Development of Novel Microencapsulation Processes

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 Engineering Arts, Sciences and Engineering School of Engineering and Applied Sciences University of Rochester Rochester, New York

2009

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Curriculum Vitae
The author was born in Susong, Anhui Province, People’s Republic of China in 1980. He attended Nanjing University from 1997 to 2004, and graduated with a Bachelor of Science degree in Polymer Science and Engineering in 2001 and a Master of Science degree in Polymer Physics and Chemistry in 2004. He came to the University of Rochester in the Fall of 2004 and began graduate studies in Chemical Engineering. He pursued his research in microencapsulation, colloids, and supercritical carbon dioxide under the direction of Professor Matthew Z. Yates and received a Master of Science degree from the University of Rochester in 2008.

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Acknowledgements
First, I would like to express my gratitude to my advisor Professor Matthew Z. Yates for his continuous support in the Ph.D. program. He guided me to the microencapsulation study and encouraged me to try new ideas. He was always there to listen and give valuable suggestions. He allowed me to have flexible schedules to arrange my experiments, while he gave strict requirements on experiments and publishing papers, which benefit me a lot. I really enjoy doing research under his direction. Besides my advisor, I would like to thank Professor Todd D. Krauss for help on the project of quantum dots (QDs) encapsulation. And I would like to thank Dr. Xiqiang Yang and Mridula Nair from Eastman Kodak Company for guide in a 2-year cooperative project of encapsulating nanoparticles for new printing processes. I gratefully acknowledge support from the National Science Foundation (CTS0343774 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

I am grateful to my friends for mutual inspiration. educated me. They gave me life. It would be difficult for me to pass hardships without their encouragement and backing. and worked hard to support me. . I would like to thank Brian McIntyre for his patient teaching and help in electron microscopy and sample preparation. the world would not be so pleasant. share experiences. To my lab-mates and other graduate students in the community.iv Levasseur helped me make hollow poly(lactide-co-glycolide) particles. At last. We exchange ideas. I want to say “thank you” for experiment discussions and equipment sharing. and talk about affairs. I want to thank my family for long-lasting support. Without them. and Graciela Mohamedi-Smith helped me encapsulate nanoparticles for new printing processes in the cooperative project with Eastman Kodak Company.

and printing. The simple method results in QDs encapsulated into the particle core without requiring chemical modification of the QDs. The results show that the particles maintain their size and morphology after exposure to CO2 . Ionic dyes were successfully encapsulated in polystyrene (PS) particles by CO2 based microencapsulation. However. The hydrophobic ion pairs were then encapsulated in preexisting size monodisperse PS particles dispersed in water. and that ion-paired dyes have significantly higher loading in the polymer particles than the original dyes. The polyisoprene is easily cross-linked. compressed carbon dioxide based microencapsulation. controlled release.v Abstract This thesis is for encapsulating additives into polymer particles using different techniques including emulsification/solvent evaporation. and the cross-linking was shown to greatly enhance the fluorescence stability of the encapsulated QDs. drug delivery. The fluorescence spectra of mixtures of two different-sized QDs change in PI as compared to their solution spectra. Addition of water- . for example bio-labeling. and encapsulation with porous polymer particles. Such microencapsulations can be applied to a vast range of areas. Fluorescent CdSe/ZnS quantum dots (QDs) were incorporated into polyisoprene (PI) particles by emulsification/solvent evaporation. suggesting energy transfer between QDs due to their aggregation during the encapsulation. indicating that the particles are suitable for multicolor coding. High-pressure carbon dioxide swelled and plasticized PS and thus facilitated mass transport of the dye into the particles. different emission peaks were clearly resolved. The water-soluble dyes were made hydrophobic by forming ion pairs with alkyl quaternary ammonium cations.

and that morphological changes are driven largely by lowering interfacial free energy. which were made by the freeze-drying and subsequent closing process. which imparts mobility to polymer chains. a water-soluble drug. surfactant. It is shown that the particle morphology is due to phase separation in the polymer emulsion droplets upon freezing in liquid nitrogen. 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. and drying in vacuum. Hollow PS particles were obtained by swelling PS latex with solvent. To encapsulate water-soluble additives. A controlled release biodegradable vehicle for drug was made by encapsulating procaine hydrochloride. amount of closing agent. The dried hollow particles were resuspended in a dispersing media and exposed to a plasticizer. exposure time. 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. The encapsulation efficiency is affected by the hollow particle morphology. into the core of poly(DL-lactide) (PLA) microcapsules. porous polymer particles were made by freeze-drying droplets of polymer solution suspended in water or from a spray. to close the surface opening and form microcapsules surrounding an aqueous core. 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. . and method of dispersing the hollow particles in water. freezing in liquid nitrogen.vi miscible cosolvents was shown to further enhance the incorporation of the hydrophobic ion pairs into the polymer colloids. Controlled release of procaine hydrochloride from the microcapsules into phosphate buffer was observed.

.4 2 Microencapsulation . .2 1. . . .4 21 Introduction . . . . . . . . . . . . . . . . . . . 24 Results and Discussion . . . .1 2. . . . . . . . . . . . . . . . .3 2. . . . . . . . . . . .3 1. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32 3 Hydrophobic Ion Pairing to Enhance Encapsulation of Water-Soluble Additives into CO2 -Swollen Polymer Microparticles 3. . . . . . . . . . . . 15 Outline of the Thesis . . . . . . . . . . . 10 Temperature Induced Phase Separation . . . . . . 19 Fluorescent Quantum Dot-Polymer Nanocomposite Particles by Emulsification/Solvent Evaporation 2. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1 33 Introduction . . . . . . . . . .vii Contents Foreword 1 Introduction 1. . . . . . . 26 Conclusions . . . . . . . . . . . . . . . . 34 . . . . .1 1. 21 Experimental Section . . . . . . .2 2. . . . . 1 2 2 Liquid and Supercritical Carbon Dioxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . .2 5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .4 74 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3 4. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 3. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 Conclusions . . . . . . . . . 47 Effect of Interfacial Free Energy on the Formation of Polymer Microcapsules by Emulsification/Freeze-Drying 4. . . .4 4 Experimental Section . . . . . . . . . . 98 101 Bibliography . . .3 5. . . . . . . . . . . . . . .1 4. . . . . . . . . . . . . . . 78 Results and Discussion . . . . 50 Experimental Section .3 3. . . . . . . . . . . . . . . . . . . . . . . 94 Future Work . . . . . . . 93 94 6 Conclusions and Future Work 6.4 49 Introduction . . . . . 55 Conclusions . . . . . . 39 Conclusions . . . . . . . . . . . . . . . .1 6. . . . .2 4. . . . . . . . . . . . . .1 5. . . . . 71 5 Encapsulation and Sustained Release from Biodegradable Microcapsules Made by Emulsification/Freeze Drying and Spray/Freeze Drying 5.viii 3. . . . . . . 80 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Experimental Section . . . . . . . . . . . . . . . 36 Results and Discussion . . . . . . 52 Results and Discussion . . . . . . . . .

. . . . . . . . . . . . . . . . . . . . . . . . . . . . .ix List of Tables 3. . tetrahexylammonium bromide (THABr). .1 g Triton X-100. . . . .1 Effect of stirring time on procaine hydrochloride encapsulation in hollow PLA particles. 45 5. . . . . . . . . .2 Effect of surfactant removal on procaine hydrochloride encapsulation in hollow PLA particles. . . . . . . . . . .2 Effect of ion pair formation on dye loading of bromothymol blue and rose bengal into polystyrene particles (0. . . . . . . . . . . 88 . . . . . . . but no cosolvent was used) . . . . . . . . . . .1 Water and toluene solubility of bromothymol blue. . . . 42 3. . . . . . . . . . . . and tetraoctylammonium bromide (TOABr) . . . . . . . . . . . rose bengal and their ion pairs with tetrabutylammonium bromide (TBABr). 87 5. . . . . .3 Cosolvent and surfactant effects on loading of bromothymol blue in polystyrene particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40 3. . .

. . . . . . . . .x List of Figures 1. . . . . . . .3 Scheme showing microencapsulation by air suspension coating. . . . . . . . . . . . .5 Phase diagram of CO2 . . . . . . . . . . . .7 1. . . . . . . .8 2. . . . . . . 15 1.1 1. . . . 12 Scheme showing the encapsulation of additives into polymer colloids with the help of compressed CO2 . . . . .4 1. . 27 2. . . . 17 Chemical structure of trans-polyisoprene. . . . . . . . . . . . .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). . 20 CdSe/ZnS quantum dots capped with trioctylphosphine oxide. . . . . . . . . .1 2. .2 Temperature induced phase separation between water and solutes. . . . . . . 11 1. . . . . . . .6 1. . 19 Chemical structure of poly(DL-lactide). . . 29 6 9 . . .2 1. . . . . . . . . . . . . . . . . . . . . . . . . . . 24 Optical microscopic images of un-cross-linked PI-QD particles: (A) dark-field scattering and (B) true-color fluorescence. . . . Scheme showing microencapsulation by emulsification/solvent evaporation. . . . . . . . Scheme showing microencapsulation by coacervation. . . . . . .

39 SEM pictures of polystyrene particles: (A) before and (B) after microencapsulation. 36 Chemical structure of Triton X-100. . .1 Scheme showing the formation of hollow polystyrene (PS) particles by emulsification/freeze-drying and the closing of such particles for encapsulation. . . . . . . .4 3. and aqueous (W) phases. . . . solvent-rich (Q). . . . . . . . . . . . . . . 37 Variable volume high pressure stainless steel vessel. . 31 3. . . . . . 37 Chemical structure of tetraalkylammonium bromide used for ion pairing. . . . . . . . . . . .5 Effect of surfactant on the hollow particle morphology. . 59 4. . . . . . . . . . . .3 Chemical structure of m-PEGMA. . . . . .6 Agglomerated polystyrene particles after the microencapsulation process in the presence of ethanol. . . . . .3 3. . .7 Effect of surfactant on interfacial free energy profile. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .2 4. . . . . . . . 46 4. . . . . . . . . . . .1 3.6 4. . . . . . . .4 Digital photographs of aqueous suspensions of 0. . . . 62 . . . . . . 56 4. . . . . . . . . . 44 3. . . . . . . . . . . . . . . .4 4. . . . . . . . . . . . 53 Polystyrene latex particles as synthesized (A) by surfactant-free emulsion polymerization and (B) by emulsion polymerization with poly(ethylene glycol) macromonomer. . . . 53 4. . . . . . . . . . . 57 Model for a biphasic emulsion droplet consisting of polymer-rich (P). . . . . . . . .xi 2. . . . . . . . . . . . . . . . . . . . . . . . . .3 wt% PI-QD particles under UV illumination. . . . . .5 Molecular structure of: (A) bromothymol blue and (B) rose bengal. . 61 Effect of volume fraction of the swelling solvent on the morphology of the freeze-dried PS particles grafted with PEG. . . . . . . .2 3. . . .

. . . . . . . 72 4. . .xii 4. .15 Confirmation of the hollow structure of closed polystyrene capsules encapsulating rose bengal. . . . . . 79 Hollow PLA particles made by emulsification/freeze-drying: (A) 0. . . . . . . . . . .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. . . . . . . . . . . . . D). . . 45. . . . . . . . .9 Freeze-dried samples with n-heptane (A) and benzene (B) as the swelling solvent. 83 . . . . .11 Effect of surfactant on closing hollow PS by CO2 (A. . . . . . . . . and γPQ to 30. . . .14 Encapsulation of rose bengal by exposure of aqueous PS suspension to toluene in the presence of dye: (A) solid PS with grafted PEG. . . . . 67 4. . . . . . . . The volume fraction of the swelling solvent was 0. . (B) hollow PS with grafted PEG. . . . . . . . . . .8 Interfacial free energy (E/ECS ) vs ΦQ with different volume fractions of solvent (νQ ) by setting γPW . . . . . . . . .4 g/ml. . . . . . . . . . . . B) and toluene (C. (B) 0. . . . 70 4. . . 66 4. . . . . . . . . . 72 5. . . . . . . . . . 69 4. . . 82 5. . γQW . . . . .1 5. . . . . . . .5 hours at room temperature. . . . . .2 g/ml PLA in dichloromethane. 64 4. . . . . . . . . . . . . . and 12. . . . . . .2 Coaxial spray nozzle.13 Schematic illustrating the hole closing process. . . . . . .3 The concentration of procaine hydrochloride in PLA particles as a function of amount of dichloromethane used during encapsulation for 1. . . . . 70 4. . . . . . . . .8. . . . .12 Effect of surfactant on closing hollow PS by toluene. . . . . . . . . . . . . . . respectively. . . . . . . . . .

85 5. . . . .2. . . . row 1. .5 hours at room temperature. column 2). . . . . . . .8 PLA microcapsules formed by exposure of aqueous hollow particle suspension to compressed carbon dioxide.7 Porous PLA particles made by coaxial spray/freeze-drying with dimethyl carbonate as the solvent. .2. . . . . . . . column 3). . . . . . . . . 90 5. . . . (B) after encapsulation. . . . . . . . . . . . . . . . . ◦ microcapsules formed without removing excess surfactant (Table 5.2 phosphate buffer: microcapsules formed with excess surfactant removed (Ta- ble 5. 87 5.9 Release of encapsulated procaine hydrochloride in pH=7. . . . . . . . . . . . . . . . . 92 . . 85 5. . . . 91 5. . . . .xiii 5. .5 Interior structure of PLA particles encapsulating procaine hydrochloride using different amounts of dichloromethane and stirring time. . (A) Before encapsulation. row 1.4 PLA particles after exposure of aqueous suspensions to different amounts of dichloromethane for 1. . . . . . . . . . . . . . . . . . .6 Scheme showing the closing process of hollow PLA particles for encapsulation. . . . . . . . . . .

Li Guo. crosslinked PI particles and studied the effect of crosslinking on fluorescence stability of QDs. synthesized QDs of different sizes. collected a transmission electron microscopy image of uncrosslinked PI-QD particles. I encapsulated two kinds of QDs into PI particles by emulsification/solvent evaporation. I collaborated with Hongwei Liu. took microscopic fluorescence images and single-particle fluorescence spectra of PI-QDs. He examined the effects of homogenizing speed and concentration of polymer solution on PI particle size. The results from these collaborators are given in a published paper (Chem. and Fengzhi Jiang. a previous postdoctoral student of Professor Krauss.1 Foreword For the project encapsulating fluorescent quantum dots (QDs) into polyisoprene (PI) particles. Hongwei Liu. did early work in the project. Mater. All data shown in Chapter 2 are from my portion of the study. . 19. Fengzhi Jiang. 2007. investigated the energy transfer between QDs in PI particles. a previous PhD student of Professor Krauss. a previous PhD student of Professor Yates. Li Guo. 2930-2936) in which they are listed as coauthors. and carried out selective binding of streptavidin-coated PI-QD particles to biotin-conjugated polystyrene spheres.

