Journal of Microencapsulation, February 2006; 23(1): 69–78

Electrospray encapsulation of water-soluble protein withpolylactide. I. Effects of formulations and process onmorphology and particle size

YIXIANG XU1, MACIEJ SKOTAK2, & MILFORD HANNA1

1Industrial Agricultural Products Center and Food Science & Technology and 2Department

of Chemical Engineering, University of Nebraska Lincoln, NE, USA

(Received 28 April 2005; accepted 20 June 2005)

AbstractBovine serum albumin (BSA) was encapsulated with poly (lactide) (PLA) using an electrospraytechnique in which a sufficiently strong electric field was applied to overcome the surface tensionof a droplet and to produce small particles. The influences of PLA solvent type, PLA solutionconcentrations; the viscosity, electrical conductivity and surface tension of PLA solutions andPLA/BSA emulsions; and the applied voltage and flow rate on the morphology and size of theBSA-loaded PLA particles were examined. 1,2-dichloroethane (DCE) was a better solvent for PLAthan dichloromethane. Spherical electrosprayed particles, with smooth surfaces, were observed withboth solvents. The electrical conductivity increased and particle size decreased when acetone wasmixed with DCE as PLA solvent. However, the particles were no longer spherical. The size of theparticles increased and shape became spherical as PLA concentration was increased from 1–3%.Increasing the concentration to 4% resulted in the formation of a mixture of beads and fibres.Particle size decreased as applied voltage was increased from 10–15 kV and increased as flow ratewas increased from 0.5–3ml h�1.

Keywords: Electrospray, encapsulation, poly(lactide)

Introduction

Drugs are orally or parenterally delivered to the body in conventional drug delivery systems.

They often are filtered out or are degraded by hydrolysis enzymes while circulating though

the body. Therefore, only a small fraction reaches the intended target and the expected

therapeutic effect is impaired (Gomez et al. 1998). Recently, considerable attention has been

given to controlled delivery of pharmacological agents to their targets at a therapeutically

optimal rate and dosage by encapsulating them with biodegradable polymers. Poly (lactide)

(PLA) is a primary polymeric candidate for such applications based on its biodegradability,

biocompatibility, high mechanical strength and good shaping and moulding properties

Correspondence: Milford Hanna, Industrial Agricultural Products Center and Food Science & Technology, University of

Nebraska, Lincoln, NE 68583-0730, USA. E-mail: mhanna1@unl.edu

ISSN 0265–2048 print/ISSN 1464–5246 online � 2006 Taylor & Francis

DOI: 10.1080/02652040500435048

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(Ouchi et al. 2004). Different encapsulation approaches have been studied. Of the methods

available, a double emulsion (water-in-oil-in-water) solvent extraction/evaporation techni-

que is used widely to encapsulate water-soluble drugs and protein into polymer matrices

(Ogawa et al. 1988, Alex and Bodmeier 1989, Langer 1998). Particles size and

polydispersity from the double emulsion method were reduced by homogenization

(Lamprecht et al. 2000). Novel techniques, including ultrasonic atomization and

electrospinning, also have been developed to encapsulate water-soluble molecules

(e.g. drugs, enzymes, growth factors and DNA) to immobilize or release them in a

controlled manner (Felder et al. 2003, Sanders et al. 2003).

Recently, electrostatic extrusion (electrospray) has attracted more interest in producing

drug/protein loaded micro/nano particles (Amsden and Goosen 1997, Loscertales et al.

2002). Its well-known applications are in ionization and characterization of protein and

DNA in mass spectrometry or respiratory drug delivery (Tang and Gomez 1994, Ijsebaert

et al. 2001). Basically, electrospraying can be implemented by applying a sufficiently large

electric field to a polymer/protein solution in a syringe with a metal needle relative to a

counter electrode a short distance away, as shown in Figure 1. In the absence of an electric

field, a droplet forming on a needle tip will grow until its mass is large enough to escape the

surface tension at the needle-droplet interface (Sanders et al. 2003). Under the action

of an electric field, the solution forms a conical meniscus from whose apex a jet is issued.

