Transcript
Development of Novel MicroencapsulationProcesses
by
Weisi Yin
Submitted in Partial Fulfillment
of the
Requirements for the Degree
Doctor of Philosophy
Supervised by
Professor Matthew Z. Yates
Department of Chemical EngineeringArts, Sciences and Engineering
School of Engineering and Applied Sciences
University of RochesterRochester, New York
2009
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Curriculum Vitae
The author was born in Susong, Anhui Province, People’s Republic of China
in 1980. He attended Nanjing University from 1997 to 2004, and graduated with
a Bachelor of Science degree in Polymer Science and Engineering in 2001 and a
Master of Science degree in Polymer Physics and Chemistry in 2004. He came to
the University of Rochester in the Fall of 2004 and began graduate studies in Chemical
Engineering. He pursued his research in microencapsulation, colloids, and supercritical
carbon dioxide under the direction of Professor Matthew Z. Yates and received a Master
of Science degree from the University of Rochester in 2008.
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Acknowledgements
First, I would like to express my gratitude to my advisor Professor Matthew
Z. Yates for his continuous support in the Ph.D. program. He guided me to the
microencapsulation study and encouraged me to try new ideas. He was always there
to listen and give valuable suggestions. He allowed me to have flexible schedules
to arrange my experiments, while he gave strict requirements on experiments and
publishing papers, which benefit me a lot. I really enjoy doing research under his
direction.
Besides my advisor, I would like to thank Professor Todd D. Krauss for help on
the project of quantum dots (QDs) encapsulation. And I would like to thank Dr.
Xiqiang Yang and Mridula Nair from Eastman Kodak Company for guide in a 2-year
cooperative project of encapsulating nanoparticles for new printing processes.
I gratefully acknowledge support from the National Science Foundation (CTS-
0343774 and CHE-0316173), the Department of Energy (DE-FC03-92SF19460), the
Alfred P. Sloan Foundation, the Camille and Henry Dreyfus Foundation, the University
of Rochester, the Laboratory for Laser Energetics, Eastman Kodak Company, and
the Center for Electronic Imaging Systems. The electron microscopy facility at the
University of Rochester is supported by NSF CTS-6571042.
A special thanks goes to all collaborators during my Ph.D. program. Hongwei Liu
did early work in QDs encapsulation and gave me suggestions when I was finishing the
project. Li Guo provided me with QDs, and Fengzhi Jiang characterized polymer/QDs
composite particles with fluorescence imaging and spectroscopy. Sherry Chen helped
me encapsulate dyes in polystyrene particles with supercritical carbon dioxide, Rob
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Levasseur helped me make hollow poly(lactide-co-glycolide) particles, and Graciela
Mohamedi-Smith helped me encapsulate nanoparticles for new printing processes in
the cooperative project with Eastman Kodak Company.
I would like to thank Brian McIntyre for his patient teaching and help in electron
microscopy and sample preparation. To my lab-mates and other graduate students in
the community, I want to say “thank you” for experiment discussions and equipment
sharing.
I am grateful to my friends for mutual inspiration. We exchange ideas, share
experiences, and talk about affairs. Without them, the world would not be so pleasant.
At last, I want to thank my family for long-lasting support. They gave me life,
educated me, and worked hard to support me. It would be difficult for me to pass
hardships without their encouragement and backing.
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Abstract
This thesis is for encapsulating additives into polymer particles using different
techniques including emulsification/solvent evaporation, compressed carbon dioxide
based microencapsulation, and encapsulation with porous polymer particles. Such
microencapsulations can be applied to a vast range of areas, for example bio-labeling,
controlled release, drug delivery, and printing.
Fluorescent CdSe/ZnS quantum dots (QDs) were incorporated into polyisoprene
(PI) particles by emulsification/solvent evaporation. The simple method results in
QDs encapsulated into the particle core without requiring chemical modification of
the QDs. The fluorescence spectra of mixtures of two different-sized QDs change in
PI as compared to their solution spectra, suggesting energy transfer between QDs due
to their aggregation during the encapsulation. However, different emission peaks were
clearly resolved, indicating that the particles are suitable for multicolor coding. The
polyisoprene is easily cross-linked, and the cross-linking was shown to greatly enhance
the fluorescence stability of the encapsulated QDs.
Ionic dyes were successfully encapsulated in polystyrene (PS) particles by CO2-
based microencapsulation. The water-soluble dyes were made hydrophobic by forming
ion pairs with alkyl quaternary ammonium cations. The hydrophobic ion pairs were
then encapsulated in preexisting size monodisperse PS particles dispersed in water.
High-pressure carbon dioxide swelled and plasticized PS and thus facilitated mass
transport of the dye into the particles. The results show that the particles maintain their
size and morphology after exposure to CO2, and that ion-paired dyes have significantly
higher loading in the polymer particles than the original dyes. Addition of water-
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miscible cosolvents was shown to further enhance the incorporation of the hydrophobic
ion pairs into the polymer colloids.
To encapsulate water-soluble additives, porous polymer particles were made by
freeze-drying droplets of polymer solution suspended in water or from a spray. Hollow
PS particles were obtained by swelling PS latex with solvent, freezing in liquid
nitrogen, and drying in vacuum. It is shown that the particle morphology is due to
phase separation in the polymer emulsion droplets upon freezing in liquid nitrogen,
and that morphological changes are driven largely by lowering interfacial free energy.
The dried hollow particles were resuspended in a dispersing media and exposed to a
plasticizer, which imparts mobility to polymer chains, to close the surface opening and
form microcapsules surrounding an aqueous core. The interfacial free energy difference
between the hydrophobic inside and hydrophilic outside surfaces is the major driving
force for closing the hole on the surface.
A controlled release biodegradable vehicle for drug was made by encapsulating
procaine hydrochloride, a water-soluble drug, into the core of poly(DL-lactide) (PLA)
microcapsules, which were made by the freeze-drying and subsequent closing process.
The encapsulation efficiency is affected by the hollow particle morphology, amount of
closing agent, exposure time, surfactant, and method of dispersing the hollow particles
in water. Controlled release of procaine hydrochloride from the microcapsules into
phosphate buffer was observed. The use of benign solvents dimethyl carbonate in
spray/freeze-drying and CO2 for closing would eliminate concerns of residual harmful
solvent in the product. The ease of separation of CO2 from the drug solution may also
enable recycling of the drug solution to increase the overall encapsulation efficiency
using these novel hollow particles.
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Contents
Foreword 1
1 Introduction 2
1.1 Microencapsulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
1.2 Liquid and Supercritical Carbon Dioxide . . . . . . . . . . . . . . . . . 10
1.3 Temperature Induced Phase Separation . . . . . . . . . . . . . . . . . . 15
1.4 Outline of the Thesis . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
2 Fluorescent Quantum Dot-Polymer Nanocomposite Particles by Emulsifi-
cation/Solvent Evaporation 21
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
2.2 Experimental Section . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
2.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3 Hydrophobic Ion Pairing to Enhance Encapsulation of Water-Soluble
Additives into CO2-Swollen Polymer Microparticles 33
3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
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3.2 Experimental Section . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4 Effect of Interfacial Free Energy on the Formation of Polymer Microcap-
sules by Emulsification/Freeze-Drying 49
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.2 Experimental Section . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
4.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 55
4.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
5 Encapsulation and Sustained Release from Biodegradable Microcapsules
Made by Emulsification/Freeze Drying and Spray/Freeze Drying 74
5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
5.2 Experimental Section . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
5.3 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
6 Conclusions and Future Work 94
6.1 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
6.2 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Bibliography 101
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List of Tables
3.1 Water and toluene solubility of bromothymol blue, rose bengal and
their ion pairs with tetrabutylammonium bromide (TBABr), tetra-
hexylammonium bromide (THABr), and tetraoctylammonium bromide
(TOABr) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
3.2 Effect of ion pair formation on dye loading of bromothymol blue and
rose bengal into polystyrene particles (0.1 g Triton X-100, but no
cosolvent was used) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3.3 Cosolvent and surfactant effects on loading of bromothymol blue in
polystyrene particles . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5.1 Effect of stirring time on procaine hydrochloride encapsulation in
hollow PLA particles. . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
5.2 Effect of surfactant removal on procaine hydrochloride encapsulation
in hollow PLA particles. . . . . . . . . . . . . . . . . . . . . . . . . . 88
x
List of Figures
1.1 Scheme showing microencapsulation by air suspension coating. . . . . 6
1.2 Scheme showing microencapsulation by coacervation. . . . . . . . . . . 9
1.3 Scheme showing microencapsulation by emulsification/solvent evapo-
ration. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.4 Phase diagram of CO2. . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.5 Scheme showing the encapsulation of additives into polymer colloids
with the help of compressed CO2. . . . . . . . . . . . . . . . . . . . . 15
1.6 Temperature induced phase separation between water and solutes. . . . 17
1.7 Chemical structure of trans-polyisoprene. . . . . . . . . . . . . . . . . 19
1.8 Chemical structure of poly(DL-lactide). . . . . . . . . . . . . . . . . . 20
2.1 CdSe/ZnS quantum dots capped with trioctylphosphine oxide. . . . . . 24
2.2 Optical microscopic images of un-cross-linked PI-QD particles: (A)
dark-field scattering and (B) true-color fluorescence. . . . . . . . . . . 27
2.3 Normalized single-particle fluorescence spectrum of un-cross-linked PI
containing two different-colored QDs (solid line) and the fluorescence
emission spectrum of the same mixture of QDs in hexane (dashed line). 29
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2.4 Digital photographs of aqueous suspensions of 0.3 wt% PI-QD parti-
cles under UV illumination. . . . . . . . . . . . . . . . . . . . . . . . . 31
3.1 Molecular structure of: (A) bromothymol blue and (B) rose bengal. . . . 36
3.2 Chemical structure of Triton X-100. . . . . . . . . . . . . . . . . . . . 37
3.3 Chemical structure of tetraalkylammonium bromide used for ion pairing. 37
3.4 Variable volume high pressure stainless steel vessel. . . . . . . . . . . . 39
3.5 SEM pictures of polystyrene particles: (A) before and (B) after
microencapsulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.6 Agglomerated polystyrene particles after the microencapsulation pro-
cess in the presence of ethanol. . . . . . . . . . . . . . . . . . . . . . . 46
4.1 Scheme showing the formation of hollow polystyrene (PS) particles
by emulsification/freeze-drying and the closing of such particles for
encapsulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.2 Chemical structure of m-PEGMA. . . . . . . . . . . . . . . . . . . . . 53
4.3 Polystyrene latex particles as synthesized (A) by surfactant-free emul-
sion polymerization and (B) by emulsion polymerization with poly(ethylene
glycol) macromonomer. . . . . . . . . . . . . . . . . . . . . . . . . . . 56
4.4 Effect of surfactant on the hollow particle morphology. . . . . . . . . . 57
4.5 Model for a biphasic emulsion droplet consisting of polymer-rich (P),
solvent-rich (Q), and aqueous (W) phases. . . . . . . . . . . . . . . . . 59
4.6 Effect of surfactant on interfacial free energy profile. . . . . . . . . . . 61
4.7 Effect of volume fraction of the swelling solvent on the morphology of
the freeze-dried PS particles grafted with PEG. . . . . . . . . . . . . . 62
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4.8 Interfacial free energy (E/ECS) vs ΦQ with different volume fractions of
solvent (νQ) by setting γPW, γQW, and γPQ to 30, 45, and 12, respectively. 64
4.9 Freeze-dried samples with n-heptane (A) and benzene (B) as the
swelling solvent. The volume fraction of the swelling solvent was 0.8. . 66
4.10 PS particles grafted with PEG and swollen with toluene after freezing
in ethylene glycol/dry ice at −10 °C and drying by a vacuum pump at
0 °C. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.11 Effect of surfactant on closing hollow PS by CO2 (A, B) and toluene
(C, D). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
4.12 Effect of surfactant on closing hollow PS by toluene. . . . . . . . . . . 70
4.13 Schematic illustrating the hole closing process. . . . . . . . . . . . . . 70
4.14 Encapsulation of rose bengal by exposure of aqueous PS suspension
to toluene in the presence of dye: (A) solid PS with grafted PEG; (B)
hollow PS with grafted PEG. . . . . . . . . . . . . . . . . . . . . . . . 72
4.15 Confirmation of the hollow structure of closed polystyrene capsules
encapsulating rose bengal. . . . . . . . . . . . . . . . . . . . . . . . . 72
5.1 Coaxial spray nozzle. . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
5.2 Hollow PLA particles made by emulsification/freeze-drying: (A) 0.4 g/ml,
(B) 0.2 g/ml PLA in dichloromethane. . . . . . . . . . . . . . . . . . . 82
5.3 The concentration of procaine hydrochloride in PLA particles as a
function of amount of dichloromethane used during encapsulation for
1.5 hours at room temperature. . . . . . . . . . . . . . . . . . . . . . . 83
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5.4 PLA particles after exposure of aqueous suspensions to different
amounts of dichloromethane for 1.5 hours at room temperature. . . . . . 85
5.5 Interior structure of PLA particles encapsulating procaine hydrochlo-
ride using different amounts of dichloromethane and stirring time. . . . 85
5.6 Scheme showing the closing process of hollow PLA particles for
encapsulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
5.7 Porous PLA particles made by coaxial spray/freeze-drying with dimethyl
carbonate as the solvent. (A) Before encapsulation; (B) after encapsu-
lation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
5.8 PLA microcapsules formed by exposure of aqueous hollow particle
suspension to compressed carbon dioxide. . . . . . . . . . . . . . . . . 91
5.9 Release of encapsulated procaine hydrochloride in pH=7.2 phosphate
buffer: � microcapsules formed with excess surfactant removed (Ta-
ble 5.2, row 1, column 3); ◦ microcapsules formed without removing
excess surfactant (Table 5.2, row 1, column 2). . . . . . . . . . . . . . . 92
1
Foreword
For the project encapsulating fluorescent quantum dots (QDs) into polyisoprene
(PI) particles, I collaborated with Hongwei Liu, Li Guo, and Fengzhi Jiang. The
results from these collaborators are given in a published paper (Chem. Mater. 2007,
19, 2930-2936) in which they are listed as coauthors. Hongwei Liu, a previous
PhD student of Professor Yates, did early work in the project. He examined the
effects of homogenizing speed and concentration of polymer solution on PI particle
size, collected a transmission electron microscopy image of uncrosslinked PI-QD
particles, and carried out selective binding of streptavidin-coated PI-QD particles to
biotin-conjugated polystyrene spheres. Li Guo, a previous PhD student of Professor
Krauss, synthesized QDs of different sizes. Fengzhi Jiang, a previous postdoctoral
student of Professor Krauss, took microscopic fluorescence images and single-particle
fluorescence spectra of PI-QDs. I encapsulated two kinds of QDs into PI particles by
emulsification/solvent evaporation, investigated the energy transfer between QDs in PI
particles, crosslinked PI particles and studied the effect of crosslinking on fluorescence
stability of QDs. All data shown in Chapter 2 are from my portion of the study.
2
Chapter 1
Introduction
1.1 Microencapsulation
The term microencapsulation refers to technologies for enveloping small droplets
of liquids, gases or fine solid particles with natural or synthetic polymers.1–4 Biological
cells are an example of natural microencapsulation. The cell wall protects the inside
cellular parts from undesirable environmental conditions and the selectively permeable
cell membrane controls influx and release of metabolites. The encapsulated material
in microcapsules is referred to as the core, internal phase, or fill and the encapsulating
matrix is called shell, coating, wall material or membrane. The core can be any form,
crystalline, amorphous, emulsion, suspension of solids, or even smaller microcapsules.
The shell can be matrix, single layer or multilayer, and can consist of one kind
or several kinds of materials.5 The capsule can be permeable, semipermeable, or
impermeable. Many materials can be used for the wall, including gums, carbohydrates,
cellulose, lipids, inorganic materials, and proteins.2 The choice of wall materials
3
depends on the physicochemical properties of the core materials, the process making
the microcapsules, and the desired properties of the product. Biodegradable polymers
have been widely used in microcapsules for drug delivery to provide controlled
release of encapsulated drugs. The size of microcapsules is typically several hundred
nanometers to a few thousand micrometers.2 The outside may appear smooth or rough,
spherical or irregular. The microcapsules can be solid as free-flowing powders or
suspended in water, depending on the applications and stability of the capsules and
the encapsulated ingredients. The properties of the microcapsules such as average size,
size distribution, surface morphology, inner structure, and ingredient distribution will
affect the subsequent release profile.6
Microencapsulation can be used to protect active ingredients, reduce nutritional
loss, mask or preserve flavors, control the release of encapsulated materials, reduce drug
dosage, deliver drugs to specific locations, and make handling encapsulated material
easier.1,2,7 Carbonless paper is an early commercial application of microencapsula-
tion.8 In the food industry, microencapsulation is widely used to encapsulate flavors,
enzymes, oils, and fats to protect the encapsulated ingredients from environmental
conditions such as light, oxygen and moisture for increasing durability, reducing
volatility, or transferring liquid to solid for dry mixing.5 Enzymes can be trapped
in microcapsules to accelerate cheese ripening and improve cheese flavor.2 Carbon
dioxide can be encapsulated in hard candies to produce a sizzling effect on the
tongue as the candy melts in the mouth.9 In feedstuff industry, vitamins, unsaturated
vegetable fats, and hormones are encapsulated to prevent oxidation, degradation or
enzymatic hydrogenation. In agriculture, water-soluble fertilizers are encapsulated by
waxes, asphalt, and polymers such as polyurethanes, which will avoid higher local
4
fertilizer concentration and reduce the number of applications. In pharmaceutical
industry, microencapsulation is used to control the release of therapeutic agents and
prevent overdose after administration. Cells can be encapsulated in a polymer matrix
surrounded by a semipermeable membrane, which protects the inner cells from the
host’s immune system while allowing the diffusion of nutrients, oxygen and waste,
which can reduce or eliminate chronic administration of immunosuppressants. This
technique can be used to treat numerous medical diseases, including diabetes, cancer,
and central nervous system diseases.10 In the cosmetic industry, microencapsulation is
used to gain sustained release of deodorants and perfumes.