2 Chapter 1 Introduction 1. gases or fine solid particles with natural or synthetic polymers. Many materials can be used for the wall. internal phase. or even smaller microcapsules. or fill and the encapsulating matrix is called shell. lipids. suspension of solids. amorphous. crystalline.1 Microencapsulation The term microencapsulation refers to technologies for enveloping small droplets of liquids. The core can be any form. carbohydrates. coating. inorganic materials. wall material or membrane. emulsion. single layer or multilayer. semipermeable. or impermeable. The shell can be matrix. 5 The capsule can be permeable. The cell wall protects the inside cellular parts from undesirable environmental conditions and the selectively permeable cell membrane controls influx and release of metabolites. and can consist of one kind or several kinds of materials. and proteins. The encapsulated material in microcapsules is referred to as the core. including gums. 1–4 Biological cells are an example of natural microencapsulation. cellulose. 2 The choice of wall materials .

The properties of the microcapsules such as average size. 9 In feedstuff industry. 5 Enzymes can be trapped in microcapsules to accelerate cheese ripening and improve cheese flavor. 8 In the food industry. size distribution. and ingredient distribution will affect the subsequent release profile. 6 Microencapsulation can be used to protect active ingredients. or transferring liquid to solid for dry mixing. unsaturated vegetable fats. control the release of encapsulated materials.3 depends on the physicochemical properties of the core materials. Biodegradable polymers have been widely used in microcapsules for drug delivery to provide controlled release of encapsulated drugs. vitamins. asphalt. reducing volatility.7 Carbonless paper is an early commercial application of microencapsulation. and fats to protect the encapsulated ingredients from environmental conditions such as light. reduce drug dosage. reduce nutritional loss. water-soluble fertilizers are encapsulated by waxes. 1. the process making the microcapsules. which will avoid higher local . enzymes. surface morphology. degradation or enzymatic hydrogenation. mask or preserve flavors. deliver drugs to specific locations. spherical or irregular. microencapsulation is widely used to encapsulate flavors. and hormones are encapsulated to prevent oxidation. and polymers such as polyurethanes. and the desired properties of the product. oils. and make handling encapsulated material easier. oxygen and moisture for increasing durability. The size of microcapsules is typically several hundred nanometers to a few thousand micrometers. In agriculture.2. 2 Carbon dioxide can be encapsulated in hard candies to produce a sizzling effect on the tongue as the candy melts in the mouth. depending on the applications and stability of the capsules and the encapsulated ingredients. inner structure. 2 The outside may appear smooth or rough. The microcapsules can be solid as free-flowing powders or suspended in water.

Alternatively. partition coefficient. oxygen and waste. given the small size and thin wall thickness of typical microcapsules. This technique can be used to treat numerous medical diseases. Complete impermeability to the encapsulated gases or liquids is hard to achieve. such as diffusivity. chewing or crushing. porosity and reactivity. 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. which protects the inner cells from the host’s immune system while allowing the diffusion of nutrients. microencapsulation is used to control the release of therapeutic agents and prevent overdose after administration. such as by shearing. melting or burning. and central nervous system diseases. microencapsulation is used to gain sustained release of deodorants and perfumes. However. such as thickness. It can be done mechanically from outside. such as by heating above the boiling point of the core material. In pharmaceutical industry. the release is regulated by the wall thickness and porosity. which can reduce or eliminate chronic administration of immunosuppressants. cancer. The wall can also be destroyed by dissolving. providing sustained release of the inside content. or from inside. 10 In the cosmetic industry. the durability of core materials can be improved by choosing suitable wall materials and capsule size. the solubility parameter of the polymer should be far from that of the core material. and of the wall material. . Cells can be encapsulated in a polymer matrix surrounded by a semipermeable membrane. 11 Generally the release profile is affected by the physicochemical properties of the core material. If the capsule wall is permeable to the core material. the capsule wall is often opened.4 fertilizer concentration and reduce the number of applications. To prevent leaking of the encapsulated materials. and vapor pressure. including diabetes. To release the core material.

5 which is desirable in therapeutic treatment. microcapsule size. such as dipping or centrifuging. such as biodegradation kinetics of the polymer. 1 . opening the microcapsule wall provides immediate burst release. the diameter of which can reach up to 8 mm. cocrystallization. 16. For drug delivery applications. spray drying. liposome entrapment. Most of them are physical techniques. application and release mechanism. The selection of a method depends on economics. The release of drugs from biodegradable polymers can be controlled by many factors. which is then hardened. properties of the core and wall material. One typical method involving chemical reaction is interfacial polymerization.13 properties of polymers and drugs. 12. 15 and the shape of the microcapsules. and biodegradability can be used to tune the release.1 Methods for Microencapsulation There are many methods for microencapsulation.17 1.1. without chemical reactions. It has been reported that kerosene was encapsulated this way using a solution of polyvinyl alcohol and sodium alginate in water/glycerol. The process can make uniform and relatively large capsules. air suspension coating or fluidized bed coating. allowing small molecules entering inside but preventing contents inside from leaving. 14 compatibility between polymers and drugs. The dipping or centrifuging technique passes core material droplets at high speed through a thin film of liquid wall material. emulsification/solvent evaporation or extraction. which can be used for encapsulating cells. The wall material was hardened in calcium chloride solution. The capsule wall can also be semipermeable. In contrast. spray chilling. interfacial polymerization. the capsule wall should be biocompatible. coacervation.

2 Figure 1.1). generally air and occasionally inert gas such as nitrogen. with a 100 µm minimum particle size. The process can be repeated to achieve the desired film thickness. 1. During spraying. 5 The resulting microcapsules are solid and free-flowing. Polymers are dissolved in a solvent containing the additives to be encapsulated. The core material may be sprayed from an inner nozzle and the encapsulating material from a concentric ring nozzle. seeds and foodstuffs. The size of the core particle for this technique is usually large. . After the evaporation of the solvent. leaving the additives surrounded by polymer.1: Scheme showing microencapsulation by air suspension coating. Spray drying is to spray an emulsion or suspension in a stream of hot gas. The technique has been applied to encapsulate pharmaceuticals. fine solid core materials are suspended by a vertical current of air and sprayed with the wall material solution (Figure 1.6 In the air suspension coating or fluidized bed coating. Smaller particles tend to aggregate or get carried away by the exhaust air. the atomized droplet shrinks as the solvent evaporates. a layer of the encapsulating material is deposited onto the core material.

and the solvent can be removed by sublimation in vacuum. It is important to control the rates of nucleation and crystallization during the process. 20. The cooling solidifies the coating material. and the syrup is mixed to induce nucleation and agglomeration. The atomized droplets can be frozen. The crystal aggregates are in the size range of 3 to 30 µm and can entrap materials. resulting in microcapsules. Spray chilling or cooling can be used to encapsulate water-soluble ingredients. then the material to be encapsulated is added. the sucrose syrup is concentrated to the point of supersaturation. it is the main method for encapsulating flavors in the food industry due to its low cost. such as water-soluble vitamins and enzymes. . and hardened at −80 °C wherein solvent extraction occurs. By this way.21 First. proteins can be encapsulated in polymer micro-particles without significant loss of biological activity. droplets of polymer/drug solution can be sprayed into liquid nitrogen containing frozen ethanol. 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. 18 An alternative to spray drying is spray chilling or cooling. freeze-drying or lyophilization. A variation of the method includes solvent extraction while cooling. 19 There is another approach to the drying process. 5 which is 30-50 times cheaper than freeze-drying. For example. Numerous types of flavor ingredients can be encapsulated by this way. such as in liquid nitrogen. monoclinic and spherical and not suitable for encapsulation.7 Although the high temperature in spray drying is problematic for some thermally labile materials. aggregates of the crystals form when they are spontaneously crystallized from a supersaturated solution. Although sucrose crystals are solid. Co-crystallization is a process to encapsulate ingredients between sucrose crystals.

The use of organic solvents is also a concern for some applications.25 Recently microfluidic techniques have been developed that allow high efficiency encapsulation into liposomes in a solvent-free continuous process. To encapsulate material in the core of liposomes. the liposome bilayer is broken and the content is released immediately. 3 The electrostatic charge. such as chloroform. 22. pH. However. ionic strength or temperature . typically around 50 °C. which is not favorable for large-scale production. permeability and stability of liposomes can be more easily controlled than for other types of microcapsules. another challenge is that the liposomes must be kept in dilute aqueous suspension. which is then hydrated in buffer solution to form liposomes. and mixed with a solution containing the material to be encapsulated.27 However. phospholipids are usually dissolved in organic solvents. Above the transition temperature. 3 Coacervation is an encapsulation technique based on polymer phase separation. 2. hormones. and the polymer is then gradually deposited onto the core material by inducing precipitation by adding non-solvent for the polymer. 26. which have been used to deliver vaccines. poor encapsulation efficiency and the lack of a continuous production process limit large-scale use. 24 The encapsulated ingredients can be released from liposomes by heating above the transition temperature of the phospholipids. The solvent is evaporated to form dry lipid film. enzymes. Both the lipid and ingredients to be encapsulated cane be lost during the solvent evaporation process. The core material is dispersed in a continuous phase in which polymer is dissolved. and vitamins.23 The size range is from 25 nm to several micrometers in diameter.8 Liposomes are hollow microcapsules of phospholipids that form spontaneously when phospholipids are dispersed in water. storage and shipping. adjusting pH. Aqueous or lipid-soluble materials can be entrapped in liposomes.

as extremely sticky coacervate droplets frequently adhere to each other before complete phase separation. However. The mixture is then emulsified into water containing emulsifier (Figure 1.30 The solvent in the emulsion is removed by evaporation at high temperature or reduced pressure or . vegetable oil. crosslinking or removing solvents by exposing to excess amount of another nonsolvent. the coating is solidified by heating. Coacervation is efficient and can produce microcapsules with a broad range of sizes.9 (Figure 1.28 Commonly used precipitants or nonsolvents include silicone oil. 7 When phase separation happens.2). Emulsification/solvent evaporation or extraction is mainly used to encapsulate hydrophobic materials through oil-in-water (o/w) emulsification process. 2 Another disadvantage of coacervation is that formation of large aggregates is hard to avoid. and they tend to coat solid dispersed particles. it is complicated and expensive. very fine coacervate droplets appear at first. and diethyl ether. Polymer is dissolved in a water-immiscible and volatile organic solvent. so it is not broadly used in industry. washed with solvents. 29. 8. Figure 1. such as hexane. The droplets coalesce until a coherent coacervate phase appears surrounding the solid particles. Finally. and the material to be encapsulated is dissolved or suspended in the polymer solution.2: Scheme showing microencapsulation by coacervation. light liquid paraffin and low molecular weight polybutadiene. The microcapsules are collected by filtration or centrifugation. and then dried. heptane.3).

However finding conditions that provide high encapsulation efficiency can be difficult. Such rate is determined by temperature. non-flammable. Carbon dioxide is non-toxic. giving rise to a w/o/w emulsion. 6 The solvent removal rate will affect the morphology of the final particles. 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. leaving solid particles. solvent. However.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. and hydrophobic oils are used as the continuous phase. the solvent is removed by evaporation or extraction. and pressure. water-miscible organic solvent is used to dissolve polymer and hydrophilic materials to be encapsulated. 31–33 The micro-particles are then collected and dried to eliminate the residual solvent. In this method. 6 For most water-soluble materials. 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.2 Liquid and Supercritical Carbon Dioxide Compressed carbon dioxide in the liquid or supercritical state is attractive as a solvent in microencapsulation processes. The primary emulsion is then transferred into water containing an emulsifier under vigorous mixing. 6 To encapsulate hydrophilic materials. 37 1. polymer solubility. 35. To obtain micro-particles. and inexpensive. oil-in-oil (o/o) emulsification is used. The high volatility of carbon dioxide allows it to be easily separated .10 extraction in a large amount of water. water-in-oil-in-water (w/o/w) methods are used.

the density can be continuously varied from gas-like to liquid-like without undergoing a phase transition.11 Figure 1. Although it is a good solvent for many non-polar volatile materials. 38.4. including the ability to dissolve solids. Generally the solubility of a substance in supercritical fluid increases with vapor pressure. from polymeric materials by lowering pressure. miscibility with permanent gases. Above the critical temperature. and low viscosity. For carbon dioxide. 40 CO2 is a poor solvent for most highly polar or high molecular weight materials under moderate pressure and temperature. high diffusivity.3: Scheme showing microencapsulation by emulsification/solvent evaporation. 38 as shown in Figure 1. the critical temperature is 31 °C and the critical pressure is 74 bar. The supercritical fluid state is reached when the temperature and pressure of a substance are above its critical temperature and pressure. the solvating power of a supercritical fluid can be fine-tuned by adjusting pressure or temperature. Supercritical fluids display a hybrid of the properties that are typical for a liquid and a gas. with gases being .39 Since the solubility parameter is dependent on density.

Its poor solvent power for most polar and high molecular weight materials has led to applications in polymer fractionation. The high solubility of fluorinated polymers in CO2 has led to applications in coatings. such as fluorocarbons and silicones. porogenic diluent for synthesizing porous polymers. 42 Figure 1. such as methanol. 43 extraction. Supercritical CO2 can be used as a medium for polymer synthesis and processing. 45 Low cohesive energy density polymers. 46 lithography.4: Phase diagram of CO2 . 38. supercritical CO2 is attractive because of the ease of solvent evaporation and reduction in use of harmful organic solvents. can be added to increase the solvent power of supercritical CO2 .12 completely miscible. . 49 Although elevated pressure and specialized equipment are required. 47 and formation of water-in-CO2 emulsions.48 Supercritical CO2 is also an excellent nonsolvating. 41 Organic solvents. purification. are the only types of polymers that have appreciable solubility in CO2 at moderate temperature and pressure. 44 and polymer particle formation by anti-solvent precipitation.

Although CO2 is a poor solvent for most polymers. or near-critical. 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 . 53 Impurities in polymers. The addition of ethanol has been shown to increase the diffusion coefficient of a dye over that with pure CO2 . 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). The reduction of Tg is a thermodynamic effect due to intermolecular interaction between CO2 and polymer. since they can . unlike the discontinuous liquid/vapor phase transition that occurs at the vapor pressure when temperature is below the critical temperature. 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. liquid CO2 exhibits many solvent and transport properties similar to those of supercritical CO2 . catalysts. The liquid state is reached when a substance is compressed above its vapor pressure at a temperature below its critical temperature. it is swollen. it has substantial solubility in many polymers. Stronger interaction will enhance the depression. 51 When polymer is exposed to supercritical CO2 . When slightly below the critical temperature and pressure. residual solvents. the vapor pressure of CO2 is about 60-70 bar.13 Liquid CO2 is also desirable for many of the same reasons as supercritical CO2 . such as unreacted monomers. and byproducts can adversely affect the use of polymer products. Near room temperature. 52 A cosolvent may promote the diffusion of additives in polymer under supercritical CO2 . even at modest pressure. which results in decrease in the glass transition temperature (Tg) of polymers or plasticization.

indicating shorter depressurization will favor extracting impurities out of polymer. and they can show significant volume change on mixing. then the solution of additives in CO2 is introduced and the solute is transferred from CO2 to polymer. which can influence the transport of impurities out of polymer. steam stripping. 50 . When pressure is reduced and supercritical CO2 diffuses out of the polymer. The rate of releasing CO2 after the impregnation is related to polymer relaxation rate. Supercritical extraction from polymers can produce high-purity. so strict regulations have been enacted to limit permissible levels of various impurities. 55 When polymer is exposed to supercritical CO2 . the rate of removing CO2 from the polymer can be fast compared to polymer relaxation to its un-swollen state. Slow release of CO2 allows for the escape of CO2 from polymer matrix before the polymer recovers from the swollen state. or solvent extraction. high-quality products with lower energy cost. Last. In some cases these methods are not sufficient to reduce the residual impurities to the permissible levels. CO2 is released and the solute is trapped in the polymer material. Usually polymers are purified by vacuum. or the entrapped CO2 may lead to foaming. Supercritical fluid/polymer systems are highly thermodynamically nonideal. Such nonideal volumetric behavior can cause convection induced by diffusion. 54 Excess impurities are not desirable for both environment and health. First. Generally there are three steps in the impregnation.14 change the taste. the diffusion of impurities out of polymer is significantly enhanced. toxicity or thermophysical properties of polymers through plasticization or depolymerization. which can lead to a coupling between the polymer relaxation and the mass transfer. color. the polymer materials are exposed to supercritical CO2 for a while. 54 Enhanced diffusivity of additives in polymers exposed to supercritical CO2 can also be used to impregnate additives into polymer.

