AND MICROCARRIER FABRICATION a Thes

A NOVEL μ-FLUIDIC CHANNEL ASSISTED ENCAPSULATION TECHNIQUE FOR

LAYER-BY-LAYER POLYMER NANO- AND MICROCARRIER FABRICATION

A Thesis

Presented to

The Graduate Faculty of The University of Akron

In Partial Fulfillment

of the Requirements for the Degree

Master of Science

Jingyu Li

August, 2015

A NOVEL μ-FLUIDIC CHANNEL ASSISTED ENCAPSULATION TECHNIQUE FOR

LAYER-BY-LAYER POLYMER NANO- AND MICROCARRIER FABRICATION

Jingyu Li

Thesis

Approved: Accepted:

Advisor Department Chair Dr. Younjin Min Dr. Sadhan C. Jana

Committee Member Dean of the College Dr. Mukerrem Cakmak Dr. Eric J. Amis

Committee Member Interim Dean of the Graduate School Dr. Hossein Tavana Dr. Rex D. Ramsier

Date

ii ABSTRACT

Layer-by-layer (LbL) assembly is a popular technique for fabricating thin multilayer films on templates by depositing alternating layers of oppositely charged polyelectrolytes with rinsing steps in between. The LbL technique fabricated capsules can be used for biomedical applications such as drug and vaccine delivery, biosensors and bioreactors. LbL assembly enables us to use various types, sizes, and shapes of particles as templates, like silica particles, calcium carbonate particles and metal particles. In addition, a suite of water-soluble polymers with different properties can be applied to LbL assembly to meet researchers` requirements. Due to accurate control on size and shape, LbL technique is capable of fabricating small particles below 200 nm, which is in the optimal size for drug carriers and small enough to avoid clogging blood capillaries. The versatility of nano- and microcapsules has captured the attention of researchers to develop fabrication methods of LbL particles.

The traditional LbL assembly method has simple process, but which is inefficient and has limitations due to repeated centrifugation steps. The long preparation time could lead to premature drug release before use. The capsules containing drugs, dyes and targeting molecules is too fragile to be centrifuged. The optimal capsule size is typically around 200 nm for many drug delivery systems. Nanoparticles under 200 nm are hard to separate from solution by centrifugation. The classical method has many drawbacks; researchers attempt to improve it for a long time. Some advanced

iii methods, like atomization techniques or electrophoretic polymer assembly, have been created to better tailor properties of multilayer capsules, but influence the uniformity of films and limit the choice of polyelectrolytes and particles.

In this study, our group aims to design a novel μ-fluidic device for layer-by-layer particle fabrication, which overcomes some of shortcomings of other methods. The device takes one seventh of time to assemble the same number of layers as the traditional method (μ-fluidic device takes 20 min per layer, the traditional method takes more than 2 h per layer). The device can easily prepare twenty layers of silica particles in 8 h, which formerly requires 2 days. Dynamic light scattering, ζ-potential analyzer and transmission electron microscopy were used to demonstrate the particle size, film thickness, surface charge and morphology. Drug-loaded poly(D,L-lactide-co-glycolide) (PLGA) particles were prepared for in vitro experiments with breast cancer cells. Imaging of treated cells and a biochemical analysis were used to determine the cell population and morphology. Our observation indicates that the positively charged particles have an advantage on cell uptake and drug delivery for MDA-MB-231 cancer cells. The LbL paclitaxel-loaded PLGA particles show a better anti-cancer effect than the free paclitaxel.

iv ACKNOWLEDGEMENTS

I am grateful to all those people who helped me to accomplish the project and master thesis.

First and foremost, let me express gratitude to my advisors, Dr. Younjin Min for her continuous guidance, support and encouragement for my research work and master study. Dr. Min not only provided me training in experimental and research skills, but also built my scientific qualities, curiosity, skepticism and writing skills.

Next I would like to thank Prof. Mukerrem Cakmak and Prof. Hossein Tavana for being my thesis committee member. Also a special thanks to Dr. Rong Bai for Atomic

Force Microscopy (AFM) training, Dr. Bojie Wang for Transmission Electron

Microscopy (TEM) training, Prof. Nicole Zacharia for providing Malvern Instruments

ZEN 3690, Prof. Hossein Tavana and his student Stephanie Lemmo for providing materials for in vitro experiment.

And I would like to thank all the group members: Mr. Yuanzhong Zhang, Mr.

Yupeng Hu, Mr. Tianxin Zhao, Miss Xiaoxu Lu, Mr. Haoran Wang, Mr. Shihao Wen,

Miss Junyan Wang and friends who once helped me in experiments.

Last, my gratitude also goes out to my parents and my boyfriend who helped me a lot in daily life. They always supported and encouraged me in all areas. I can achieve nothing without them.

v TABLE OF CONTENTS

Page

LIST OF FIGURES ...... viii

LIST OF TABLES...... xi

CHAPTER

I. INTRODUCTION ...... 1

1.1 Layer-by-layer Assembly ...... 1

1.2 Fabrication methods of LbL particles ...... 3

1.2.1 Spray-assisted LbL deposition...... 3

1.2.2 Membrane filtration...... 6

1.2.3 Electrophoretic polymer assembly (EPA) ...... 7

1.2.4 μ-fluidic systems ...... 9

1.3 Biomedical Applications of LbL Nano- and Microcapsules ...... 11

1.4 Mechanism of Cell Internalizing Particles ...... 12

1.5 Mechanism of Drug Release From Drug-loaded Particles in Vitro ...... 13

1.6 Physicochemical Characterizations ...... 14

1.6.1 Transmission Electron Microscopy (TEM) ...... 14

1.6.2 Dynamic Light Scattering (DLS) ...... 15

1.6.3 ζ-potential Analyzer ...... 16

1.6.4 Atomic Force Microscope (AFM) ...... 17

1.6.5 High Performance Liquid Chromatography (HPLC) ...... 19

1.6.6 Fluorescence Microscope ...... 20 vi 1.6.7 Microplate Reader ...... 21

II. MATERIAL SPECIFICATION AND EXPERIMENTAL TECHNIQUES...... 23

2.1 Introduction ...... 23

2.2 Experiment Section ...... 26

2.2.1 Material………………………………………………………………………………………………….….. .. 26

2.2.2 Layer-by-layer Assembly on Silica Particle...... 28

2.2.3 Preparation of Nile Red PLGA Nanoparticles and Drug-Loaded PLGA Nanoparticles……………………………………………………………………………………………….…………28

2.2.4 Layer-by-layer Assembly on Drug Loaded and Nile Red PLGA Nanoparticles. ... 29

2.2.5 In Vitro Experimentation...... 29

2.2.6 Characterization of Particles...... 30

2.3 Configuration and Work Principle of μ-Fluidic Device ...... 27

III. RESULT AND DISCUSSION...... 31

3.1 The Optimum Experimental Condition of μ-fluidic Device ...... 31

3.2 LbL Silica Particles ...... 37

3.3 In Vitro Experiment ...... 40

3.3.1 Fabrication of LbL Blank and LbL Drug-loaded PLGA Particles ...... 41

3.3.2 Cytotoxicity of LbL Blank PLGA Particles ...... 42

3.3.3 MDA-MD-231 Cell Co-culturing With Free Drug ...... 45

3.3.4 Effect of Drug-loaded LbL Particles ...... 48

IV. CONCLUSION AND FUTURE WORK ...... 56

REFERENCES ...... 58

vii LIST OF FIGURES

Figure Page

1.1. The schematic of traditional film-deposition process ...... 3

1.2. Illustration of the PRINT process...... 4

1.3. SEM images of particles prepared by PRINT process...... 5

1.4. Schematic of Spray-LbL on PRINT nanoparticles ...... 6

1.5. Sketch of the membrane filtration procedure ...... 7

1.6. The schematic of EPA process fabricating LbL particles ...... 8

1.7. Microscopy images of SiO2/(PAH/PSS)4 capsules with different size (3.08 μm, 1.10 μm, 585 nm, 35 nm) prepared by EPA ...... 9

1.8. Schematic of the microchip design with images of the comb withdrawal channels ...... 10

1.9. Overview of ‘microfluidic pinball’ device...... 10

1.10. Illustration of three strategies of cellular internalization ...... 13

1.11. Schematic of dynamic light scattering system...... 16

1.12. Schematic of ionic distribution and electric potential as the distance increase from the particle’s surface to a dispersed medium ...... 17

1.13. Schematic of an atomic force microscope...... 18

1.14.Schematic of HPLC...... 20

1.15. Simple schematic of fluorescence microscope ...... 21

2.1. Schematic of Poiseuille flow radial distributions of velocity ...... 25

2.2. Schematic illustration of μ-fluidic derive...... 27

3.1. ζ-potential of LbL silica particles which were fabricated in different experimental conditions ...... 33

viii

3.2. TEM iamges of (a) SiO2/(PAH/PSS)2/PAH particle under 5 min incubation time with ultrasonic, without salt. (b) Bare particle (scale bar: 100 nm) ...... 35

