0.5 ml Tween 80, 100 mg PLGA 1.5mL Tween 80, 100 mg PLGA 2.0mL Tween 80, 100 mg PLGA
100 80 60 40 20
100 80 60 40 20 0
0 0
2
24
168
336
504
672
0
2
24
Time in Hours
168
336
504
672
Time in Hours
Figure 4. Cumulative release profile from microparticles prepared using (a) PVA and (b) Tween 80.
suspension stabilizer (Figure 2). The EE% of microparticles produced using Tween 80 exhibited the same trend, however very low EE%s were observed. The low EE% observed by microparticles prepared with Tween 80 combined with large pores sizes support the findings of Schlicher et al.38. The increased EE% observed with PVA suggest improved emulsion stability due to an interaction between PVA and BSA at the interface preventing diffusion to the external phase35. This may have contributed to the higher EE% observed compared to Tween 80. Based on the data generated oleic acid appears to achieve higher EE% than PVA providing the formulation is optimized (Table 1). It is worth noting that BSA is an efficient surfactant31 improving protein and antigen stability when preparing microparticles30,44. This suggests that BSA and PVA compete at the interfaces however, with oleic acid there is no such competition. Based on the data generated oleic acid appears that to achieve higher EE% than PVA suggesting oleic acid is able to prevent the migration of BSA towards the external interface. However, the formulation requires further optimization. In-vitro release from microparticles All porous microsphere formulations assessed for their in-vitro release behavior show a continuous pattern of release as the BSA escapes through the porous network. Based on the surface morphology illustrating a reduced porous morphology with increasing internal aqueous phase volume, there is a corresponding reduction in burst effect observed. A burst effect of 60.54% (±4.82) compared to 44.53% (±9.70) was observed after 2 h when comparing porous versus nonporous microparticles obtained using PVA and 100 mg PLGA (Figure 4a). Data obtained on the highly porous microparticles adopting a primary emulsion comprising of 1.5 ml Tween 80 and 100 mg PLGA suggest the BSA encapsulated was surface bound. In this instance a burst effect of 97.76% (±1.34) after 2 h was observed (Figure 4b). The high burst effect correlates with the low EE observed suggesting that Tween 80 is ineffective at preventing the migration of BSA towards the external aqueous phase. A similar effect was observed for formulations prepared using oleic acid. Although a porous morphology and narrow size distribution was obtained, the burst effect is indicative of surface bound material, a burst effect of 76.47% (±6.32) with a corresponding EE% of 32.61% (±3.56). There is a direct correlation between the surface morphology of the microparticles and the release profiles exhibited when comparing formulations which had a narrow size distribution. The surface morphology increased in porosity in the following
120
% cummulative Release
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0.5 mL PVA, 100 mg PLGA 1.5 mL PVA, 100 mg PLGA 2.0 mL PV, 100 PLGA
(b) 120 % cumulative Release
% cumulative Release
(a) 120
5
100 80 60
0.5 ml oleic acid
40
1.5 mL oleic acid
20
1.5 mL oleic acid (100mg PLGA)
0 0
2
24
168
336
504
672
Time in Hours
Figure 5. Cumulative release profile from microparticles prepared using oleic acid.
order; PVA, oleic acid and Tween 80 (Figures 4 and 5) with a corresponding increase in burst effect 60.54% (±4.82), 76.47% (±6.32) and 97.76% (±1.34), respectively. The increased porosity of the microparticles has resulted in increased surface area exposed to the release media, and thus a higher burst effect45. The data may also suggest that although Tween 80 and oleic acid are suitable suspension stabilizers to achieve porous microsphere morphology they do not appear to prevent the migration of BSA towards the external aqueous phase and thus achieve the desired release characteristics. This is supported by the low EE% observed, particularly following the inclusion of Tween 80.
Conclusions Microparticles exhibiting a porous morphology may be produced by adopting various suspension stabilizers providing a low internal aqueous phase is used. The variation in surface morphology may be attributed to the size of the water droplets in the internal aqueous phase. Although porous microparticles with large pores and thus small aerodynamic diameters may be desirable for pulmonary delivery they may not possess suitable encapsulation or release characteristics. However, the research shows the potential for successfully employing Tween 80 in the production of porous microparticles. The use of Tween 80 is beneficial when developing pulmonary based formulations as it one of a limited number of excipients that have FDA approval for pulmonary use. Therefore, formulation of microparticles employing Tween as a stabilizer may offer a potential for pulmonary drug
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delivery of vaccines. Further optimization of the formulation parameters is required with the alternative suspension stabilizers to ensure suitable EE% and particle sizes can be achieved.
Declaration of interest
Drug Dev Ind Pharm, Early Online: 1–7
20. 21.
This work completed as part of a PhD studentship funded by 3M Healthcare Ltd, Morley Street, Loughborough, Leicestershire. 22.
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