Porous or hollow microparticles can potentially be used in producing novel microcapsules. 57.3 Temperature Induced Phase Separation Porous polymers can be made from phase-separated polymer/solvent systems by evaporating.60 Freeze drying usually consists of freezing an aqueous solution or suspension and .15 When suspensions of polymer particles in water are exposed to supercritical CO2 with the presence of additives in water. 56 as shown in Figure 1. or sublimating the solvent-rich phase. 57 porogen leaching. such as phase separation. There are several techniques for making porous polymers.5: Scheme showing the encapsulation of additives into polymer colloids with the help of compressed CO2 . After releasing CO2 . Figure 1. 59 To some extent. these techniques rely on thermally induced phase separation and freeze-drying to remove solvent. and create porous structures. the transport of the additive into polymer particles can also be enhanced.5. Although the presence of CO2 may require some appropriate surfactants to stabilize polymer particles in water. 58 emulsion/freeze-drying. additives can be trapped in colloidal polymer particles. it does not require harmful organic solvents used in conventional microencapsulation and may remove residual solvents in the premade polymer particles. 1.

62–65 To produce large ice crystals or pores. such as cooling rate. To produce small crystals or pores. ice crystal growth. 60 During crystallization. 61 During the freezing. ice nucleation temperature. such as polymers. The subsequent drying process. Actually the adsorption and desorption at the ice surface are in dynamic equilibrium. and porosity can be tuned by cryogenic parameters. 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. and solute concentration. Very low crystal growth speed leads to a pure crystal and highly enriched solution. while complete desorption would allow ice crystals growing freely. While at high crystal growth speed. The growing ice crystals exclude solute molecules. The size of ice crystals is also related to solute concentration and the size of solute. the freezing temperature should be high and the time for crystallization should be long. The effectiveness of a solute in inhibiting . from its freezing front to the boundaries between adjacent ice crystals. Irreversible adsorption will stop ice crystal growth. leads to macroporous structures in which the empty spaces are occupied originally by ice crystals. 66 The ability of ice crystal growth depends on the adsorption/desorption balance of the solute at the ice crystal surface. The pore size. 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. and solute cryo-concentration take place.6. during which ice crystal sublimation and desorption of the bound water occur.16 subsequent drying under vacuum. supercooling degree. as shown in Figure 1. pore geometry. unless the solute possesses crystallographic similarities with the growing crystal that favors adsorption. phase separation between water and solute. temperature gradient. the solute is unable to diffuse away from the growing crystal and is completely engulfed among crystals.

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. In the region closest to the initial contact with the cold bath. Therefore large solutes or high solute concentration leads to small crystals. 65. by lowering a vessel containing the solution into a cold bath. at a controlled rate. for example. such as liquid nitrogen. Figure 1. either in the form of three-dimensional porous structure or twodimensional oriented surface pattern.17 crystal growth depends on the extent of ice crystals’ surface that is covered by such a solute.67 The directional freezing can be simply done. while small solutes or low solute concentration leads to large ones. With crystal growth aligned under directional freezing. 61 When pure . well-defined porous structures can be obtained. no porosity is observed after the subsequent freeze-drying process and the material is dense. 60 The structure is heterogeneous in the freezing direction.6: Temperature induced phase separation between water and solutes.

impurities in water affect ice crystallization. 57. amorphous ice forms in the immersed portion. Away from the immersion level. a freezing rate of about 106 °C/s is required. ice crystallization determines solute redistribution and leads to porous structure after freeze-drying. At last the temperature is not low enough for supercooling. leading to porous structures. When an aqueous solution or suspension is immersed partially into liquid nitrogen. 67 . The pore size depends on the distance from the immersion level in liquid nitrogen. Aligned porous materials by directional freezing can be used in micro-fluidics.69 gelatin. such as poly(DL-lactic-co-glycolic acid). 64 and poly(vinyl alcohol). Homogeneous porous structure can be made by continuous immersion at a constant rate into liquid nitrogen. 72 In addition to aqueous solution or suspension. the more temperature increases. The further the ice front moves away from the immersion level in liquid nitrogen. 68 However. A lot of porous materials have been made by this way. an ice front runs along the non-immersed portion. 73–75 the process can be applied to organic solution or suspension. The temperature at the ice front is higher than that of liquid nitrogen. 70 molecular filtration. 61 The temperature induced phase separation is a simple and generic way for preparing porous structure. and no matter segregation happens. 71 and tissue engineering. amorphous ice does not form.18 water is frozen in liquid nitrogen. It has also been reported that CO2 can be used as the solvent for the process to make porous polymers. and so crystalline rather than amorphous ice is formed. 65 The process avoids chemical reaction and further purification. A solution of sugar acetate in liquid CO2 was frozen in liquid nitrogen and the solid CO2 was removed by sublimation. so that porous structure does not form. Meanwhile. To get amorphous ice from pure water.

encapsulating aqueous solution inside. The oil-in-water emulsification as described in Chapter 2 is not effective for encapsulating hydrophilic materials. improving the stability of QDs. Chapter 3 describes a method with liquid and supercritical carbon dioxide as a processing solvent.7) can be reacted further to crosslink the particle. hollow polystyrene microparticles were made by temperature induced phase separation as described in Chapter 4. And the resulting hydrophobic ion-paired dyes diffused into polystyrene particles plasticized by CO2 . procaine hydrochloride. The double bonds in polyisoprene (Figure 1.19 1. Figure 1. Chapter 5 describes the use of the novel method developed in Chapter 4 to encapsulate a water-soluble drug. in biodegradable poly(DL- . The hollow particles were subsequently converted to microcapsules.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 hydrophilic species were made hydrophobic by ion pairing without covalent bond formation.7: Chemical structure of trans-polyisoprene. To encapsulate hydrophilic materials without hydrophobicity conversion such as by ion pairing. For encapsulating hydrophilic species.

and the drug release from the microcapsules were measured.8) microcapsules.20 lactide) (Figure 1. Chapter 6 gives overall conclusions of the thesis and describes potential future work building upon the current study. Figure 1. . Effect of experimental conditions on drug encapsulation was investigated.8: Chemical structure of poly(DL-lactide).

Yates. Liu. M. Mater. W. 2. H. The resulting PI-QD nanocomposite particles form as colloidally stable suspensions in water that exhibit stable fluorescence for months. Yin. Du. Emulsification/solvent evaporation results in QDs encapsulated into the particle core without requiring chemical modification of the as-prepared QDs. Z. D. 2007. and inexpensive. The PI can be easily cross-linked after encapsulation to enhance the fluorescence stability of the QDs. L. Krauss. robust. ∗ . a technique that is facile. 19. Guo..1 Introduction Semiconductor quantum dots (QDs) are ideal fluorophores for biological imaging: 76... H. Chem. T.... Jiang.21 Chapter 2 Fluorescent Quantum Dot-Polymer Nanocomposite Particles by Emulsification/Solvent Evaporation∗ CdSe/ZnS quantum dots (QDs) were incorporated into biocompatible polyisoprene (PI) particles by microencapsulation through emulsification/solvent evaporation. 2930-2936.77 their emission wavelength can be tuned by simply changing the particle size. F.

as synthesized. the surface of QDs.97 Encapsulation of QDs through emulsification/solvent evaporation has several potential advantages over other encapsulation methods. 79–81 making them unsuitable for direct use in biological environments. we chose to investigate QD/polymer nanocomposite formation through emulsification/solvent evaporation. 88 covalently bonding QDs with polymerizable ligands to polystyrene during suspension polymerization.91 entrapment through strong noncovalent interactions such as hydrogen bonding and ionic attraction. 89. a technique commonly used to create pharmaceutical/polymer composite particles for drug delivery. 76. Examples of strategies used to make QDs more biocompatible include exchanging the organic ligands on the QD surface with a more polar species. 92–94 and physically entrapping QDs into particles formed by emulsion polymerization 90 or sol-gel synthesis. 78 allowing facile multicolor coding for high-throughput assays.77. none of these methods completely prevent individual QDs from photooxidation. 96.82 coating with a silica layer. 85 Recently. and thus fluorescence efficiencies and the shelf life of biocompatible QDs are typically reduced several fold relative to their organic-capped counterparts. Emulsification/solvent evaporation can encapsulate QDs without chemical modification of the QD surface. unlike methods that copolymerize . 86 entrapping QDs in cross-linked reverse micelles. 83 and encapsulating the hydrophobic QD in lipid micelles. is hydrophobic. 95 In the present study. 84 However. However. 86–90 Several different approaches have been taken including solvent swelling of aqueous polymer colloids.22 and simultaneous excitation of different-sized QDs can be achieved using only a single wavelength. 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.

98 Finally. nontoxic. so it is expected that polyisoprene (PI) should interact favorably with the alkane-coated quantum dots. ensuring that the QDs remain entrapped after encapsulation. 99 . Then. In the present study. leaving a polymer/additive composite colloid stabilized by surfactant. transPolyisoprene was chosen because it is soluble in many of the same solvents as the QDs. PI is also highly desirable because it is biocompatible. In the emulsification/solvent evaporation technique. Control of surface chemistry is important to allow subsequent bioconjugation to the polymer-QD particles. To form QD/polymer composite particles by emulsification/solvent evaporation. Methods that incorporate QDs by solvent swelling of preformed aqueous polymer colloids 86 generally lead to a large fraction of surfaceadsorbed QDs.23 reactive QDs. the polymer/additive solution is emulsified into an aqueous surfactant solution and the polymer solvent is evaporated. with a relatively small fraction actually encapsulated in the core of the polymer particles. the additive to be encapsulated is CdSe QDs coated with alkane ligands.91 The reactive QDs can also adversely affect the nucleation and growth of polymer particles during copolymerization. the polymer must dissolve in a solvent compatible with the QDs. and noncarcinogenic. leading to a widening of the particle size distribution. 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. the polymer and desired additive are first dissolved in a mutual solvent. 91 The QDs are encapsulated in the core of the polymer particles during emulsification/solvent evaporation. The polymer chain is unsaturated so that it is easy to chemically cross-link after processing. including alkanes. 89. The alkane ligands stabilize the QDs in hydrophobic solvents such as chloroform and hexane.

and sodium hydroxide(NaOH) were purchased from Aldrich. Baker. methanol was added to precipitate the QDs. The precipitated QDs were separated by centrifugation and then dried in a vacuum oven. Hexane (AR grade) and methanol (SpectrAR grade) were obtained from Mallinckrodt Chemicals. 100 The core CdSe QDs were synthesized in various sizes. and capped with tri-n-octylphosphine and trioctylphosphine oxide following procedures similar to those in the literature. Figure 2. Deionized water was used in the experiments. lauric acid.101 The capped QDs were stored in hexane. Prior to microencapsulation.80. coated with a thin shell of ZnS.24 2. The CdSe/ZnS quantum dots (Figure 2. The dried QDs were dissolved in 5 mL of chloroform to make a QD solution. 2. Microencapsulation of QDs in Un-cross-linked PI Particles.2 Experimental Section Acrylic acid. T. Sodium dodecyl sulfate (SDS) was purchased from J.1) were provided by Professor Krauss’ group. and its concentration .2 -azobisiso- Materials. butyronitrile (AIBN). trans-polyisoprene (PI). 79.1: CdSe/ZnS quantum dots capped with trioctylphosphine oxide.

Particle Size Measurement. The QD solution and the polymer solution were mixed together. A polymer solution containing QDs and an aqueous surfactant solution were made as stated above. Microencapsulation of QDs in Cross-linked PI Particles. The hydrodynamic particle diameter and polydispersity were determined by dynamic light scattering (Brookhaven Instruments model 90 Plus) using cumulant analysis for polydispersity determination.15 g of lauric acid and 0. The flask was sealed and purged with Ar for 15 min and then put into an oil bath at 70 °C.050 g of SDS had been added. Then 0. The solution thus obtained was poured into the aqueous surfactant solution and bubbled with Ar for 20 min. In a separate flask. Then 0. 0. The cross-linked latex produced was transferred to a beaker after cooling and stirred overnight under Ar to allow the organic solvent to evaporate. During the experiment.25 was determined by absorption spectroscopy. The mixture was homogenized at 24000 rpm for 1 min and then transferred to a round-bottom flask into which 0. polymer particles formed and entrapped the QDs. The mixture was homogenized at 24000 rpm for 1 min (IKA WORKS.020 g acrylic acid were added into the polymer solution.15 g of PI was dissolved in 5 mL of chloroform to form a polymer solution. . T25 basic ULTRA-TURRAX homogenizer) and then stirred using a magnetic stirrer for 5 h under Ar to evaporate the organic solvent chloroform. The solution of polyisoprene and QDs in chloroform was then poured into the aqueous surfactant solution and purged with argon (Ar) for 20 min.045 g of sodium hydroxide were dissolved in 50 mL of water to produce an aqueous surfactant solution. the system was shaded from light to avoid any possible photobleaching of QDs. After solvent evaporation.020 g AIBN and 0. The polymerization was carried out for 6 h.

Particle fluorescence spectra were acquired by coupling an Acton spectrometer (SpectraPro 300i) to the output port of the microscope. 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. and detected with either a cooled CCD camera (Versarry 512B. The effective diameter determined by dynamic light scattering is about 400 nm. 2. Princeton Instruments) or a digital camera (Nikon Coolpix 995). 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. and the half width of the size distribution curve is about 200 nm. respectively.26 Fluorescence Imaging and Spectroscopy. 103–105 or microchannel emulsification.3 Results and Discussion The colloidal particles prepared by the emulsification/solvent evaporation have broad size distribution. Uniform polymer particles can be made by emulsification/solvent evaporation starting from a monodisperse emulsion created by microfluidic flow focusing. 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. Colloidal QD fluorescence spectra were taken with a modular Acton Research fluorometer. 102 membrane emulsification. 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. The resulting nanocomposite particles were 403 ± 10 nm in diameter as measured by dynamic light .