3.3. TEM iamges of (a) 5 min incubation time, with ultrasonic, with salt, 5-layer sample; (b) 5min incubation time, without ultrasonic, with salt, 5-layer sample ...... 35

3.4. The counterions (ions with opposite charge to the surface co-ions) will accumulate and deplete near to a charged surface. The bottom schematic shows graphically for a 1:1 electrolyte (e.g NaCl, KCl), where ∞ is the electrolyte concentration at x=∞.49 ...... 36

3.5. TEM images of LbL silica capsules/particles are obtained from PAH/PSS-coated 300 nm SiO2 particles. The silica particles were removed from polyelectrolytes films by 10% hydrogen fluoride solution for n=10 (b) and 20 (c) samples. (Scale bar: 100 nm) ...... 38

3.6. Physicochemical characterizations of LbL silica nanoparticles: Particle sizes (a), ζ-potential values (b), and TEM images (c) (scale bar: 100 nm). ζ-potential analysis were implemented in Millipore water at 25 ℃ . ζ-potential of bare SiO2 is (-49.87±4.62) mV...... 39

3.7. schematic illustration of blank LbL PLGA nanoparticles (a) and drug-loaded LbL PLGA nanoparticles (b), AFM images of bare PLGA particles (c), ζ-potential of drug-loaded LbL PLGA nanoparticles (d), and micrograph of PLGA/PLL/DSS/PLL particles (e). (Scale bar of micrograph is 2 μm)...... 41

3.8. MDA-MB-231 triple negative breast cancer cells were treated with blank PLGA particles for 24h and 48h...... 43

3.9. Micrographs of MDA-MB-231 cancer cells co-cultured with LbL blank PLGA particles for 24 h and 48 h. (n is the number of layer, scale bar is 100 μm) The green signal in blue channel is from FITC-PLL layer(s) of particles...... 45

3.10. Cell viability of MDA-MB-231 treated with 1 nM, 5 nM, 10 nM and 20 nM of free drug. The free drug was removed at 48 h when renewed the media...... 46

3.11. Micrographs of MDA-MB-231 treated with different concentrations` paclitaxel for 24 h, 48 h, 72 h and 96 h...... 48

3.12. MDA-MB-231 triple negative breast cancer cells were treated with LbL drug-loaded PLGA particles for 4 days. (Drug loading: 2 nM, red dash line is 100%)...... 49

3.13. Micrographs of MDA-MB-231 triple negative breast cancer cells co-cultured with LbL paclitaxel-loaded PLGA particles. (n is the number of layer, scale bar is 100 um) ...... 52

3.14. Micrographs of MDA-MB-231 triple negative breast cancer cells co-cultured with LbL paclitaxel-loaded PLGA particles. The green signal denotes live cells which were dyed by Calcein AM. The red signal indicates dead cells which were dyed by EthD-1. (The scale bare is 100 μm. n is the number of layer) 55

LIST OF TABLES

Table Page

1. The Thicknesses of (PAH/PSS)N films...... 31

2. The experimental conditions of LbL silica nanoparticles ...... 32

3. The optimum experimental conditions ...... 37

CHAPTER I

INTRODUCTION

1.1 Layer-by-layer Assembly

Layer-by-layer (LbL) assembly is a popular technique for fabricating thin multilayers on templates, which has attracted increasing attention due to its mertis in biomedicine. The films are built by depositing alternating layers of oppositely charged polyelectrolytes with washing steps in between. In 1966, R. K. Iler, for the first time, successfully carried this technique out using colloidal microparticles.1

However, the method did not attract a lot of attention until Prof. Gero Decher discovered that it could be applied to numerous polyelectrolytes.2 LbL offers several advantages over other thin film deposition methods. LbL can be applied to a wide range of materials, such as metals, polyions, ceramics, and nanoparticles. Another important feature of LbL, which makes it a desirable method in biomedicine, is the increased control of film thickness in 1 nm resolution.

The LbL fabricated microcapsules can be used for biomedical application, such as drug and vaccine delivery3-5, biosensors6-9 and bioreactors10-12. LbL assembly is applicable to varied types, sizes, and shapes of particles as templates, like silica particles, calcium carbonate particles and metal particles. LbL can easily fabricate particles smaller than 200 nm for drug delivery, i.e. small enough to avoid clogging of blood capillaries. Furthermore, LbL process can be implemented in nontoxic aqueous

1 solutions. The versatility of nano- and microcapsules has greatly attracted researchers’ attention for developing fabrication methods of LbL particles.

A significant method in this field of LbL-engineered particles is traditional film-deposition process (Figure 1.1) which typically requires numerous rinsing and centrifugation steps to separate the residual polyelectrolytes from coated particles.

This method results in non-aggregated multilayer-coated microparticles. However, strategy is time-consuming. Commonly, three rinsing and centrifugation cycles are required after one adsorption step to prevent residual polyelectrolytes from aggregating in subsequent layer, and one rinsing and centrifugation cycle takes more than half hour. Therefore, the typical method takes more than 2 hours to assemble only one layer. Also, the capsule containing drugs, dyes and targeting molecules is too fragile to centrifuge. Moreover, it is generally difficult to separate nanoparticles less than 200 nm by centrifugation in the traditional methods, which limits its use for drug delivery applications since the optimal carrier size is about 200 nm.13, 14 The aggregation of particles, especially for small particles, is also a problem researchers are working on.

Figure 1.1. The schematic of traditional film-deposition process

1.2 Fabrication methods of LbL particles

Since the classical method has many shortcomings, researchers have attempted to improve it for a long time. Various new methods, such as spray-assisted LbL deposition,15 microfluidic systems,16, 17 membrane filtration18 and electrophoretic polymer assembly,19 have been attempted to overcome certain drawbacks and assemble high quality LbL particles.

1.2.1 Spray-assisted LbL deposition

Particle Replication in Non-wetting Templates (PRINT) process20-22 is an essential element for this method due to scalability and high yield of fabricating nanoparticles.

PRINT is a powerful nano-molding technique which enables people to fabricate particles with precise control over the size, morphology and composition. The process of fabricating particles is quite similar to lithographing books. People print particles on the substrate but not words on the paper. The process of PRINT is shown

3 in Figure 1.2. The non-wetting nature of fluorinated materials and surfaces (shown in green) confines the liquid precursor inside the features of the mold, allowing for the generation of isolated particles. Figure 1.3 illustrates the breadth of shapes, sizes, and compositions possible for nanoparticle fabrication using the PRINT process.

(A) A true solution (red) is casted on a poly (ethylene terephthalate) substrate using a Mayer rod. A solid-state solution polymer film (solid state solution is a crystalline material in which two or more elements or compounds share a common lattice) is generated under heat referred to the delivery sheet to deliver the composition to the mold. (B) A perfluoropolyether elastomeric mold (green) is placed on a delivery sheet (red) and passed through a heated nip (gray) to separate. The cavities of the mold are filled. (C) A filled mold is placed on with a high energy film or excipient layer (yellow) and passed through the heated nip without splitting. The mold is removed to reveal an array of particles after cooling on the high-energy film or excipient layer.

Figure 1.3. SEM images of particles prepared by PRINT process: (a) Hydrogel rods containing antisense oligonucleotide; (b) crosslinked degradable matrix cubes containing doxorubicin HCl; (c) Abraxane harvested onto medical adhesive; (d) Insulin particles harvested onto a medical adhesive; (e) Hydrogel ‘boomerangs’ containing 15 wt% iron oxide; (f) Hydrogel cylinders containing 10 wt% Omniscan. Reprinted with permission from reference 21. Copyright 2009, John Wiley & Sons, Inc.

PRINT particles were fabricated and stored on the harvesting layer. The substrate of arrays was PVA which could be easily dissolved in the first spray and lost most particles. To avoid PVA dissolution, the PVA adhesion layer was crosslinked in the vapor-phase containing glutaraldehyde (50%) and concentrated acid (10%) to make it insoluble. Spray-LbL application was applied as follow: sequential deposition of polycation/wash/polyanion/wash to comprise one bilayer (Figure 1.4).15

Functionalized particles were collected by sonication of the arrays in water and purified by filtration and ultracentrifugation.

Despite the advantages of PRINT, there is a significant problem that one side of the particle always fixes on the high-energy film or excipient layer cannot be coated, which influences the homogeneousness of the particles. Moreover, the heating step in PRINT process and the chemicals used in the vapor-phase crosslinking may limit the types of materials which can be applied for the particle.