Complete impermeability to the encapsulated gases or liquids is hard to achieve,
given the small size and thin wall thickness of typical microcapsules. However, the
durability of core materials can be improved by choosing suitable wall materials and
capsule size. To release the core material, the capsule wall is often opened. It can be
done mechanically from outside, such as by shearing, chewing or crushing, or from
inside, such as by heating above the boiling point of the core material. The wall
can also be destroyed by dissolving, melting or burning. To prevent leaking of the
encapsulated materials, the solubility parameter of the polymer should be far from that
of the core material. Alternatively, controlled release of the encapsulated materials
can be achieved through permeation through the shell wall and the permeability of
the wall can be modified by crosslinking.11 Generally the release profile is affected
by the physicochemical properties of the core material, such as diffusivity, partition
coefficient, and vapor pressure, and of the wall material, such as thickness, porosity and
reactivity. If the capsule wall is permeable to the core material, the release is regulated
by the wall thickness and porosity, providing sustained release of the inside content,
5
which is desirable in therapeutic treatment. In contrast, opening the microcapsule
wall provides immediate burst release. The capsule wall can also be semipermeable,
allowing small molecules entering inside but preventing contents inside from leaving,
which can be used for encapsulating cells. For drug delivery applications, the capsule
wall should be biocompatible, and biodegradability can be used to tune the release.
The release of drugs from biodegradable polymers can be controlled by many factors,
such as biodegradation kinetics of the polymer,12,13 properties of polymers and drugs,14
compatibility between polymers and drugs,15 and the shape of the microcapsules.16,17
1.1.1 Methods for Microencapsulation
There are many methods for microencapsulation, such as dipping or centrifuging,
air suspension coating or fluidized bed coating, spray drying, spray chilling, co-
crystallization, liposome entrapment, coacervation, emulsification/solvent evaporation
or extraction, interfacial polymerization. Most of them are physical techniques, without
chemical reactions. One typical method involving chemical reaction is interfacial
polymerization. The selection of a method depends on economics, properties of the
core and wall material, microcapsule size, application and release mechanism.
The dipping or centrifuging technique passes core material droplets at high speed
through a thin film of liquid wall material, which is then hardened. The process can
make uniform and relatively large capsules, the diameter of which can reach up to
8 mm. It has been reported that kerosene was encapsulated this way using a solution
of polyvinyl alcohol and sodium alginate in water/glycerol. The wall material was
hardened in calcium chloride solution.1
6
In the air suspension coating or fluidized bed coating, fine solid core materials
are suspended by a vertical current of air and sprayed with the wall material solution
(Figure 1.1). After the evaporation of the solvent, a layer of the encapsulating material
is deposited onto the core material. The process can be repeated to achieve the desired
film thickness. The size of the core particle for this technique is usually large, with a
100 µm minimum particle size. Smaller particles tend to aggregate or get carried away
by the exhaust air. The technique has been applied to encapsulate pharmaceuticals,
seeds and foodstuffs.1,2
Figure 1.1: Scheme showing microencapsulation by air suspension coating.
Spray drying is to spray an emulsion or suspension in a stream of hot gas, generally
air and occasionally inert gas such as nitrogen. Polymers are dissolved in a solvent
containing the additives to be encapsulated. During spraying, the atomized droplet
shrinks as the solvent evaporates, leaving the additives surrounded by polymer.5 The
resulting microcapsules are solid and free-flowing. The core material may be sprayed
from an inner nozzle and the encapsulating material from a concentric ring nozzle.
7
Although the high temperature in spray drying is problematic for some thermally labile
materials, it is the main method for encapsulating flavors in the food industry due to its
low cost,5 which is 30-50 times cheaper than freeze-drying.18
An alternative to spray drying is spray chilling or cooling. A heated emulsion
or suspension containing molten wall material and aqueous or solid core material
is sprayed into a chamber containing cool air or liquid. The cooling solidifies the
coating material, resulting in microcapsules. Spray chilling or cooling can be used
to encapsulate water-soluble ingredients, such as water-soluble vitamins and enzymes.
A variation of the method includes solvent extraction while cooling. For example,
droplets of polymer/drug solution can be sprayed into liquid nitrogen containing frozen
ethanol, and hardened at −80 °C wherein solvent extraction occurs. By this way,
proteins can be encapsulated in polymer micro-particles without significant loss of
biological activity.19 There is another approach to the drying process, freeze-drying
or lyophilization. The atomized droplets can be frozen, such as in liquid nitrogen, and
the solvent can be removed by sublimation in vacuum.
Co-crystallization is a process to encapsulate ingredients between sucrose crystals.
Although sucrose crystals are solid, monoclinic and spherical and not suitable for
encapsulation, aggregates of the crystals form when they are spontaneously crystallized
from a supersaturated solution. The crystal aggregates are in the size range of 3 to
30 µm and can entrap materials.20,21 First, the sucrose syrup is concentrated to the
point of supersaturation, then the material to be encapsulated is added, and the syrup
is mixed to induce nucleation and agglomeration. It is important to control the rates of
nucleation and crystallization during the process. Numerous types of flavor ingredients
can be encapsulated by this way.
8
Liposomes are hollow microcapsules of phospholipids that form spontaneously
when phospholipids are dispersed in water. To encapsulate material in the core of
liposomes, phospholipids are usually dissolved in organic solvents, such as chloroform,
and mixed with a solution containing the material to be encapsulated. The solvent is
evaporated to form dry lipid film, which is then hydrated in buffer solution to form
liposomes.22,23 The size range is from 25 nm to several micrometers in diameter.
Aqueous or lipid-soluble materials can be entrapped in liposomes, which have been
used to deliver vaccines, hormones, enzymes, and vitamins.24 The encapsulated
ingredients can be released from liposomes by heating above the transition temperature
of the phospholipids, typically around 50 °C. Above the transition temperature, the
liposome bilayer is broken and the content is released immediately.3 The electrostatic
charge, pH, permeability and stability of liposomes can be more easily controlled
than for other types of microcapsules. However, poor encapsulation efficiency and
the lack of a continuous production process limit large-scale use. The use of organic
solvents is also a concern for some applications. Both the lipid and ingredients to be
encapsulated cane be lost during the solvent evaporation process.2,25 Recently micro-
fluidic techniques have been developed that allow high efficiency encapsulation into
liposomes in a solvent-free continuous process.26,27 However, another challenge is that
the liposomes must be kept in dilute aqueous suspension, which is not favorable for
large-scale production, storage and shipping.3
Coacervation is an encapsulation technique based on polymer phase separation. The
core material is dispersed in a continuous phase in which polymer is dissolved, and the
polymer is then gradually deposited onto the core material by inducing precipitation
by adding non-solvent for the polymer, adjusting pH, ionic strength or temperature
9
(Figure 1.2).8,28 Commonly used precipitants or nonsolvents include silicone oil,
vegetable oil, light liquid paraffin and low molecular weight polybutadiene.7 When
phase separation happens, very fine coacervate droplets appear at first, and they tend to
coat solid dispersed particles. The droplets coalesce until a coherent coacervate phase
appears surrounding the solid particles. Finally, the coating is solidified by heating,
crosslinking or removing solvents by exposing to excess amount of another nonsolvent,
such as hexane, heptane, and diethyl ether. The microcapsules are collected by filtration
or centrifugation, washed with solvents, and then dried. Coacervation is efficient and
can produce microcapsules with a broad range of sizes. However, it is complicated and
expensive, so it is not broadly used in industry.2 Another disadvantage of coacervation
is that formation of large aggregates is hard to avoid, as extremely sticky coacervate
droplets frequently adhere to each other before complete phase separation.
Figure 1.2: Scheme showing microencapsulation by coacervation.
Emulsification/solvent evaporation or extraction is mainly used to encapsulate
hydrophobic materials through oil-in-water (o/w) emulsification process. Polymer
is dissolved in a water-immiscible and volatile organic solvent, and the material to
be encapsulated is dissolved or suspended in the polymer solution. The mixture
is then emulsified into water containing emulsifier (Figure 1.3).29,30 The solvent in
the emulsion is removed by evaporation at high temperature or reduced pressure or
10
extraction in a large amount of water, leaving solid particles.6 The solvent removal
rate will affect the morphology of the final particles. Such rate is determined by
temperature, polymer solubility, solvent, and pressure.31–33 The micro-particles are then
collected and dried to eliminate the residual solvent. However, this method is only
effective for hydrophobic materials as hydrophilic materials may not be dissolved in
the organic solvent and diffuse out or partition from the dispersed oil phase into the
aqueous phase.6 To encapsulate hydrophilic materials, oil-in-oil (o/o) emulsification
is used. In this method, water-miscible organic solvent is used to dissolve polymer
and hydrophilic materials to be encapsulated, and hydrophobic oils are used as the
continuous phase.34 A cosolvent can be added into the dispersed phase to help dissolve
the hydrophilic material or the hydrophilic material can be dispersed as fine particles in
the dispersed phase.6 For most water-soluble materials, water-in-oil-in-water (w/o/w)
methods are used.35,36 An aqueous solution of hydrophilic materials is emulsified into
organic solvent in which polymer is dissolved to form the primary water-in-oil (w/o)
emulsion. The primary emulsion is then transferred into water containing an emulsifier
under vigorous mixing, giving rise to a w/o/w emulsion. To obtain micro-particles,
the solvent is removed by evaporation or extraction. However finding conditions that
provide high encapsulation efficiency can be difficult.37
1.2 Liquid and Supercritical Carbon Dioxide
Compressed carbon dioxide in the liquid or supercritical state is attractive as a
solvent in microencapsulation processes. Carbon dioxide is non-toxic, non-flammable,
and inexpensive. The high volatility of carbon dioxide allows it to be easily separated
11
Figure 1.3: Scheme showing microencapsulation by emulsification/solvent evapora-tion.
from polymeric materials by lowering pressure. The supercritical fluid state is reached
when the temperature and pressure of a substance are above its critical temperature
and pressure. For carbon dioxide, the critical temperature is 31 °C and the critical
pressure is 74 bar,38 as shown in Figure 1.4. Above the critical temperature, the
density can be continuously varied from gas-like to liquid-like without undergoing
a phase transition. Supercritical fluids display a hybrid of the properties that are
typical for a liquid and a gas, including the ability to dissolve solids, miscibility with
permanent gases, high diffusivity, and low viscosity.38,39 Since the solubility parameter
is dependent on density, the solvating power of a supercritical fluid can be fine-tuned
by adjusting pressure or temperature. Although it is a good solvent for many non-polar
volatile materials,40 CO2 is a poor solvent for most highly polar or high molecular
weight materials under moderate pressure and temperature. Generally the solubility
of a substance in supercritical fluid increases with vapor pressure, with gases being
12
completely miscible.41 Organic solvents, such as methanol, can be added to increase
the solvent power of supercritical CO2.42
Figure 1.4: Phase diagram of CO2.
Supercritical CO2 can be used as a medium for polymer synthesis and processing.
Its poor solvent power for most polar and high molecular weight materials has led to
applications in polymer fractionation,43 extraction, purification,44 and polymer particle
formation by anti-solvent precipitation.45 Low cohesive energy density polymers, such
as fluorocarbons and silicones, are the only types of polymers that have appreciable
solubility in CO2 at moderate temperature and pressure. The high solubility of
fluorinated polymers in CO2 has led to applications in coatings,46 lithography,47 and
formation of water-in-CO2 emulsions.38,48 Supercritical CO2 is also an excellent non-
solvating, porogenic diluent for synthesizing porous polymers.49 Although elevated
pressure and specialized equipment are required, supercritical CO2 is attractive because
of the ease of solvent evaporation and reduction in use of harmful organic solvents.
13
Liquid CO2 is also desirable for many of the same reasons as supercritical CO2.
The liquid state is reached when a substance is compressed above its vapor pressure at
a temperature below its critical temperature. Near room temperature, the vapor pressure
of CO2 is about 60-70 bar. The transition from liquid to supercritical is continuous when
it is heated above its critical temperature (31 °C) while pressure is maintained above
its critical pressure (74 bar), unlike the discontinuous liquid/vapor phase transition that
occurs at the vapor pressure when temperature is below the critical temperature. When
slightly below the critical temperature and pressure, or near-critical, liquid CO2 exhibits
many solvent and transport properties similar to those of supercritical CO2.
Although CO2 is a poor solvent for most polymers, it has substantial solubility
in many polymers, which results in decrease in the glass transition temperature (Tg)
of polymers or plasticization, even at modest pressure. The reduction of Tg is a
thermodynamic effect due to intermolecular interaction between CO2 and polymer.
Stronger interaction will enhance the depression.50 The glass transition temperature
of polystyrene was reduced by up to 50 °C when exposed to CO2 under the pressure
of only 25 bar.51 When polymer is exposed to supercritical CO2, it is swollen, and the
free volume in the polymer is increased so that the diffusion of additives in polymers is
significantly enhanced. The diffusivity of dimethyl phthalate in poly(vinyl chloride) is
6 orders of magnitude higher under supercritical CO2 than without CO2.52 A cosolvent
may promote the diffusion of additives in polymer under supercritical CO2. The
addition of ethanol has been shown to increase the diffusion coefficient of a dye over
that with pure CO2.53
Impurities in polymers, such as unreacted monomers, residual solvents, catalysts,
and byproducts can adversely affect the use of polymer products, since they can
14
change the taste, color, toxicity or thermophysical properties of polymers through
plasticization or depolymerization.54 Excess impurities are not desirable for both
environment and health, so strict regulations have been enacted to limit permissible
levels of various impurities. Usually polymers are purified by vacuum, steam stripping,
or solvent extraction. In some cases these methods are not sufficient to reduce the
residual impurities to the permissible levels. Supercritical extraction from polymers can
produce high-purity, high-quality products with lower energy cost.55 When polymer is
exposed to supercritical CO2, the diffusion of impurities out of polymer is significantly
enhanced. Supercritical fluid/polymer systems are highly thermodynamically nonideal,
and they can show significant volume change on mixing. Such nonideal volumetric
behavior can cause convection induced by diffusion, which can influence the transport
of impurities out of polymer. When pressure is reduced and supercritical CO2 diffuses
out of the polymer, the rate of removing CO2 from the polymer can be fast compared
to polymer relaxation to its un-swollen state, which can lead to a coupling between the
polymer relaxation and the mass transfer, indicating shorter depressurization will favor
extracting impurities out of polymer.54
Enhanced diffusivity of additives in polymers exposed to supercritical CO2 can also
be used to impregnate additives into polymer. Generally there are three steps in the
impregnation. First, the polymer materials are exposed to supercritical CO2 for a while;
then the solution of additives in CO2 is introduced and the solute is transferred from
CO2 to polymer. Last, CO2 is released and the solute is trapped in the polymer material.
The rate of releasing CO2 after the impregnation is related to polymer relaxation rate.
Slow release of CO2 allows for the escape of CO2 from polymer matrix before the
polymer recovers from the swollen state, or the entrapped CO2 may lead to foaming.50
15
When suspensions of polymer particles in water are exposed to supercritical CO2 with
the presence of additives in water, the transport of the additive into polymer particles
can also be enhanced. After releasing CO2, additives can be trapped in colloidal
polymer particles,56 as shown in Figure 1.5. Although the presence of CO2 may require
some appropriate surfactants to stabilize polymer particles in water, it does not require
harmful organic solvents used in conventional microencapsulation and may remove
residual solvents in the premade polymer particles.
Figure 1.5: Scheme showing the encapsulation of additives into polymer colloids withthe help of compressed CO2.
1.3 Temperature Induced Phase Separation
Porous polymers can be made from phase-separated polymer/solvent systems by
evaporating, or sublimating the solvent-rich phase. Porous or hollow microparticles
can potentially be used in producing novel microcapsules. There are several tech-
niques for making porous polymers, such as phase separation,57 porogen leaching,58
emulsion/freeze-drying.59 To some extent, these techniques rely on thermally induced
phase separation and freeze-drying to remove solvent, and create porous structures.57,60
Freeze drying usually consists of freezing an aqueous solution or suspension and
16
subsequent drying under vacuum.61 During the freezing, phase separation between
water and solute, ice crystal growth, and solute cryo-concentration take place, as
shown in Figure 1.6. The growing ice crystals exclude solute molecules, such as
polymers, from its freezing front to the boundaries between adjacent ice crystals. The
subsequent drying process, during which ice crystal sublimation and desorption of
the bound water occur, leads to macroporous structures in which the empty spaces
are occupied originally by ice crystals. The pore size, pore geometry, and porosity
can be tuned by cryogenic parameters, such as cooling rate, supercooling degree,
ice nucleation temperature, temperature gradient, and solute concentration.62–65 To
produce large ice crystals or pores, the freezing temperature should be high and the
time for crystallization should be long. To produce small crystals or pores, the freezing
should be done under low temperature and/or high pressure conditions and the freezing
rate should be high to reduce the time available for ice crystals to grow.60 During
crystallization, redistribution of solute is determined by its solubility in the crystal
and its ability to diffuse away from the crystal interface before it is overgrown.66 The
ability of ice crystal growth depends on the adsorption/desorption balance of the solute
at the ice crystal surface. Irreversible adsorption will stop ice crystal growth, while
complete desorption would allow ice crystals growing freely. Actually the adsorption
and desorption at the ice surface are in dynamic equilibrium. Very low crystal growth
speed leads to a pure crystal and highly enriched solution, unless the solute possesses
crystallographic similarities with the growing crystal that favors adsorption. While at
high crystal growth speed, the solute is unable to diffuse away from the growing crystal
and is completely engulfed among crystals. The size of ice crystals is also related to
solute concentration and the size of solute. The effectiveness of a solute in inhibiting
17
crystal growth depends on the extent of ice crystals’ surface that is covered by such
a solute. Therefore large solutes or high solute concentration leads to small crystals,
while small solutes or low solute concentration leads to large ones.