25. and not simply adsorbed on the surface. The fluorescence intensity of particles can be adjusted by changing QD concentration before solvent evaporation. It appears that QDs were distributed evenly inside the particles. (A) (B) Figure 2. the relative brightness of each nanocomposite particle. .2: Optical microscopic images of un-cross-linked PI-QD particles: (A) darkfield scattering and (B) true-color fluorescence. The fluorescence image shows that the location of the QD fluorescence corresponds to the location of the large PI-QD nanocomposite particles. White light scattering and fluorescence images of the hybrid particles are shown in Figure 2. However.2B is the corresponding true color fluorescence image obtained for the same sample. and Figure 2. Figure 2. as expected.27 scattering with a polydispersity index of 0.2A is a white-light dark-field scattering intensity image obtained by the CCD. 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.2. was highly nonuniform due to the particle size polydispersity. 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.

as demonstrated recently. Compared to the fluorescence spectrum of a hexane solution of yellow and red QDs with the same molar ratio of 2:1. 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. the average separation would be ∼30 nm. The spectrum has two sharp peaks resulting from yellow QDs (fluorescence maximum = 560 nm) and red QDs (fluorescence maximum = 620 nm). However.28 Figure 2. Even for samples in which the fluorescence spectrum of the QDs is altered within the polymer. much too far apart for efficient energy transfer.2. 91 The spatial distribution of QDs inside the PI-QD particles is important for multicolor optical labeling. respectively. it is still possible to achieve multicolor coding by adjusting the number ratio of QDs.) The changes in the fluorescence spectrum are consistent with nonradiative (F¨ rster) energy transfer from the yellow to the red QDs. (Note that the yellow QDs have a fluorescence quantum yield (QY) of 12% compared to only 1% for the red QDs. the relative fluorescence intensity of the larger (red) QDs was enhanced relative to the smaller (yellow) QDs when entrapped in the PI-QD particles. Because of the changes in fluorescence emission caused by micro-phase separation in the polymer. 107 If the QDs were uniformly o distributed in the PI. the resulting multicolor fluorescence spectrum from the particle will not represent the relative concentrations of the QDs inside. Local close-packing was also recently observed for QDs with polymerizable ligands incorporated into polystyrene during dispersion polymerization. Therefore.3 shows the fluorescence spectrum (solid line) of a single PI-QD particle from the same sample shown in Figure 2. 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. .

29 Figure 2. Lower mobility will inhibit QD aggregation and prevent migration to the surface of the polymer particles. Polymerization can optionally be employed after microencapsulation to cross-link the polyisoprene to reduce QD mobility. crosslinking may also improve the barrier properties of polyisoprene with respect to oxygen. it is likely that crosslinking the polymer will result in increased stability to oxidation and longer shelf life. 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. 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. In addition to preventing QD migration.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). .

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 remaining fluorescent QDs were stable for extended periods.3 wt% aqueous suspensions of PI-QD nanocomposite particles under UV light illumination. A series of images are shown for both vials. The upper vial contained cross-linked PI-QD particles and the lower one contained un-cross-linked particles. It is apparent that the crosslinked samples display enhanced stability against photooxidation when compared to the un-cross-linked sample. Others have also reported that free radical polymerization with AIBN is detrimental to QD fluorescence. While the cross-linked sample is much more stable after preparation. resulting in increased energy transfer to the red QDs. The un-cross-linked sample is orange just after preparation.4 shows digital photographs of two vials. Figure 2.2. The images of fluorescence decay are consistent with visual observations of the samples. each containing 0. taken immediately after preparation. possibly due to higher polymer chain mobility that allows QDs to aggregate during the evaporation process. The same mixture of yellow and red QDs in hexane looks similar in color to the cross-linked sample.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. 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. 45 days later. 110 Even though some QDs were quenched by the free radical polymerization. To increase overall fluorescence from the cross- . and 261 days later. we observed an approximately 10 fold decrease in the fluorescence intensity of the PI-QD nanocomposite caused by the cross-linking reaction. Both types of PI-QD particles were loaded with the same yellow and red QDs.

respectively. (B) 45 days after preparation.4: Digital photographs of aqueous suspensions of 0. and (C) 261 days after preparation. the concentration of QDs incorporated into the PI-QD particles could be increased. Pictures were taken (A) immediately after preparation. . Both samples have yellow and red QDs encapsulated in a number ratio of 2:1.31 linked samples. The upper sample is cross-linked. while the lower sample is un-cross-linked.3 wt% PI-QD particles under UV illumination. (A) (B) (C) Figure 2.

Cross-linking was shown to greatly enhance the long-term fluorescence stability of the encapsulated QDs. These fluorescent hybrid nanocomposite particles have potential application in genomics.4 Conclusions Fluorescent CdSe(ZnS) quantum dots (QDs) were successfully encapsulated into biocompatible polyisoprene (PI) particles using an emulsification/solvent evaporation method. indicating that the particles are suitable for multicolor coding. The fluorescence spectra of mixtures of two differentsized QDs change in PI as compared to their solution spectra. The PI can be surface-modified with carboxyl groups during encapsulation to facilitate subsequent bioconjugation and selective binding.32 2. The simple encapsulation method results in QDs encapsulated into the core of the PI particles without requiring surface modification of the QDs. different emission peaks were clearly resolved. drug discovery. suggesting energy transfer between QDs due to their aggregation during the encapsulation. The cross-linked PI-QD nanocomposite particles displayed strong and stable fluorescence emission after months in aqueous suspension. and other applications that could exploit the high-throughput afforded by multicolor coding. 100 The polyisoprene is easily cross-linked by adding a free radical initiator during encapsulation. Even though there appears to be some QD aggregation. .

41. The effects of cosolvent and surfactant are also discussed. The results show that the particles maintain their size and morphology after exposure to CO2 . ∗ Yin. W. Z. Chen. N. Supercrit. M.33 Chapter 3 Hydrophobic Ion Pairing to Enhance Encapsulation of Water-Soluble Additives into CO2-Swollen Polymer Microparticles∗ Ionic dyes were successfully encapsulated in colloidal polymer particles by a CO2 based microencapsulation technique. 293-298. The water-soluble dyes were made hydrophobic by forming ion pairs with several types of alkyl quaternary ammonium cations. Yates. J. High-pressure carbon dioxide swelled and plasticized polystyrene and thus facilitated mass transport of the dye into the particles.. Dong.. X.. Finn. The hydrophobic ion pairs were then encapsulated in preexisting size monodisperse polystyrene particles dispersed in water. and that ion-paired dyes have significantly higher loading in the polymer particles than the original dyes. Fluids 2007. .. Z.

In many applications. 111–113 The composite particles can be used in a wide range of applications. when used for drug delivery. cell labeling. 116. 96 particle coating. and controlled release properties.1 Introduction Functional additives can be incorporated into polymer colloids to form composite particles with adjustable optical. 115 coacervation.34 3. The size of the particles influences the drug release rate and also can affect the location of accumulation of particles in the body. The additive must be hydrophobic to partition into the polymer from the . precise control of particle size and size distribution is required to decrease side-effects of the drugs for better performance. coating. Most techniques also require harmful solvents that are difficult to remove. composition. The CO2 -based process eliminates the need for harmful organic solvents and decouples the particle formation and microencapsulation. For example.We have recently developed a carbon dioxide-based microencapsulation technique in which additives are impregnated into a preformed aqueous latex. and coating. and surface characteristics must be carefully controlled. 56 Compressed carbon dioxide is used to swell the aqueous latex particles and facilitate transport of additives into the particles. offering improved control over particle size and size polydispersity. The CO2 is also easy to separate from the particles due to its high volatility. particle properties such as size. such as drug delivery. 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. 114 The most widely used encapsulation methods such as emulsification/solvent evaporation. catalytic.117 and spray drying 118 provide limited control of particle size and polydispersity. magnetic. size distribution. However.

119. a hydrophilic counter-ion. Therefore. Two anionic dyes.35 aqueous phase. with bromothymol blue having one sodium cation and rose bengal having two. 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. The molecular structures of the two dyes are shown in Figure 3. 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. such as a sodium cation. 122.121 Ion pairing is only an ionic interaction and there is no new covalent bond produced. 124.1. Ion pairs were formed by replacing the sodium cations with hydrophobic quaternary ammonium cations. 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.Water-soluble additives tend to remain in the aqueous phase surrounding the polymer particles.123 for enhancing drug absorption. This technique has been widely used for pharmaceuticals in drug extraction. The transport of the hydrophobic ion pairs into polystyrene colloids swollen with CO2 was investigated. were examined as model ionic compounds for encapsulation.125 for analysis by HPLC. .120 In the ion pairing process. 56 Ionic additives are particularly difficult to encapsulate using the CO2 -based process because of their strong interaction with water. resulting in very low encapsulation efficiency. such as a long alkyl quaternary ammonium cation. Both are sodium salts. it does not affect other properties of the original additives. is replaced by a hydrophobic counter-ion. The results illustrate the promise of ion pairing as one route to enhance microencapsulation of water-soluble drugs by CO2 -based microencapsulation. bromothymol blue and rose bengal. 119.

Chloroform (SpectrAR) was from Mallinckrodt Chemicals.7 -tetraiodofluorescein sodium salt.4 .5 . Baker. bromothymol blue (3 . 128. All chemicals were used as received.36 (A) (B) Figure 3.9%) was purchased from Fisher Chemicals.1: Molecular structure of: (A) bromothymol blue and (B) rose bengal. Deionized water was used during the experiments. Dichloromethane (99. Triton X-100 (octyl phenol ethoxylate. dye content 90%). 99. Polystyrene (PS) microparticles were prepared by surfactant free emulsion polymerization.7-tetrachloro.2) was obtained from J.99%). dye content 90%). Acetone (HPLC grade) was from Burdick & Jackson. 1-Decanol (>97%) was from TCI. Preparation of microspheres.3 -dibromothymolsulfonephthalein sodium salt.5+%) were purchased from Aldrich. tetrabutylammonium bromide (TBABr.2 ml . rose bengal (4. Carbon dioxide (SFC/SFE grade) was obtained from Air Products and Chemicals. potassium persulfate (KPS. certified.129 Ninety millilitres of water and 2. 99%).2 Experimental Section Materials.6. 3. 98%) and toluene (99.2 . T. tetrahexylammonium bromide (THABr. Figure 3.5. tetraoctylammonium bromide (TOABr. Styrene (99+%). Absolute ethanol (200 proof) was purchased from Pharmco Products. 99%).

the reaction was stopped. The flask gradually became turbid due to polymer particle nucleation.3: Chemical structure of tetraalkylammonium bromide used for ion pairing. The flask containing monomer was heated to 70 °C while stirred magnetically. which was then collected and evaporated to collect the ion pairs. The dye was first dissolved in 5 ml water.04 g KPS. As the ion pair formed. an amount of tetraalkylammonium bromide (Figure 3.3) equimolar to the amount of sodium counter ions was added along with 5 ml of chloroform. Figure 3.9 wt%. The original dye had no solubility in chloroform. Both flasks were sealed and purged with argon for 10 min. Preparation of ion pair. the dye partitioned from the aqueous phase into the chloroform phase. After 24 h. and then the KPS solution was injected into the flask containing monomer by a syringe. styrene were added to a 250 ml round-bottom flask.37 Figure 3. . The solid content of this latex was 1. Then. The ion pairs were dried overnight in a vacuum oven prior to use.2: Chemical structure of Triton X-100. Five millilitres of water was added to another smaller flask containing 0.

3. 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.4.5 g CO2 was added to the vessel by a computer-controlled high pressure syringe pump (ISCO model 260D). 60 mg dye (weight of different ion pairs adjusted to give 60 mg dye molecules).15 g cosolvent in specified cases were transferred to a variable volume high pressure stainless steel vessel described previously. The particles were dried in a vacuum oven for 24 h at room temperature prior to characterization. The vessel was then pressurized to 310 bar using CO2 as a hydraulic fluid to compress the volume-variable vessel. The dye-loaded polymer particles were collected by centrifuge and washed by ethanol to remove any surface adsorbed dye.4 g) was added to 10 g of the PS latex. Ethanol washes were repeated until the supernatant appeared clear. was added to maintain the stability of PS latex during encapsulation and enhance emulsification of compressed CO2 into the aqueous latex. The morphology of the PS particles was observed by scanning electron microscope (LEO 982 FE-SEM). and it was stirred gently for 24 h. and 0. 130 The latex containing Triton X-100.38 Microencapsulation. The vessel was then depressurized very slowly over about 20 h to minimize foaming of the polymer particles. Particle characterization. The vessel was sealed and placed in a water bath at 35 °C. Dynamic light scattering (Brookhaven Instruments model 90 Plus) was used to investigate the size and size polydispersity of the obtained sample in water. 131 as shown in Figure 3. Prior to encapsulation. Triton X-100 (0. Triton X100. A magnetically coupled stir bar was used to emulsify CO2 into the aqueous latex. and the calculation for the dye loading was based on .1 g or 0. 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 surfactant.

The solubility of the dyes and ion pairs in water and toluene .4: Variable volume high pressure stainless steel vessel. The absorbance peak for the ion pair is in the visible region. 3. and tetraoctylammonium bromide (TOABr) was qualitatively measured in toluene and water by visual observation. tetrahexylammonium bromide (THABr). Ion pairing had only a slight effect on the absorbance peak. rose bengal. standard absorbance curve of the ion-paired dye in chloroform. at which there is no absorption of polystyrene in chloroform. while bromothymol blues absorbance maximum is near 400 nm. 132 It is expected that the trend of solubility of the dyes in toluene will parallel that in polystyrene.39 Figure 3. and their ion pairs with tetrabutylammonium bromide (TBABr). Toluene is an aromatic solvent compatible with polystyrene and was chosen because toluene has a solubility parameter similar to that of polystyrene.3 Results and Discussion The solubility of the dyes bromothymol blue. Rose bengal has a maximum absorbance near 564 nm.

but the ion pair with bromothymol blue was soluble. rose bengal and their ion pairs with tetrabutylammonium bromide (TBABr). while rose bengal has two. when TOABr was used. both ion pairs were soluble in toluene. the solubility of the ion pair is lower in water and higher in toluene than that of the original dye.134 The enthalpy decrease is due not only to the ionic . The data also show that rose bengal is more hydrophilic than bromothymol blue. soluble in water and insoluble in toluene. 133.1. Table 3. As a result. 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. As shown in Figure 3. bromothymol blue has one sodium cation per molecule. and tetraoctylammonium bromide (TOABr) Ion pairing salt None TBABr THABr TOABr Bromothymol blue Water solubility Soluble Slightly soluble Insoluble Insoluble Rose bengal Water solubility Soluble Slightly soluble Insoluble Insoluble Toluene solubility Insoluble Slightly soluble Soluble Soluble Toluene solubility Insoluble Slightly soluble Slightly soluble Soluble In studies of the thermodynamics of hydrophobic ion pairing.1: Water and toluene solubility of bromothymol blue. When THABr was used. the ion pair of rose bengal was slightly soluble in toluene. The hydrophobic ion pair was formed by replacing the hydrophilic sodium cation with a tetraalkylammonium cation. 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.40 at room temperature is shown in Table 3. tetrahexylammonium bromide (THABr). Both bromothymol blue and rose bengal are sodium salts.1.