The meniscus deforms further and breaks into droplets with small particle sizes and narrow

size distribution due to the pull of the electrostatic force. Coulombic repulsion between the

highly charged droplets resulted in self-dispersed particles and no coalescence.

It was found that many parameters including applied voltage, solution flow rate, solvent

types and solution properties (i.e. viscosity, electrical conductivity and surface tension)

played important roles in electrospraying (Tang and Gomez 1996, Amsden and

Figure 1. Schematic representation of the electrospray apparatus: (a) pump; (b) feeding line;(c) needle; (d) electrospray; (e) collector plate (OD¼ 15 cm); and (f) high voltage power supply.

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Goosen 1997). In order to gain a better understanding of the potential of electrospraying

for encapsulation, the objective of this study was to investigate the effects of processing

conditions on properties of encapsulated particles with an emphasis on particle size and

morphology. Bovine serum albumin (BSA) was chosen as the model drug because it has

a high molecular weight, is a globular protein that is well characterized and is soluble

in water (Thimma and Tannishetti 2001).

Materials and methods

Materials

Poly-L-lactide (Mw 175 000Da), in the form of 2–4mm spherical granules, was purchased

from Cargill Inc. (Minneapolis, MN, USA). Bovine serum albumin with a mean

molecular weight of 65 000Da was purchased from Sigma Aldrich and used as provided.

Dichloromethane (DCM), 1,2-dichloroethane (1,2-DCE), acetone and

N, N-dimethylformamide (DMF) (reagent grade) were purchased from Fisher Scientific

(Pittsburgh, PA, USA).

Microparticles preparation

PLA solutions were prepared by dissolving PLA in 10ml of different solvents and solvent

mixtures and stirring for 8 h at room temperature. Specific amounts of BSA, previously

dissolved in 0.5ml of distilled water, were mixed with the PLA solutions and emulsified

by sonication for 10min. The ratio of PLA to BSA was at 5 : 1. The emulsion was drawn

into a 5ml syringe, with a blunt tip and 18 gauge metal needle. The syringe was placed

in a syringe pump (Cole-Parmer 74900–00, Vernon Hills, IL) and a voltage in the range of

0–30 kV and a limiting current of 166 mA (Gamma High Voltage Research ES30P-5W/PRG,

Ormond Beach, FL) were applied to the needle. The solution was sprayed vertically

downward, at different voltages and flow rates, toward a circular-shaped collector 10 cm

away from the needle tip. The factors tested included solvent type (DCM, 1,2-DCE,

1,2-DCE/acetone (1 : 1) and 1,2-DCE/DMF (1 : 1) v/v), PLA concentration (1%, 2%, 3%

and 4% w/v), applied voltage (10 kV, 12.5 kV and 15 kV), flow rate (0.5, 1, 2 and 3ml h�1)

and emulsion physical properties.

Viscosity, electrical conductivity and surface tension measurements

The viscosity of the PLA solutions and PLA/BSA emulsions were measured with

a Brookfield DV-IIþ programmable viscometer (Brookfield Engineering Lab. Inc.,

Middleboro, MA) at 25�C with SC-18 spindle speed of 200 rpm. The electrical conductivity

was determined using a digital conductivity meter with a gold dip cell (Model 2052, VWR

Scientific, West Chester, PA). The cell was dipped into the container holding the solution

and was moved gently up and down a few times to dislodge air bubbles. The measurement

was recorded after the cell temperature was equilibrated with the solution. Surface tension

was measured with a CSC-DuNouy tensiometer (Model 70545, CSC Scientific Inc.,

Fairfax, VA). The clean ring attached to the lever arm was placed into a container holding

the solution whose surface tension was to be measured. The surface tension was the force

of the pull exerted on the ring at the breaking point of the film.

Electrospray encapsulation of water-soluble protein with polylactide 71

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Morphology, size and structure characterization of particles

The morphology and size of BSA-loaded PLA particles were measured using scanning

electron microscopy (SEM) at a voltage of 15 kV ((Hitachi S-3000N, Tokyo, Japan). Before

testing, the samples were mounted on SEM stubs with double-sided adhesive tape and

then coated under vacuum with platinum to make the sample conductive.