Figure 1.6: Temperature induced phase separation between water and solutes.
With crystal growth aligned under directional freezing, well-defined porous struc-
tures can be obtained, either in the form of three-dimensional porous structure or two-
dimensional oriented surface pattern.65,67 The directional freezing can be simply done,
for example, by lowering a vessel containing the solution into a cold bath, such as
liquid nitrogen, at a controlled rate.60 The structure is heterogeneous in the freezing
direction. In the region closest to the initial contact with the cold bath, no porosity is
observed after the subsequent freeze-drying process and the material is dense. The next
region to the dense part has a cellular morphology, and the next one is lamellar with
long parallel pores aligned in the direction of movement of the ice front.61 When pure
18
water is frozen in liquid nitrogen, amorphous ice does not form. To get amorphous ice
from pure water, a freezing rate of about 106 °C/s is required.68 However, impurities in
water affect ice crystallization. When an aqueous solution or suspension is immersed
partially into liquid nitrogen, amorphous ice forms in the immersed portion, and no
matter segregation happens, so that porous structure does not form. Meanwhile, an ice
front runs along the non-immersed portion. The temperature at the ice front is higher
than that of liquid nitrogen. The further the ice front moves away from the immersion
level in liquid nitrogen, the more temperature increases. At last the temperature is not
low enough for supercooling, and so crystalline rather than amorphous ice is formed.
Away from the immersion level, ice crystallization determines solute redistribution and
leads to porous structure after freeze-drying. The pore size depends on the distance
from the immersion level in liquid nitrogen. Homogeneous porous structure can be
made by continuous immersion at a constant rate into liquid nitrogen.61
The temperature induced phase separation is a simple and generic way for preparing
porous structure. A lot of porous materials have been made by this way, such as
poly(DL-lactic-co-glycolic acid),57,69 gelatin,64 and poly(vinyl alcohol).65 The process
avoids chemical reaction and further purification. Aligned porous materials by
directional freezing can be used in micro-fluidics,70 molecular filtration,71 and tissue
engineering.72 In addition to aqueous solution or suspension,73–75 the process can be
applied to organic solution or suspension. It has also been reported that CO2 can be
used as the solvent for the process to make porous polymers. A solution of sugar
acetate in liquid CO2 was frozen in liquid nitrogen and the solid CO2 was removed by
sublimation, leading to porous structures.67
19
1.4 Outline of the Thesis
Chapter 2 describes encapsulating fluorescent CdSe/ZnS quantum dots (QDs) into
trans-polyisoprene (PI) particles by emulsification/solvent evaporation to create novel
optically functional particles useful for bio-labeling and sensing. The double bonds in
polyisoprene (Figure 1.7) can be reacted further to crosslink the particle, improving the
stability of QDs.
Figure 1.7: Chemical structure of trans-polyisoprene.
The oil-in-water emulsification as described in Chapter 2 is not effective for
encapsulating hydrophilic materials. For encapsulating hydrophilic species, Chapter 3
describes a method with liquid and supercritical carbon dioxide as a processing solvent.
The hydrophilic species were made hydrophobic by ion pairing without covalent bond
formation. And the resulting hydrophobic ion-paired dyes diffused into polystyrene
particles plasticized by CO2.
To encapsulate hydrophilic materials without hydrophobicity conversion such as
by ion pairing, hollow polystyrene microparticles were made by temperature induced
phase separation as described in Chapter 4. The hollow particles were subsequently
converted to microcapsules, encapsulating aqueous solution inside.
Chapter 5 describes the use of the novel method developed in Chapter 4 to
encapsulate a water-soluble drug, procaine hydrochloride, in biodegradable poly(DL-
20
lactide) (Figure 1.8) microcapsules. Effect of experimental conditions on drug
encapsulation was investigated, and the drug release from the microcapsules were
measured.
Figure 1.8: Chemical structure of poly(DL-lactide).
Chapter 6 gives overall conclusions of the thesis and describes potential future work
building upon the current study.
21
Chapter 2
Fluorescent Quantum Dot-PolymerNanocomposite Particles byEmulsification/Solvent Evaporation∗
CdSe/ZnS quantum dots (QDs) were incorporated into biocompatible polyisoprene
(PI) particles by microencapsulation through emulsification/solvent evaporation, a tech-
nique that is facile, robust, and inexpensive. Emulsification/solvent evaporation results
in QDs encapsulated into the particle core without requiring chemical modification of
the as-prepared QDs. The PI can be easily cross-linked after encapsulation to enhance
the fluorescence stability of the QDs. The resulting PI-QD nanocomposite particles
form as colloidally stable suspensions in water that exhibit stable fluorescence for
months.
2.1 Introduction
Semiconductor quantum dots (QDs) are ideal fluorophores for biological imag-
ing:76,77 their emission wavelength can be tuned by simply changing the particle size,
∗Yin, W.; Liu, H.; Yates, M. Z.; Du, H.; Jiang, F.; Guo, L.; Krauss, T. D. Chem. Mater. 2007, 19,2930-2936.
22
and simultaneous excitation of different-sized QDs can be achieved using only a single
wavelength,78 allowing facile multicolor coding for high-throughput assays. However,
the surface of QDs, as synthesized, is hydrophobic,79–81 making them unsuitable for
direct use in biological environments. Examples of strategies used to make QDs more
biocompatible include exchanging the organic ligands on the QD surface with a more
polar species,76,77,82 coating with a silica layer,83 and encapsulating the hydrophobic
QD in lipid micelles.84 However, none of these methods completely prevent individual
QDs from photooxidation, and thus fluorescence efficiencies and the shelf life of
biocompatible QDs are typically reduced several fold relative to their organic-capped
counterparts.85
Recently, the encapsulation of QDs into polymer colloids has seen growing interest
as a route to improve photostability and for development of colorimetric optical bar
codes for biological sensing.86–90 Several different approaches have been taken includ-
ing solvent swelling of aqueous polymer colloids,86 entrapping QDs in cross-linked
reverse micelles,88 covalently bonding QDs with polymerizable ligands to polystyrene
during suspension polymerization,89,91 entrapment through strong noncovalent interac-
tions such as hydrogen bonding and ionic attraction,92–94 and physically entrapping
QDs into particles formed by emulsion polymerization90 or sol-gel synthesis.95 In
the present study, we chose to investigate QD/polymer nanocomposite formation
through emulsification/solvent evaporation, a technique commonly used to create
pharmaceutical/polymer composite particles for drug delivery.96,97 Encapsulation of
QDs through emulsification/solvent evaporation has several potential advantages over
other encapsulation methods. Emulsification/solvent evaporation can encapsulate QDs
without chemical modification of the QD surface, unlike methods that copolymerize
23
reactive QDs.89,91 The reactive QDs can also adversely affect the nucleation and growth
of polymer particles during copolymerization, leading to a widening of the particle size
distribution.91 The QDs are encapsulated in the core of the polymer particles during
emulsification/solvent evaporation. Methods that incorporate QDs by solvent swelling
of preformed aqueous polymer colloids86 generally lead to a large fraction of surface-
adsorbed QDs, with a relatively small fraction actually encapsulated in the core of the
polymer particles.98 Finally, emulsification/solvent evaporation also allows the surface
chemistry of the nanocomposite particles to be easily controlled through the choice of
surfactant used to form the emulsion. Control of surface chemistry is important to allow
subsequent bioconjugation to the polymer-QD particles.
In the emulsification/solvent evaporation technique, the polymer and desired
additive are first dissolved in a mutual solvent. Then, the polymer/additive solution
is emulsified into an aqueous surfactant solution and the polymer solvent is evaporated,
leaving a polymer/additive composite colloid stabilized by surfactant. In the present
study, the additive to be encapsulated is CdSe QDs coated with alkane ligands.
The alkane ligands stabilize the QDs in hydrophobic solvents such as chloroform
and hexane. To form QD/polymer composite particles by emulsification/solvent
evaporation, the polymer must dissolve in a solvent compatible with the QDs. trans-
Polyisoprene was chosen because it is soluble in many of the same solvents as the QDs,
including alkanes, so it is expected that polyisoprene (PI) should interact favorably with
the alkane-coated quantum dots. The polymer chain is unsaturated so that it is easy to
chemically cross-link after processing, ensuring that the QDs remain entrapped after
encapsulation. PI is also highly desirable because it is biocompatible, nontoxic, and
noncarcinogenic.99
24
2.2 Experimental Section
Materials. Acrylic acid, trans-polyisoprene (PI), lauric acid, 2,2′
-azobisiso-
butyronitrile (AIBN), and sodium hydroxide(NaOH) were purchased from Aldrich.
Hexane (AR grade) and methanol (SpectrAR grade) were obtained from Mallinckrodt
Chemicals. Sodium dodecyl sulfate (SDS) was purchased from J. T. Baker. Deionized
water was used in the experiments.
The CdSe/ZnS quantum dots (Figure 2.1) were provided by Professor Krauss’
group.100 The core CdSe QDs were synthesized in various sizes, coated with a thin shell
of ZnS, and capped with tri-n-octylphosphine and trioctylphosphine oxide following
procedures similar to those in the literature.79,80,101 The capped QDs were stored in
hexane. Prior to microencapsulation, methanol was added to precipitate the QDs. The
precipitated QDs were separated by centrifugation and then dried in a vacuum oven.
Figure 2.1: CdSe/ZnS quantum dots capped with trioctylphosphine oxide.
Microencapsulation of QDs in Un-cross-linked PI Particles. The dried QDs
were dissolved in 5 mL of chloroform to make a QD solution, and its concentration
25
was determined by absorption spectroscopy. Then 0.15 g of PI was dissolved in
5 mL of chloroform to form a polymer solution. The QD solution and the polymer
solution were mixed together. In a separate flask, 0.15 g of lauric acid and 0.045 g of
sodium hydroxide were dissolved in 50 mL of water to produce an aqueous surfactant
solution. The solution of polyisoprene and QDs in chloroform was then poured into the
aqueous surfactant solution and purged with argon (Ar) for 20 min. The mixture was
homogenized at 24000 rpm for 1 min (IKA WORKS, T25 basic ULTRA-TURRAX
homogenizer) and then stirred using a magnetic stirrer for 5 h under Ar to evaporate the
organic solvent chloroform. After solvent evaporation, polymer particles formed and
entrapped the QDs. During the experiment, the system was shaded from light to avoid
any possible photobleaching of QDs.
Microencapsulation of QDs in Cross-linked PI Particles. A polymer solution
containing QDs and an aqueous surfactant solution were made as stated above. Then
0.020 g AIBN and 0.020 g acrylic acid were added into the polymer solution. The
solution thus obtained was poured into the aqueous surfactant solution and bubbled
with Ar for 20 min. The mixture was homogenized at 24000 rpm for 1 min and
then transferred to a round-bottom flask into which 0.050 g of SDS had been added.
The flask was sealed and purged with Ar for 15 min and then put into an oil bath at
70 °C. The polymerization was carried out for 6 h. The cross-linked latex produced
was transferred to a beaker after cooling and stirred overnight under Ar to allow the
organic solvent to evaporate.
Particle Size Measurement. The hydrodynamic particle diameter and polydisper-
sity were determined by dynamic light scattering (Brookhaven Instruments model 90
Plus) using cumulant analysis for polydispersity determination.
26
Fluorescence Imaging and Spectroscopy. Particle fluorescence and dark field
scattering images were acquired on a Nikon inverted confocal microscope (Eclipse
TE300) illuminated with 488 nm (Ar laser) and white light, respectively, and detected
with either a cooled CCD camera (Versarry 512B, Princeton Instruments) or a digital
camera (Nikon Coolpix 995). Colloidal QD fluorescence spectra were taken with a
modular Acton Research fluorometer. Particle fluorescence spectra were acquired by
coupling an Acton spectrometer (SpectraPro 300i) to the output port of the microscope.
2.3 Results and Discussion
The colloidal particles prepared by the emulsification/solvent evaporation have
broad size distribution. The effective diameter determined by dynamic light scattering
is about 400 nm, and the half width of the size distribution curve is about 200 nm.
For this initial study, all particles were used as made by simple emulsification with a
homogenizer to investigate the encapsulation of QDs and no effort was made to reduce
the particle size distribution. The broad particle size distribution is a result of the
broad size distribution of the precursor emulsion and is not an inherent limitation of the
emulsification/solvent evaporation technique. Uniform polymer particles can be made
by emulsification/solvent evaporation starting from a monodisperse emulsion created
by microfluidic flow focusing,102 membrane emulsification,103–105 or microchannel
emulsification.106
Emulsification/solvent evaporation was carried out to encapsulate a mixture of
yellow and red quantum dots with a yellow:red number ratio of 2:1. The resulting
nanocomposite particles were 403 ± 10 nm in diameter as measured by dynamic light
27
scattering with a polydispersity index of 0.25. The estimated theoretical QD loading
was approximately 1270 QDs per PI-QD polymer particle assuming a particle size of
403 nm and that all of the QDs added were incorporated evenly into the particles.
The polymer colloid was diluted 10 fold by water and the particles were placed onto
a glass slide by spin-coating one drop (∼20 µL) of the resulting suspension. White
light scattering and fluorescence images of the hybrid particles are shown in Figure 2.2.
Figure 2.2A is a white-light dark-field scattering intensity image obtained by the CCD,
and Figure 2.2B is the corresponding true color fluorescence image obtained for the
same sample. The fluorescence image shows that the location of the QD fluorescence
corresponds to the location of the large PI-QD nanocomposite particles. However, the
relative brightness of each nanocomposite particle, as expected, was highly nonuniform
due to the particle size polydispersity. It appears that QDs were distributed evenly
inside the particles, and not simply adsorbed on the surface. The fluorescence intensity
of particles can be adjusted by changing QD concentration before solvent evaporation.
(A) (B)
Figure 2.2: Optical microscopic images of un-cross-linked PI-QD particles: (A) dark-field scattering and (B) true-color fluorescence.
28
Figure 2.3 shows the fluorescence spectrum (solid line) of a single PI-QD particle
from the same sample shown in Figure 2.2. The spectrum has two sharp peaks resulting
from yellow QDs (fluorescence maximum = 560 nm) and red QDs (fluorescence
maximum = 620 nm). Compared to the fluorescence spectrum of a hexane solution
of yellow and red QDs with the same molar ratio of 2:1, respectively, the relative
fluorescence intensity of the larger (red) QDs was enhanced relative to the smaller
(yellow) QDs when entrapped in the PI-QD particles. (Note that the yellow QDs
have a fluorescence quantum yield (QY) of 12% compared to only 1% for the red
QDs.) The changes in the fluorescence spectrum are consistent with nonradiative
(Forster) energy transfer from the yellow to the red QDs.107 If the QDs were uniformly
distributed in the PI, the average separation would be ∼30 nm, much too far apart
for efficient energy transfer. Therefore, our observation of inter-QD energy transfer
suggests aggregation of the QDs in the polymer particle likely due to micro-phase
separation during the encapsulation process. Local close-packing was also recently
observed for QDs with polymerizable ligands incorporated into polystyrene during
dispersion polymerization.91 The spatial distribution of QDs inside the PI-QD particles
is important for multicolor optical labeling. Because of the changes in fluorescence
emission caused by micro-phase separation in the polymer, the resulting multicolor
fluorescence spectrum from the particle will not represent the relative concentrations
of the QDs inside. Even for samples in which the fluorescence spectrum of the QDs is
altered within the polymer, it is still possible to achieve multicolor coding by adjusting
the number ratio of QDs, as demonstrated recently.108
The emulsification/solvent evaporation technique is a facile route to encapsulate the
QDs into the core of the polymer particle without free radical polymerization. However,
29
Figure 2.3: Normalized single-particle fluorescence spectrum of un-cross-linked PIcontaining two different-colored QDs (solid line) and the fluorescence emissionspectrum of the same mixture of QDs in hexane (dashed line).
the PI particle is in the rubber state at room temperature (glass transition temperature
of trans-polyisoprene is −68 °C) and thus the QDs have potentially high mobility
in the polymer that would allow them to aggregate. Polymerization can optionally
be employed after microencapsulation to cross-link the polyisoprene to reduce QD
mobility. Lower mobility will inhibit QD aggregation and prevent migration to the
surface of the polymer particles. In addition to preventing QD migration, cross-
linking may also improve the barrier properties of polyisoprene with respect to oxygen.
Previous studies have demonstrated that highly cross-linked polymers display improved
barrier properties to oxygen when compared to un-cross-linked polymers.109 Since
oxygen plays a major role in degradation of QD fluorescence, it is likely that cross-
linking the polymer will result in increased stability to oxidation and longer shelf life.
30
Cross-linked PI-QD particles were made containing a mixture of yellow and red
QDs with the same yellow:red number ratio as in the un-cross-linked sample shown in
Figure 2.2. Figure 2.4 shows digital photographs of two vials, each containing 0.3 wt%
aqueous suspensions of PI-QD nanocomposite particles under UV light illumination.