1 shows that there are a variety of polar functional groups including hydroxy. The amount of bromothymol blue incorporated into polystyrene is increased for hydrophobic ion pairs. greater than a five-fold increase when compared to the unmodified dye. 134 We also examined solubility in several solvents and found that all of the hydrophobic ion pairs in Table 3.41 interaction. For rose bengal.11 wt%. Table 3. the dye incorporation is very low (<0.02 wt%) for both rose bengal and bromothymol blue when the unmodified dye is used. For the bromothymol blue ion paired with TBABr. carbonyl. chloroform. and ethanol. As expected. the amount of dye increases to 0. 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. having the shortest alkyl chain investigated. ion pairing did not enhance the encapsulation of dye in the polystyrene. there is no measurable difference in dye incorporation when compared with the unmodified dye. 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. acetone. The longer alkyl chains of THABr and TOABr allow the ion pair to be hydrophobic enough to increase partitioning into the polymer particles.1 were soluble in polar solvents dichloromethane. and ether as well as attached halogens that can provide favorable interaction with polar solvents. Examination of the molecular structure of the dyes in Figure 3. None . For bromothymol blue ion pairs with THABr and TOABr.2 shows the weight percent of dyes and their hydrophobic ion pairs incorporated into the polystyrene latex through carbon dioxide-based microencapsulation. but all other intermolecular forces between ion pair and solvent.

136 Other work has shown that colloidal particles can be more swollen than bulk polymers due to interfacial adsorption of CO2 .11 0. but no cosolvent was used) Ion pairing salt None TBABr THABr TOABr Dye concentration in PS particles (wt%) Bromothymol blue Rose bengal < 0.02 < 0.2: Effect of ion pair formation on dye loading of bromothymol blue and rose bengal into polystyrene particles (0. if the particles lose colloidal stability they will agglomerate and coalesce in the presence of compressed CO2 .02 < 0. since two bulky tetraalkyl cations associate with each rose bengal molecule. In addition.02 < 0. Table 3. 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.11 < 0. 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. 137.02 < 0. The poor dye incorporation with rose bengal can be attributed to its relatively low solubility. 130 The surfactant .138 As a result. and to the fact that hydrophobic ion pairs are more bulky.1 g Triton X-100. The bulky molecules are expected to diffuse more slowly into the polymer. 135.02 0.42 of the rose bengal ion pairs investigated had a measurable difference in encapsulated amount compared to the unmodified dye.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.

5. as shown in Figure 3. Figure 3. Swelling of the particles by CO2 is nearly completely reversible.5B. There was no measurable change in the polydispersity as determined by DLS using the method of cumulants. ethanol.43 Triton X-100 stabilized the polymer particles during the microencapsulation process. Since both of these ionic complexes are insoluble in water. Particle size analysis by dynamic light scattering (DLS) gave an average particle size of 312 nm before microencapsulation and 315 nm after microencapsulation. Some particles appeared larger after the encapsulation.2. as these were the two ionic complexes that showed enhanced encapsulation in Table 3. and acetone. We hypothesized that the addition of a polar. The ion pairs of bromothymol blue with THABr and TOABr were chosen for study. chloroform. as shown in Figure 3. 130 The particle size and morphology was examined before and after encapsulation by SEM. However. The results demonstrate that the microencapsulation process results in very little change in particle size. morphology. the particles were still spherical and did not agglomerate. the aqueous phase surrounding the polymer particles acts as a barrier for transport of the hydrophobic ion pair. After the encapsulation.5A shows that the particles were spherical with a narrow particle size distribution before the encapsulation. probably due to slight foaming of the polymer during depressurization. water- . However. and the morphology of the original latex particles is maintained after microencapsulation. It also aided in emulsifying CO2 into water. and particle size distribution. In an effort to further enhance the incorporation of bromothymol blue into polystyrene. the ion pairs are soluble in some polar solvents such as dichloromethane. the fraction of larger particles was small and the average size did not change significantly. the effect of cosolvents and additional Triton X-100 surfactant were investigated. improving encapsulation kinetics.

For the TOABr ion pair.11 wt% loading without acetone. Other cosolvents were investigated as well. 0. 1-decanol.41 wt% compared to 0.15 g of acetone was added to 10 ml of aqueous latex. and the microencapsulation process was repeated. loading is increased to 0.3 shows that the added acetone enhances the loading of the ion pairs into polystyrene significantly.5: SEM pictures of polystyrene particles: microencapsulation. toluene. The addition of toluene and dichloromethane resulted in a loss of colloidal stability. so these solvents further swell the polymer particles. Table 3. For the THABr ion pair. and dichloromethane. the loading was 0.44 (A) (B) Figure 3. Toluene and dichloromethane are both good solvents for the polystyrene particles. thus improving the kinetics of transport through the aqueous phase and into the polymer.46 wt%. The added acetone did not measurably alter the latex stability or particle size when compared to the experiments without acetone. including ethanol. Particle aggregation in the presence of toluene and dichloromethane resulted in rapid . (A) before and (B) after miscible cosolvent could allow some small level of solubility of the ion pair in the aqueous phase.

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. individual polystyrene spheres were clearly discernible with necking between the particles apparent. as shown in Figure 3. in some cases >2 wt%.1 g Triton X-100 0. Again.11 0.4 g Triton X-100 0. 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. 56 The surfactant aids transport by facilitating the formation of an emulsion of compressed carbon dioxide in water. The measured dye loading in these cases was spuriously high. The loading is not reported in Table 3.50 coalescence.45 Table 3.3 because the high values are the result of entrapment of solid particles of hydrophobic ion pairs in the aggregates of polystyrene. However. After microencapsulation in the presence of alcohols. Ethanol and decanol similarly destabilized the latex.3: Cosolvent and surfactant effects on loading of bromothymol blue in polystyrene particles Ion pairing salt THABr TOABr Cosolvent None Acetone None Acetone Dye concentration in PS particles (wt%) 0.63 0.08 0.46 0. coalescence was less pronounced.11 0. The surfactant also disperses .09 0. and not uniform loading in the polystyrene spheres. Individual polystyrene particles were no longer discernible after microencapsulation in the presence of dichloromethane and toluene. since the alcohols are not good solvents for polystyrene.6.41 0.

the microencapsulation experiments were repeated using 0. For the THABr ion pair. When acetone cosolvent is used.46 Figure 3.63 wt% as the surfactant amount is increased from 0. Without cosolvent. It is possible that the additional surfactant solubilizes the hydrophobic ion pairs in water. thus limiting the transport to the polymer phase.4 g.08 wt% and for the TOABr ion pair it decreases from 0. 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. the additional surfactant actually appears to slightly decrease the dye loading. For the THABr ion pair with acetone cosolvent. the additional surfactant has very little effect on the amount of dye incorporated into the polystyrene particles. .1 g surfactant.1 g to 0. To investigate the role of surfactant.11 wt% to 0. the hydrophobic ion pairs in water.09 wt%. the loading increases from 0.4 g of surfactant in 10 ml of latex instead of 0. the additional surfactant enhances the dye loading.3. For both types of ion pairs.6: Agglomerated polystyrene particles after the microencapsulation process in the presence of ethanol. The results are shown in Table 3.11 wt% to 0. measured dye loading decreases from 0.41 wt% to 0.

4 Conclusions Hydrophobic ion pairing was successful in allowing ionic dyes to be encapsulated into polymer colloids. solvents usage can be minimized or completely eliminated in some cases. Unfortunately. In cases where colloidal stability was lost. 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. Future research in heterocoagulation or other techniques that kinetically entrap hydrophilic additives in . For bromothymol blue. In the encapsulation process. the results show that hydrophobic ion pairing is limited in its effectiveness. 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. The ion pairs with rose bengal showed no enhanced loading in the polymer. Solvent elimination is particularly attractive for drug delivery applications. it requires compatibility between polymer and ion pair and depends on the relatively slow diffusion kinetics in the plasticized polymer phase. Since the method reported here relies on spontaneous diffusion of the ion pair into the polymer. in these cases irreversible and uncontrolled coalescence led to loss of polymer particle integrity. Addition of water-miscible cosolvents was shown to further enhance the incorporation of the hydrophobic ion pairs into the polymer colloids.47 3. However. As a result. presumably due to its lower solubility in nonpolar solvents and the larger molecular size of the hydrophobic ion pair when compared with bromothymol blue. very high dye loading was observed due to heterocoagulation between the polymer colloid and dispersed particles of ion-paired dye. compressed carbon dioxide is used in place of volatile organic solvents.

48 a hydrophobic polymer environment could potentially obviate problems due to slow diffusion and thermodynamically limited loading in the polymer phase. .

The interfacial free energy difference between the inside and outside surfaces is the main driving force for closing the hole on the surface. Langmuir 2008. The effects of added surfactant. The emulsification/freeze-drying technique can be used to encapsulate hydrophilic ∗ Yin. M. volume fraction of solvent in the polymer emulsion droplet. W. 24. The results show that the particle morphology is due to phase separation in the polymer emulsion droplets upon freezing in liquid nitrogen. 701-708.. and that morphological changes are driven largely by lowering interfacial free energy. Yates. type of solvent swelling polymer colloids. .49 Chapter 4 Effect of Interfacial Free Energy on the Formation of Polymer Microcapsules by 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 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. Z. and processing conditions on the particle morphology were examined and compared to theoretical predictions.

149.1 Introduction Hollow polymer particles or microcapsules are lightweight.148 and controlled phase separation in multicomponent polymer emulsions.150 Many of these current techniques suffer from complexity and are limited in the types of polymers that can be synthesized or processed. As a result of these attractive properties. 139–143 A variety of methods have been reported to synthesize microcapsules. enzymatic microreactors. Recently. 144 polymerization of double emulsions. 4. including polymerization of shells around oil emulsion droplets. microcapsules are used in a number of applications. reported a simple method to prepare polymer microcapsules by freeze-drying aqueous latex particles swollen with solvent. 145 layerby-layer assembly of polyelectrolytes around a sacrificial core.50 additives in the core of the microcapsules. Im et al. We refer to this method as “emulsification/freeze-drying” in analogy to the emulsification/solvent evaporation process. and can act as semipermeable barriers for encapsulated substances. 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. 147. 96 The emulsification/freeze drying process is attractive due to its relative . electrophoretic displays. demonstrating the potential of the technique in controlled release applications. 146 assembly and fusion of polymer particles around emulsion droplets. strongly scatter light and ultrasonic waves. 151 The resulting dried particles were hollow with a single open hole on the surface. deformable. such as functional paper coatings. and microencapsulation for controlled release.

The equilibrium morphology of biphasic emulsion droplets is controlled by minimization of the interfacial free energy. the mechanism of microcapsule formation by this method has not been investigated in detail. 152–154 It was initially thought that the structure was formed in situ during the early stages of emulsion polymerization. 156–160 However. were the first to show that the hole can be closed to form microcapsules by redispersing the hollow particles and . Hollow particles with typically a single hole on each particle are obtained after freeze-drying. were first described by Wilkinson and co-workers as “anomalous” particles that form during certain emulsion polymerizations. moved toward the surface during the solidification in liquid nitrogen. Equilibrium morphology is welldescribed theoretically and depends on the interfacial free energy between each phase and relative volume fraction of each phase. the swelling solvent.51 simplicity and potential ability to form microcapsules from a wide variety of polymeric materials. Hollow polymer particles with the morphology observed by Im et al. Im et al. kinetics also plays a role in the freeze-drying process. leaving particles with a hole in the surface of each particle. 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. however. leaving a void (vacuum) at the center of the particle. as freezing of the solvent and polymer can lock in the morphology in a nonequilibrium state. However. that the structure is caused by polymer/solvent phase separation during freezing and subsequent interfacial free energy minimization. Im proposed that toluene. the solvent must migrate to the core of the polymer particle as a separate phase prior to its evaporation or sublimation. and polymer chains migrated toward the surface driven by the flux of toluene when the system was warming up. We propose.

151 Again. Styrene (99+%). n-heptane (99%).1 can be used to encapsulate water-soluble or water-dispersible compounds.99%) were purchased from Aldrich. and dichloromethane (99. the process illustrated in Figure 4. As shown in Figure 4. Methanol (99. 4. toluene (99. Vincent and co-workers formed microcapsules around oil droplets via internal phase separation and characterized the controlled release of encapsulated oil-soluble compounds.9%).2). Better understanding 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. and potassium persulfate (KPS. Mn =2080. 161.5+%). Rose Bengal. Absolute ethanol and 95% ethanol (ACS/USP grade) were from Pharmco. 50 wt% in water.2 Experimental Section Materials. the process can be used to encapsulate desired additives in the hollow polymer shells.9%) were from FisherChemicals.162 Unlike these earlier studies. 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. Baker. 99.1. 98%). T.AAPER. AIBN was recrystallized from . Figure 4.52 subjecting them to a plasticizing solvent. interfacial free energy minimization is the expected driving force for closing the holes to form microcapsules. poly(ethylene glycol) methyl ether methacrylate (m-PEGMA. benzene (Spectranalyzed). 2.1. The entire process for forming hollow polymer particles and subsequent closing open holes to form microcapsules is illustrated in Figure 4. Octyl phenol ethoxylate (Triton X-100) was obtained from J.2 -azobisisobutyronitrile (AIBN.

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. Synthesis of Polystyrene Latex. Fifteen minutes later.607 g styrene was added. Styrene was passed through an inhibitor remover column (Aldrich) prior to use. . The flask was purged with argon for 30 min.53 Figure 4.2: Chemical structure of m-PEGMA. Figure 4.1: Scheme showing the formation of hollow polystyrene (PS) particles by emulsification/freeze-drying and the closing of such particles for encapsulation. methanol. after which 13.

04 Pa).012 g KPS in 2 mL water was charged into the flask. 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. 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 ). was dried by a vacuum pump.54 a solution of 0.5 h. PS grafted with PEG was synthesized by the same method except that 4 h after initiation.000) and dialyzed in water for one week with the water replaced every day.73 Pa) and toluene (0. The swollen latex was rapidly frozen by dripping into liquid nitrogen.166 The amount of solvent soluble in water was subtracted from the total solvent added.006 g KPS in 1. 165. it was assumed that the polymer and solvent form an ideal solution so that pure component densities could be used for volume fraction calculation. Approximately 65 mg of dichloromethane and 3 mg of toluene will dissolve in 5 grams of pure water near room temperature. a solution of 0. The reaction was maintained at 70 °C for 28. Formation of Microcapsules. MWCO=12-14. the drying process was via sublimation. which is below the triple point pressure for both water (611. The frozen sample. The morphology of resulting particles was observed under a scanning electron microscope (SEM.36 g m-PEGMA aqueous solution (50 wt%) was added to the flask. during which the swelling solvent and ice were removed. In addition. To make hollow PS particles. Microcapsules were formed from selected freezedried particles through a second solvent swelling step to close the open hole on the . 167 Therefore. LEO 982 FE-SEM). kept at 0 °C by ice/water. 164 Preparation of Hollow Polystyrene Particles. The resulting latex was placed in dialysis tubing (Spectra/Por. The vacuum was maintained at 4×10−4 Pa.