1.2.2 Membrane filtration

This method utilizes filter membranes to separate particles from coating and rinsing solutions (Figure 1.5). Vacuum filtration, pressure filtration, and atmospheric filtration are manipulated in different steps. Particles and polyelectrolyte solutions are added firstly and incubated under stir for a certain time. During the polyelectrolyte adsorption period, a weak underpressure should be maintained to the incubation chamber in relation to the lower filtrate chamber, which prevents loss of the incubation medium with a simultaneous decrease of the polyelectrolyte to particle number ratio. Several rinsing steps are needed between each deposition step.

The method is very effective with no need of numerous rinsing-centrifugation cycles between deposition steps. However, the filter membranes must have appropriate pore size and good property to support repeated vacuuming and keep unclogging. A certain amount of polyelectrolyte solution is needed to immerse stirrer, and a certain distance between the membrane and stirrer increases the material consumption. The force from stirrer should be kept in a range to keep discrete particles and no harm to the fragile nanocapsules.

1.2.3 Electrophoretic polymer assembly (EPA)

In this approach, LbL particles were generated by electrophoresis and followed core removal. The particles are fixed in a porous hydrogel which is referred as the biologically derived polysaccharide agarose. Agarose gelating under ambient conditions forms various tunable pore sizes with low reactivity. The immobilized particles are deposited and rinsed in electrophoresis (Figure 1.6).

1) Polycation solution is loaded into the wells adjacent to the anode and electrophoresed moving toward the cathode coats the particles. 2) Polyanion solution is loaded into the wells adjacent to the cathode and electrophoresed; it coats the cationic polymer-coated particles as it moves toward the anode. Repeat step 1 and 2 until forming the desired layers. 3) The agarose is heated, and the EPA products are recovered.

This technique reduces handling time and minimizes the chance of aggregation, especially for small particles. Polymer multilayer capsules can be fabricated automatically without novel apparatus. The method works promisingly in a wild particle size range from 35 nm to 3.8 μm (Figure 1.7). But the heating step that is required for collecting particles from the agarose leads to consistent negative

ζ-potential of particles, which may limit the choice of polyelectrolytes, particles and further application.

Figure 1.7. Microscopy images of SiO2/(PAH/PSS)4 capsules with different size (3.08 μm, 1.10 μm, 585 nm, 35 nm) prepared by EPA. a–d) Fluorescence microscopy images of the capsules (scale bars: 5 μm). e–h) TEM images of the capsules (scale bars: 1 μm). i–l) SEM images of the capsules (scale bars: 500 nm). Reprinted with permission from reference 19. Copyright 2013, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

1.2.4 Microfluidic systems

This type of method utilizes micro-fluidic channels as a container for particles and solutions. The streaming medium drives particles to move to polyelectrolyte solutions or rinsing solutions alternatively to assemble LbL particles. Various designs of the channel make it hard to summarize a model for this method, while the principles they followed are the same—incubating and rinsing.17, 23 Two successful examples of microfluidic device are demonstrated below (Figure 1.8 and Figure 1.9).

Figure 1.8. Schematic of the microchip design with images of the comb withdrawal channels. Sodium dodecyl sulfate (SDS) is added to avoid the liquid crystal (LC) spontaneously coalesces prior to adsorption of the first polymer layer. Reprinted with permission from reference 23. Copyright 2008, The Royal Society of Chemistry.

Figure 1.9. Overview of ‘microfluidic pinball’ device. (a) Schematic (not to scale) of the configuration of the device which requires inputs and outputs for the deposition of six layers of polyelectrolyte. (b) Expanded view of pillars in zigzag arrangement. (c 1) and (c 2) Micrographs of droplet getting incubated in the first PE and changing direction following the polydimethylsiloxane ladder; (c 3) and (c 4)droplet entering

10 the washing solution which removes the residual PE; (c 5) and (c 6) droplet entering into the second PE for deposition after the wash solution; (c 7) and (c 8) droplet incubated in second PE and changed the PDMS ladder. Scale bar 200 um. The mold pattern used Cadence Vitruoso software to print on a plastic photomask for photolithograph. Reprinted with permission from reference 17. Copyright 2011, The Royal Society of Chemistry.

Sequential adsorption of interacting polymers is automatic under continuous flow conditions. The size of the droplets can be controlled via precise adjustment of the flow rates of the dispersed and continuous phases to avoid aggregation.

Although the authors claim that particle size can be adjusted, the particle sizes implemented in the device and samples for AFM, TEM or SEM are all in micrometer range.17, 23-25 None of them can be applied in vitro or in vivo experiments because particles in the micrometer range cannot be uptake by cells and may clog blood vessel.

Many new techniques to fabricating multilayer particles have been developed and demonstrated because of the advantages of LbL particles and drawbacks of existing fabrication methods. Efficiency is always a topic for fabricating LbL particles, and all new methods are attempted to avoid centrifuging between rinsing steps.

Aggregation is the second problem impeding researchers from producing perfect multilayer particles. Every method owns its strengths and drawbacks as mentioned above.

1.3 Biomedical Applications of LbL Nano- and Microcapsules

The LbL mirocapsules and nanoparticles are one of the ideal tools for sensing application because of diverse protocols for integration and capability of microcapsules and nanoparticles. The fluorochromes and analyte-sensitive can be simultaneously loaded into the same capsule to become a precise biosensor.

McShane’s group first reported biosensors based on LbL microcapsules which targeted on glucose. Nowadays, this technique has been extended to ions6 and pH sensing7, 8.

The LbL assembled capsules are also particularly suitable for catalysis, transportation and protection26 due to its hollow controllable structure. The LbL microcapsules are firstly applied to encapsulate enzymes for enzymecatalyzed polymerization27, 28, bio-mineralization29, 30 and bienzyme reaction31, 32.

Drug delivery and biotherapeutics also can be performed by LbL assembled capsules, since the unique colloidal structure can offer a versatile function for storage, encapsulation and delivery of diverse chemotherapeutics. Biodegradable and non-cytotoxic polymers, like poly(lactide-co-glcolide) (PLGA)33-35, are mostly investigated polymers for subcutaneous and intravenous administration of various therapeutic agents such as anticancer agents. To enhance the intracellular delivery capability in the tumor site, some researchers modified the surface of nanoparticles by tethering targeting ligands. Since the drug carriers can concentrate in tumor site, the dosage of drugs can be reduced to achieve same therapeutic effect, meanwhile, minimize the cytotoxic side effects.

1.4 Mechanism of Cell Internalizing Particles

The drug-loaded particles can be internalized by cells through diverse pathways, including phagocytosis, pinocytosis and receptor-mediated endocytosis (Figure

1.10).36

Phagocytosis normally refers to the uptake of solids such as microparticles and bacteria into cells, which occurs dependently of actin polymerization and is usually independent of clathrin.37 Phagocytosis is also a approach for some cells to acquire

12 nutrients. Phagocytosis can engulf whole microparticles, which will be eroded and degraded by enzymes, and absorbed into the cells. The majority of cells have evolved some phagocytic capacity to phagocytose particles in culture.

Pinocytosis refers to the uptake of extracellular fluid and solute. In contrast to receptor-mediated endocytosis, pinocytosis is nonspecific selection for absorbing substances. Selective uptake of substances is called receptor-mediated endocytosis.

Pinocytosis and receptor-mediated endocytosis share clathrin-dependent mechanism and are usually independent of actin polymerization. A wide variety of drug delivery system is based on pinocytosis and receptor-mediated endocytosis delivery.38, 39

Figure 1.10. Illustration of three strategies of cellular internalization

1.5 Mechanism of Drug Release From Drug-loaded Particles in Vitro

In this study, drug-loaded particles refer to matrix systems in which the drug is dispersed throughout the nanoparticle. For LbL drug-loaded particles, the drug only exists in its core. There are four major possible mechanisms which should be considered for drug release from drug-loaded particles in vitro40-42: (1) pulsed release initiated by the application of ultrosonic to well disperse particles in solvent,

(2) release from the surface of particle, (3) self-diffusion through pores, (4) release due to polymer degradation.

In general, some of these mechanisms may coexist in whole in vitro experiment.

Before treating cells with particles, the particles may be ultrasonic for several minutes to disperse homogeneously in the media, which can initiate pulsed release.

The initial drug release originates from free drug near or present on the particles surface, especially for bare particles. For the self-diffusion process, a minimum drug loading is required for the drug through multilayer outside the particles. This mechanism may not occur if the encapsulation efficiency is too low. Drug release caused by polymer degradation is a common process when degradable polymeric nanoparticles were utilized. The polymer chain degradate and particles are eroded in media or in cells, and the drug liberates out. All these processes affect the liberation of the drug from particles, and the distinction of them is significant sometimes.

1.6 Physicochemical Characterizations

To characterize the fabricated LbL particles, transmission electron microscopy

(TEM), dynamic light scattering (DLS), ζ-potential analyzer, and atomic force microscope (AFM) are used to determine their size, surface charge and morphology.

High performance liquid chromatography (HPLC), fluorescence microscope and microplate reader are applied for anlysis of drug loading of particles, cell observation and quantitation of cell viability respectivly in in vitro experiments.