The upper vial contained cross-linked PI-QD particles and the lower one contained
un-cross-linked particles. Both types of PI-QD particles were loaded with the same
yellow and red QDs. A series of images are shown for both vials, taken immediately
after preparation, 45 days later, and 261 days later. It is apparent that the cross-
linked samples display enhanced stability against photooxidation when compared to
the un-cross-linked sample. The un-cross-linked sample appears to completely lose
fluorescence after 261 days in aqueous suspension and also appears to change color
from orange to red over time. The same mixture of yellow and red QDs in hexane
looks similar in color to the cross-linked sample. The un-cross-linked sample is
orange just after preparation, possibly due to higher polymer chain mobility that
allows QDs to aggregate during the evaporation process, resulting in increased energy
transfer to the red QDs. The shift over time to red color for the un-cross-linked
sample is likely due to differences in the rates of oxidation for the yellow versus red
QDs. The images of fluorescence decay are consistent with visual observations of the
samples. While the cross-linked sample is much more stable after preparation, we
observed an approximately 10 fold decrease in the fluorescence intensity of the PI-QD
nanocomposite caused by the cross-linking reaction. Others have also reported that free
radical polymerization with AIBN is detrimental to QD fluorescence.110 Even though
some QDs were quenched by the free radical polymerization, the remaining fluorescent
QDs were stable for extended periods. To increase overall fluorescence from the cross-
31
linked samples, the concentration of QDs incorporated into the PI-QD particles could
be increased.
(A) (B) (C)
Figure 2.4: Digital photographs of aqueous suspensions of 0.3 wt% PI-QD particlesunder UV illumination. Both samples have yellow and red QDs encapsulated in anumber ratio of 2:1, respectively. The upper sample is cross-linked, while the lowersample is un-cross-linked. Pictures were taken (A) immediately after preparation, (B)45 days after preparation, and (C) 261 days after preparation.
32
2.4 Conclusions
Fluorescent CdSe(ZnS) quantum dots (QDs) were successfully encapsulated into
biocompatible polyisoprene (PI) particles using an emulsification/solvent evaporation
method. The simple encapsulation method results in QDs encapsulated into the core
of the PI particles without requiring surface modification of the QDs. The PI can be
surface-modified with carboxyl groups during encapsulation to facilitate subsequent
bioconjugation and selective binding.100 The polyisoprene is easily cross-linked by
adding a free radical initiator during encapsulation. Cross-linking was shown to greatly
enhance the long-term fluorescence stability of the encapsulated QDs. The cross-linked
PI-QD nanocomposite particles displayed strong and stable fluorescence emission after
months in aqueous suspension. The fluorescence spectra of mixtures of two different-
sized QDs change in PI as compared to their solution spectra, suggesting energy transfer
between QDs due to their aggregation during the encapsulation. Even though there
appears to be some QD aggregation, different emission peaks were clearly resolved,
indicating that the particles are suitable for multicolor coding. These fluorescent
hybrid nanocomposite particles have potential application in genomics, drug discovery,
and other applications that could exploit the high-throughput afforded by multicolor
coding.
33
Chapter 3
Hydrophobic Ion Pairing to EnhanceEncapsulation of Water-SolubleAdditives into CO2-Swollen PolymerMicroparticles∗
Ionic dyes were successfully encapsulated in colloidal polymer particles by a CO2-
based microencapsulation technique. The water-soluble dyes were made hydrophobic
by forming ion pairs with several types of alkyl quaternary ammonium cations.
The hydrophobic ion pairs were then encapsulated in preexisting size monodisperse
polystyrene particles dispersed in water. High-pressure carbon dioxide swelled and
plasticized polystyrene and thus facilitated mass transport of the dye into the particles.
The results show that the particles maintain their size and morphology after exposure to
CO2, and that ion-paired dyes have significantly higher loading in the polymer particles
than the original dyes. The effects of cosolvent and surfactant are also discussed.
∗Yin, W.; Dong, Z.; Chen, X.; Finn, N.; Yates, M. Z. J. Supercrit. Fluids 2007, 41, 293-298.
34
3.1 Introduction
Functional additives can be incorporated into polymer colloids to form composite
particles with adjustable optical, magnetic, catalytic, coating, and controlled release
properties.111–113 The composite particles can be used in a wide range of applications,
such as drug delivery, cell labeling, and coating. In many applications, particle
properties such as size, size distribution, composition, and surface characteristics must
be carefully controlled. For example, when used for drug delivery, precise control of
particle size and size distribution is required to decrease side-effects of the drugs for
better performance. The size of the particles influences the drug release rate and also
can affect the location of accumulation of particles in the body.114
The most widely used encapsulation methods such as emulsification/solvent evapo-
ration,96 particle coating,115 coacervation,116,117 and spray drying118 provide limited
control of particle size and polydispersity. Most techniques also require harmful
solvents that are difficult to remove.We have recently developed a carbon dioxide-based
microencapsulation technique in which additives are impregnated into a preformed
aqueous latex.56 Compressed carbon dioxide is used to swell the aqueous latex
particles and facilitate transport of additives into the particles. The CO2-based
process eliminates the need for harmful organic solvents and decouples the particle
formation and microencapsulation, offering improved control over particle size and
size polydispersity. The CO2 is also easy to separate from the particles due to its
high volatility. However, the drawback of the CO2-based approach is that it relies on
spontaneous diffusion of the additive to be encapsulated into the CO2-swollen polymer
particles. The additive must be hydrophobic to partition into the polymer from the
35
aqueous phase.Water-soluble additives tend to remain in the aqueous phase surrounding
the polymer particles, resulting in very low encapsulation efficiency.56
Ionic additives are particularly difficult to encapsulate using the CO2-based process
because of their strong interaction with water. One possible way to overcome the
limitation in encapsulation efficiency of ionic compounds is to increase the solubility
of the ionic compound in a nonaqueous environment by forming a hydrophobic ion
pair.119,120 In the ion pairing process, a hydrophilic counter-ion, such as a sodium cation,
is replaced by a hydrophobic counter-ion, such as a long alkyl quaternary ammonium
cation.119,121 Ion pairing is only an ionic interaction and there is no new covalent
bond produced. Therefore, it does not affect other properties of the original additives.
This technique has been widely used for pharmaceuticals in drug extraction,122,123 for
enhancing drug absorption,124,125 for analysis by HPLC.126 Ion pairing has also been
used to enhance encapsulation of drugs into poly(L-lactide) microspheres made by
precipitation with a compressed antisolvent process.127
The objective of this study is to determine if hydrophobic ion pairing will allow
water-soluble ionic additives to be incorporated into aqueous polymer colloids through
CO2-based microencapsulation. Two anionic dyes, bromothymol blue and rose bengal,
were examined as model ionic compounds for encapsulation. The molecular structures
of the two dyes are shown in Figure 3.1. Both are sodium salts, with bromothymol
blue having one sodium cation and rose bengal having two. Ion pairs were formed
by replacing the sodium cations with hydrophobic quaternary ammonium cations. The
transport of the hydrophobic ion pairs into polystyrene colloids swollen with CO2 was
investigated. The results illustrate the promise of ion pairing as one route to enhance
microencapsulation of water-soluble drugs by CO2-based microencapsulation.
36
(A) (B)
Figure 3.1: Molecular structure of: (A) bromothymol blue and (B) rose bengal.
3.2 Experimental Section
Materials. Styrene (99+%), potassium persulfate (KPS, 99.99%), bromothymol
blue (3′
,3′′
-dibromothymolsulfonephthalein sodium salt, dye content 90%), rose bengal
(4,5,6,7-tetrachloro- 2′
,4′
,5′
,7′
-tetraiodofluorescein sodium salt, certified, dye content
90%), tetrabutylammonium bromide (TBABr, 99%), tetrahexylammonium bromide
(THABr, 99%), tetraoctylammonium bromide (TOABr, 98%) and toluene (99.5+%)
were purchased from Aldrich. Dichloromethane (99.9%) was purchased from Fisher
Chemicals. Chloroform (SpectrAR) was from Mallinckrodt Chemicals. Acetone
(HPLC grade) was from Burdick & Jackson. 1-Decanol (>97%) was from TCI. Triton
X-100 (octyl phenol ethoxylate, Figure 3.2) was obtained from J. T. Baker. Absolute
ethanol (200 proof) was purchased from Pharmco Products. Carbon dioxide (SFC/SFE
grade) was obtained from Air Products and Chemicals. Deionized water was used
during the experiments. All chemicals were used as received.
Preparation of microspheres. Polystyrene (PS) microparticles were prepared by
surfactant free emulsion polymerization.128,129 Ninety millilitres of water and 2.2 ml
37
Figure 3.2: Chemical structure of Triton X-100.
styrene were added to a 250 ml round-bottom flask. Five millilitres of water was added
to another smaller flask containing 0.04 g KPS. Both flasks were sealed and purged
with argon for 10 min. The flask containing monomer was heated to 70 °C while
stirred magnetically, and then the KPS solution was injected into the flask containing
monomer by a syringe. The flask gradually became turbid due to polymer particle
nucleation. After 24 h, the reaction was stopped. The solid content of this latex was 1.9
wt%.
Preparation of ion pair. The dye was first dissolved in 5 ml water. Then,
an amount of tetraalkylammonium bromide (Figure 3.3) equimolar to the amount of
sodium counter ions was added along with 5 ml of chloroform. The original dye had no
solubility in chloroform. As the ion pair formed, the dye partitioned from the aqueous
phase into the chloroform phase, which was then collected and evaporated to collect
the ion pairs. The ion pairs were dried overnight in a vacuum oven prior to use.
Figure 3.3: Chemical structure of tetraalkylammonium bromide used for ion pairing.
38
Microencapsulation. Prior to encapsulation, Triton X-100 (0.1 g or 0.4 g) was
added to 10 g of the PS latex, and it was stirred gently for 24 h. The surfactant, Triton X-
100, was added to maintain the stability of PS latex during encapsulation and enhance
emulsification of compressed CO2 into the aqueous latex.130 The latex containing Triton
X-100, 60 mg dye (weight of different ion pairs adjusted to give 60 mg dye molecules),
and 0.15 g cosolvent in specified cases were transferred to a variable volume high
pressure stainless steel vessel described previously,131 as shown in Figure 3.4. The
vessel was sealed and placed in a water bath at 35 °C. 3.5 g CO2 was added to the
vessel by a computer-controlled high pressure syringe pump (ISCO model 260D). The
vessel was then pressurized to 310 bar using CO2 as a hydraulic fluid to compress the
volume-variable vessel. A magnetically coupled stir bar was used to emulsify CO2 into
the aqueous latex. The vessel was maintained at constant temperature and pressure
for 24 h with continuous stirring to impregnate the ion-paired dye into the polymer
particles. The vessel was then depressurized very slowly over about 20 h to minimize
foaming of the polymer particles. The dye-loaded polymer particles were collected
by centrifuge and washed by ethanol to remove any surface adsorbed dye. Ethanol
washes were repeated until the supernatant appeared clear. The particles were dried in
a vacuum oven for 24 h at room temperature prior to characterization.
Particle characterization. Dynamic light scattering (Brookhaven Instruments
model 90 Plus) was used to investigate the size and size polydispersity of the obtained
sample in water. The morphology of the PS particles was observed by scanning electron
microscope (LEO 982 FE-SEM). The dye content in the particles was determined
by measuring the UV-vis absorbance of the sample in chloroform with Perkin-Elmer
Lambda 900 spectrometer, and the calculation for the dye loading was based on
39
Figure 3.4: Variable volume high pressure stainless steel vessel.
standard absorbance curve of the ion-paired dye in chloroform. The absorbance peak
for the ion pair is in the visible region, at which there is no absorption of polystyrene in
chloroform. Rose bengal has a maximum absorbance near 564 nm, while bromothymol
blues absorbance maximum is near 400 nm. Ion pairing had only a slight effect on the
absorbance peak.
3.3 Results and Discussion
The solubility of the dyes bromothymol blue, rose bengal, and their ion pairs
with tetrabutylammonium bromide (TBABr), tetrahexylammonium bromide (THABr),
and tetraoctylammonium bromide (TOABr) was qualitatively measured in toluene
and water by visual observation. Toluene is an aromatic solvent compatible with
polystyrene and was chosen because toluene has a solubility parameter similar to that
of polystyrene.132 It is expected that the trend of solubility of the dyes in toluene will
parallel that in polystyrene. The solubility of the dyes and ion pairs in water and toluene
40
at room temperature is shown in Table 3.1. Both bromothymol blue and rose bengal
are sodium salts, soluble in water and insoluble in toluene. As shown in Figure 3.1,
bromothymol blue has one sodium cation per molecule, while rose bengal has two.
The hydrophobic ion pair was formed by replacing the hydrophilic sodium cation with
a tetraalkylammonium cation. As a result, the solubility of the ion pair is lower in
water and higher in toluene than that of the original dye. Table 3.1 shows qualitatively
that the solubility of ion pairs in toluene increases and that in water decreases as the
length of the alkyl chains in the hydrophobic counter-ion is increased. The data also
show that rose bengal is more hydrophilic than bromothymol blue. When THABr was
used, the ion pair of rose bengal was slightly soluble in toluene, but the ion pair with
bromothymol blue was soluble; when TOABr was used, both ion pairs were soluble in
toluene.
Table 3.1: Water and toluene solubility of bromothymol blue, rose bengal and theirion pairs with tetrabutylammonium bromide (TBABr), tetrahexylammonium bromide(THABr), and tetraoctylammonium bromide (TOABr)
Ion pairing salt Bromothymol blue Rose bengalWater solubility Toluene solubility Water solubility Toluene solubility
None Soluble Insoluble Soluble InsolubleTBABr Slightly soluble Slightly soluble Slightly soluble Slightly solubleTHABr Insoluble Soluble Insoluble Slightly solubleTOABr Insoluble Soluble Insoluble Soluble
In studies of the thermodynamics of hydrophobic ion pairing, it has been shown
that enthalpy change is the driving force for a hydrophobic ion pair to leave an aqueous
phase and transfer to an oil phase because ionic attraction between cation and anion
is stronger in the oil phase.133,134 The enthalpy decrease is due not only to the ionic
41
interaction, but all other intermolecular forces between ion pair and solvent. A previous
study investigated ion pair formation with pharmaceuticals in various solvents and
found that polar solvents such as chloroform had more favorable enthalpic interaction
with hydrophobic ion pairs than nonpolar solvents such as carbon tetrachloride.134 We
also examined solubility in several solvents and found that all of the hydrophobic ion
pairs in Table 3.1 were soluble in polar solvents dichloromethane, chloroform, acetone,
and ethanol. Examination of the molecular structure of the dyes in Figure 3.1 shows
that there are a variety of polar functional groups including hydroxy, carbonyl, and
ether as well as attached halogens that can provide favorable interaction with polar
solvents. Dispersion forces become more dominant as the length of the alkane chains
on the hydrophobic counter ions are increased, allowing the ion pairs to dissolve more
favorably in toluene.
Table 3.2 shows the weight percent of dyes and their hydrophobic ion pairs incor-
porated into the polystyrene latex through carbon dioxide-based microencapsulation.
As expected, the dye incorporation is very low (<0.02 wt%) for both rose bengal and
bromothymol blue when the unmodified dye is used. The amount of bromothymol
blue incorporated into polystyrene is increased for hydrophobic ion pairs. For the
bromothymol blue ion paired with TBABr, having the shortest alkyl chain investigated,
there is no measurable difference in dye incorporation when compared with the
unmodified dye. For bromothymol blue ion pairs with THABr and TOABr, the amount
of dye increases to 0.11 wt%, greater than a five-fold increase when compared to the
unmodified dye. The longer alkyl chains of THABr and TOABr allow the ion pair to
be hydrophobic enough to increase partitioning into the polymer particles. For rose
bengal, ion pairing did not enhance the encapsulation of dye in the polystyrene. None
42
of the rose bengal ion pairs investigated had a measurable difference in encapsulated
amount compared to the unmodified dye. The poor dye incorporation with rose bengal
can be attributed to its relatively low solubility, and to the fact that hydrophobic ion
pairs are more bulky, since two bulky tetraalkyl cations associate with each rose bengal
molecule. The bulky molecules are expected to diffuse more slowly into the polymer.
In addition, the mass transport rate of the ion pair into polystyrene depends on how
well the ion pair is dispersed in the CO2/water emulsion so that the ion pair can be in
contact with the polymer particles.
Table 3.2: Effect of ion pair formation on dye loading of bromothymol blue and rosebengal into polystyrene particles (0.1 g Triton X-100, but no cosolvent was used)
Ion pairing salt Dye concentration in PS particles (wt%)Bromothymol blue Rose bengal
None < 0.02 < 0.02TBABr < 0.02 < 0.02THABr 0.11 < 0.02TOABr 0.11 < 0.02
The polystyrene particles are highly swollen by CO2 at the pressures used for
microencapsulation, and the glass transition temperature of PS is depressed below
the operating conditions.135,136 Other work has shown that colloidal particles can be
more swollen than bulk polymers due to interfacial adsorption of CO2.137,138 As a
result, if the particles lose colloidal stability they will agglomerate and coalesce in the
presence of compressed CO2. Our previous work has demonstrated that an aqueous
PS latex made by surfactant free emulsion polymerization will lose stability when
exposed to compressed CO2 due to changes in pH and ionic strength.130 The surfactant
43
Triton X-100 stabilized the polymer particles during the microencapsulation process.
It also aided in emulsifying CO2 into water, improving encapsulation kinetics.130 The
particle size and morphology was examined before and after encapsulation by SEM,
as shown in Figure 3.5. Figure 3.5A shows that the particles were spherical with a
narrow particle size distribution before the encapsulation. After the encapsulation,
as shown in Figure 3.5B, the particles were still spherical and did not agglomerate.
Some particles appeared larger after the encapsulation, probably due to slight foaming
of the polymer during depressurization. However, the fraction of larger particles
was small and the average size did not change significantly. Particle size analysis
by dynamic light scattering (DLS) gave an average particle size of 312 nm before
microencapsulation and 315 nm after microencapsulation. There was no measurable
change in the polydispersity as determined by DLS using the method of cumulants. The
results demonstrate that the microencapsulation process results in very little change in
particle size, morphology, and particle size distribution. Swelling of the particles by
CO2 is nearly completely reversible, and the morphology of the original latex particles
is maintained after microencapsulation.