When compressed carbon dioxide was used as the plasticizing solvent. Additional stabilizing groups can optionally be grafted on the surface during the polymerization. After the closing process.55 surface of the particles. and dried in vacuum.3 Results and Discussion Figure 4.3A) were synthesized by surfactant-free emulsion polymerization. particles synthesized for this study.3 shows the two types of polystyrene Hollow Particle Formation. the particles were collected by centrifugation. The freeze-dried PS particles were dispersed into water or water/ethanol mixtures by sonication. These particles are electrostatically stabilized in water due to sulfate groups on the surface from the potassium persulfate initiator. The particles on the right (Figure 4. Both particles have negative zeta potential from the surface grafted potassium persulfate initiator fragments. The zeta potential was −51 mV for the surfactant-free particles and −40 mV for the particles with grafted PEG. the suspension was loaded into a variable-volume highpressure stainless steel vessel. the suspension was exposed to a plasticizing solvent to close the holes. Rose Bengal dye was added to the suspension to examine encapsulation. washed repeatedly with 95% ethanol. 131 In some cases. Then. Those on the left (Figure 4.3B) have grafted poly(ethylene glycol) (PEG) on the surface to provide additional steric stabilization. 4. 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 magnitude of the zeta potential is lower for the PEG-grafted particles due to electrostatic charge shielding by the uncharged PEG layer covering . Both particles are uniform in size.

then freeze-dried.7). By comparison with Figure 4. The .3A. The PS without grafted stabilizer allows the investigation of the effects of surface adsorption of added surfactant.3A were swollen with toluene (νQ = 0. The surfactant-free PS particles shown in Figure 4. Figure 4.4A shows that the resulting particles appear hollow with a single opening on the surface.3: Polystyrene latex particles as synthesized (A) by surfactant-free emulsion polymerization and (B) by emulsion polymerization with poly(ethylene glycol) macromonomer. (A) (B) Figure 4. a second experiment was conducted in which 4 mg of Triton X100 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. To examine the role of interfacial free energy in determining particle morphology.56 the surface.4B). it can be seen that the swelling/freeze-drying steps increase the particle size and size distribution significantly. but the broader size distribution suggests that some of the polymer particles coalesce during the solvent swelling step. The size is increased because the particles are hollow. The grafted PEG layer enhances latex stability during the swelling and subsequent closing process.

The morphology is described by ΦQ . The model also assumes that the solvent-rich phase is always spherical. Hollow particles were made by freeze drying 5 g PS latex (1 wt%) swollen with 0. (B) 4 mg Triton X-100. 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. while the polymer-rich phase can have changing morphology to partially or completely engulf the solvent-rich phase. The changes in particle morphology observed in Figure 4.1 g toluene (νQ = 0. and W represent polymer-rich phase.7): (A) no additional surfactant. (A) (B) Figure 4. and water phase. lowering interfacial tension. To apply the model to the freeze-drying process. represented in Figure 4.5. is used to compare the observed changes in particle morphology to that predicted by interfacial free energy minimization. P.4 strongly suggest that interfacial free energy plays a significant role in the process. A simple model of a biphasic emulsion droplet. it is assumed that the morphology is not altered by compression from the surrounding ice. which ranges from 0 ◦ . solvent-rich phase. Q.57 surfactant is expected to adsorb at both the toluene/water and polymer/water interfaces. respectively.4: Effect of surfactant on the hollow particle morphology.

if Q is very hydrophobic (high value for γQW ) or P is very hydrophilic (low value for γPW ).1) where νQ is the volume fraction of Q in the total volume of P and Q.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.2 158 sin2 ΦQ π + γQW (1 + cos ΦQ ) + γPQ (1 − cos ΦQ ) E = ( )1/3 (3νQ )2/3 γPW (1 + cos ΦP ) 2 2 sin ΦP (4. For a given system. and P and Q. E.1 158 4(1 − νQ ) 2 + 3 cos ΦP − cos3 ΦP 2 − 3 cos ΦQ + cos3 ΦQ = + sin3 ΦP sin3 ΦQ νQ sin3 ΦQ (4. 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.2) where γPW . Generally. respectively. the preferred morphology is described by the angle ΦQ that gives the minimum overall free energy. Taking the total volume of P and Q as unity. . 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 4.58 when P and Q form separate spherical droplets to 180 ◦ when Q is engulfed by P in a core-shell structure. 2/3 ECS = 32/3 (4π)1/3 (γPW + νQ γPQ ) (4. Q and W. coreshell structure is preferred. 158 However. γQW . the total interfacial free energy of the biphasic emulsion droplet is given by eq 4. ΦP is related to ΦQ through eq 4.3. and γPQ are the interfacial free energy per unit area between phases P and W.

170 The degree of lowering of interfacial tension due to the surfactant or surface grafted groups is unknown. the monomer unit of PS.3 to the experimental system.5 dynes/cm between styrene. the interfacial tension is 36. the polystyrene particles are covered with hydrophilic surface stabilizing groups. it is necessary to know the values of interfacial tension for the three interfaces during the process.59 Figure 4. Interfacial tension changes with temperature and generally increases as temperature is lowered.2 and 4.1 dynes/cm between toluene and water. either potassium sulfate groups or grafted PEG. since they form a solution (zero interfacial tension) at room temperature. both of which will lower the PS/water interfacial tension relative to pure PS. The interfacial tension between toluene and polystyrene is also unknown. The interfacial tension . 169 In addition.5: Model for a biphasic emulsion droplet consisting of polymer-rich (P). The polymer morphology after removing solvent is determined by ΦQ . and 35. solvent-rich (Q). 168 Unfortunately. the interfacial tension values are unknown during and after freezing in liquid nitrogen. Triton X-100 will also lower the interfacial tension between oil and water. and water. At room temperature. Rough estimates of interfacial tension were made to enable the calculation of E/ECS versus ΦQ . and aqueous (W) phases. To apply eqs 4.

In these experiments. The added surfactant Triton X-100 is expected to adsorb at both the toluene/water and PS/water interfaces. 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. and 12. as toluene is a solvent for PS at room temperature. and between PS and toluene (γPQ ) was set to 30. γQW .4. Bowlshaped particles were obtained when νQ = 0.2. The hole . it is assumed that the surfactant lowers both γPW and γQW . and γPQ should be low. The free energy minimum shifts to ΦQ = 56 ◦ with surfactant. Figure 4. Figure 4.7 shows the freeze-dried particles produced from a series of experiments with increasing νQ .8. as PS particles as synthesized are hydrophilic. When Triton X-100 is added. 45. for calculation of the free energy without surfactant.60 between PS and water or ice (γPW ). As a result. These estimates were made by keeping in mind that γPW should not be very high. 27. respectively. The effect of volume fraction of swelling solvent (νQ ) was examined using both toluene and dichloromethane. γPQ were set to 27. there was just one small dent on the particle when νQ was 0. Core-shell-like hollow particles with a small opening on the surface were formed when νQ was increased to 0.6 shows that with these assumptions the free energy without surfactant is at a minimum when ΦQ is 180 ◦ . γPW . which means the bowl-shaped structure is preferred at equilibrium. For the samples swollen with dichloromethane. and 12 dynes/cm. between toluene and water or ice (γQW ). PS particles with surface-grafted PEG were used to enhance stability during swelling and dispersibility of freeze-dried particles. While the theoretical predictions are only a crude estimate. Surface-grafted PEG also partly eliminates the effects of surfactant adsorption on changing particle morphology.4. which means the core-shell structure is favored at equilibrium. respectively.

When no surfactant is used.61 Figure 4. respectively. γPW .7. . respectively. and 12. The inset images represent the cross section of the favored morphology corresponding to the minimum free energy. they are set to 27. γQW . E/ECS is plotted vs ΦQ with νQ = 0. 27.6: Effect of surfactant on interfacial free energy profile. and γPQ are set to 30. When a surfactant is used. 45. and 12.

E/ECS is plotted versus ΦQ with different νQ as shown in Figure 4. Dichloromethane was used in the samples in the upper images and toluene was used for those shown in the lower images. as was done for the surfactant-free case in Figure 4. and γPQ were set to 30.7. However. a typical hole had a diameter of 579 nm. The minimum free energy in all cases corresponds to the core-shell structure (ΦQ = 180 ◦ ). and its volume fraction was 0.2 (A). 0.72. when toluene was used. The hole size can be estimated from SEM images. The volume fraction of the swelling solvent (νQ ) was 0. 45.6. A similar trend was observed when toluene was used as the swelling solvent. The white scale bar represents 2 µm. (A) (B) (C) (D) Figure 4. respectively.68.7: Effect of volume fraction of the swelling solvent on the morphology of the freeze-dried PS particles grafted with PEG.8. when dichloromethane was used. and its volume fraction was 0. and 0. To study the effect of increasing the swelling solvent as shown in Figure 4. γQW .8 (D). 0.8. the typical hole has a diameter of 539 nm. However. a greater solvent volume fraction was needed to obtain core-shell structured particles.6 (C). it shows that the slope of E/ECS versus ΦQ near the .4 (B). and 12.62 became larger. and it tended to embed into the particle upon increasing the amount of swelling solvent. For νQ = 0. γPW . with toluene.

and α is the thermal diffusivity of the droplet. Even so.2 to 0. By assuming α to be the thermal diffusivity of ice at 0 °C.7 °C) can be estimated by a Heissler chart. especially when low volume fractions of swelling solvent are used. the morphology change may stop at a point farther from the core-shell structure than that with higher slope. 171 αt/r2 = 0. it is −93 °C).63 minimum in free energy is different with different values of νQ .0114 cm2 /s. First. in which r is the radius.7. 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. in liquid nitrogen (−195. but indicate that the particles are likely not at equilibrium morphology. the solvents have different interfacial free energy that will result in slightly different free energy profiles. Figure 4. the predicted free energy driving force to equilibrium is consistent with the observations shown in Figure 4. With such rapid cooling.8. The slope increases with increasing νQ from 0. The frozen morphology will be affected by the driving force to reach equilibrium or the free energy slope. the emulsion droplets maybe frozen before reaching the morphology with minimum energy. The slope can be regarded as the driving force to the minimum free energy.7 also shows that there are differences in morphology for particles produced using toluene versus dichloromethane as the swelling solvent. Second. If the slope is small. The theoretical predictions are only crude estimates. the freezing temperature of dichloromethane (for toluene. 168 the time is just 0.14. 0.7 mm. .365 s for the center of the droplet to reach the freezing point of pure solvent. There are several factors that can play a role in the differences between the two swelling solvents. the phase separation may stop at different times due to the difference in freezing temperature between toluene and dichloromethane. The time for the center of the droplet to cool from room temperature to −97 °C.

and 12.64 Figure 4. . respectively. γQW . and γPQ to 30. 45.8: Interfacial free energy (E/ECS ) vs ΦQ with different volume fractions of solvent (νQ ) by setting γPW .

will be influenced by the type of swelling solvent. This is possibly due to the relatively high melting temperature of benzene (5 °C). leaving an irregular hollow structure with multiple holes after the freeze-drying. Since dichloromethane has higher diffusivity in water than toluene. n-heptane and benzene. Toluene and dichloromethane.7.9A. Finally. The amount of solvent in the polymer-rich phase depends on the kinetics and thermodynamics of polymer phase separation during cooling. as well as the time required to reach equilibrium. it should be emphasized that phase separation results in polymer-rich and solvent-rich phases. However. voided particles were obtained. . The equilibrium amount of solvent in the polymer-rich phase at low temperature.9B). Two other swelling solvents. In order for the polymer-rich phase to have mobility. 172 it is possible that particles with dichloromethane have a higher relative volume fraction of solvent than those with toluene. complete transfer of solvent to PS may not have occurred after 24 h. The result is expected. it must contain a significant fraction of solvent.9. When n-heptane. It was assumed that all of the solvent not soluble in water was in the polymer phase after the 24 h swelling period. and the resulting particles are shown in Figure 4. a nonsolvent of PS. was used. but the morphology was irregular with several openings on individual particles (Figure 4.8. The particle morphology with benzene is quite different from that observed with toluene and dichloromethane as shown is Figure 4. since the amount of n-heptane in PS is low and phase separation probably does not occur during freezing. there was no morphology change in the particles (Figure 4. it is possible that different amounts of solvent are in the polymer phase even though the calculated volume fraction is the same. Some benzene may be frozen at several loci in the particle early during the process. were also examined with νQ = 0. When benzene was used.65 Third.

The latex particles may separate from the media as water is crystallizing during the slow freezing.7).8). but the majority of the sample had coalesced (Figure 4. the frozen latex was dried by a vacuum pump while maintaining the sample at 0 °C in an ice bath. hollow particles produced from toluene and dichloromethane have a single hole (Figure 4. and then the vial containing the swollen latex was immersed into a cooling bath of ethylene glycol/dry ice (around −10 °C). respectively). Hollow particles were also obtained. Two hours later.8. However. As a result. so that there is a longer time for morphology changes after phase separation to lower interfacial free energy. The volume fraction of the swelling solvent was 0. (A) (B) Figure 4. have much lower melting temperatures (−93 and −97 °C.9: Freeze-dried samples with n-heptane (A) and benzene (B) as the swelling solvent. .66 however. and the swollen PS particles will coalesce while toluene is still liquid at that temperature. The effect of the freezing process was also examined. The latex of PS grafted with PEG was swollen with toluene (νQ = 0. rapid freezing in liquid nitrogen appears to prevent coalescence.10).

67

Figure 4.10: PS particles grafted with PEG and swollen with toluene (νQ = 0.8) after freezing 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 and 27.9 MPa for 67 h: (A) no additional surfactant; (B) 0.2 wt% Triton X-100. With toluene, the suspension was mixed for 1.5 h at room temperature: (C) no additional surfactant; (D) 0.004 wt% Triton X-100.

To demonstrate the feasibility of the process in microencapsulation.12: Effect of surfactant on closing hollow PS by toluene. As a result. decreasing the interfacial tension difference. 20 mg of the particles shown in Figure 4.2 wt% Triton X-305. Higher interfacial tension on the interior particle surface tends to pull the hole closed upon exposure to a plasticizing solvent. a water-soluble dye was added during the holeclosing process.5 h at room temperature. The emulsification/freeze-drying process has potential to be used as a microencapsulation process for hydrophilic compounds. The suspension of hollow PS particles in water was mixed with toluene for 1. (B) 0. Many particles had small unclosed opening after the process. (A) 0. Figure 4.70 (A) (B) Figure 4.7D (with dichloromethane .2 wt% Brij 35. the hole-closing process is slowed significantly in the presence of surfactant.13: Schematic illustrating the hole closing process.