1.6.1 Transmission Electron Microscopy (TEM)

The transmission electron microscope (TEM) is one of the most powerful techniques for analysis of the morphology and size at nanoscale, which is usually extensively used in observing nanoparticles. The illumination source is a beam of

14 electrons at a very short wavelength which is emitted from a tungsten filament at the top of the TEM. The electron beam is transmitted through an ultra-thin specimen and scattered by its internal structure. The whole optical system must work in vacuum to avoid scattering of electrons and colliding between electrons and air molecules.

The bright field imaging mode is the most common mode of operation which is used in this project. In this mode, the contrast formation is determined directly by absorption and occlusion of electrons of the sample. The image appears as dark region when the sample is thicker or has a higher atomic number in this region.

Hence, the particles form black rounds, polymer films are gray circles coated on the particles and other regions are bright fields.

1.6.2 Dynamic Light Scattering (DLS)

Dynamic light scattering is one of the most popular methods to measure the size of colloids, micelles, proteins, and nanoparticles. The working principle is that the sample is illuminated by a laser beam and the fluctuations of the scattered light are detected at a known scattering angle by a fast photon detector (Figure 1.11).

When a laser beam hits small particles, the monochromatic light scatters in all directions if the particle size is smaller than the light wavelength. The particles in solution undergo Brounian motion. The distance between particles and the position of particles are constantly changing, which lead to the scattered light undergoing newly-created or disappeared surface of surrounding particles. Hence, the scattering intensity in a certain scattering angle will fluctuate over time.

Figure 1.11. Schematic of dynamic light scattering system. The laser beam hits a moving particle and scatters in all directions. A fast photon detector is placed at a certain angle to detect the light intensity of scattering light.

1.6.3 ζ-potential Analyzer

In colloidal systems, researchers usually use ζ-potential to demonstrate the electrokinetic potential on the slipping plane (Figure 1.12).43 Theoretically, a charged particle is coated by oppositely charged ions. As the distance increases, the force from the particle to hold ions decays and the ions do not move with the particle on the slipping plane. After connecting with bulk solute, the force normally reduces to zero at a sufficient distance. The value of electric potential at the slipping plane is ζ-potential.

Figure 1.12. Schematic of ionic distribution and electric potential as the distance increase from the particle’s surface to a dispersed medium

To measure ζ-potential, the devices have two common points: (1) can create relative movement between solid surface and liquid for disturbing double-layer; (2) can monitor generated signals. The first point can be achieved by the mechanical pressure field (syringe pump) or the external electric field (electrophoresis). The second point can be induced by optical methods (DLS) or electrical methods

(streaming potential device). In this thesis, we use the external electric field and DLC to measure ζ-potential of the particles.

1.6.4 Atomic Force Microscope (AFM)

As a high-resolution type of scanning probe microscopy, AFM can characterize a specimen with resolution in the range of fractions of nanometers. It can be used to

17 study the size and morphology of the particles. The working principle of AFM is demonstrated in Figure 1.13. A beam emitted by laser reaches the cantilever of the

AFM tip. The beam is reflected by the cantilever and mirror, and then reaches to a position sensitive photo-detector (PSD). In the tapping mode, the cantilever which is driven by a small piezoelectric element in the AFM tip holder oscillates up and down near its resonance frequency in a certain range of amplitude. The amplitude of this oscillation will induce as the tip comes closer to the surface due to the interaction of forces acting on the cantilever and surface. To maintain a constant interactional force between the tip and surface, the height position of cantilever is adjusted by a piezoelectric actuator. The constant interactional force between the tip and the sample can be controlled by a feedback loop.

Figure 1.13. Schematic of an atomic force microscope. A beam is emitted by a laser and reflected from the back of the catilever to a mirror. The reflected beam reflected again from the mirror and collected by a position sensitive photo-detector. The signal goes into a feedback loop to adjust the height between tip and sample surface by controling the position of piezoelectric scanner. 18

1.6.5 High Performance Liquid Chromatography (HPLC)

HPLC is a common technique applied to quantify each component, to identify each component and to separate the components in a mixture. In this project, HPLC is an efficient method to test the drug loading (paclitaxel) in particles. The liquid sample is pressurized with pumps and through a column filled with a sorbent (Figure

1.14). Each compound in the sample undergoes slightly different absorption force with the sorbent, which causes different flow rates for different components and leads to different retention times for flowing out of the column. The sorbent is normally a granular material with 2—50 micrometer size. Reversed-phase HPLC is a common method to analyse the drug which has a non-polar column and an aqueous, moderately polar mobile phase. One common sorbent in the column is silica particles which have been surface-modified by R-Me2SiCl, where R is a linear alkyl group such as C8H17 or C18H37. With such stationary phases, retention time is longer for less polar molecules than polar molecules.

Figure 1.14. Schematic of HPLC. A mobile phase (e.g. water and acetonitrile) is constantly flowing in the equipment when analysis samples. The liquid sample is injected into the equipment with autosampler, pressurized with pumps and through a HPLC column. The detector collectes the signal and sends it to computer.

1.6.6 Fluorescence Microscope

The fluorescence microscope refers to any microscope which utilizes fluorescence to generate an image.It could be a simple device like an optical microscope with a dichromatic filter, or a complicated set up like confocal microscope.

This technique has become an important tool in the field of biomedicine since fluorescent labeling of biomolecules and compounds has been widely used in research.

When the specimen is exposed under a specific wavelength light, the fluorescent groups in the specimen will absorb energy and emit a light with a longer wavelength (different color from absorbed light). Because the illumination light is much stronger than emitted fluorescence, it needs to be separated from the weak signal through a spectral emission filter. Typical constitution of a fluorescence

20 microscope includes a microscope with a specific light source (e.g. mercury-vapor lamp), an excitation filter, a dichroic mirror and an emission filter (Figure 1.15). The filters and the dichroic mirror need to match the spectral excitation and emission wavelength of the fluorophore in the specimen.

Figure 1.15. Simple schematic of fluorescence microscope

1.6.7 Microplate Reader

The microplate reader is widly used to detect and monitor the sample in microtiter plates in biological, chemical and physical research. The versatile detecting events are based on different detection methods and detection reagents.

Common detection methods of a microplate reader are absorbance, fluorescence, and luminescence. In this thesis, fluorescence intensity detection of a PrestoBlue®

Cell Viability Reagent is used to detect cell viability.

The working principle of fluorescence intensity detection is similar to fluorescence microscope’s. In this detection mode, there are two optical systems.

The first optical system is to illuminate the sample with a light at a specific wavelength as excitation light. The second optical system is to seprate the emitting light of the sample from the excitation light, collecte it and measure the intensity of the signal.

PrestoBlue® Cell Viability Reagent is a fluorescent dye manufactured and sold by

Life Technologies. PrestoBlue® utilizes the reducing power of living cells to quantitatively indicate cell viability. The cell permeable resazurin-based solution in

PrestoBule® is metabolized by living cells to a highly fluorescent form and can be monitored by fluorescence or absorbance measurments.

퐼푠푎푚푝푙푒 Cell viability % = × 100% 퐼푐표푛푡푟표푙

Where Isample and Icontrol denote the fluorescence intensity detected for cells treated with LbL nanoparticles/free drug, and for negative control cells (nontreated), respectively.

CHAPTER II

MATERIAL SPECIFICATION AND EXPERIMENTAL TECHNIQUES

2.1 Introduction

Layer-by-layer (LbL) assembly is a popular technique for fabricating thin multilayer films on templates due to its merits in biomedicine. LbL assembly enables us to use various types, sizes, and shapes of particles as templates, like inorganic nonmetal particles (silica particles and calcium carbonate particles), precious metal particles (Au particles and Ag particles), and biodegradable particles (PLGA particles).

In addition, a suite of water-soluble polymers with different biological properties can be applied to LbL assembly to meet researchers` requirements.19 Due to the well control on size and shape, LbL technique enables to fabricate particles smaller than

200 nm as drug carriers which are small enough to avoid clogging the blood capillaries. These advantages of the LbL assembly have greatly attracted researchers’ attention.

The traditional film deposition method is time-consuming due to numerous centrifugation steps which are also limited the particle size (well over 200 nm). The overlong preparation time could lead to premature drug release before use.

Moreover, some capsules containing drugs, dyes and targeting molecules are too fragile to be centrifuged. The aggregation of particles is also a challenge, especially for small particles. Various alternative methods have been created on account of the drawbacks of classical method. Voigt et al. 18 demonstrated an effective method of

23 membrane filtration to solve the aggregation problem, while the filter membranes must have an appropriate pore size and good property to support repeated vacuuming. A scalable and high throughput method suggested by Morton et al.15 utilized Particle Replication in Non-wetting Templates (PRINT) process21 to fabricate arrays of particles and spray PEs and rinsing solutions on it. Homogeneousness of films is affected during spray as one side of the particle fixing on the high-energy film or excipient layer. The PRINT process and the chemicals used in the vapor-phase crosslinking may limit the types of materials which can be applied for particle.