In an effort to further enhance the incorporation of bromothymol blue into
polystyrene, the effect of cosolvents and additional Triton X-100 surfactant were
investigated. The ion pairs of bromothymol blue with THABr and TOABr were chosen
for study, as these were the two ionic complexes that showed enhanced encapsulation in
Table 3.2. Since both of these ionic complexes are insoluble in water, the aqueous phase
surrounding the polymer particles acts as a barrier for transport of the hydrophobic ion
pair. However, the ion pairs are soluble in some polar solvents such as dichloromethane,
chloroform, ethanol, and acetone. We hypothesized that the addition of a polar, water-
44
(A) (B)
Figure 3.5: SEM pictures of polystyrene particles: (A) before and (B) aftermicroencapsulation.
miscible cosolvent could allow some small level of solubility of the ion pair in the
aqueous phase, thus improving the kinetics of transport through the aqueous phase
and into the polymer. 0.15 g of acetone was added to 10 ml of aqueous latex, and
the microencapsulation process was repeated. Table 3.3 shows that the added acetone
enhances the loading of the ion pairs into polystyrene significantly. For the THABr
ion pair, loading is increased to 0.41 wt% compared to 0.11 wt% loading without
acetone. For the TOABr ion pair, the loading was 0.46 wt%. The added acetone did not
measurably alter the latex stability or particle size when compared to the experiments
without acetone.
Other cosolvents were investigated as well, including ethanol, 1-decanol, toluene,
and dichloromethane. The addition of toluene and dichloromethane resulted in a
loss of colloidal stability. Toluene and dichloromethane are both good solvents
for the polystyrene particles, so these solvents further swell the polymer particles.
Particle aggregation in the presence of toluene and dichloromethane resulted in rapid
45
Table 3.3: Cosolvent and surfactant effects on loading of bromothymol blue inpolystyrene particles
Ion pairing salt Cosolvent Dye concentration in PS particles (wt%)0.1 g Triton X-100 0.4 g Triton X-100
THABr None 0.11 0.08Acetone 0.41 0.63
TOABr None 0.11 0.09Acetone 0.46 0.50
coalescence. The measured dye loading in these cases was spuriously high, in some
cases >2 wt%. The loading is not reported in Table 3.3 because the high values are
the result of entrapment of solid particles of hydrophobic ion pairs in the aggregates of
polystyrene, and not uniform loading in the polystyrene spheres. Individual polystyrene
particles were no longer discernible after microencapsulation in the presence of
dichloromethane and toluene. Ethanol and decanol similarly destabilized the latex.
However, since the alcohols are not good solvents for polystyrene, coalescence was
less pronounced. After microencapsulation in the presence of alcohols, individual
polystyrene spheres were clearly discernible with necking between the particles
apparent, as shown in Figure 3.6. Again, dye loading is not reported for the alcohol
cosolvent due to spurious values caused by physical entrapment of ion pair particles in
the polystyrene aggregates.
Our previous research has shown that the amount of surfactant added during the
microencapsulation process can enhance the kinetics of transport into the aqueous
colloidal polymer particles.56 The surfactant aids transport by facilitating the formation
of an emulsion of compressed carbon dioxide in water. The surfactant also disperses
46
Figure 3.6: Agglomerated polystyrene particles after the microencapsulation process inthe presence of ethanol.
the hydrophobic ion pairs in water. To investigate the role of surfactant, the
microencapsulation experiments were repeated using 0.4 g of surfactant in 10 ml of
latex instead of 0.1 g surfactant. The results are shown in Table 3.3. Without cosolvent,
the additional surfactant has very little effect on the amount of dye incorporated into
the polystyrene particles. For both types of ion pairs, the additional surfactant actually
appears to slightly decrease the dye loading. For the THABr ion pair, measured dye
loading decreases from 0.11 wt% to 0.08 wt% and for the TOABr ion pair it decreases
from 0.11 wt% to 0.09 wt%. It is possible that the additional surfactant solubilizes the
hydrophobic ion pairs in water, thus limiting the transport to the polymer phase. When
acetone cosolvent is used, the additional surfactant enhances the dye loading. For the
THABr ion pair with acetone cosolvent, the loading increases from 0.41 wt% to 0.63
wt% as the surfactant amount is increased from 0.1 g to 0.4 g. An slight increase in
dye loading is also observed for the TOABr ion pair when the surfactant amount is
increased in the presence of acetone cosolvent.
47
3.4 Conclusions
Hydrophobic ion pairing was successful in allowing ionic dyes to be encapsulated
into polymer colloids. For bromothymol blue, there was at least a five-fold increase in
dye loading in the polymer particles when hydrophobic ion pairs were encapsulated
as compared to the encapsulation of unmodified dye. Addition of water-miscible
cosolvents was shown to further enhance the incorporation of the hydrophobic ion
pairs into the polymer colloids. It was demonstrated that up to a 30-fold increase in
the concentration of ionic compounds in the polymer could be achieved by ion pairing
and addition of acetone as a cosolvent. In the encapsulation process, compressed
carbon dioxide is used in place of volatile organic solvents. As a result, solvents
usage can be minimized or completely eliminated in some cases. Solvent elimination
is particularly attractive for drug delivery applications. However, the results show
that hydrophobic ion pairing is limited in its effectiveness. The ion pairs with rose
bengal showed no enhanced loading in the polymer, presumably due to its lower
solubility in nonpolar solvents and the larger molecular size of the hydrophobic ion
pair when compared with bromothymol blue. Since the method reported here relies
on spontaneous diffusion of the ion pair into the polymer, it requires compatibility
between polymer and ion pair and depends on the relatively slow diffusion kinetics in
the plasticized polymer phase. In cases where colloidal stability was lost, very high
dye loading was observed due to heterocoagulation between the polymer colloid and
dispersed particles of ion-paired dye. Unfortunately, in these cases irreversible and
uncontrolled coalescence led to loss of polymer particle integrity. Future research in
heterocoagulation or other techniques that kinetically entrap hydrophilic additives in
48
a hydrophobic polymer environment could potentially obviate problems due to slow
diffusion and thermodynamically limited loading in the polymer phase.
49
Chapter 4
Effect of Interfacial Free Energy on theFormation of Polymer Microcapsulesby Emulsification/Freeze-Drying∗
Hollow polymer microparticles with a single opening on the surface were formed
by freeze-drying aqueous polymer colloids swollen with solvent. The results show that
the particle morphology is due to phase separation in the polymer emulsion droplets
upon freezing in liquid nitrogen, and that morphological changes are driven largely by
lowering interfacial free energy. The effects of added surfactant, volume fraction of
solvent in the polymer emulsion droplet, type of solvent swelling polymer colloids,
and processing conditions on the particle morphology were examined and compared to
theoretical predictions. The dried hollow particles were resuspended in a dispersing
media and exposed to a second swelling solvent or plasticizer to close the surface
opening and form microcapsules. The interfacial free energy difference between the
inside and outside surfaces is the main driving force for closing the hole on the surface.
The emulsification/freeze-drying technique can be used to encapsulate hydrophilic
∗Yin, W.; Yates, M. Z. Langmuir 2008, 24, 701-708.
50
additives in the core of the microcapsules, demonstrating the potential of the technique
in controlled release applications.
4.1 Introduction
Hollow polymer particles or microcapsules are lightweight, deformable, strongly
scatter light and ultrasonic waves, and can act as semipermeable barriers for encapsu-
lated substances. As a result of these attractive properties, microcapsules are used in a
number of applications, such as functional paper coatings, electrophoretic displays, en-
zymatic microreactors, and microencapsulation for controlled release.139–143 A variety
of methods have been reported to synthesize microcapsules, including polymerization
of shells around oil emulsion droplets,144 polymerization of double emulsions,145 layer-
by-layer assembly of polyelectrolytes around a sacrificial core,146 assembly and fusion
of polymer particles around emulsion droplets,147,148 and controlled phase separation
in multicomponent polymer emulsions.149,150 Many of these current techniques suffer
from complexity and are limited in the types of polymers that can be synthesized or
processed.
Recently, Im et al. reported a simple method to prepare polymer microcapsules
by freeze-drying aqueous latex particles swollen with solvent.151 The resulting dried
particles were hollow with a single open hole on the surface. The size of the hole
could be adjusted by the processing conditions and the hole could be closed by
a second solvent swelling step to form a microcapsule. We refer to this method
as “emulsification/freeze-drying” in analogy to the emulsification/solvent evaporation
process.96 The emulsification/freeze drying process is attractive due to its relative
51
simplicity and potential ability to form microcapsules from a wide variety of polymeric
materials. However, the mechanism of microcapsule formation by this method has
not been investigated in detail. Im proposed that toluene, the swelling solvent, moved
toward the surface during the solidification in liquid nitrogen, leaving a void (vacuum)
at the center of the particle, and polymer chains migrated toward the surface driven by
the flux of toluene when the system was warming up, leaving particles with a hole
in the surface of each particle. We propose, however, that the structure is caused
by polymer/solvent phase separation during freezing and subsequent interfacial free
energy minimization. Hollow polymer particles with the morphology observed by Im
et al. were first described by Wilkinson and co-workers as “anomalous” particles that
form during certain emulsion polymerizations.152–154 It was initially thought that the
structure was formed in situ during the early stages of emulsion polymerization, but
a more detailed later investigation by Cox attributed the hollow structure to residual
monomer that caused phase separation during cooling and left a hole after drying.155
To form hollow particles during emulsification/freeze drying, the solvent must
migrate to the core of the polymer particle as a separate phase prior to its evaporation or
sublimation. The equilibrium morphology of biphasic emulsion droplets is controlled
by minimization of the interfacial free energy. Equilibrium morphology is well-
described theoretically and depends on the interfacial free energy between each phase
and relative volume fraction of each phase.156–160 However, kinetics also plays a role
in the freeze-drying process, as freezing of the solvent and polymer can lock in the
morphology in a nonequilibrium state. Hollow particles with typically a single hole
on each particle are obtained after freeze-drying. Im et al. were the first to show that
the hole can be closed to form microcapsules by redispersing the hollow particles and
52
subjecting them to a plasticizing solvent.151 Again, interfacial free energy minimization
is the expected driving force for closing the holes to form microcapsules. The objective
of the present study is to investigate the mechanism of hollow particle formation by
examining the role interfacial free energy plays in determining particle morphology.
The entire process for forming hollow polymer particles and subsequent closing
open holes to form microcapsules is illustrated in Figure 4.1. As shown in Figure 4.1,
the process can be used to encapsulate desired additives in the hollow polymer
shells. Vincent and co-workers formed microcapsules around oil droplets via internal
phase separation and characterized the controlled release of encapsulated oil-soluble
compounds.161,162 Unlike these earlier studies, the process illustrated in Figure 4.1 can
be used to encapsulate water-soluble or water-dispersible compounds. Better under-
standing of the mechanism of hollow particle formation and the factors controlling their
conversion to microcapsules will be helpful in developing emulsification/freeze-drying
as a route to encapsulating hydrophilic substances for controlled release.
4.2 Experimental Section
Materials. Styrene (99+%), 2,2′
-azobisisobutyronitrile (AIBN, 98%), toluene
(99.5+%), n-heptane (99%), Rose Bengal, poly(ethylene glycol) methyl ether meth-
acrylate (m-PEGMA, Mn =2080, 50 wt% in water, Figure 4.2), and potassium
persulfate (KPS, 99.99%) were purchased from Aldrich. Methanol (99.9%), benzene
(Spectranalyzed), and dichloromethane (99.9%) were from FisherChemicals. Absolute
ethanol and 95% ethanol (ACS/USP grade) were from Pharmco- AAPER. Octyl phenol
ethoxylate (Triton X-100) was obtained from J. T. Baker. AIBN was recrystallized from
53
Figure 4.1: Scheme showing the formation of hollow polystyrene (PS) particles byemulsification/freeze-drying and the closing of such particles for encapsulation.
methanol. Styrene was passed through an inhibitor remover column (Aldrich) prior to
use.
Figure 4.2: Chemical structure of m-PEGMA.
Synthesis of Polystyrene Latex. Polystyrene (PS) latex was synthesized by
surfactant-free emulsion polymerization as reported elsewhere.163 148 milliliters of
water was added to a three-neck round-bottom flask equipped with a condenser and
a magnetic stirring bar and maintained at 70 °C in an oil bath. The flask was purged
with argon for 30 min, after which 13.607 g styrene was added. Fifteen minutes later,
54
a solution of 0.012 g KPS in 2 mL water was charged into the flask. The reaction
was maintained at 70 °C for 28.5 h. The resulting latex was placed in dialysis tubing
(Spectra/Por, MWCO=12-14,000) and dialyzed in water for one week with the water
replaced every day. PS grafted with PEG was synthesized by the same method except
that 4 h after initiation, a solution of 0.006 g KPS in 1.36 g m-PEGMA aqueous solution
(50 wt%) was added to the flask.164
Preparation of Hollow Polystyrene Particles. To make hollow PS particles, 5
grams of 1 wt% PS latex in water was mixed with a swelling solvent (toluene or
dichloromethane) and in selected cases additional surfactant for 24 h under magnetic
stirring. Approximately 65 mg of dichloromethane and 3 mg of toluene will dissolve in
5 grams of pure water near room temperature.165,166 The amount of solvent soluble
in water was subtracted from the total solvent added, and it was assumed that all
excess solvent was dissolved in the polymer-rich phase for calculating volume fraction
of solvent in the polymer (νQ). In addition, it was assumed that the polymer and
solvent form an ideal solution so that pure component densities could be used for
volume fraction calculation. The swollen latex was rapidly frozen by dripping into
liquid nitrogen. The frozen sample, kept at 0 °C by ice/water, was dried by a vacuum
pump, during which the swelling solvent and ice were removed. The vacuum was
maintained at 4×10−4 Pa, which is below the triple point pressure for both water (611.73
Pa) and toluene (0.04 Pa).167 Therefore, the drying process was via sublimation. The
morphology of resulting particles was observed under a scanning electron microscope
(SEM, LEO 982 FE-SEM).
Formation of Microcapsules. Microcapsules were formed from selected freeze-
dried particles through a second solvent swelling step to close the open hole on the
55
surface of the particles. The freeze-dried PS particles were dispersed into water
or water/ethanol mixtures by sonication. Then, the suspension was exposed to a
plasticizing solvent to close the holes. When compressed carbon dioxide was used
as the plasticizing solvent, the suspension was loaded into a variable-volume high-
pressure stainless steel vessel.131 In some cases, Rose Bengal dye was added to the
suspension to examine encapsulation. After the closing process, the particles were
collected by centrifugation, washed repeatedly with 95% ethanol, and dried in vacuum.
4.3 Results and Discussion
Hollow Particle Formation. Figure 4.3 shows the two types of polystyrene
particles synthesized for this study. Both particles are uniform in size. Those on
the left (Figure 4.3A) were synthesized by surfactant-free emulsion polymerization.
These particles are electrostatically stabilized in water due to sulfate groups on the
surface from the potassium persulfate initiator. Additional stabilizing groups can
optionally be grafted on the surface during the polymerization. The particles on the
right (Figure 4.3B) have grafted poly(ethylene glycol) (PEG) on the surface to provide
additional steric stabilization. Dynamic light scattering (Brookhaven 90 Plus/BI-MAS)
gave an average hydrodynamic diameter of 571 nm for the surfactant-free particles
and 513 nm for the particles with grafted PEG. The zeta potential was −51 mV for
the surfactant-free particles and −40 mV for the particles with grafted PEG. Both
particles have negative zeta potential from the surface grafted potassium persulfate
initiator fragments. The magnitude of the zeta potential is lower for the PEG-grafted
particles due to electrostatic charge shielding by the uncharged PEG layer covering
56
the surface. The grafted PEG layer enhances latex stability during the swelling and
subsequent closing process. The PS without grafted stabilizer allows the investigation
of the effects of surface adsorption of added surfactant.
(A) (B)
Figure 4.3: Polystyrene latex particles as synthesized (A) by surfactant-free emulsionpolymerization and (B) by emulsion polymerization with poly(ethylene glycol)macromonomer.
The surfactant-free PS particles shown in Figure 4.3A were swollen with toluene
(νQ = 0.7), then freeze-dried. Figure 4.4A shows that the resulting particles appear
hollow with a single opening on the surface. By comparison with Figure 4.3A, it
can be seen that the swelling/freeze-drying steps increase the particle size and size
distribution significantly. The size is increased because the particles are hollow, but the
broader size distribution suggests that some of the polymer particles coalesce during
the solvent swelling step. To examine the role of interfacial free energy in determining
particle morphology, a second experiment was conducted in which 4 mg of Triton X-
100 surfactant was added during the swelling process. The particles produced in the
presence of Triton X-100 were bowl-shaped with a wider opening (Figure 4.4B). The
57
surfactant is expected to adsorb at both the toluene/water and polymer/water interfaces,
lowering interfacial tension. The changes in particle morphology observed in Figure 4.4
strongly suggest that interfacial free energy plays a significant role in the process.
(A) (B)
Figure 4.4: Effect of surfactant on the hollow particle morphology. Hollow particleswere made by freeze drying 5 g PS latex (1 wt%) swollen with 0.1 g toluene (νQ = 0.7):(A) no additional surfactant; (B) 4 mg Triton X-100.