Statistical analysis

The diameters of the particles were analysed as general linear models (GLM) in SAS

analysis program 8.0 (SAS Institute Inc., Cary, NC) and reported as the mean of 100–150

individual particles on a random sampling basis to minimize potential selection bias

(Kuo et al. 2004). Duncan’s multiple range tests were conducted to check for significant

(p<0.05) differences between treatment groups.

Results and discussion

Effect of solvent types

The solvent types and consequential solution physical properties played important roles in

determining the morphology and size of the electrosprayed particles. The physical properties

of the 3% PLA solutions and PLA/BSA emulsions and the diameters of BSA-loaded

PLA particles from the different solvents are given in Table I. The voltage and flow rate

during electrospraying were fixed at 12.5 kV and 1.0ml h�1, respectively. The solvent most

commonly used with PLA in encapsulation is DCM. PLA solutions in different solvents

were Newtonian fluids. The viscosity, electrical conductivity and surface tension of the

3% PLA in DCM were 4.77mPa s, 0.039 mS cm�1 and 35.8mNm�1, respectively. After

emulsifying with BSA aqueous solution, the emulsions became non-Newtonian fluid but the

viscosity was reported after stirring for 3min. The viscosity and electrical conductivity of the

PLA/BSA emulsion increased after emulsifying the BSA water phase with the PLA solution.

BSA-loaded PLA particles had an average diameter of 4.68� 1.35 mm. They were spherical

with smooth surfaces (Figure 2(a)). However, the needle tip clogged in this case. This may

have been due to the low boiling point (39.8�C) of DCM resulting in rapid evaporation

during electrospraying. Therefore, a higher boiling point (83.4�C) 1,2-DCE was chosen.

Table I. Effects of the solvent type on physical properties of PLA solutions and PLA/BSA emulsions and

diameter of the particles.

Solvent

Solution

(3%)

Viscosity

(mPa s)

Conductivity

(mS cm�1)

Surface tension

(mNm�1)

Particle diameter

(mm)

Dichloromethane PLA 4.77 0.039 35.8 4.68�1.35b

PLA/BSAa 5.38 0.204 35.9

1,2-Dichloroethane PLA 7.05 0.058 39.1 4.77�1.93b

PLA/BSAa 7.60 0.201 39.4

1,2-Dichloroethane/acetone PLA 5.26 0.412 33.4 1.64�0.51c

PLA/BSAa 5.89 1.24 33.7

1,2-Dichloroethane/ PLA 7.02 2.26 39.4

N,N-Dimethylformamide PLA/BSAa 10.9 15.96 42.5 –

awater phase/oil phase (v/v)¼ 20:1 and PLA/BSA(w/w)¼ 5:1; means no particle;b–cmeans with same letter within a column indicate no significant (p>0.05) difference by Ducan multiple range test.

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The viscosity and surface tension of the PLA solution and PLA/BSA emulsion increased

when the solvent type was changed from DCM to 1,2-DCE. The electrical conductivity

of the PLA solution with 1,2-DCE was higher than its counterpart with DCM. However,

after emulsifying with BSA water phase, the properties of two emulsions were similar.

The BSA-loaded particles from 1,2-DCE also were spherical with smooth surfaces

(Figure 2(b)). They had an average diameter of 4.77� 1.97 mm, which was close to that

obtained with DCM. This suggested that the electrical conductivity was the most important

physical property in determining the size of the electrosprayed particles. This was consistent

with the results of Tang and Gomez (1996). Therefore, acetone and DMF, two water

miscible organic solvents, having relatively high polarities, were chosen to mix with 1,2-DCE

at a 1 : 1 volume ratio as PLA solvents, to increase the electrical conductivity of liquid. The

electrical conductivities of PLA solutions were increased �7 times when acetone was added

to 1,2-DCE and 40 times when DMF was added to 1,2-DCE. After emulsifying with BSA

water phase, the conductivity of the emulsions increased �6–78 times compared to their

counterparts in DCM and 1,2-DCE, respectively. The viscosity and surface tension of the

Figure 2. SEM micrographs of BSA-loaded PLA particles with solvents: (a) dichloromethane;(b) 1,2-dichloroethane; (c) 1,2-dicloroethane/acetone(1 : 1); and (d) 1,2-dichloroethane/N,N-dimethylformamide (1 : 1) (3% PLA concentration, applied voltage of 12.5 kV and flow rate of1.0ml h�1).