Microfluidic systems reported by Priest et al.23 and Kantak et al.17 are effective in adsorbing sequential interacting polymers under continuous flow conditions.

Although the size of the droplets can be controlled via precise adjustment of the flow rates of the dispersed and continuous phases to avoid aggregation, the samples for physicochemical characterizations are all at micro scale and lack in vitro or in vivo experiments. Since each layer requires its own microfluidic unit, the enlargement of the overall system size and structure complexity is proportional to the increasing number of layers depositing on particles.

In this thesis, our group aims to design an efficient μ-fluidic system to fabricate nanoscale particles in a fixed-size device with gentle separation steps. Compared to classical film deposition methods, this device is efficient without numerous centrifugations and avoids premature drug release as well as particle degradation.

Moreover, the gentle separation steps are desirable for fabricating fragile cell encapsulations. The silica particles were coated by multilayer films to demonstrate the efficiency and capacity of the device. The LbL drug loaded PLGA nanoparticles were tested with breast cancer cells to illustrate their practicality.

There are various choices to design a device for LbL particle fabrication. Our group designed a μ-fluidic device for the following reasons.

The Reynolds number of μ-fluidic device

Re=ρVD/η

D: diameter of channel is 0.762 mm

V: fluid velocity (V=Q/A) is 3.7 mm/s (0.1 ml/min) or 7.4 mm/s (0.2 ml/min)

ρ: fluid density is about 1000 kg/m3

η: fluid viscosity is about 0.001 Pa·s

Reynolds number is quite small (Re=3—6) in the laminar flow range. For laminar flow, the fluid flows in parallel layers without diffusion between layers, which means its convection is much faster than diffusive mixing. The particles and fluid near the wall never co-mingle with fluid elements in the center of the channel (Figure 2.1)44.

However, if we increase the velocity of flows to approach better mixing, the films will break under strong force and the particles will escape from the shells. The definition of Péclet number (Pe) shows another way to get better mixing in the channel.

Figure 2.1. Schematic of Poiseuille flow radial distributions of velocity. (a) (d): initial bolus of fluid. (b): fluid after elapsed time t in the absence of diffusive mixing. (c): fluid after elapsed time t with convection. (e): fluid after elapsed time t in the absence of convection. (f): fluid after elapsed time t with diffusion and convection.

Péclet number (Pe)

Pe = Uh/D = tD/tC

U is the mean value of velocity in radial direction.

h is the diameter of the channel. D is diffusion coefficient.

tD is the diffusion time. tC is convection time.

If diffusive mixing is faster (tD < tC) than convection time, mixing is rapid for multiple samples.45 Péclet number reduces as the dimension (channel width, h) gets smaller. That is the reason why we must use μ-fluidic channel.44

2.2 Experiment Section

In this section, we introduce the process of fabricating LbL particles by μ-fluidic device and characterization methods of LbL particles.

2.2.1 Material

Poly (sodium 4-styrenesulfonate) (PAH) (Mw 70 kDa, Sigma-Aldrich),

FITC-poly-L-lysine hydrochloride (FITC-PLL) (Mw 20 kDa — 40 kDa, Novel Poly-L-lysine) were used as the polycations; poly (allylamine hydrochloride) (PSS) (Mw 65 kDa,

Sigma-Aldrich) and dextran sulfate sodium salt (DSS, Mw 36 kDa — 50 kDa, MP) were used as polyanions. Hydrochloric acid (1.0 M, VWR) and sodium hydroxide (1.0 M,

VWR) were used for adjusting pH of solutions. Poly (D,L-lactide-co-glycolide) (PLGA,

Mw 24 KDa — 38 KDa, 50:50, sigma-Aldrich), Nile red (TCI), dichloromethane (DCM)

(Sigma-Aldrich), phosphate buffered saline (pH 7.4, Sigma-Aldrich), paclitaxel

(Calbiochem), Dulbecco’s Modified Eagle’s Medium (DMEM) (Sigma-Aldrich), fetal bovine serum (FBS) (Sigma Aldrich), dulbecco’s PBS (Sigma), antibiotic-antimycotic

(Life Technologies), glutamine (Life Technologies). PrestoBlue (Life Technologies), calcein AM (Life Technologies) and EthD-1 (Life Technologies) were used for assessing

26 cell viability. All polyelectrolytes were used as received. All solutions for LbL particle fabrication were filtered with 200 nm filters before use.

2.2.2 Configuration and Working Principle of μ-Fluidic Device

In this study, we utilized silica particles and PLGA particles which both had negatively charged surface. The odd layer samples (n = 1, 3, 5…) refer to positively charged polyelectrolytes (PAH, FITC-PLL) coated particles. The even layer samples (n

= 2, 4, 6…) denote negatively charged polyelectrolytes (PSS, DSS) coated particles.

Figure 2.2. Schematic illustration of μ-fluidic derive.

The μ-fluidic system consists of two filters, micro channels, three four-way valves, four syringes and four syringe pumps. The bare particles or LbL particles can be injected or collected from the middle port. Each side of the device has one valve with three outlets intended for injecting PE (+/-) solutions, injecting rinsing solutions and flowing out waste. Two filters are installed at junctions of the micro channels and the valves to protect particles from flowing out from the waste valves.

The bare particles (silica particles or PLGA particles) were injected from the middle port. For odd layers, PE+ solutions were added and incubated (turn on 1+ and

2-) for a certain time, and rinsed for several double times (turn on 3- and 2+, or 3+ 27 and 2-). For even layers, same steps as odd layers were applied in opposite direction.

PE- solutions were added and incubated (turn on 1- and 2+) for a certain time, and rinsed for even number of times (turn on 3+ and 2-, or 3- and 2+). These steps were repeated for each additional bilayer until forming the desired number of layers.

Eventually, the sample was collected for characterization or further use through the middle port.

2.2.3 Layer-by-layer Assembly on Silica Particle.

The deposition of the first layer of positively charged polyelectrolyte resembles the traditional procedure previously mentioned in introduction. In brief, a certain amount of silica particles (3.67 mg for n = 20) and 3 ml PAH solution (1 mg/mL,

Millipore water) were mixed and magnetically stirred at 1500 rpm for 60 min to build up the first layer. The particles were collected by centrifuging at 4000 rpm for 30 min, injected in to the right portion of the device and rinsed with Millipore water for 4 times. PSS (2mg/mL, Millipore water) or PAH (1 mg/mL, Millipore water) solutions were alternatively injected in to channels, and incubated with particles for 5 min

(except n=2 for 10 min) with four times rinsing steps for each layer until desired layers were achieved. The volume and the rate of injection are summarized in Table

2.2.4 Preparation of Blank PLGA Nanoparticles and Drug-loaded PLGA

Nanoparticles.

30 mg PLGA (and 10 μL Nile red, 5 μg/mL, DMC) or 30mg PLGA, 150 μL paclitaxel (10 mg/mL, DMC) (and 10 μL Nile red, 5 μg/mL, DMC) were co-dissolved in

1 mL of DCM and stirred for 60 min. 200 μL of PBS buffer (pH 7.4) was added into the

DCM solution. The emulsion was magnetically stirred at 1500 rpm for 1 min. Then,

28 the emulsion was added into 4 mL of Millipore water (pH 7.4) dropwise and magnetically stirred overnight at 25 °C to evaporate DCM. The dye-loaded PLGA nanoparticles were purified by centrifuging at 1000 rpm for 5 min to remove oversized particles. The nanoparticles were collected by centrifuging in centrifugal filter units (10000 NMWL) at 4000 rpm for 20 min. The particles were re-dispersed in

Millipore water (pH 7.4). The whole process was performed under aseptic condition.

2.2.5 Layer-by-layer Assembly on Blank and Drug-loaded PLGA Nanoparticles.

A certain amount of blank PLGA particle (272.4 ± 4.9 nm in hydrodynamic size;

-53.0 ± 1.1 mV) and 2 mL PLL (5 mg/mL, Millipore water pH 7.4) were mixed and magnetically stirred at 1000 rpm for 90 min. PLGA/PLL particles were centrifuged and collected at 4000 rpm for 20 min. PLGA/PLL particles were re-dispersed in Millipore water (pH 7.4) and injected into the device which had been sterilized with 70% ethanol. After 4 times rinsing steps, DSS (5 mg/mL, Millipore water pH 7.4) solution was injected into the device and incubated for 5 min. PLL and DSS were alternatively injected into the device with 4 rinsing steps in between until desired layers were achieved.