A simple model of a biphasic emulsion droplet, represented in Figure 4.5, is used
to compare the observed changes in particle morphology to that predicted by interfacial
free energy minimization. P, Q, and W represent polymer-rich phase, solvent-rich
phase, and water phase, respectively. To apply the model to the freeze-drying process,
it is assumed that the morphology is not altered by compression from the surrounding
ice. Density changes and possible cracks in ice during its vitrification in liquid nitrogen
may compensate for the geometrical confinement of particles from the surrounding
ice. The model also assumes that the solvent-rich phase is always spherical, while the
polymer-rich phase can have changing morphology to partially or completely engulf
the solvent-rich phase. The morphology is described by ΦQ, which ranges from 0 ◦
58
when P and Q form separate spherical droplets to 180 ◦ when Q is engulfed by P in a
core-shell structure. ΦP is related to ΦQ through eq 4.1158
2 + 3 cos ΦP − cos3 ΦP
sin3 ΦP=
2 − 3 cos ΦQ + cos3 ΦQ
sin3 ΦQ+
4(1 − νQ)
νQ sin3 ΦQ(4.1)
where νQ is the volume fraction of Q in the total volume of P and Q. Taking the total
volume of P and Q as unity, the total interfacial free energy of the biphasic emulsion
droplet is given by eq 4.2158
E = (π
2)1/3(3νQ)2/3
[γPW(1 + cos ΦP)
sin2 ΦQ
sin2 ΦP+ γQW(1 + cos ΦQ) + γPQ(1 − cos ΦQ)
](4.2)
where γPW, γQW, and γPQ are the interfacial free energy per unit area between phases
P and W, Q and W, and P and Q, respectively. For a given system, the preferred
morphology is described by the angle ΦQ that gives the minimum overall free energy,
E. A dimensionless overall free energy can be defined as E/ECS where ECS is the
interfacial free energy of core-shell morphology (when ΦQ = 180 ◦) represented by
eq 4.3.
ECS = 32/3(4π)1/3(γPW + ν2/3Q γPQ) (4.3)
Plots of E/ECS versus ΦQ can be used to determine the morphology with minimum free
energy that will be the preferred morphology at equilibrium. Generally, if Q is very
hydrophobic (high value for γQW) or P is very hydrophilic (low value for γPW), core-
shell structure is preferred.158 However, any value of ΦQ between 0 ◦ and 180 ◦ can be
the preferred structure depending on the interfacial tension between the three phases
and volume ratio between P and Q.
59
Figure 4.5: Model for a biphasic emulsion droplet consisting of polymer-rich (P),solvent-rich (Q), and aqueous (W) phases. The polymer morphology after removingsolvent is determined by ΦQ.
To apply eqs 4.2 and 4.3 to the experimental system, it is necessary to know the
values of interfacial tension for the three interfaces during the process. At room
temperature, the interfacial tension is 36.1 dynes/cm between toluene and water, and
35.5 dynes/cm between styrene, the monomer unit of PS, and water.168 Unfortunately,
the interfacial tension values are unknown during and after freezing in liquid nitrogen.
Interfacial tension changes with temperature and generally increases as temperature is
lowered.169 In addition, the polystyrene particles are covered with hydrophilic surface
stabilizing groups, either potassium sulfate groups or grafted PEG, both of which will
lower the PS/water interfacial tension relative to pure PS. Triton X-100 will also lower
the interfacial tension between oil and water.170 The degree of lowering of interfacial
tension due to the surfactant or surface grafted groups is unknown. The interfacial
tension between toluene and polystyrene is also unknown, since they form a solution
(zero interfacial tension) at room temperature. Rough estimates of interfacial tension
were made to enable the calculation of E/ECS versus ΦQ. The interfacial tension
60
between PS and water or ice (γPW), between toluene and water or ice (γQW), and
between PS and toluene (γPQ) was set to 30, 45, and 12 dynes/cm, respectively, for
calculation of the free energy without surfactant. These estimates were made by
keeping in mind that γPW should not be very high, as PS particles as synthesized are
hydrophilic, and γPQ should be low, as toluene is a solvent for PS at room temperature.
The added surfactant Triton X-100 is expected to adsorb at both the toluene/water
and PS/water interfaces. As a result, it is assumed that the surfactant lowers both
γPW and γQW. When Triton X-100 is added, γPW, γQW, γPQ were set to 27, 27,
and 12, respectively. Figure 4.6 shows that with these assumptions the free energy
without surfactant is at a minimum when ΦQ is 180 ◦, which means the core-shell
structure is favored at equilibrium. The free energy minimum shifts to ΦQ = 56 ◦ with
surfactant, which means the bowl-shaped structure is preferred at equilibrium. While
the theoretical predictions are only a crude estimate, they do reveal that the expected
lowering of interfacial free energy by surfactant results in equilibrium morphology
changes that are consistent with the observations shown in Figure 4.4.
The effect of volume fraction of swelling solvent (νQ) was examined using both
toluene and dichloromethane. In these experiments, PS particles with surface-grafted
PEG were used to enhance stability during swelling and dispersibility of freeze-dried
particles. Surface-grafted PEG also partly eliminates the effects of surfactant adsorption
on changing particle morphology. Figure 4.7 shows the freeze-dried particles produced
from a series of experiments with increasing νQ. For the samples swollen with
dichloromethane, there was just one small dent on the particle when νQ was 0.2. Bowl-
shaped particles were obtained when νQ = 0.4. Core-shell-like hollow particles with
a small opening on the surface were formed when νQ was increased to 0.8. The hole
61
Figure 4.6: Effect of surfactant on interfacial free energy profile. E/ECS is plotted vsΦQ with νQ = 0.7. When no surfactant is used, γPW, γQW, and γPQ are set to 30, 45, and12, respectively. When a surfactant is used, they are set to 27, 27, and 12, respectively.The inset images represent the cross section of the favored morphology correspondingto the minimum free energy.
62
became larger, and it tended to embed into the particle upon increasing the amount of
swelling solvent. A similar trend was observed when toluene was used as the swelling
solvent. However, with toluene, a greater solvent volume fraction was needed to obtain
core-shell structured particles. The hole size can be estimated from SEM images. For
νQ = 0.8, when dichloromethane was used, a typical hole had a diameter of 579 nm, and
its volume fraction was 0.72; when toluene was used, the typical hole has a diameter of
539 nm, and its volume fraction was 0.68.
(A) (B) (C) (D)
Figure 4.7: Effect of volume fraction of the swelling solvent on the morphology ofthe freeze-dried PS particles grafted with PEG. The volume fraction of the swellingsolvent (νQ) was 0.2 (A), 0.4 (B), 0.6 (C), and 0.8 (D). Dichloromethane was used inthe samples in the upper images and toluene was used for those shown in the lowerimages. The white scale bar represents 2 µm.
To study the effect of increasing the swelling solvent as shown in Figure 4.7, E/ECS
is plotted versus ΦQ with different νQ as shown in Figure 4.8. γPW, γQW, and γPQ
were set to 30, 45, and 12, respectively, as was done for the surfactant-free case
in Figure 4.6. The minimum free energy in all cases corresponds to the core-shell
structure (ΦQ = 180 ◦). However, it shows that the slope of E/ECS versus ΦQ near the
63
minimum in free energy is different with different values of νQ. The slope increases
with increasing νQ from 0.2 to 0.8. The slope can be regarded as the driving force to
the minimum free energy. If the slope is small, the morphology change may stop at a
point farther from the core-shell structure than that with higher slope, especially during
the fast freezing process. It is estimated from the pipet diameter used that the droplets
added to liquid nitrogen have a radius of approximately 1.7 mm. The time for the
center of the droplet to cool from room temperature to −97 °C, the freezing temperature
of dichloromethane (for toluene, it is −93 °C), in liquid nitrogen (−195.7 °C) can be
estimated by a Heissler chart.171 αt/r2 = 0.14, in which r is the radius, and α is the
thermal diffusivity of the droplet. By assuming α to be the thermal diffusivity of ice at
0 °C, 0.0114 cm2/s,168 the time is just 0.365 s for the center of the droplet to reach the
freezing point of pure solvent. With such rapid cooling, the emulsion droplets maybe
frozen before reaching the morphology with minimum energy. The frozen morphology
will be affected by the driving force to reach equilibrium or the free energy slope.
The theoretical predictions are only crude estimates, but indicate that the particles are
likely not at equilibrium morphology, especially when low volume fractions of swelling
solvent are used. Even so, the predicted free energy driving force to equilibrium is
consistent with the observations shown in Figure 4.7.
Figure 4.7 also shows that there are differences in morphology for particles
produced using toluene versus dichloromethane as the swelling solvent. There are
several factors that can play a role in the differences between the two swelling solvents.
First, the solvents have different interfacial free energy that will result in slightly
different free energy profiles. Second, the phase separation may stop at different times
due to the difference in freezing temperature between toluene and dichloromethane.
64
Figure 4.8: Interfacial free energy (E/ECS) vs ΦQ with different volume fractions ofsolvent (νQ) by setting γPW, γQW, and γPQ to 30, 45, and 12, respectively.
65
Third, it is possible that different amounts of solvent are in the polymer phase even
though the calculated volume fraction is the same. It was assumed that all of the
solvent not soluble in water was in the polymer phase after the 24 h swelling period.
However, complete transfer of solvent to PS may not have occurred after 24 h. Since
dichloromethane has higher diffusivity in water than toluene,172 it is possible that
particles with dichloromethane have a higher relative volume fraction of solvent than
those with toluene. Finally, it should be emphasized that phase separation results in
polymer-rich and solvent-rich phases. In order for the polymer-rich phase to have
mobility, it must contain a significant fraction of solvent. The amount of solvent in
the polymer-rich phase depends on the kinetics and thermodynamics of polymer phase
separation during cooling. The equilibrium amount of solvent in the polymer-rich phase
at low temperature, as well as the time required to reach equilibrium, will be influenced
by the type of swelling solvent.
Two other swelling solvents, n-heptane and benzene, were also examined with νQ =
0.8, and the resulting particles are shown in Figure 4.9. When n-heptane, a nonsolvent
of PS, was used, there was no morphology change in the particles (Figure 4.9A. The
result is expected, since the amount of n-heptane in PS is low and phase separation
probably does not occur during freezing. When benzene was used, voided particles
were obtained, but the morphology was irregular with several openings on individual
particles (Figure 4.9B). The particle morphology with benzene is quite different from
that observed with toluene and dichloromethane as shown is Figure 4.7. This is possibly
due to the relatively high melting temperature of benzene (5 °C). Some benzene may be
frozen at several loci in the particle early during the process, leaving an irregular hollow
structure with multiple holes after the freeze-drying. Toluene and dichloromethane,
66
however, have much lower melting temperatures (−93 and −97 °C, respectively), so that
there is a longer time for morphology changes after phase separation to lower interfacial
free energy. As a result, hollow particles produced from toluene and dichloromethane
have a single hole (Figure 4.7).
(A) (B)
Figure 4.9: Freeze-dried samples with n-heptane (A) and benzene (B) as the swellingsolvent. The volume fraction of the swelling solvent was 0.8.
The effect of the freezing process was also examined. The latex of PS grafted with
PEG was swollen with toluene (νQ = 0.8), and then the vial containing the swollen
latex was immersed into a cooling bath of ethylene glycol/dry ice (around −10 °C).
Two hours later, the frozen latex was dried by a vacuum pump while maintaining the
sample at 0 °C in an ice bath. Hollow particles were also obtained, but the majority
of the sample had coalesced (Figure 4.10). The latex particles may separate from the
media as water is crystallizing during the slow freezing, and the swollen PS particles
will coalesce while toluene is still liquid at that temperature. However, rapid freezing
in liquid nitrogen appears to prevent coalescence.
67
Figure 4.10: PS particles grafted with PEG and swollen with toluene (νQ = 0.8) afterfreezing in ethylene glycol/dry ice at −10 °C and drying by a vacuum pump at 0 °C.Most of the sample had coagulated, but some hollow particles were observed.
Microcapsule Formation from Hollow Particles. The holes on the surface of
the hollow particles could be closed to form microcapsules by exposure to a second
swelling solvent. To examine microcapsule formation, hollow PS particles similar
to those shown in Figure 4.4A were resuspended in water and exposed to a swelling
solvent. Two swelling solvents were examined: toluene and compressed carbon
dioxide. Compressed CO2 can depress the glass transition temperature of polystyrene
below the operating temperature,135,136 and polymer chains are able to move to close
the hole. Carbon dioxide is a nontoxic solvent that is easy to separate from the polymer
suspension, making it attractive for microencapsulation processes.56 A suspension of
hollow particles was made with 10 mg of dry particles and 5 mL water. When
compressed carbon dioxide was used, 4 grams CO2 were added and the suspension
was maintained at 27.9 MPa and 35 °C for 67 h. Figure 4.11A shows that the holes
of the particl were closed after exposure to compressed CO2. Figure 4.11B shows the
68
particles after the same process except additional surfactant, 9 mg Triton X-100, had
been added. With additional surfactant, the opening decreased in size, but was not
completely closed after exposure to compressed CO2. A similar result was observed
when toluene was used as the swelling solvent. When toluene was used as the swelling
solvent, 0.1 g toluene was added to the suspension, and it was maintained at room
temperature for 1.5 h. Figure 4.11C shows that toluene exposure closes the openings
on the surface of the particles. When additional Triton X-100 was present, the opening
was not completely closed (Figure 4.11D). Similar results were obtained when other
surfactants were used as shown in Figure 4.12.
The results shown in Figure 4.11 suggest that interfacial free energy plays a
significant role in closing the holes to form microcapsules. A proposed mechanism
for microcapsule formation is illustrated in Figure 4.13. The exterior surfaces of
the polymer particles are hydrophilic due to grafted potassium persulfate initiator
fragments or PEG stabilizing groups. During emulsification/freeze drying, the interior
surface forms around the hydrophobic solvent-rich phase. There are likely some
sulfate groups embedded in the core of the polymer particle that become exposed
on the interior surface that forms during emulsification/freeze-drying. However, the
concentration of sulfate groups is enhanced on the exterior surface of the particle.154,173
It is therefore expected that the interior surface of the hollow polymer particles is
more hydrophobic than the exterior surface. As a result, there is a difference in
interfacial tension between the interior and exterior surfaces when the particles are
resuspended in water. When the particles are exposed to a plasticizing solvent that
makes PS chains mobile, the higher interfacial tension in the particle interior pulls the
hole closed. Added surfactant can adsorb on both the exterior and interior surfaces,
69
(A) (B)
(C) (D)
Figure 4.11: Effect of surfactant on closing hollow PS by CO2 (A, B) and toluene (C,D). The suspension of hollow PS particles in water was mixed with CO2 at 35 °C and27.9 MPa for 67 h: (A) no additional surfactant; (B) 0.2 wt% Triton X-100. Withtoluene, the suspension was mixed for 1.5 h at room temperature: (C) no additionalsurfactant; (D) 0.004 wt% Triton X-100.
70
(A) (B)
Figure 4.12: Effect of surfactant on closing hollow PS by toluene. The suspension ofhollow PS particles in water was mixed with toluene for 1.5 h at room temperature.(A) 0.2 wt% Brij 35; (B) 0.2 wt% Triton X-305. Many particles had small unclosedopening after the process.
decreasing the interfacial tension difference. As a result, the hole-closing process is
slowed significantly in the presence of surfactant.
Figure 4.13: Schematic illustrating the hole closing process. Higher interfacial tensionon the interior particle surface tends to pull the hole closed upon exposure to aplasticizing solvent.
The emulsification/freeze-drying process has potential to be used as a microen-
capsulation process for hydrophilic compounds. To demonstrate the feasibility of
the process in microencapsulation, a water-soluble dye was added during the hole-
closing process. 20 mg of the particles shown in Figure 4.7D (with dichloromethane
71
as the swelling solvent) were resuspended in 2 mL of water/0.5 mL ethanol containing
40 mg of Rose Bengal dye. Microcapsules were formed by exposing the particles
to 60 mg of toluene for 1.5 h at room temperature. In addition, a parallel control
experiment was done following the same procedure, except the solid particles shown in
Figure 4.3B were used. After exposure to toluene, both types of particles were collected
by centrifugation and washed with 95% ethanol until the supernatant was clear, then
dried in vacuum. After washing, the solid particles appeared almost white in color,
while the microcapsules remained red. Figure 4.14 shows that the hollow particles
were closed, and the inset images show the color difference between the two samples.
It was confirmed by SEM and a transmission electron microscope (TEM, JEOL 200EX)
that such closed particles are hollow inside (Figure 4.15). It was also observed
that such particles encapsulating dye remained colored after dispersing in ethanol for
months. The emulsification/freeze-drying technique is therefore potentially applicable
for microencapsulation and controlled release. We plan additional experiments with
biodegradable microcapsules to demonstrate controlled release.
4.4 Conclusions
Hollow polystyrene (PS) particles were obtained by swelling PS latex with solvent,
freezing in liquid nitrogen, and drying in vacuum. The morphology of the resulting
hollow particles can be controlled by the amount of swelling solvent and surfactant. The
core-shell structure was favored at high solvent content and without added surfactant.
A bowl-shaped morphology was favored with added surfactant and at lower solvent
content. The experimental results can be interpreted in terms of interfacial free energy
72
(A) (B)
Figure 4.14: Encapsulation of rose bengal by exposure of aqueous PS suspension totoluene in the presence of dye: (A) solid PS with grafted PEG; (B) hollow PS withgrafted PEG. The inset images are photographs of the dried samples.
(A) (B)
Figure 4.15: Confirmation of the hollow structure of closed polystyrene capsulesencapsulating rose bengal (Figure 4.14B). (A) SEM image of capsules embedded inepoxy, cut by a microtome; (B) TEM image of an intact capsule.
73
minimization. It can be concluded that phase separation occurred, as swollen aqueous
latex particles were frozen by liquid nitrogen. Subsequent morphological changes in
the biphasic emulsion droplets are driven by the minimization of interfacial free energy.
After removing the solvent-rich phase by drying under vacuum, hollow particles were
obtained with an open hole on the surface.