Electrospray encapsulation of water-soluble protein with polylactide 73

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PLA solution and PLA/BSA emulsion decreased when acetone was added, while viscosity

did not change. Surface tension increased with addition of DMF. The average diameter of

the electrosprayed particles with 1,2-DCE/acetone was 1.64� 0.51 mm and was significantly

lower than that with 1,2-DCE. This was due to the combined effects of high electrical

conductivity, low viscosity and low surface tension when acetone was added to 1,2-DCE.

However, the particles were no longer spherical and were accompanied by the formation of

fibres (Figure 2(c)). Particles were not formed with 1,2-DCE/DMF mixed solvents in spite

of the high electrical conductivity, possibly because DMF had a destructive influence on

the PLA. It also is possible that the applied voltage was too high in the case when fluid had

high electrical conductivity.

Effects of PLA concentrations

PLA concentration was a key factor in determining the size and morphology of particles. The

effects of PLA concentration on the physical properties of PLA solutions and PLA/BSA

emulsions and the particle size are summarized in Table II. 1,2-DCE was used as the solvent

with voltage of 12.5 kV and flow rate of 1.0ml h�1. As the PLA concentration increased from

1–4%, the viscosity and surface tension of the PLA solution increased from 2.19–12.1mPa s

and 37.4–38.9mNm�1, respectively, while the electrical conductivity decreased from

0.065–0.053 mS cm�1 since PLA solutions, in general, showed intrinsically very low

electrical conductivities (Jun et al. 2003). After emulsifying with BSA water phase, the

emulsions were non-Newtonian fluid but viscosity was reported after stirring for 3min.

The viscosity and surface tension of the emulsions did not increase significantly with

concentration level. However, the electrical conductivity of the emulsions increased

a minimum of two times for 4% concentration and a maximum of seven times for 1%

concentration with addition of high conductivity water phase. The average diameter of

the particles increased as the PLA concentration increased from 1–3%, followed by

a decrease at 4%. The increase in the particle size was attributed to high surface tension,

high viscosity and low electrical conductivity. In electrospray experiments, an electric force

is applied to overcome surface tension and form small droplet. However, when the electric

force was held constant (12.5 kV), increasing the surface tension resulted in the formation of

larger droplets on the tip of the needle (Amsden and Goosen 1997). In addition, viscosity

played an important role in determining the size of the particles since the jet break-up

Table II. Effects of PLA concentrations in 1,2-dichloroethane on physical properties of PLA solutions and

PLA/BSA emulsions and diameter of the particles.

Concentration

(wt)

Viscosity

(mPa s)

Conductivity

(mS cm�1)

Surface tension

(mNm�1)

Particle diameter

(mm)

1% PLA 2.19 0.065 37.4 3.19�0.98b

1% PLA/BSAa 2.23 0.446 37.6

2% PLA 4.23 0.061 37.8 3.48�0.88b

2% PLA/BSAa 4.24 0.251 38.5

3% PLA 7.05 0.058 38.7 4.77�1.97c

3% PLA/BSAa 7.60 0.201 39.2

4% PLA 12.1 0.053 38.9 2.72�1.17b

4% PLA/BSAa 12.6 0.108 39.5

awater phase/oil phase (v/v)¼ 20:1 and PLA/BSA(w/w)¼ 5:1;b–cmeans with same letter within a column indicate no significant (p>0.05) difference by Ducan multiple range test.

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process in electrospraying was controlled by a natural surface wave instability, where

the wavelength with the maximum growth rate along the liquid ligament depended on the

viscosity (Tang and Gomez 1996). The morphologies of BSA-loaded particles, at different

concentrations, are shown in Figure 3. Spherical particles were not observed for 1%

emulsion since it was difficult for PLA to form a film from a diluted and less viscous

solution. Instead, PLA was extended easily by the electric force during solvent evaporation.

