thesis

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

ii

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.

iii

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

iv

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.

v

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-

vi

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.

vii

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

viii

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

ix

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

xi

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

xii

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

xiii

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

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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

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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

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(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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