The dry hollow particles can be redispersed into water or water/ethanol mixtures by
mild sonication. When exposed to a solvent or plasticizer, such as compressed CO2,
the open hole on the hollow PS particles can be closed. It is likely that the interior
surface of the hollow particles is more hydrophobic than the exterior surface. The
experimental evidence shows that the hole-closing process is driven by the difference
in interfacial tension between the interior and exterior surfaces. When surfactant is
added to the suspension of hollow particles, it interferes with closing of the holes. The
surfactant adsorbs onto both the inside and outside surfaces of the hollow particles to
lower the interfacial tension difference and thus reduces the driving force for closing
the hollow particles. The emulsification/freeze drying process has potential application
in microencapsulation for controlled release. A water-soluble dye was encapsulated
into hollow PS particles, and the dye remained trapped in the microcapsule colloidal
suspension for months. A controlled-release vehicle can be achieved by use of
biodegradable polymer with tunable release characteristics.
74
Chapter 5
Encapsulation and Sustained Releasefrom Biodegradable MicrocapsulesMade by Emulsification/Freeze Dryingand Spray/Freeze Drying∗
Hollow biodegradable poly(DL-lactide) (PLA) particles with porous shell walls
were prepared by freeze drying small droplets of PLA solution formed by emul-
sification or spraying. The hollow freeze-dried particles were dispersed in water,
and the resulting aqueous suspensions were exposed to plasticizing solvents, either
dichloromethane or compressed carbon dioxide. The plasticizing solvent causes the
pores in the shell wall to close, forming microcapsules surrounding an aqueous core.
A water soluble drug, procaine hydrochloride, was successfully encapsulated in the
microcapsule core. The encapsulation efficiency is affected by the hollow particle
morphology, amount of solvent used, solvent exposure time, surfactant, and method of
dispersing the freeze-dried particles in water. The encapsulation process is explained in
terms of interfacial free energy of the hollow particles and mobility of the plasticized
∗Yin, W.; Yates, M. Z. J. Colloid Interface Sci. 2009, accepted.
75
polymer. Controlled release of procaine hydrochloride from the microcapsules into
phosphate buffer solution was observed. The microcapsules had a small burst release,
with the remainder of encapsulated drug slowly released over 9 days. The novel hollow
PLA particles produced by emulsification/freeze drying and spray/freeze drying can
potentially be used as vehicles for controlled release.
5.1 Introduction
Sustained and targeted release of therapeutics can be achieved by microen-
capsulation inside biodegradable colloidal particles of poly(lactic acid) (PLA) and
poly(lactic-co-glycolic acid) (PLGA).174–177 The polymer degradation rate can be tuned
by adjusting particle size,178 molar ratio between lactic acid and glycolic acid,176
molecular weight, and porosity.29 The particle surface may be functionalized to target
specific tissues, or magnetically functional particles may be created to allow targeting
specific locations in the body by an applied magnetic field.100,179–187 There are three
main techniques for microencapsulation: spray-drying, coacervation (precipitation),
and emulsification/solvent evaporation.188 Each of these techniques produces solid PLA
or PLGA particles with a therapeutic agent distributed in the particle. The spray drying
technique requires high temperatures that can destroy thermally labile compounds,189
while the coacervation and emulsification/solvent evaporation techniques suffer from
the use of large quantities of harmful organic solvents that are undesirable in drug
delivery applications.96,116,190 One promising alternative is the use of compressed
carbon dioxide as an alternative solvent for microencapsulation.56,191 Unfortunately,
carbon dioxide-based microencapsulation is much less effective with water soluble
drugs.192
76
In order to encapsulate hydrophilic additives into the hydrophobic polymer, ef-
fective techniques kinetically entrap the additive in the polymer, rather than relying
on the unfavorable thermodynamically driven diffusion of additives into the polymer.
The most common route to encapsulate water-soluble compounds is through solvent
evaporation from water/oil/water double emulsions, but finding conditions that provide
high encapsulation efficiency can be difficult.37 An alternative method is to create
polymer microcapsules to surround the hydrophilic additive with a polymer shell. A
number of novel encapsulation schemes have been reported using hollow colloidal
polymer particles.139,193–198 The hollow particles can be filled to encapsulate species
in the core, thereby decoupling the particle formation and encapsulation steps. Hollow
particles potentially allow labile species to be protected from harsh operating conditions
during conventional microencapsulation and give greater flexibility in controlling
particle properties. A few types of polymers can be formed as hollow particles
through novel polymerization processes.139,144,145 A facile alternative to polymerization
is creating hollow polymer particles through confined phase separation in emulsion
droplets of polymer solution.149,156,161 Since phase separation processes are non-
reactive, they can be applied to a wide range of solution processable polymers to create
hollow particles.
An “emulsification/freeze drying” process has recently been described, in which
a polymer solution is emulsified to form droplets in water, then rapidly frozen and
dried under vacuum to produce hollow particles.151,199 Temperature-induced phase
separation inside the droplets causes structured polymer phase domains to form. Upon
drying, the solvent-rich phase is removed, leaving a hollow region in its place. The
resulting polymer particles can be porous (with numerous small holes) or hollow
77
(with a single large hole), depending on the kinetics of freezing and the interfacial
free energy between the solvent, polymer, and aqueous phases. Under appropriate
conditions, a single hole is produced that is open to the outside surface of the particle.
When the hollow particles are dispersed in water, exposure to a plasticizing solvent
causes the hole to close, forming a core-shell structured microcapsule. It has been
demonstrated using poly(methyl methacrylate) and polystyrene particles that water-
soluble additives may be encapsulated inside the microcapsules.151,199 In principle,
emulsification/freeze drying can be employed to create hollow polymer particles and
microcapsules from nearly any solution processable polymer given the right interfacial
free energy and freezing kinetics. In the present study, we demonstrate the production
of hollow particles of biodegradable PLA via freeze drying droplets of polymer solution
formed either by emulsification or spraying. The hollow PLA particles have pores in
the shell wall, allowing them to be filled with an aqueous phase. The hollow porous
PLA particles are converted to microcapsules entrapping a model water soluble drug
by exposure to a plasticizing solvent. It is demonstrated that the entire process can
be carried out using only nontoxic and biodegradable materials. Dimethyl carbonate,
a “green” biodegradable solvent is effective for forming hollow PLA particles with
porous shell walls via freeze drying. Compressed carbon dioxide, a nontoxic solvent
easily separated from the polymer, can be used as the plasticizing agent to close pores
in the polymer shell for encapsulation. The release kinetics of the encapsulated drug
is investigated to explore the application of the novel microcapsules as vehicles for
controlled release.
78
5.2 Experimental Section
Materials. Poly(DL-lactide) (PLA, inherent viscosity = 0.65 dl/g in chloroform at
30 °C) was purchased from DURECT Corporation. Acetonitrile (ChromAR® HPLC)
was from Mallinckrodt. Procaine hydrochloride (99%), phosphate buffer solution in
water (pH = 7.2), dimethyl carbonate (Reagent Plus®, 99%), and poly(vinyl alcohol)
(PVA, 87-89% hydrolyzed, Mw = 13000-23000 g/mol) were from Sigma-Aldrich.
Dichloromethane (99.9%) was from Fisher Scientific. Pluronic P123 was donated by
BASF. De-ionized water was used in all experiments.
Emulsification/Freeze Drying Process. A solution of PLA in dichloromethane
was emulsified into aqueous PVA solution using a homogenizer (IKA Ultra-Turrax
T25 basic) at 21500 rpm for 1 minute. The emulsion was then immediately added
dropwise into liquid nitrogen. Water and dichloromethane were removed by placing
the frozen sample under vacuum at 0 °C, leaving dry hollow PLA particles. In a typical
experiment, the oil phase was 0.2 g/ml PLA in dichloromethane, the aqueous phase was
0.01 g/ml PVA, and the volume ratio of water to dichloromethane was 5:1.
Spray/Freeze Drying Process. A solution of PLA (0.05 g/ml) and Pluronic P123
surfactant (0.01 g/ml) in dimethyl carbonate was atomized by a coaxial spray nozzle
shown in Figure 5.1. The liquid flow rate was 15 ml/h. Compressed air was fed to
the outer annular tubing at a pressure of 50 psig. The atomized spray was collected in
liquid nitrogen and then dried under vacuum at room temperature.
Microencapsulation Using Hollow PLA Particles. The hollow PLA particles
were dispersed into an aqueous solution of procaine hydrochloride by placing in an
ultrasonic bath for several seconds. Dichloromethane or compressed carbon dioxide
79
Figure 5.1: Coaxial spray nozzle.
was added to the aqueous suspension to close the hollow particles and encapsulate
procaine hydrochloride. The typical colloidal suspension was 0.02 g/ml PLA dispersed
in aqueous solution of 0.2 g/ml procaine hydrochloride. Microcapsules were formed
after stirring for 1.5 hours with the plasticizing solvent (either dichloromethane at
room temperature or excess carbon dioxide at 207 bar and 32 °C). After stirring, the
sample was washed by centrifugation/redispersion with water three times, and dried
in vacuum. The amount of encapsulated material was measured with UV spectrometry
(PerkinElmer Lambda 900) by dissolving the resulting particles in acetonitrile, a mutual
solvent for PLA and procaine hydrochloride.
Measuring Release of Encapsulated Drug. The particles encapsulating procaine
hydrochloride (without drying in vacuum) were dispersed at a concentration of about
0.005 g/ml in phosphate buffer (pH = 7.2) maintained at room temperature. The drug
80
concentration in the buffer solution versus time was measured using UV spectrometry.
Samples taken for spectroscopy were first passed through a filter (0.2 µm) to remove
the particles.
5.3 Results and Discussion
The morphology of the polymer particles obtained by emulsification/freeze drying
depends upon the kinetics of freezing and phase separation, as well the interfacial
free energy between the aqueous, solvent-rich, and polymer-rich phases that form.199
The interfacial free energy is affected strongly by the surfactant used to stabilize the
polymer emulsion. The kinetics of the freezing and phase separation processes are
affected strongly by the freezing point of the solvent used to form the polymer emulsion
and the polymer concentration. Previous investigations of emulsification/freeze drying
made particles of polystyrene and poly(methyl methacrylate) using the solvents
toluene, dichloromethane, and styrene monomer.151,199 For the emulsification/freeze
drying of PLA, we chose dichloromethane as the solvent and PVA as the emulsifier.
Dichloromethane is commonly used with PLA in the traditional emulsification/solvent
evaporation microencapsulation process. We investigated a variety of emulsifiers, but
chose PVA for detailed investigation since it is effective in stabilizing PLA emulsions
and most easily allowing freeze dried PLA particles to be redispersed in water.
Hollow PLA particles were readily obtained by freeze-drying oil-in-water emul-
sions. Figure 5.2 shows that the resulting PLA particles are hollow, with thin-walled
shells and a single opening on the surface. The polymer precipitates and separates
from dichloromethane upon cooling the emulsion droplets. The hollow interior is
81
from the dichloromethane-rich phase that was removed in the drying process.199 The
average particle diameter and polydispersity index (weight average diameter divided
by number average diameter) were estimated by measuring the diameter of over 500
particles from scanning electron microscopy (SEM) (LEO 982 FE-SEM) images. The
average size could be adjusted by altering the polymer concentration. Decreasing the
polymer concentration decreases oil phase viscosity and thus makes the oil droplets
smaller. With a polymer concentration of 0.4 g/ml PLA in dichloromethane, the number
average particle diameter is approximately 10.86 µm (Figure 5.2A). Decreasing the
polymer concentration to 0.2 g/ml results in a decrease of the number average particle
diameter to 3.86 µm (Figure 5.2B). The size distribution of the hollow particles is
broad, with a typical polydispersity index of 1.6. The broad size distribution of the
hollow particles is correlated to the size distribution of precursor emulsion droplets.
When monodisperse polystyrene particles were swollen with solvent and freeze-dried,
hollow particles with a narrow size distribution were obtained.199 By applying novel
emulsification techniques to create uniform precursor emulsion droplets, more uniform
hollow particles may be obtained by phase separation.194
When the freeze dried particles are redispersed in water, exposure to a plasticizing
solvent can induce the hollow particles to convert to microcapsules. The plasticizing
solvent gives the polymer mobility, allowing the hollow polymer particles to change
shape to decrease the interfacial free energy. The interfacial free energy is often higher
in the hydrophobic interior of the hollow particle than on the exterior surface that is
coated with surfactant.199 As a result, the interfacial tension can pull the hole closed
to form a microcapsule encapsulating a portion of the aqueous phase. The hollow
PLA particles were converted to microcapsules in the presence of aqueous solutions of
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(A) (B)
Figure 5.2: Hollow PLA particles made by emulsification/freeze-drying: (A) 0.4 g/ml,(B) 0.2 g/ml PLA in dichloromethane.
a model water-soluble drug, procaine hydrochloride, to investigate encapsulation and
controlled release from the microcapsules.
The encapsulation of procaine hydrochloride was dependent on the amount of
dichloromethane that the particles were exposed to under a fixed time of 1.5 hours
at room temperature. Figure 5.3 shows that the amount of procaine hydrochloride
encapsulated first increased and then decreased as the amount of dichloromethane used
was increased. The data indicate an optimal amount of dichloromethane to achieve
maximum encapsulation. A control experiment was also done with solid PLA particles
under the conditions corresponding to the highest point in Figure 5.3. The solid
particles were prepared by evaporating dichloromethane in the emulsion for 24 h at
room temperature, rather than freeze-drying, and the number average diameter is about
4.79 µm. The drug content was just 0.21 wt% when using solid PLA particles, while
it was 2.1 wt% with the hollow particles, confirming the encapsulation capacity of the
hollow particles. The hollow particles have higher surface area than the solid particles
83
so some of the increase in encapsulation amount measured using hollow particles could
be partially attributed to surface adsorption. However, based on SEM images of average
particle size and interior hole volume, the increased surface area is not enough to
account for the ten fold increase in amount encapsulated using hollow particles. Based
on the average particle diameters and morphology measured by SEM, it is estimated
that a minimum of 80% of the procaine hydrochloride is encapsulated in the hollow
core.
Figure 5.3: The concentration of procaine hydrochloride in PLA particles as a functionof amount of dichloromethane used during encapsulation for 1.5 hours at roomtemperature.
The particle morphology after encapsulation changed with the amount of dichloro-
methane, as shown in Figure 5.4. When there was only 0.40% v/v dichloromethane
in water, most of the resulting particles still had holes open to the hollow interior,
resulting in poor encapsulation (0.06 wt% drug content). Increasing the amount of
84
dichloromethane resulted in a higher fraction of the particles with the holes closed.
When there was 1.2% v/v dichloromethane, nearly all particles were completely
closed, appearing spherical with a smooth surface, giving rise to a higher encapsulated
drug content (1.57 wt%). The core-shell morphology was confirmed with scanning
transmission electron microscopy (STEM) (SUPRA 40VP, 30 kV). The inner hollow
region appears lighter than the surrounding polymer shell in the STEM images shown
in Figure 5.5. The STEM images also reveal that the inside hole volume decreases
if too much dichloromethane is added (Figure 5.5A, B). Particles exposed to 1.38%
v/v dichloromethane/water for 1.5 hours are microcapsules with a large hollow core.
Increasing the dichloromethane concentration to 1.98% v/v, results in a decrease in
the size of the hollow core. The STEM images are consistent with the encapsulation
results which show that too much dichloromethane reduces the amount encapsulated
due to the decreased volume of the inside cavity. The core-shell structure has a higher
interfacial free energy than solid particles. Therefore, it is expected that all particles will
revert to the solid morphology given enough polymer mobility and time. The additional
mobility imparted on the polymer by increasing dichloromethane amount can allow
some microcapsules to re-shape so that the interior cavity is reduced or eliminated in a
shorter time.
The method used to disperse the hollow PLA particles in water is critical for drug
encapsulation. Generally the hollow particles were dispersed in water by placing in
an ultrasonic bath. With 1.58% v/v dichloromethane/water, the drug content after
encapsulation was 1.6 wt% when particles were dispersed by the ultrasonic bath.
However, the drug content was just 0.13 wt% if the hollow particles were not dispersed
in water by sonication but instead by mild stirring for 30 min. Since the interior surface
85
(A) (B) (C)
Figure 5.4: PLA particles after exposure of aqueous suspensions to different amountsof dichloromethane for 1.5 hours at room temperature: (A) 0.40%, (B) 0.80%, (C) 1.2%v/v dichloromethane/water.
(A) (B) (C)
Figure 5.5: Interior structure of PLA particles encapsulating procaine hydrochlorideusing different amounts of dichloromethane and stirring time: (A) 1.38% v/vdichloromethane, 1.5 h; (B) 1.98% v/v dichloromethane, 1.5 h; (C) 1.98% v/vdichloromethane, 3 h.
86
of hollow polymer particles prepared by emulsification/freeze-drying is hydrophobic,
the hollow particles tend to hold air inside and float above water even though the density
of PLA (specific gravity = 1.25) is larger than that of water. So the hollow PLA particles
can not be dispersed well in water by mild stirring, even with surfactant present. Drug
solution does not enter the hollow core easily, resulting in poor encapsulation. However,
sonication can remove the trapped air, allowing the hollow particles to be dispersed
well and the aqueous solution to be encapsulated. The encapsulated drug content
decreased when the mixing time with dichloromethane was extended from 1.5 h to
3 h (Table 5.1). Longer exposure to dichloromethane allows the core-shell structured
polymer particles enough time to revert to solid particles with lower interfacial free
energy, and reduces the amount of encapsulated drug. The STEM images in Figure 5.5
(B and C) confirm that the particle size is reduced and the hollow core is no longer
visible on most particles as the exposure time is increased from 1.5 hours to 3 hours.
The result also confirms that drug trapping is not mainly by diffusion into polymer shell
wall, but by encapsulation inside microcapsules. Otherwise the longer exposure to the
plasticizing solvent would increase the amount of encapsulated drug. The driving force
for the hole closing is the difference in interfacial free energy between the hydrophobic
core and the exterior surface that is coated with hydrophilic PVA (Figure 5.6). Table 5.2
shows that the amount of drug encapsulated is increased by washing off excess PVA.