2.2.6 In Vitro Experimentation.

MDA-MB-231 breast cancer cells were used in our experiments, and the cells grew in Dulbecco’s Modified Eagle’s Medium (DMEM) media supplemented with 10% fetal bovine serum, 1% glutamine and 1% antibiotic. The cells (7500 cells per well) were cultured for at least 24 h in 5% CO2 at 37 ℃ before using. For cytotoxicity test, the cancer cells were treated with a certain amount of blank LbL PLGA particles for

48 h in 96-well plate. The cells were imaged using a fluorescence microscope and a microplate reader at 24 h and 48 h after rinsing with Dulbecco’s PBS. For drug effect

29 of the drug-loaded LbL particles test, the cancer cells were treated with 2 nM paclitaxel-loaded LbL PLGA particle for 48 h in 96-well plate. The cells were observed by the optical and fluorescence microscope and the microplate reader every 24 h for four days. Cells were rinsed with Dulbecco’s PBS before microscopic observation. For free drug test, the cancer cells were treated with 1 nM, 5 nM, 10 nM and 20 nM paclitaxel for 48 h in 96-well plates. The cells were observed by the optical microscope and the microplate reader at 24 h, 48 h, 72 h and 96 h. All cell viability data were obtained using PrestoBlue (excitation / emission: 560 nm / 590 nm) treatment for 15 min and measuring the fluorescent signal with microplate reader.

Calcein AM and EthD-1 were used for Live / Dead two-color fluorescence assay.

2.2.7 Characterization of Particles.

The particles were characterized by DLS and ζ-potential to demonstrate the particles size and the surface charge by Malvern Instruments ZEN 3690. For TEM imaging, the particles or the capsules were dropped on the carbon coated copper grids without any further process and dried overnight. The samples were placed under vacuum for more than 1 h before being tested. For AFM scanning, the samples were dropped in the plasma-cleaned-glass slider and dried overnight. The drug loading of drug-loaded LbL PLGA particles were tested by HPLC (Agilent 1100).

CHAPTER III

RESULT AND DISCUSSION

3.1 The Optimum Experimental Condition of μ-fluidic Device

As an unprecedented device, the primary step is to seek for the optimal parameters for the experiment. The parameters needed to be determined first were the concentration and pH of the polyelectrolyte solutions. PAH and PSS were used as polyelectrolytes to coat silica particles. To test whether PAH/PSS layers could be built up on the silica surface, plasma-cleaned silicon substrate (has silica layer on the substrate) was used as the template to assemble LbL films. The film thicknesses were tested by ellipsometry.

Table 1. The Thicknesses of (PAH/PSS)N films

Layer Thickness(nm) PAH-PSS 800 5 59.56±0.38

600 10 116.90±1.57

15 167.20±1.46 400

20 224.46±0.95 200 Thickness d(nm) Thickness

25 478.76±2.9 0 5 10 15 20 25 30 30 754.64±3.76 Layer n

The reasonable increasing film thicknesses confirm that the LbL films were adsorbed on silica substrate (Table 1). The thickness measured as a function of the number of deposited layers demonstrates that the LbL films grow linearly at a rate of about 11 nm/per layer over the first twenty deposited layers. After that the thickness

31 increased significantly which is approximately 55 nm per deposited layer. The driving force of the deposition process is the alternate overcompensation of the surface charge.46 The film thickness can increase exponentially due to interdiffusion of positively and negatively charged layers above a critical thickness. 46, 47

To explore an optimal experimental condition for the μ-fluidic device, several experimental parameters, such as incubation time, ultrasonic and pH, have been taken into account (Table 2). To be efficient, the minimum incubation time was investigated, and 5 min and 10 min were attempted. The ultrasonic was taken into consideration on different steps, which can enhance the particles distribution in the solutions and also offer extra energy for the reaction between polyelectrolytes. The

LbL particles were analyzed by ζ-potential and TEM.

Table 2. The experimental conditions of LbL silica nanoparticles

Sample 1 inject PE(+/-) Incubation time washing 1 washing 2—washing 4 time(min) 5:00 5:00/10:00 2:30 2:30 ultrasonic Yes/No Yes/No Yes/No No

2 mg/mL aqueous solutions of PAH and PSS were prepared using Millipore water to prepare, which have pH of 11.26 (initial pH) and pH 6.73 (initial pH) respectively; or adjusted the pH of PAH and PSS solutions to pH 9.3 with hydrochloric acid and sodium hydroxide.

Figure 3.1. ζ-potential of LbL silica particles which were fabricated in different experimental conditions

As shown in figure 3.1, 5 min incubation time sample ( ) showed higher

ζ-potential values than that of 10 min`s ( ), which is because the ultrasonic not only prevented particle aggregations as well as accelerated the reaction between

PAH and PSS, but also offered energy to weaken the electrostatic interaction between PAH and PSS. As can be anticipated, prolonged exposure of LbL nanoparticles in ultrasonic irradiation caused deterioration or morphological changes of multilayers, especially for nanoparticles films with PSS. The odd layer samples showed higher ζ-potential values when the ultrasonic was applied during injection, incubation and the first rinsing step ( ) compared to the one without ultrasonic

33 during the whole experiment ( ). On the contrary, the ζ-potential values of even layer samples were lower than that without ultrasonic during the whole experiment

( ), which demonstrates the ultrasonic could strengthen the adsorption of PAH on the particles but weaken that of PSS. Shon et al.48 studied the stability of PAH and PSS films under ultrasonic by monitoring the change in absorbance of films by UV-vis spectrophotometer. Five hours of ultrasonic irradiation of PAH films did not cause any visible change in UV-vis spectra, while, the PSS films showed a clear visible red-shift. This indicated that the morphology of PSS films changed under ultrasonic irradiation. To avoid the morphological changes of PSS films, the ultrasonic treatment should be minimized during depositing PSS layer and 10 min incubation time is unnecessary in this case.

By comparing LbL nanoparticles prepared by polyelectrolyte solutions with initial pH values ( ) (PSS pH 6.7, PAH pH 11.26) to those prepared by polyelectrolyte solutions with pH value of 9.3 ( ), the latter one showed more balanced ζ-potential values between the odd layers and the even layers, i.e. lower values of the odd layers. The possible reason is that the salts from hydrochloric acid and sodium hydroxide balanced the surface charge of particles and reduced Debye length (Figure 3.4) of charged particles, which resulted lower ζ-potential.

Figure 3.2. (a) SiO2/(PAH/PSS)2/PAH particle under 5 min incubation time with ultrasonic, without salt. (b) Bare particle (scale bar: 100 nm)

The surface of 5-layer particles is rougher (Figure 3.2a) than the bare particle’s

(Figure 3.2b), which indicates that the multilayer structure adsorbed on the particle.

This phenomenon coincides with the reversal of ζ-potential values.

(a) (b)

Figure 3.3. (a) 5 min incubation time, with ultrasonic, with salt, 5-layer sample; (b) 5 min incubation time, without ultrasonic, with salt, 5-layer sample

The ultrasonic afforded extra energy to set up thicker films. Interestingly, there exist many stick-like junctions between the particles (Figure 3.3). The hypothesis is that during the first deposition step, the particles are aggregated and coated together.

And then, because of the ultrasonic, the particles tended to separate and the polymer between the particles was stretched to form a stick-like junctions. These

35 sticks did not show in initial pH samples, which are because more salts are induced in the solutions after pH adjustment, which balance the surface charge of bare silica particles. The bare silica particles have negatively charged surface, so they cannot keep close by charges repelling.

Charged surfaces are balanced by counterions in solution (Fig. 3.4)49:

2 2 1/2  (/) iie z r  kT i k-1 is Debye length, ρ is ionic concentration, e is elementary charge, z is charge on the ion, εr and ε are constants, k is Boltzmann’s constant, T is absolute temperature in kelvin.

Figure 3.4. The counterions (ions with opposite charge to the surface co-ions) will accumulate and deplete near to a charged surface. The bottom schematic shows graphically for a 1:1 electrolyte (e.g NaCl, KCl), where ∞ is the electrolyte concentration at x=∞.49

-1 1/2 k = 0.304/([M1:1]) for 1:1 electrolytes, such as HCI and NaOH used to adjust pH 9.3. Hence, as the increasing concentration of salt (ρ), the Debye length (k-1) decreases and the particles are able to keep close to be coated together.

The connections between particles are undesirable for drug delivery system.

The multilayer films can be built up by polyelectrolytes solutions with different pH.

10 min incubation time reduces efficiency of the device and fails to show better

ζ-potential values or more uniform polymeric film deposited on the particles. The ultrasonic can prevent nanoparticles form aggregations and only show positive effect for odd layers. Hence, the optimum experimental conditions for LbL silica particles are shown in Table 3.

Table 3. The optimum experimental conditions

washing 2 inject PE(+/-) Incubation time washing 1 —washing 4 time(min) 5:00 5:00 2:30 2:30 odd layer Yes Yes Yes No ultrasonic Even layer Yes No No No

1 mg/mL of PAH aqueous solution (pH 11.26, the initial pH) and 2 mg/mL of PSS aqueous solutions (pH 6.73, the initial pH) were used as polyelectrolyte solutions.