71 as the swelling solvent) were resuspended in 2 mL of water/0. It was confirmed by SEM and a transmission electron microscope (TEM.3B were used. The core-shell structure was favored at high solvent content and without added surfactant. Microcapsules were formed by exposing the particles to 60 mg of toluene for 1.5 h at room temperature. After exposure to toluene. 4. then dried in vacuum. and drying in vacuum. The morphology of the resulting hollow particles can be controlled by the amount of swelling solvent and surfactant. It was also observed that such particles encapsulating dye remained colored after dispersing in ethanol for months. freezing in liquid nitrogen. JEOL 200EX) that such closed particles are hollow inside (Figure 4. Figure 4.4 Conclusions Hollow polystyrene (PS) particles were obtained by swelling PS latex with solvent. The emulsification/freeze-drying technique is therefore potentially applicable for microencapsulation and controlled release. After washing. both types of particles were collected by centrifugation and washed with 95% ethanol until the supernatant was clear. the solid particles appeared almost white in color. We plan additional experiments with biodegradable microcapsules to demonstrate controlled release. except the solid particles shown in Figure 4. a parallel control experiment was done following the same procedure. The experimental results can be interpreted in terms of interfacial free energy . and the inset images show the color difference between the two samples. A bowl-shaped morphology was favored with added surfactant and at lower solvent content. while the microcapsules remained red. In addition.15).5 mL ethanol containing 40 mg of Rose Bengal dye.14 shows that the hollow particles were closed.

14B).72 (A) (B) Figure 4. (B) hollow PS with grafted PEG. (B) TEM image of an intact capsule. cut by a microtome. .15: Confirmation of the hollow structure of closed polystyrene capsules encapsulating rose bengal (Figure 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. (A) (B) Figure 4. (A) SEM image of capsules embedded in epoxy. The inset images are photographs of the dried samples.

as swollen aqueous latex particles were frozen by liquid nitrogen. 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. When surfactant is added to the suspension of hollow particles. The emulsification/freeze drying process has potential application in microencapsulation for controlled release. It is likely that the interior surface of the hollow particles is more hydrophobic than the exterior surface. When exposed to a solvent or plasticizer. and the dye remained trapped in the microcapsule colloidal suspension for months. the open hole on the hollow PS particles can be closed.73 minimization. A controlled-release vehicle can be achieved by use of biodegradable polymer with tunable release characteristics. such as compressed CO2 . . hollow particles were obtained with an open hole on the surface. it interferes with closing of the holes. After removing the solvent-rich phase by drying under vacuum. A water-soluble dye was encapsulated into hollow PS particles. It can be concluded that phase separation occurred. The experimental evidence shows that the hole-closing process is driven by the difference in interfacial tension between the interior and exterior surfaces. Subsequent morphological changes in the biphasic emulsion droplets are driven by the minimization of interfacial free energy. The dry hollow particles can be redispersed into water or water/ethanol mixtures by mild sonication.

and method of dispersing the freeze-dried particles in water. The encapsulation efficiency is affected by the hollow particle morphology. accepted. was successfully encapsulated in the microcapsule core. M. forming microcapsules surrounding an aqueous core. Z. Yates. J. amount of solvent used. Colloid Interface Sci. 2009. The hollow freeze-dried particles were dispersed in water. W.74 Chapter 5 Encapsulation and Sustained Release from Biodegradable Microcapsules Made by Emulsification/Freeze Drying and 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 emulsification or spraying. procaine hydrochloride. . The encapsulation process is explained in terms of interfacial free energy of the hollow particles and mobility of the plasticized ∗ Yin. and the resulting aqueous suspensions were exposed to plasticizing solvents.. surfactant. solvent exposure time. The plasticizing solvent causes the pores in the shell wall to close. A water soluble drug. either dichloromethane or compressed carbon dioxide.

176 molecular weight.190 One promising alternative is the use of compressed carbon dioxide as an alternative solvent for microencapsulation. 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.179–187 There are three main techniques for microencapsulation: spray-drying. and emulsification/solvent evaporation. carbon dioxide-based microencapsulation is much less effective with water soluble drugs.116.1 Introduction Sustained and targeted release of therapeutics can be achieved by microencapsulation inside biodegradable colloidal particles of poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA). Controlled release of procaine hydrochloride from the microcapsules into phosphate buffer solution was observed. The novel hollow PLA particles produced by emulsification/freeze drying and spray/freeze drying can potentially be used as vehicles for controlled release. 96. coacervation (precipitation). The microcapsules had a small burst release. 100. 192 . The spray drying technique requires high temperatures that can destroy thermally labile compounds. 178 molar ratio between lactic acid and glycolic acid.75 polymer. 188 Each of these techniques produces solid PLA or PLGA particles with a therapeutic agent distributed in the particle. or magnetically functional particles may be created to allow targeting specific locations in the body by an applied magnetic field.191 Unfortunately. 29 The particle surface may be functionalized to target specific tissues. with the remainder of encapsulated drug slowly released over 9 days. and porosity. 174–177 The polymer degradation rate can be tuned by adjusting particle size. 5. 56.

Upon drying. 139. 149. 37 An alternative method is to create polymer microcapsules to surround the hydrophilic additive with a polymer shell.161 Since phase separation processes are nonreactive. thereby decoupling the particle formation and encapsulation steps. the solvent-rich phase is removed. in which a polymer solution is emulsified to form droplets in water.199 Temperature-induced phase separation inside the droplets causes structured polymer phase domains to form.144.193–198 The hollow particles can be filled to encapsulate species in the core. An “emulsification/freeze drying” process has recently been described. but finding conditions that provide high encapsulation efficiency can be difficult. Hollow particles potentially allow labile species to be protected from harsh operating conditions during conventional microencapsulation and give greater flexibility in controlling particle properties. 151. The resulting polymer particles can be porous (with numerous small holes) or hollow .156. The most common route to encapsulate water-soluble compounds is through solvent evaporation from water/oil/water double emulsions.76 In order to encapsulate hydrophilic additives into the hydrophobic polymer.145 A facile alternative to polymerization is creating hollow polymer particles through confined phase separation in emulsion droplets of polymer solution. A few types of polymers can be formed as hollow particles through novel polymerization processes. then rapidly frozen and dried under vacuum to produce hollow particles. leaving a hollow region in its place. A number of novel encapsulation schemes have been reported using hollow colloidal polymer particles. rather than relying on the unfavorable thermodynamically driven diffusion of additives into the polymer. effective techniques kinetically entrap the additive in the polymer. they can be applied to a wide range of solution processable polymers to create hollow particles. 139.

. When the hollow particles are dispersed in water. exposure to a plasticizing solvent causes the hole to close. Under appropriate conditions. a “green” biodegradable solvent is effective for forming hollow PLA particles with porous shell walls via freeze drying. we demonstrate the production of hollow particles of biodegradable PLA via freeze drying droplets of polymer solution formed either by emulsification or spraying. can be used as the plasticizing agent to close pores in the polymer shell for encapsulation. depending on the kinetics of freezing and the interfacial free energy between the solvent. allowing them to be filled with an aqueous phase. Dimethyl carbonate. The release kinetics of the encapsulated drug is investigated to explore the application of the novel microcapsules as vehicles for controlled release. forming a core-shell structured microcapsule. Compressed carbon dioxide. a nontoxic solvent easily separated from the polymer. a single hole is produced that is open to the outside surface of the particle. 151. It has been demonstrated using poly(methyl methacrylate) and polystyrene particles that watersoluble additives may be encapsulated inside the microcapsules. The hollow PLA particles have pores in the shell wall. polymer. and aqueous phases. The hollow porous PLA particles are converted to microcapsules entrapping a model water soluble drug by exposure to a plasticizing solvent. In the present study. It is demonstrated that the entire process can be carried out using only nontoxic and biodegradable materials. 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.199 In principle.77 (with a single large hole).

2). and the volume ratio of water to dichloromethane was 5:1. leaving dry hollow PLA particles. Spray/Freeze Drying Process. Water and dichloromethane were removed by placing the frozen sample under vacuum at 0 °C. Emulsification/Freeze Drying Process. Acetonitrile (ChromAR® HPLC) was from Mallinckrodt. De-ionized water was used in all experiments.65 dl/g in chloroform at 30 °C) was purchased from DURECT Corporation.01 g/ml) in dimethyl carbonate was atomized by a coaxial spray nozzle shown in Figure 5.05 g/ml) and Pluronic P123 surfactant (0. Dichloromethane (99.2 g/ml PLA in dichloromethane.2 Experimental Section Materials.9%) was from Fisher Scientific. Compressed air was fed to the outer annular tubing at a pressure of 50 psig. A solution of PLA (0. 87-89% hydrolyzed. Poly(DL-lactide) (PLA. phosphate buffer solution in water (pH = 7. the aqueous phase was 0.1. the oil phase was 0. 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 liquid flow rate was 15 ml/h. The hollow PLA particles were dispersed into an aqueous solution of procaine hydrochloride by placing in an ultrasonic bath for several seconds. The atomized spray was collected in liquid nitrogen and then dried under vacuum at room temperature.01 g/ml PVA. Pluronic P123 was donated by BASF. In a typical experiment. Microencapsulation Using Hollow PLA Particles. inherent viscosity = 0. Mw = 13000-23000 g/mol) were from Sigma-Aldrich. Procaine hydrochloride (99%). Dichloromethane or compressed carbon dioxide . dimethyl carbonate (Reagent Plus®. The emulsion was then immediately added dropwise into liquid nitrogen. 99%).78 5. and poly(vinyl alcohol) (PVA.

79 Figure 5. a mutual solvent for PLA and procaine hydrochloride. was added to the aqueous suspension to close the hollow particles and encapsulate procaine hydrochloride.2) maintained at room temperature. The amount of encapsulated material was measured with UV spectrometry (PerkinElmer Lambda 900) by dissolving the resulting particles in acetonitrile. After stirring. The drug .02 g/ml PLA dispersed in aqueous solution of 0.5 hours with the plasticizing solvent (either dichloromethane at room temperature or excess carbon dioxide at 207 bar and 32 °C). The typical colloidal suspension was 0. The particles encapsulating procaine hydrochloride (without drying in vacuum) were dispersed at a concentration of about 0. Measuring Release of Encapsulated Drug. and dried in vacuum.2 g/ml procaine hydrochloride.005 g/ml in phosphate buffer (pH = 7. the sample was washed by centrifugation/redispersion with water three times. Microcapsules were formed after stirring for 1.1: Coaxial spray nozzle.

with thin-walled shells and a single opening on the surface. Hollow PLA particles were readily obtained by freeze-drying oil-in-water emulsions.80 concentration in the buffer solution versus time was measured using UV spectrometry.2 µm) to remove the particles. 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.2 shows that the resulting PLA particles are hollow. 199 The interfacial free energy is affected strongly by the surfactant used to stabilize the polymer emulsion. The hollow interior is . The polymer precipitates and separates from dichloromethane upon cooling the emulsion droplets. Previous investigations of emulsification/freeze drying made particles of polystyrene and poly(methyl methacrylate) using the solvents toluene. 5. 151. 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. Dichloromethane is commonly used with PLA in the traditional emulsification/solvent evaporation microencapsulation process. dichloromethane. Figure 5. and styrene monomer. We investigated a variety of emulsifiers. Samples taken for spectroscopy were first passed through a filter (0. and polymer-rich phases that form.3 Results and Discussion The morphology of the polymer particles obtained by emulsification/freeze drying depends upon the kinetics of freezing and phase separation. solvent-rich.199 For the emulsification/freeze drying of PLA. as well the interfacial free energy between the aqueous. we chose dichloromethane as the solvent and PVA as the emulsifier.

81 from the dichloromethane-rich phase that was removed in the drying process. The plasticizing solvent gives the polymer mobility. allowing the hollow polymer particles to change shape to decrease the interfacial free energy.6. The hollow PLA particles were converted to microcapsules in the presence of aqueous solutions of . The average size could be adjusted by altering the polymer concentration. more uniform hollow particles may be obtained by phase separation. When monodisperse polystyrene particles were swollen with solvent and freeze-dried.2A). 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.4 g/ml PLA in dichloromethane. Decreasing the polymer concentration to 0. With a polymer concentration of 0.86 µm (Figure 5. 199 As a result. 199 By applying novel emulsification techniques to create uniform precursor emulsion droplets.86 µm (Figure 5. The size distribution of the hollow particles is broad. 194 When the freeze dried particles are redispersed in water.2B). the number average particle diameter is approximately 10.2 g/ml results in a decrease of the number average particle diameter to 3. exposure to a plasticizing solvent can induce the hollow particles to convert to microcapsules. the interfacial tension can pull the hole closed to form a microcapsule encapsulating a portion of the aqueous phase. hollow particles with a narrow size distribution were obtained. with a typical polydispersity index of 1. The broad size distribution of the hollow particles is correlated to the size distribution of precursor emulsion droplets. 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. Decreasing the polymer concentration decreases oil phase viscosity and thus makes the oil droplets smaller.

3.82 (A) (B) Figure 5. A control experiment was also done with solid PLA particles under the conditions corresponding to the highest point in Figure 5. The data indicate an optimal amount of dichloromethane to achieve maximum encapsulation.21 wt% when using solid PLA particles. while it was 2.2 g/ml PLA in dichloromethane.3 shows that the amount of procaine hydrochloride encapsulated first increased and then decreased as the amount of dichloromethane used was increased. rather than freeze-drying. The drug content was just 0.2: Hollow PLA particles made by emulsification/freeze-drying: (A) 0. a model water-soluble drug.4 g/ml. The solid particles were prepared by evaporating dichloromethane in the emulsion for 24 h at room temperature.5 hours at room temperature. procaine hydrochloride. confirming the encapsulation capacity of the hollow particles. to investigate encapsulation and controlled release from the microcapsules. The hollow particles have higher surface area than the solid particles . The encapsulation of procaine hydrochloride was dependent on the amount of dichloromethane that the particles were exposed to under a fixed time of 1. Figure 5.1 wt% with the hollow particles. and the number average diameter is about 4.79 µm. (B) 0.

Figure 5.3: The concentration of procaine hydrochloride in PLA particles as a function of amount of dichloromethane used during encapsulation for 1. most of the resulting particles still had holes open to the hollow interior. When there was only 0. Based on the average particle diameters and morphology measured by SEM.83 so some of the increase in encapsulation amount measured using hollow particles could be partially attributed to surface adsorption. Increasing the amount of .5 hours at room temperature. the increased surface area is not enough to account for the ten fold increase in amount encapsulated using hollow particles.06 wt% drug content).40% v/v dichloromethane in water. The particle morphology after encapsulation changed with the amount of dichloromethane. based on SEM images of average particle size and interior hole volume. it is estimated that a minimum of 80% of the procaine hydrochloride is encapsulated in the hollow core.4. However. resulting in poor encapsulation (0. as shown in Figure 5.

nearly all particles were completely closed. The core-shell morphology was confirmed with scanning transmission electron microscopy (STEM) (SUPRA 40VP.38% v/v dichloromethane/water for 1.5A. B). Since the interior surface .5.5 hours are microcapsules with a large hollow core. However. The method used to disperse the hollow PLA particles in water is critical for drug encapsulation. it is expected that all particles will revert to the solid morphology given enough polymer mobility and time. the drug content after encapsulation was 1. Generally the hollow particles were dispersed in water by placing in an ultrasonic bath. The STEM images also reveal that the inside hole volume decreases if too much dichloromethane is added (Figure 5. Therefore. With 1.84 dichloromethane resulted in a higher fraction of the particles with the holes closed. appearing spherical with a smooth surface. 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.13 wt% if the hollow particles were not dispersed in water by sonication but instead by mild stirring for 30 min.6 wt% when particles were dispersed by the ultrasonic bath. The core-shell structure has a higher interfacial free energy than solid particles. giving rise to a higher encapsulated drug content (1. 30 kV). Increasing the dichloromethane concentration to 1. The inner hollow region appears lighter than the surrounding polymer shell in the STEM images shown in Figure 5.2% v/v dichloromethane. When there was 1.98% v/v.57 wt%). 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. Particles exposed to 1. results in a decrease in the size of the hollow core. the drug content was just 0.58% v/v dichloromethane/water.