The excess PVA could coat the particle interior upon sonication, lowering the interfacial
free energy difference and slowing the hole-closing process. Removing the excess PVA
likely increases the kinetics of the hole closing process. Effective microencapsulation
requires a balance between the kinetics of polymer mobility and the interfacial free
energy driving the process. A strong driving force is required to close the open holes,
87
but the process must be stopped before there is enough time for the core-shell structured
particles to revert to solid particles with the lowest interfacial free energy, as shown in
Figure 5.6.
Table 5.1: Effect of stirring time on procaine hydrochloride encapsulation in hollowPLA particles.
Dichloromethane/water Encapsulated Concentration Encapsulated ConcentrationConcentration (% v/v) (wt%) after 1.5 hr stirring (wt%) after 3 hr stirring
1.58 1.6 1.21.98 0.80 0.40
Figure 5.6: Scheme showing the closing process of hollow PLA particles forencapsulation. Minimization of interfacial free energy is the driving force to closehollow PLA particles. To have effective encapsulation, the closing process should bestopped before the microcapsules revert to solid particles having the lowest free energy.
If all drug solution filled in the void of hollow particles were encapsulated, the
drug content could reach up to about 38 wt%, which is much higher than the results
obtained. The gap is due to shrinking of hollow particles, ineffective closing of holes,
and loss during washing by centrifugation and sonication. In an effort to increase
the encapsulated drug content, porous PLA particles were prepared by a spray/freeze
88
Table 5.2: Effect of surfactant removal on procaine hydrochloride encapsulation inhollow PLA particles. Excess surfactant was removed by centrifugation (3500 rpm,5 min) with water 3 times.
Dichloromethane/water Encapsulated Concentration Encapsulated ConcentrationConcentration (% v/v) (wt%) using Original (wt%) Removing excess
hollow PLA surfactant
1.38 (a) 2.1 3.91.58 (b) 1.2 2.21.98 (b) 0.40 1.2
(a) 1.5 h stirring; (b) 3 h stirring.
drying process. In this method, a solution of PLA and surfactant Pluronic P123 in
dimethyl carbonate was sprayed through a coaxial nozzle directly into liquid nitrogen
and dried under vacuum. The surfactant facilitates the dispersion of the freeze-dried
particles in water. Dimethyl carbonate is attractive because it is a nontoxic and
biodegradable solvent.200 Figure 5.7A shows that the particles obtained were hollow
inside, but the shell was porous. Dimethyl carbonate has a higher melting point
(∼2 °C) compared to dichloromethane (−97 °C). The higher melting point causes
the solvent-rich phase to be frozen in many small domains before having enough
time to migrate into a single solvent-rich phase domain. For the dichloromethane
solutions, the particles are formed by freeze drying aqueous emulsion droplets that
are ∼1.7 mm in diameter (based on the average weight per droplet). The time for
the center of a water droplet with a radius of 1.7 mm to cool from room temperature
to −97 °C, the freezing temperature of dichloromethane, in liquid nitrogen is about
0.4 seconds.199 For the dimethyl carbonate solutions, the particles are produced by
freeze drying the sprayed droplets of polymer solution that are less than 100 microns
89
in diameter. The smaller droplets with higher freezing point are expected to freeze
much faster than the aqueous dichloromethane emulsions. A previous investigation has
shown that porous polymer particles are produced by freeze drying droplets of polymer
solutions in benzene, a solvent with a melting point of 5 °C.199 The porosity observed
in Figure 5.7 can also be partly attributed to the Pluronic P123 surfactant that can act
as a porogen. Previous studies have shown that porous PLGA particles were obtained
by emulsification/solvent evaporation when Pluronic F127, a water-soluble porogen,
was added to the dichloromethane oil phase.201,202 As the dichloromethane evaporated,
PLGA-rich and Pluronic F127-rich phases formed.201,203 When the Pluronic F127 was
removed by washing in water, porous PLGA particles were obtained.
Compared to the emulsification/solvent evaporation method, spray/freeze-drying
takes less time to obtain porous particles, applies to a broader range of polymers,
and gives porous particles that are hollow inside. The porous particles made by
spray/freeze-drying were subjected to the same procedure for encapsulating procaine
hydrochloride as the hollow particles made by emulsification/freeze-drying. During
the encapsulation, the excess surfactant in the porous particles was not washed off. The
amount of dichloromethane was 1.38% v/v in water, and the mixture was stirred for
1.5 h. Figure 5.7B shows the porous particles were closed and the inside was hollow,
with a number of large hollow domains in the interior of the particles. The encapsulated
drug content was 4.0 wt% using the porous particles, while it was just 2.1 wt% for
the hollow particles under the same conditions. It is assumed that the porous shell
allows the drug to enter more easily, and the smaller pores in the shell can be closed
more rapidly and more effectively than a single large pore, allowing more drug to be
encapsulated.
90
(A) (B)
Figure 5.7: Porous PLA particles made by coaxial spray/freeze-drying with dimethylcarbonate as the solvent. (A) Before encapsulation; (B) after encapsulation. The insetis the corresponding STEM image showing the inside cavity.
To avoid using detrimental organic solvents, compressed carbon dioxide was
investigated to close hollow PLA particles to form microcapsules. An aqueous
suspension of ∼1 wt% hollow particles was loaded into a variable volume high-
pressure stainless steel vessel.131 Carbon dioxide at 207 bar and 32 °C was added to the
headspace above the suspension for 1.5 h. After venting carbon dioxide, an aqueous
suspension of PLA microcapsules was obtained. Figure 5.8 shows that the hollow
particles were closed, appearing spherical with a smooth surface. The results using
compressed carbon dioxide show that the hollow particles can potentially be used for
microencapsulation while using only the benign solvents carbon dioxide and water.
The release kinetics of encapsulated procaine hydrochloride into phosphate buffer
solution at pH 7.2 was measured as shown in Figure 5.9. The solid line is the release
profile from microcapsules formed by exposure to 1.38% v/v dichloromethane for
1.5 hours and excess surfactant removed (the sample in Table 5.2, row 1, column
91
Figure 5.8: PLA microcapsules formed by exposure of aqueous hollow particlesuspension to compressed carbon dioxide.
3). The dashed line is the release profile from microcapsules formed under the same
conditions except the excess surfactant remained (the sample in Table 5.2, row 1,
column 2). The microcapsules were washed with water prior to release study by
centrifugation/redispersion three times. The release profile displays a slight burst
release with about 5% of the drug released within the first 30 minutes for microcapsules
formed with excess surfactant removed and about 12% for microcapsules formed
without removing excess surfactant. The release rate decreased gradually with time
(Figure 5.9). After ∼9 days, all drug was released. The release profiles from
the two kinds of microcapsules are similar to each other. By fitting the data with
exponential decay N(t) = C + N0e−t/τ, τ, the exponential time constant or mean
lifetime, can be calculated. Microcapsules with excess surfactant removed have a
mean lifetime of 3.11 days, close to the mean lifetime of 3.21 days for microcapsules
formed without removing excess surfactant. There are many factors affecting the
92
release from biodegradable delivery systems, such as type of polymer, type of drug,
size and shape.204 The two kinds of microcapsules shown in Figure 5.9 have similar
composition, size, and morphology, so it is expected that the release dynamics would
also be similar. Compared to the release of procaine hydrochloride encapsulated in
PLGA nanoparticles by nano-precipitation,205 the release was slower and lasted longer,
which is likely due to the larger size of the microcapsules. The freeze-drying processes
therefore have potential for producing biodegradable particles suitable for controlled,
extended release of water-soluble therapeutic agents.
Figure 5.9: Release of encapsulated procaine hydrochloride in pH=7.2 phosphatebuffer: � microcapsules formed with excess surfactant removed (Table 5.2, row 1,column 3); ◦microcapsules formed without removing excess surfactant (Table 5.2, row1, column 2).
93
5.4 Conclusions
Emulsification/freeze drying and spray/freeze drying are simple, effective processes
for creating hollow biodegradable PLA particles. The particle morphology can be
controlled by adjusting the type of solvent, solvent concentration, and surfactant to
control interfacial free energy. The hollow particles can be converted to microcapsules
through short exposure to plasticizing solvents, including non-toxic compressed carbon
dioxide, making the particles potentially useful for drug encapsulation under mild con-
ditions. The variation of encapsulation efficiency with processing conditions depends
on changes in interfacial free energy and polymer mobility that drive microcapsule
formation. The highest encapsulation amount was obtained with porous hollow
polymer shells obtained via spray drying PLA/dimethyl carbonate solution. The use of
the benign solvents dimethyl carbonate and carbon dioxide for the microencapsulation
process would eliminate concerns of residual solvent remaining in the product for drug
delivery applications. The ease of separation of carbon dioxide from the drug solution
may also enable recycling of the drug solution to increase the overall efficiency of
encapsulation using these novel hollow particles.
94
Chapter 6
Conclusions and Future Work
6.1 Conclusions
Emulsification/solvent evaporation was applied successfully to encapsulate fluores-
cent CdSe(ZnS) quantum dots (QDs) into biocompatible polyisoprene (PI) particles.
This method is simple and robust. The QDs were encapsulated into the core rather
than the surface layer of the PI particles without additional surface modification of
QDs. The polymer particles can be surface-modified with carboxyl groups during
encapsulation to facilitate subsequent bio-conjugation and selective binding.100 The
polyisoprene particles are easily cross-linked by adding a free radical initiator during
emulsification and then heating. Cross-linking was shown to greatly enhance the long-
term fluorescence stability of the encapsulated QDs. The cross-linked PI-QD nano-
composite particles displayed strong and stable fluorescence emission after months in
aqueous suspension. Polyisoprene is in rubber state at room temperature so oxygen can
diffuse into PI particles readily and quench QDs, while crosslinking gives rise to tight
95
polymer matrix inhibiting the diffusion of oxygen into PI particles and protects QDs.
Crosslinking also likely prevents the migration of encapsulated QD in the polyisoprene.
The fluorescence spectra of mixtures of two different-sized QDs change in the PI-QD
particle as compared to their solution spectra, suggesting energy transfer between QDs
due to their aggregation during the encapsulation. As solvent evaporates, QDs are
not distributed evenly in the polymer matrix due to micro-separation. Even though
there appears to be some energy transfer between QDs, different emission peaks were
clearly resolved, indicating that the particles are suitable for multicolor coding. These
fluorescent hybrid nano-composite particles have potential application in genomics,
drug discovery, and other applications that could exploit the high-throughput afforded
by multicolor coding.
Supercritical carbon dioxide was deployed to impregnate dyes into premade
monodisperse polystyrene (PS) particles. Water-soluble ionic dyes were made hy-
drophobic by ion-pairing with alkyl quaternary ammonium cations. The hydrophobic
ion-paired dye can be encapsulated more efficiently into PS particles. For bromothymol
blue, there was at least a five-fold increase in dye loading in the polymer particles
when hydrophobic ion pairs were encapsulated as compared to the encapsulation of
unmodified dye. Addition of water-miscible cosolvents was shown to further enhance
the incorporation of the hydrophobic ion pairs into the polymer colloids. It was
demonstrated that up to a 30-fold increase in the concentration of ionic compounds
in the polymer could be achieved by ion pairing and addition of acetone as a
cosolvent. In the encapsulation process, supercritical carbon dioxide is used in place of
volatile organic solvents, which is particularly attractive for drug delivery applications.
However, the results show that hydrophobic ion pairing is limited in its effectiveness.
96
The ion pairs with rose bengal showed no enhanced loading in the polymer, presumably
due to its lower solubility in nonpolar solvents and the larger molecular size of the
hydrophobic ion pair when compared with bromothymol blue. Since the method
reported here relies on spontaneous diffusion of the ion pair into the polymer, it
requires compatibility between polymer and ion pair and depends on the relatively slow
diffusion kinetics in the plasticized polymer phase. In cases where colloidal stability
was lost, very high dye loading was observed due to heterocoagulation between the
polymer colloid and dispersed particles of ion-paired dye. Unfortunately, in these cases
irreversible and uncontrolled coalescence led to loss of polymer particle integrity.
Hollow PS particles were obtained by thermally induced phase separation. The
PS latex was swollen with solvent, frozen in liquid nitrogen, and dried in vacuum.
The morphology of the resulting hollow particles can be controlled by the amount
of swelling solvent and surfactant. The core-shell structure was favored at high
solvent content and without added surfactant. A bowl-shaped morphology was favored
with added surfactant and at lower solvent content. The experimental results can
be interpreted in terms of interfacial free energy minimization. It can be concluded
that phase separation occurred, as swollen aqueous latex particles were frozen by
liquid nitrogen. Subsequent morphological changes in the biphasic emulsion droplets
are driven by the minimization of interfacial free energy. The thermodynamical
nonequilibrium during the quenching is also considered. After removing the solvent-
rich phase by drying under vacuum, hollow particles were obtained with an open hole
on the surface. The dry hollow particles can be re-dispersed into water or water/ethanol
mixtures by mild sonication. When exposed to a plasticizer, such as compressed CO2,
the open hole on the hollow PS particles can be closed. It is likely that the interior
97
surface of the hollow particles is more hydrophobic than the exterior surface. The
experimental evidence shows that the hole-closing process is also driven by interfacial
free energy minimization, especially the difference in interfacial tension between the
interior and exterior surfaces. When surfactant is added to the suspension of hollow
particles, it interferes with closing of the holes. The surfactant adsorbs onto both
the inside and outside surfaces of the hollow particles to lower the interfacial tension
difference and thus reduces the driving force for closing the hollow particles. The
emulsification/freeze drying process has potential application in microencapsulation
for controlled release. A water-soluble dye was encapsulated into hollow PS particles,
and the dye remained trapped in the microcapsule colloidal suspension for a long time.
Hollow biodegradable poly(DL-lactide) (PLA) particles were also prepared by the
thermally induced phase separation. The tiny droplets of PLA solution suspended in
water as emulsion or directly from a spray nozzle were frozen in liquid nitrogen and
then freeze-dried, giving rise to porous structures. The particle morphology can be
controlled by adjusting the type of solvent, solvent concentration, and surfactant to
control interfacial free energy. The hollow particles can be converted to microcapsules
through short exposure to plasticizing solvents, including non-toxic compressed carbon
dioxide, making the particles potentially useful for drug encapsulation under mild con-
ditions. The variation of encapsulation efficiency with processing conditions depends
on changes in interfacial free energy and polymer mobility that drive microcapsule
formation. Interfacial free energy minimization drives closing the hollow particles if
the polymer chains have mobility. However, core-shell structures or microcapsules are
not the minimal in terms of free energy, while solid particles are. So the hollow particles
will eventually convert to solid particles giving enough time, which is not favorable for
98
encapsulation. To achieve optimized encapsulation efficiency, the exposure time to the
plasticizer should be modest. The particle morphology also affects the encapsulation
efficiency. The highest encapsulation amount was obtained with porous hollow polymer
shells obtained via spray drying PLA/dimethyl carbonate solution. The use of the
benign solvents dimethyl carbonate and carbon dioxide for the microencapsulation
process would eliminate concerns of residual solvent remaining in the product for drug
delivery applications. The ease of separation of carbon dioxide from the drug solution
may also enable recycling of the drug solution to increase the overall efficiency of
encapsulation using these novel hollow particles.
6.2 Future Work
6.2.1 Stabilizing Porous Particles in CO2
Making porous PLA particles by freeze-drying spray of PLA solution in dimethyl
carbonate, a green solvent, is simple, economical, and environmental benign. Such
porous PLA particles can be converted to microcapsules for microencapsulation by
exposure to compressed carbon dioxide for a short time, which is nontoxic and easy to
separate from the suspension, making it attractive for drug encapsulation. Porous PLA
particles without any surfactant could not be suspended effectively in water. Pluronic
P123 surfactant was dissolved in dimethyl carbonate with PLA, and the resulting
porous particles can be suspended in water. However, the particles lost stability and
coalesced in a short time when exposed to compressed CO2. Stabilizing the particles
under CO2 is necessary for CO2-based microencapsulation. Different biodegradable
99
polymers soluble in dimethyl carbonate, such as poly(lactic-co-glycolic acid), can be
tested if they have better stability under CO2. A more promising way is to find a
surfactant soluble in dimethyl carbonate which can stabilize the porous particles under
CO2. It would be desirable that such surfactant is biocompatible. Block copolymer
surfactants,206 such as poly(dimethylsiloxane)-b-poly(methacrylic acid),131 may be a
good choice. The surfactant can also be adsorbed onto the resulting porous particles
for stabilization in CO2. The particles would be stabilized if the surfactant can adsorb
to the polymer surface and make the polymer particle less sensitive to pH and ionic
strength changes when exposed to CO2.130
6.2.2 Controlling Particle Size Distribution by Spraying
The size distribution of the resulting porous PLA particles prepared by coaxial
spray/freeze-drying as described in chapter 5 is broad. It would be highly desirable
for many application to achieve narrow particle size distribution with good control
of the average particle size. The broad size distribution of the particles is due to the
broad size distribution of polymer solution droplets from the spray nozzle. It is not
easy to obtain narrowly distributed droplets by breaking up the polymer solution with
high-speed annular air jet.207,208 Drop-on-demand devices209 for making monodisperse
droplets would be promising to combine with freeze-drying to make uniform porous
polymer particles. In addition, eliminating the high-speed air stream in these devices
would reduce the usage of liquid nitrogen.
100
6.2.3 Making Porous Polymer Particles by Freeze-drying Emul-
sions of CO2 in Water
It has been reported that a solution of sugar acetate in CO2 was frozen and CO2
was sublimated at low temperature to produce porous scaffolds.67 It is possible to
make porous polymer particles by freezing emulsions of polymer/CO2 in water and
then sublimating CO2 at low temperature and under normal atmospheric pressure. This
will not require removing large quantities of water by vacuum pump as conventional
freeze-drying does. Biodegradable porous polymer particles can potentially be made
this way from the recently developed biodegradable non-fluorous polymers soluble in
CO2.210
101
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