Although spherical particles were found with the PLA concentration of 2%, some were

shrunk during solvent evaporation. Fully spherical particles were produced at 3%. Further

increasing the concentration to 4% resulted in the formation of a mixture of beads

and fibres.

Effects of the applied voltage and the flow rate

Voltage and flow rate control the morphology and size of the electrosprayed particles.

High voltage is required to overcome the surface tension of the droplet at the tip of the

Figure 3. SEM micrographs of BSA-loaded particles with PLA concentrations: (a) 1%; (b) 2%;(c) 3%; and (d) 4% (1,2-dichloroethane as solvents, voltage of 12.5 kV, and flow rate of 1.0ml h�1).

Electrospray encapsulation of water-soluble protein with polylactide 75

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needle and to form small particles. The formation of a continuous and stable spray is the key

for selection of the voltage range. Three voltage levels (10, 12.5 and 15 kV) were chosen,

based on preliminary test results, and their influences on the morphologies of BSA-loaded

PLA particles were investigated while holding the other conditions constant (1,2-DCE as

solvent, 3% PLA concentration and the flow rate of 0.5ml h�1). Some particles were found

to have tails when sprayed at 10 kV (Figure 4(a)), indicating that the electric force was not

strong enough to overcome the surface tension of the droplet. The tails gradually

disappeared as the voltage increased to 12.5 kV and 15 kV (Figure 4(b) and (c)). Further

increasing the voltage above 15 kV resulted in an unstable spray. The influence of the

applied voltages on the size of the particles was investigated using different flow rates

(Figure 5). The average diameters of the particles decreased when the voltage was increased

from 10–15 kV for each flow rate. This was because the strong electric field at the high

voltage forced the conical meniscus at the tip of the needle to deform and to break into small

droplets. Further, the flow rate affected the rate of decrease in particle size when the voltage

was changed. The particle’s average diameter decreased 55% at the flow rate of 0.5ml h�1,

while there was a 70% decrease for the flow rate of 3ml h�1, as the voltage increased

from 10–15 kV.

Figure 4. SEM micrographs of BSA-loaded particles at applied voltages: (a) 10 kV; (b) 12.5 kV;and (c) 15 kV (3% PLA in 1,2-dichloroethane and flow rate of 0.5ml h�1).

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The changes in the particle sizes with the flow rates of the emulsions at different voltages

are shown at Figure 5. In general, the particle sizes increased with increasing flow rate for

each voltage. This can be explained by the fact that the uneven spread of the emulsion at the

tip of the needle caused by high flow rate resulted in uncontrolled atomization and formation

of large particles (Freitas et al. 2004). However, different rates of increase were observed for

different voltages. The increases in the average diameter were 49% at the voltage of 10 kV,

44% at 12.5 kV and 36% at 15 kV, respectively, as the flow rate increased from 0.5–3ml h�1.

The higher the voltage was, the smaller the increase in the particle size was.

Conclusion

Solvent type, solution concentration, the physical properties of poly(lactide) (PLA) solutions

and PLA/bovine serum albumin (BSA) emulsions, applied voltage and flow rate played

important roles in determining the morphology and size of the BSA-loaded PLA particles

produced by electrospray technique. 1,2-dichloroethane was a better PLA solvent than

dichloromethane. Electrical conductivity was the most effective physical property in

describing particle size. Particle size decreased with addition of acetone to increase the

electrical conductivity of solvents. Particle size increased with increasing PLA concentra-

tions and flow rate and decreased as applied voltage increased. Fully spherical particles were

observed in the PLA concentration range of 2–3%. Higher concentrations resulted in the

formation of a mixture of beads and fibres.

0.51

23

15

12.5

10

0

1

2

3

4

5

6

7

Ave

rag

e d

iam

ter

(um

)

Flow rate (ml/hr)

Vo

ltag

e (k

V)

Figure 5. The average diameters of BSA-loaded PLA particles as the functions of the applied voltageand flow rate (3% PLA in 1,2-dichloroethane).