85

(A)

(B)

(C)

Figure 5.4: PLA particles after exposure of aqueous suspensions to different amounts of 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 hydrochloride using different amounts of dichloromethane and stirring time: (A) 1.38% v/v dichloromethane, 1.5 h; (B) 1.98% v/v dichloromethane, 1.5 h; (C) 1.98% v/v dichloromethane, 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 hollow PLA particles. Dichloromethane/water Concentration (% v/v) 1.58 1.98 Encapsulated Concentration (wt%) after 1.5 hr stirring 1.6 0.80 Encapsulated Concentration (wt%) after 3 hr stirring 1.2 0.40

Figure 5.6: Scheme showing the closing process of hollow PLA particles for encapsulation. Minimization of interfacial free energy is the driving force to close hollow PLA particles. To have effective encapsulation, the closing process should be stopped 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

Dimethyl carbonate is attractive because it is a nontoxic and biodegradable solvent.58 (b) 1. the particles are produced by freeze drying the sprayed droplets of polymer solution that are less than 100 microns Encapsulated Concentration (wt%) Removing excess surfactant 3. Dichloromethane/water Concentration (% v/v) 1. In this method. 199 For the dimethyl carbonate solutions. For the dichloromethane solutions. 5 min) with water 3 times.5 h stirring. but the shell was porous.1 1.2 1. 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. drying process. The time for the center of a water droplet with a radius of 1. the freezing temperature of dichloromethane.2 0. (b) 3 h stirring.7A shows that the particles obtained were hollow inside.9 2.4 seconds.7 mm to cool from room temperature to −97 °C. Dimethyl carbonate has a higher melting point (∼2 °C) compared to dichloromethane (−97 °C).88 Table 5. the particles are formed by freeze drying aqueous emulsion droplets that are ∼1. 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.2: Effect of surfactant removal on procaine hydrochloride encapsulation in hollow PLA particles.2 .98 (b) Encapsulated Concentration (wt%) using Original hollow PLA 2. in liquid nitrogen is about 0.38 (a) 1. Excess surfactant was removed by centrifugation (3500 rpm.7 mm in diameter (based on the average weight per droplet). 200 Figure 5.40 (a) 1.

201. allowing more drug to be encapsulated. was added to the dichloromethane oil phase. while it was just 2. The smaller droplets with higher freezing point are expected to freeze much faster than the aqueous dichloromethane emulsions. Figure 5. with a number of large hollow domains in the interior of the particles. PLGA-rich and Pluronic F127-rich phases formed. a water-soluble porogen. and the smaller pores in the shell can be closed more rapidly and more effectively than a single large pore. and the mixture was stirred for 1.89 in diameter. During the encapsulation. 199 The porosity observed in Figure 5.7B shows the porous particles were closed and the inside was hollow. spray/freeze-drying takes less time to obtain porous particles. Previous studies have shown that porous PLGA particles were obtained by emulsification/solvent evaporation when Pluronic F127. applies to a broader range of polymers. A previous investigation has shown that porous polymer particles are produced by freeze drying droplets of polymer solutions in benzene. 201. The encapsulated drug content was 4. the excess surfactant in the porous particles was not washed off. The amount of dichloromethane was 1.0 wt% using the porous particles.38% v/v in water. porous PLGA particles were obtained.203 When the Pluronic F127 was removed by washing in water.1 wt% for the hollow particles under the same conditions. 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. It is assumed that the porous shell allows the drug to enter more easily. Compared to the emulsification/solvent evaporation method.5 h.202 As the dichloromethane evaporated. a solvent with a melting point of 5 °C. .7 can also be partly attributed to the Pluronic P123 surfactant that can act as a porogen. and gives porous particles that are hollow inside.

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.90 (A) (B) Figure 5.5 h.8 shows that the hollow particles were closed. Figure 5.7: Porous PLA particles made by coaxial spray/freeze-drying with dimethyl carbonate as the solvent. The release kinetics of encapsulated procaine hydrochloride into phosphate buffer solution at pH 7. appearing spherical with a smooth surface. (B) after encapsulation.38% v/v dichloromethane for 1. The inset is the corresponding STEM image showing the inside cavity. To avoid using detrimental organic solvents. column . compressed carbon dioxide was investigated to close hollow PLA particles to form microcapsules.2 was measured as shown in Figure 5. The solid line is the release profile from microcapsules formed by exposure to 1. (A) Before encapsulation. an aqueous suspension of PLA microcapsules was obtained.2. After venting carbon dioxide.5 hours and excess surfactant removed (the sample in Table 5. row 1. An aqueous suspension of ∼1 wt% hollow particles was loaded into a variable volume highpressure stainless steel vessel.9. 131 Carbon dioxide at 207 bar and 32 °C was added to the headspace above the suspension for 1.

By fitting the data with exponential decay N(t) = C + N0 e−t/τ . row 1. The release rate decreased gradually with time (Figure 5. τ.8: PLA microcapsules formed by exposure of aqueous hollow particle suspension to compressed carbon dioxide. the exponential time constant or mean lifetime. The dashed line is the release profile from microcapsules formed under the same conditions except the excess surfactant remained (the sample in Table 5. Microcapsules with excess surfactant removed have a mean lifetime of 3.11 days.21 days for microcapsules formed without removing excess surfactant. column 2). all drug was released. can be calculated. close to the mean lifetime of 3. There are many factors affecting the . The microcapsules were washed with water prior to release study by centrifugation/redispersion three times.91 Figure 5. 3).9). After ∼9 days.2. 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 profiles from the two kinds of microcapsules are similar to each other.

2 phosphate buffer: microcapsules formed with excess surfactant removed (Table 5. column 3). Figure 5. The freeze-drying processes therefore have potential for producing biodegradable particles suitable for controlled. row 1. extended release of water-soluble therapeutic agents. so it is expected that the release dynamics would also be similar. ◦ microcapsules formed without removing excess surfactant (Table 5. which is likely due to the larger size of the microcapsules.9 have similar composition. Compared to the release of procaine hydrochloride encapsulated in PLGA nanoparticles by nano-precipitation.92 release from biodegradable delivery systems. and morphology. . size.9: Release of encapsulated procaine hydrochloride in pH=7.2. size and shape. 204 The two kinds of microcapsules shown in Figure 5.2. row 1. type of drug. such as type of polymer. column 2). 205 the release was slower and lasted longer.

The hollow particles can be converted to microcapsules through short exposure to plasticizing solvents. . making the particles potentially useful for drug encapsulation under mild conditions. 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.93 5. The particle morphology can be controlled by adjusting the type of solvent. The variation of encapsulation efficiency with processing conditions depends on changes in interfacial free energy and polymer mobility that drive microcapsule formation. solvent concentration.4 Conclusions Emulsification/freeze drying and spray/freeze drying are simple. 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. effective processes for creating hollow biodegradable PLA particles. The highest encapsulation amount was obtained with porous hollow polymer shells obtained via spray drying PLA/dimethyl carbonate solution. including non-toxic compressed carbon dioxide. and surfactant to control interfacial free energy.

Cross-linking was shown to greatly enhance the longterm 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 QDs were encapsulated into the core rather than the surface layer of the PI particles without additional surface modification of QDs. 100 The polyisoprene particles are easily cross-linked by adding a free radical initiator during emulsification and then heating. The polymer particles can be surface-modified with carboxyl groups during encapsulation to facilitate subsequent bio-conjugation and selective binding. while crosslinking gives rise to tight .94 Chapter 6 Conclusions and Future Work 6. Polyisoprene is in rubber state at room temperature so oxygen can diffuse into PI particles readily and quench QDs. This method is simple and robust.1 Conclusions Emulsification/solvent evaporation was applied successfully to encapsulate fluorescent CdSe(ZnS) quantum dots (QDs) into biocompatible polyisoprene (PI) particles.

These fluorescent hybrid nano-composite particles have potential application in genomics. indicating that the particles are suitable for multicolor coding. Supercritical carbon dioxide was deployed to impregnate dyes into premade monodisperse polystyrene (PS) particles. different emission peaks were clearly resolved. QDs are not distributed evenly in the polymer matrix due to micro-separation.95 polymer matrix inhibiting the diffusion of oxygen into PI particles and protects QDs. As solvent evaporates. Water-soluble ionic dyes were made hydrophobic by ion-pairing with alkyl quaternary ammonium cations. However. 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. Even though there appears to be some energy transfer between QDs. supercritical carbon dioxide is used in place of volatile organic solvents. 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. For bromothymol blue. . which is particularly attractive for drug delivery applications. The hydrophobic ion-paired dye can be encapsulated more efficiently into PS particles. suggesting energy transfer between QDs due to their aggregation during the encapsulation. The fluorescence spectra of mixtures of two different-sized QDs change in the PI-QD particle as compared to their solution spectra. and other applications that could exploit the high-throughput afforded by multicolor coding. the results show that hydrophobic ion pairing is limited in its effectiveness. Addition of water-miscible cosolvents was shown to further enhance the incorporation of the hydrophobic ion pairs into the polymer colloids. In the encapsulation process. drug discovery. Crosslinking also likely prevents the migration of encapsulated QD in the polyisoprene.

and dried in vacuum. When exposed to a plasticizer. as swollen aqueous latex particles were frozen by liquid nitrogen. It can be concluded that phase separation occurred. The morphology of the resulting hollow particles can be controlled by the amount of swelling solvent and surfactant. presumably due to its lower solubility in nonpolar solvents and the larger molecular size of the hydrophobic ion pair when compared with bromothymol blue. In cases where colloidal stability was lost. such as compressed CO2 . Subsequent morphological changes in the biphasic emulsion droplets are driven by the minimization of interfacial free energy. in these cases irreversible and uncontrolled coalescence led to loss of polymer particle integrity. Since the method reported here relies on spontaneous diffusion of the ion pair into the polymer. The PS latex was swollen with solvent. It is likely that the interior . 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 open hole on the hollow PS particles can be closed. Unfortunately. The experimental results can be interpreted in terms of interfacial free energy minimization. The thermodynamical nonequilibrium during the quenching is also considered.96 The ion pairs with rose bengal showed no enhanced loading in the polymer. it requires compatibility between polymer and ion pair and depends on the relatively slow diffusion kinetics in the plasticized polymer phase. hollow particles were obtained with an open hole on the surface. very high dye loading was observed due to heterocoagulation between the polymer colloid and dispersed particles of ion-paired dye. frozen in liquid nitrogen. After removing the solventrich phase by drying under vacuum. The dry hollow particles can be re-dispersed into water or water/ethanol mixtures by mild sonication. Hollow PS particles were obtained by thermally induced phase separation.

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. Hollow biodegradable poly(DL-lactide) (PLA) particles were also prepared by the thermally induced phase separation.97 surface of the hollow particles is more hydrophobic than the exterior surface. Interfacial free energy minimization drives closing the hollow particles if the polymer chains have mobility. 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. especially the difference in interfacial tension between the interior and exterior surfaces. and surfactant to control interfacial free energy. it interferes with closing of the holes. When surfactant is added to the suspension of hollow particles. The variation of encapsulation efficiency with processing conditions depends on changes in interfacial free energy and polymer mobility that drive microcapsule formation. while solid particles are. giving rise to porous structures. making the particles potentially useful for drug encapsulation under mild conditions. and the dye remained trapped in the microcapsule colloidal suspension for a long time. which is not favorable for . A water-soluble dye was encapsulated into hollow PS particles. core-shell structures or microcapsules are not the minimal in terms of free energy. The particle morphology can be controlled by adjusting the type of solvent. The hollow particles can be converted to microcapsules through short exposure to plasticizing solvents. So the hollow particles will eventually convert to solid particles giving enough time. The experimental evidence shows that the hole-closing process is also driven by interfacial free energy minimization. However. The emulsification/freeze drying process has potential application in microencapsulation for controlled release. solvent concentration. including non-toxic compressed carbon dioxide.

98 encapsulation. and the resulting porous particles can be suspended in water. Different biodegradable . However. which is nontoxic and easy to separate from the suspension. the particles lost stability and coalesced in a short time when exposed to compressed CO2 .1 Future Work Stabilizing Porous Particles in CO2 Making porous PLA particles by freeze-drying spray of PLA solution in dimethyl carbonate. Such porous PLA particles can be converted to microcapsules for microencapsulation by exposure to compressed carbon dioxide for a short time. a green solvent. 6. 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. Stabilizing the particles under CO2 is necessary for CO2 -based microencapsulation. To achieve optimized encapsulation efficiency. The particle morphology also affects the encapsulation efficiency. Pluronic P123 surfactant was dissolved in dimethyl carbonate with PLA.2. The highest encapsulation amount was obtained with porous hollow polymer shells obtained via spray drying PLA/dimethyl carbonate solution. Porous PLA particles without any surfactant could not be suspended effectively in water.2 6. and environmental benign. 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. is simple. making it attractive for drug encapsulation. the exposure time to the plasticizer should be modest. economical.

It would be highly desirable for many application to achieve narrow particle size distribution with good control of the average particle size. eliminating the high-speed air stream in these devices would reduce the usage of liquid nitrogen. Block copolymer surfactants. The broad size distribution of the particles is due to the broad size distribution of polymer solution droplets from the spray nozzle. In addition. . 207. can be tested if they have better stability under CO2 . 131 may be a good choice.99 polymers soluble in dimethyl carbonate. 206 such as poly(dimethylsiloxane)-b-poly(methacrylic acid). It is not easy to obtain narrowly distributed droplets by breaking up the polymer solution with high-speed annular air jet.208 Drop-on-demand devices 209 for making monodisperse droplets would be promising to combine with freeze-drying to make uniform porous polymer particles. 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. 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 . such as poly(lactic-co-glycolic acid). 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. The surfactant can also be adsorbed onto the resulting porous particles for stabilization in CO2 .

100 6. Biodegradable porous polymer particles can potentially be made this way from the recently developed biodegradable non-fluorous polymers soluble in CO2 .2.3 Making Porous Polymer Particles by Freeze-drying Emulsions 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. This will not require removing large quantities of water by vacuum pump as conventional freeze-drying does. 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. 210 .

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