3.2 LbL Silica Particles

Initial work focused on exploring the optimum experimental conditions. Next, to demonstrate the capacity of the μ-fluidic device, we employed uniformly sized negatively charged silica particles as model nanoparticle core to perform twenty layers of nanoparticles with the optimum experimental conditions. Nanoparticles coated by PAH/PSS layers were visualized by TEM are displayed in Figure 3.5 and

Figure 3.6c. The TEM images revealed the sore-shell structure of LbL silica particles, which confirm that the film was successfully adsorbed on the particles (Figure 3.6a). 37

The collapsed nanocapsules (Figure 3.5b and c) were fabricated by removing the silica particles using hydrogen fluoride to observe the films directly.

Figure 3.5. TEM images of LbL silica capsules/particles are obtained from PAH/PSS-coated 300 nm SiO2 particles. The silica particles were removed from polyelectrolytes films by 10% hydrogen fluoride solution for n=10 (b) and 20 (c) samples. (Scale bar: 100 nm)

Figure 3.6. Physicochemical characterizations of LbL silica nanoparticles: Particle sizes (a), ζ-potential values (b), and TEM images (c) (scale bar: 100 nm). ζ-potential analysis were implemented in Millipore water at 25 ℃ . ζ-potential of bare SiO2 is (-49.87±4.62) mV.

Self-assembling PAH and PSS on the silica particles was corroborated by the steady increasing particle sizes (Figure 3.6a and c) of approximately 3—5 nm each layer and reversal of the surface charge for each additional layer, as demonstrated by

ζ-potential analysis (Figure 3.6b). The film thickness became thinner after the etching

39 process because of the extreme pH of fluoride hydrogen solution and films folding.

The average individual layer thickness is about triple that typically observed (around

1.5 nm) for PAH/PSS nanoparticles assembled by the traditional LbL deposition method,50-52 which is possibly because the different assembly conditions, i.e. concentrations and pH of electrolyte solutions.

3.3 In Vitro Experiments

Nowadays, because many women suffer from the breast cancer, an effective pharmacotherapy is urgently needed, which also has captured the attention of researchers. After demonstrating the feasibility of the μ-fluidic system, our group utilized it to fabricate anticancer drug-loaded nano-capsules, and examined the practicability of drug-loaded LbL nanoparticles using MDA-MB-231 triple-negative cancer cells.

PLGA (core), PLL (PE+) and DSS (PE-) are very common non-cytotoxic polymers used for drug delivery system.33-35 PLGA-based nanoparticles offer a variety of advantages, such as superior biocompatible property, complete degradation, and the possibility to accurately control the drug release rate over prolonged periods.40

Paclitaxel is a medication used to treat many types of cancers, such as lung cancer, head and neck carcinomas, breast cancer, and advanced ovarian carcinoma, which can inhibit cell proliferation by stabilizing the microtubule polymer and provent it from disassembling.53, 54 However, the clinical application of paclitaxel is limited in view of its low therapeutic index and low solubility in water and many other pharmaceutical solvents for subcutaneous administration.55 In our study, we incorporated paclitaxel into LbL PLGA nano-capsules, which has been shown to a promising carrier for anticancer pharmaceuticals.33, 56-58 In this study, we used the

MDA-MB-231 triple-negative cancer cell, which is a popular cell line for researching the effect of chemotherapeutic drugs. 58 53, 59, 60

3.3.1 Fabrication of LbL Blank and LbL Drug-loaded PLGA Particles

First of all, blank LbL PLGA particles and LbL paclitaxel-loaded particles were fabricated by μ-fluidic device and characterized by AFM, ζ-potential analyzer and fluorescent microscope.

Figure 3.7. Schematic illustration of blank LbL PLGA nanoparticles (a) and drug-loaded LbL PLGA nanoparticles (b), AFM images of bare PLGA particles (c), ζ-potential of drug-loaded LbL PLGA nanoparticles (d), and micrograph of PLGA/PLL/DSS/PLL particles (e). (Scale bar of micrograph is 2 μm).

The diameter of the bare blank PLGA particles (Figure 3.7a) shown in AFM images (Figure 3.7c) are about 200 nm and increased to about 400 nm after 41 encapsulation of paclitaxel and Nile red (dye) (Figure 3.7 b). PLGA as the core of drug-loaded LbL nanoparticles (ζ-potential= -55 mV) contains paclitaxel (drug) and

Nile red (dye), and was coated by FITC-PLL and DSS iteratively (Figure 3.7 b).

ζ-potential analysis showed that the surface charges of two neighboring layers are reversed (±40-60 mV in Millipore water under 25 ℃), which demonstrate that the films are layered in stepwise fashion driven by electrostatic interaction (Figure 3.7 d).

The micrographs of PLGA/PLL/DSS/PLL particles (Figure 3.7 e) are corresponding to the ζ-potential analysis where Nile red and FITC signals are sent from one particle and exist in core (red) and shell (green) respectively. Nile red was only used for characterizing the particles under fluorescent microscope. In case of cytotoxicity of

Nile red influenced the results, and the excitation and emission wavelengths of Nile red are close to PrestoBlue`s, the particle did not contain it in vitro experiment.

3.3.2 Potential Cytotoxicity of LbL Blank PLGA Particles

The blank PLGA particles were added to MDA-MD-231 cancer cells in culture to detect potential cytotoxicity of PLGA particles. We assessed the LbL PLGA particles by adding it to MDA-MD-231 cells for 24 h and 48 h. The cells were observed by microscope and the cell viability was tested using microplate reader with PrestoBlue.

PrestoBlue is a fluorescent dye which utilizes the reducing power of living cells to quantitatively indicate cell viability. PrestoBlue is metabolized by living cells to a fluorescent form which can be monitored by the microplate reader.

According to the data of cell viability, the MDA-MB-231 cells retained more than

90% viability when treated with different number of layers of PLGA nanoparticles

(Figure 3.8), which successfully demonstrates the non-cytotoxicity of PLGA particles as well as the observations by Semete et al.61 This result is confirmed by the

42 micrographs (Figure 3.9) where the morphology and population of cells in the untreated group and particle treated groups were consistent. Based on the result, we can conclude that the cytotoxicity of drug-loaded nanoparticles was induced by the paclitaxel released from the nanoparticles.

140 24h 48h 120

100

20 %normalized cell viability 0

n=0 n=1 n=2 n=3 n=4

Figure 3.8. MDA-MB-231 triple negative breast cancer cells were treated with blank PLGA particles for 24 h and 48 h.

According to literature publications, the cellular internalization of particles can be influenced by many factors, such as temperature, incubation time, particle size and concentration, cell lines and cell densities, surface properties of particle (surface hydrophobicity/hydrophilicity, surface charge).62 63-66 In this study, surface charge is considered to be the dominated factor for cellular uptake efficiency. Mailander et al.66 reported that polymeric micro- and nanoparticles whose surfaces were functionalized with positively charged polyelectrolytes could enhance the cellular uptake into HeLa cells and mesenchymal stem cells compared to uncharged particles,

43 which is consistent with our observation. More FITC signals (green) are shown in n=1 and 3 samples, which indicated the positive charged particles (n=1 and 3) are more likely internalized by cells, which is because the negatively charged cell membrane has a tendency to absorb positive charged or neutral nanoparticles.62, 63 Micrographs

(48 h) could manifest that cancer cells internalized more particles as time increasing

(Figure 3.9). The micrographs of bare PLGA particles (n=0) display no FITC signal

(green) which is because the FITC group only exists in the PLL layer.

Figure 3.9. Micrographs of MDA-MB-231 cancer cells co-cultured with LbL blank PLGA particles for 24 h and 48 h. (n is the number of layer, scale bar is 100 μm) The green signal in blue channel is from FITC-PLL layer(s) of particles.

3.3.3 MDA-MB-231 Cells with Free Paclitaxel

A type of cell death pathway is mitotic catastrophe, which can be triggered by

DNA damage, microtubule destabilizing (e.g. Vinca alkaloids) and stabilizing agents

(e.g. taxanes).67 Paclitaxel is a common microtubule stabilizing agent which can stabilize microtubules by inhibiting their disassembly resulting in imperfect cell cycle

45 checkpoints and the development of aneuploid cells.68 Many researchers used paclitaxel to treat MDA-MB-231 Cells. Vuori et al.54 treated MDA-MB-231 cells with

50 nM paclitaxel for 24 h, which induced cell death in 50%. Herbert et al.69 added 2 nM paclitaxel o MDA-MB-231 cells in culture for 5 days, which caused about 60% cell death. Nakshatri et al.60 reported that because of some unknown reasons, only 30% of MDA-MB-231 cells were killed when incubated with 30 nM or higher concentration of paclitaxel for 18 h. From these reports, we can conclude that the effect of free paclitaxel on treatment is highly dependent on culturing time, concentration of drug and maybe manufacturers. To evaluate the cytotoxic activity of free paclitaxel in our study, MDA-MB-231 cells were incubated with various concentrations of free paclitaxel (1, 5, 10 and 20 nM, respectively) for two days, and continuously observed for two days after removing the free paclitaxel.