Electrospray encapsulation of water-soluble protein with polylactide 77

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Acknowledgements

The authors are grateful to Dr Gustavo Larsen, Chemical Engineering Department, for

the use of the electrospray apparatus and other equipment in his lab and to Dr Kit Lee

in the School of Biological Science for the use of SEM. This research was supported by

funds provided through the Nebraska Research Initiative and the Agricultural Research

Division at the University of Nebraska. A contribution of the University of Nebraska

Agricultural Research Division, Lincoln, NE 68583. Journal Series No. 14523. This study

was conducted at the Industrial Agricultural Products Centre.

References

Alex R, Bodmeier R. 1989. Encapsulation of water-soluble drugs by a modified solvent evaporation method. I.

Effect of process and formulation variable on drug entrapment. Journal of Microencapsulation 7:347–355.

Amsden BG, Goosen MFA. 1997. An examination of factors affecting the size, distribution and release

characteristics of polymer microbeads made using electrostatics. Journal of Controlled Release 43:183–196.

Felder CHB, Blanco-Prieto MJ, Heizmann J, Merkle HP, Gander B. 2003. Ultrasonic atomization and subsequent

polymer desolvation for peptide and protein microencapsulation into biodegradable polyesters. Journal of

Microencapsulation 20:553–567.

Freitas S, Merkle HP, Gander B. 2004. Ultrasonic atomization into reduced pressure atmosphere-envisaging

aseptic spray-drying for microencapsulation. Journal of Controlled Release 95:185–195.

Gomez A, Bingham D, de Juan L, Tang K. 1998. Production of protein nanoparticles by electrospray drying.

Journal of Aerosol Science 29:561–574.

Ijsebaert JC, Geerse KB, Marijnissen JM, Lammers J-WJ, Zanen P. 2001. Electro-hydrodynamic atomization

of drug solutions for inhalation purpose. Journal of Applied Physiological 91:2735–2741.

Jun Z, Hou H-Q, Schaper A, Wendorff JH, Greiner A. 2003. Poly-L-lactide nanofibers by electrospinning-influence

of solution viscosity and electrical conductivity on fiber diameter and fiber morphology. e-Polymers 9:1–9.

Kuo S-M, Niu G, Chang S-J, Kuo C-H, Bair M-S. 2004. A one-step method for fabricating chitosan microspheres.

Journal of Applied Polymer Science 94:2150–2157.

Lamprecht A, Ubrich N, Hombreiro Perez M, Leh C-M, Hoffman M, Maincent P. 2000. Influences of process

parameters on nanoparticle preparation performed by a double emulsion pressure homogenization technique.

International Journal of Pharmaceutics 196:177–182.

Langer R. 1998. Drug delivery and targeting. Nature 392:5–10.

Loscertales IG, Barrero A, Guerrero I, Cortijo R, Marques M, Ganan-Calvo AM. 2002. Micro/nano encapsulation

via electrified coaxial liquid jets. Science 295:1695–1698.

Ogawa Y, Yamamoto M, Okada H, Yashiki T, Shimamoto TA. 1988. New technique to efficiently entrap

leuprolide acetate in microcapsules of copoly(lactic/glycolic acid). Chemical Pharmacy Bulletin 36:1095–1103.

Ouchi T, Saito T, Kontani T, Ohya Y. 2004. Encapsulation and/or release behavior of bovine serum albumin

within and from polylactide-grafted dextran microspheres. Macromolecules Bioscience 4:458–463.

Sanders EH, Kloefkorn R, Bowlin GL, Simpson DG, Wnek GE. 2003. Two-phase electrospinning from a

single electrified jet: Microencapsulation of aqueous reservoirs in poly(ethylene-co-vinyl acetate) fibers.

Macromolecules 36:3803–3805.

Tang K, Gomez A. 1994. Generation by electrospray of monodisperse water droplets for targeted drug delivery

by inhalation. Journal of Aerosol Science 25:1237–1249.

Tang K, Gomez A. 1996. Monodisperse electrospray of low electric conductivity liquids in the cone-jet mode.

Journal of Colloid Interface Science 184:500–511.

Thimma R, Tannishetti S. 2001. Barium chloride crosslinked carboxymethyl guar gum beads for gastrointestinal

drug delivery. Journal of Applied Polymer Science 82:3084–3090.

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