Figure 3.10. Cell viability of MDA-MB-231 treated with 1 nM, 5 nM, 10 nM and 20 nM of free drug. The free drug was removed at 48 h when renewed the media. 46

According to the results (Figure 3.10), MDA-MB-231 is not sensitive to paclitaxel at tested concentrations. After 2 days treatment with 20 nM paclitaxel, an increase in cell death to 20% was observed. Cells showed increased viability once the free drug was removed (72 h) and decreased again at 96 h. It is possible that part cells of paclitaxel treated groups are altered by paclitaxel and cannot proliferate as much as the cells in control group after 48 h. The longer the cells have been cultured, the bigger the difference appears between treated groups and control group, which leads to lower normalized cell viability in 96 h than 72 h. From the micrographs

(Figure 3.11), the morphology of cancer cells was not altered by paclitaxel, except for

20 nM treatment. Interestingly, 1 nM free drug group showed over 100% cell viability in the first 24 h. Other reports also show similar result when treating cancer cells with small amounts of the drug.70-72 This might because a small amount of drug stimulate the proliferation of cancer cell to fight against the drug.

Figure 3.11. Micrographs of MDA-MB-231 treated with different concentrations` paclitaxel for 24 h, 48 h, 72 h and 96 h.

3.3.4 Effect of Drug-loaded LbL Particles

We next assessed the efficiency of drug-loaded LbL particles (drug loading 2 nM) by adding it to MDA-MB-231 cells in culture for 48 h and refreshing the media without fresh drug or particles, and observing it in next two days. The cell death in the drug-loaded particle experiment was absolutely attributed to paclitaxel since we already confirmed the potential cytotoxicity of blank LbL PLGA particles.

Figure 3.12. MDA-MB-231 triple negative breast cancer cells were treated with LbL drug-loaded PLGA particles for 4 days. (Drug loading: 2 nM, red dash line is 100%).

Cell viability and micrographs indicate that the bare particle showed better drug effect than multilayer particles (Figure 3.12 and 3.13). It might be counterintuitive that the negatively charged particles have difficulty in being internalized by cells because the negatively charged surface is not beneficial for cell uptake. However,

Xing et al.73 and Slowing et al.74 found that the cells can internalize non-functionalized, negatively charged materials through a nonspecific adsorptive endocytosis. Hammond et al. 75 successfully utilized negatively charged LbL particles

(about -40 mV) for codelivering siRNA and a chemotherapy drug into tumor cells.

These results are in agreement with the weak and vague green signals in n=2 and n=4 samples in Figure 3.9. According to reports, uncoated PLGA particles showed 100% drug release, and LbL particles showed 60% release in 48 h.76 Moreover, PLGA particles can easily degrade without the polyelectrolyte films` protection under

37 ℃. Hence, there were more drugs released in 48 h and remained in cells after refreshing the media at 48 h. In vivo experiment, the subcutaneous-administrated particles have to take some time to reach and be internalized by target cells. Bare

PLGA particles decompose quickly in plasma, and the drug will be released into plasma. The free drug and decomposed-particles will be metabolized in one day.76

For cytotoxic chemotherapeutics, it is important to minimize systemic toxicity while the particles are en route to the target location. Hence, multilayer particles work better than bare particles.

The cell viability increased as the increasing number of layers of particles at the first 24 h. There are two major mechanism for drug release from LbL drug-loaded particles. One is the self-diffusion process from the core passing to the shell. More layers coated on the PLGA particles, longer time is needed for drug release. The other is that drug release is caused by core degradation. The polymer chains degrade and particles are eroded in the media or in the cells, and the drug liberates out. The coated films also play an important role in slowing down the degradation of PLGA cores.

The difference of cell viability of positively charged particles and negatively charged particles is shown from 48 h (Figure 3.12). The micrographs show a clear trend that cells preferred internalizing positively charged particles from 24 h observation (Figure 3.13). n=1 and n=3 samples caused more cell death than n=2 and n=4 samples in next three days. The difference of n=1, 3 and n=2, 4 is because that, as previously mentioned, the negatively charged cell membrane has a tendency to absorb positively charged or neutral nanoparticles. If we compare the cell viability at 24 h and 48 h, we can learn the odd-layer samples killed more cells

(n=1 and 3 show 13.3% and 17.3% decrease of cell viability respectively) than even-layer samples (n=2 and 4 display 6.7% increase and 6% decrease of cell viability respectively). That is because cell uptake and drug release from multilayer films take time. 24 h incubation time is not enough to show the advantage of positively charged particles in terms of cell uptake.

paclitaxel 3. Figure

- 13

loaded PLGA isof particles. (nthe number layer, s

Mcorps f MDA of Micrographs .

3 til ngtv bes cne cls co cells cancer breast negative triple 231

cale bar is cale100 bar

- cultured with LbL LbL with cultured

In most cases, the cells showed increased cell viability after removing drug-loaded particles or free drug (72 h) (Figure 3.10). However, for n=0, n=1 and n=3 groups, the cell viability decreased again at 96 h, which were increase for n=2, n=4 and free drug groups. This is because most cancer cells in n=0, n=1 and n=3 groups lost the ability to proliferation. Even removing the particles, the cancer cells still cannot grow normally, which also indicates the positively charged particles have an advantage for drug delivery system.

Drug-loaded particles are much more effective than free drug, since 2 nM free drug showed only 10% cell death at 24 h and continually increased in the next 3 days

(Figure 3.12). The morphology and population of cells demonstrate that 2 nM of free drug has no negative effect on MBA-MB-231 cancer cells (Figure 3.13), and 20 nM of free drug only killed 12% of cells in 24 h (Figure 3.10). Nevertheless, 2 nM of drug-loaded positively charged particles inhibited more than 25% cell growth. The great change of cell morphology in the particle-treated groups demonstrates that the cells were significantly affected by the treatment (Figure 3.13 and Figure 3.14). In

Figure 3.14, the living cells were dyed using Calcein AM, which were highly green fluorescence under fluorescent microscope. Calcein AM helps us to observe the population of living cells and the cell morphology directly. The cells treated with drug-loaded particles showed evident rounded and aggregated morphology as compared with cells in negative control group from the first 24 h observation. The cell densities in drug-loaded particle treated groups were much lower than those of negative and positive (free drug) control groups, where the cells adhered to the bottom of the 96 well plate. The cells morphology became unhealthier as treated time increase, which corroborated the long term drug effect of LbL paclitaxel-loaded

53 particles. These confirmed that 2 nM of paclitaxel-loaded particles worked more effectively compared with 2 nM of free paclitaxel.

signal indicates dead which cells were dyed by EthD paclitaxel 3. Figure

loaded PLGA particles. The green signal denotes live cells which were dyed by Calcein AM. The red The AM. Calcein by dyed were which cells live denotes signal green The particles. PLGA loaded

Mcorps f MDA of Micrographs .

231 triple negative breast cancer cells cells cancer breast negative triple 231

1. 1. (The scale isbare 100

m. n is the number of layer)nnumber isof the m.

cultured with LbL LbL with cultured

CHAPTER IV

CONCLUSION AND FUTURE WORK

The μ-fluidic system fabricates nanoscale particles in a fixed-size device with gentle separation steps. Compared to the classical film deposition methods, this device is efficient to finish one layer in 20 min, which took two hours before, and easily to finish 20 layers in one day. Short fabrication time avoids premature drug release and particle degradation before treating cancer cells. Moreover, the gentle separation steps are desirable for fabricating breakable encapsulations. It applies to most mechanisms of LbL, like hydrogen-bonded, covalent bonding and bio-specific interactions. Hence, the device has a broad scope in LbL technique. All of the components can be easily fabricated in machine shop. The common parts and straightforward work principal of the μ-fluidic device we used makes it easy to set up and operate comparing to the complicated standard photolithography. The filters and length of the channel can be changed according to different requirements and purposes, and the size and the complexity of device is independent of the number of depositing layers.

In the in vitro tests, we found that a small amount of the drug is a positive stimulus for cancer cell proliferation to fight the drug. Positively charged particles have an advantage on cell uptake and drug delivery for MDA-MB-231 cancer cells.

There is no doubt that the particles, no matter if positively charged or negatively charged, worked much more effectively than the free drug.

56 Our next goal is to automatize and computerize the device. Now, switch ports and valves are manual operations. Although 20 min per layer is efficient, our group will be able to modify the μ-fluidic device more conveniently and smartly. Present automation techniques are fully capable to achieve the task we want.

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